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Archive for the ‘MicroEngineering Cell-Tissue & Systems’ Category


3-D Printed Liver

Curator: Larry H. Bernstein, MD, FCAP

 

 

3D-printing a new lifelike liver tissue for drug screening

Could let pharmaceutical companies quickly do pilot studies on new drugs
February 15, 2016    http://www.kurzweilai.net/3d-printing-a-new-lifelike-liver-tissue-for-drug-screening

Images of the 3D-printed parts of the biomimetic liver tissue: liver cells derived from human induced pluripotent stem cells (left), endothelial and mesenchymal supporing cells (center), and the resulting organized combination of multiple cell types (right). (credit: Chen Laboratory, UC San Diego)

 

University of California, San Diego researchers have 3D-printed a tissue that closely mimics the human liver’s sophisticated structure and function. The new model could be used for patient-specific drug screening and disease modeling and could help pharmaceutical companies save time and money when developing new drugs, according to the researchers.

The liver plays a critical role in how the body metabolizes drugs and produces key proteins, so liver models are increasingly being developed in the lab as platforms for drug screening. However, so far, the models lack both the complex micro-architecture and diverse cell makeup of a real liver. For example, the liver receives a dual blood supply with different pressures and chemical constituents.

So the team employed a novel bioprinting technology that can rapidly produce complex 3D microstructures that mimic the sophisticated features found in biological tissues.

The liver tissue was printed in two steps.

  • The team printed a honeycomb pattern of 900-micrometer-sized hexagons, each containing liver cells derived from human induced pluripotent stem cells. An advantage of human induced pluripotent stem cells is that they are patient-specific, which makes them ideal materials for building patient-specific drug screening platforms. And since these cells are derived from a patient’s own skin cells, researchers don’t need to extract any cells from the liver to build liver tissue.
  • Then, endothelial and mesenchymal supporting cells were printed in the spaces between the stem-cell-containing hexagons.

The entire structure — a 3 × 3 millimeter square, 200 micrometers thick — takes just seconds to print. The researchers say this is a vast improvement over other methods to print liver models, which typically take hours. Their printed model was able to maintain essential functions over a longer time period than other liver models. It also expressed a relatively higher level of a key enzyme that’s considered to be involved in metabolizing many of the drugs administered to patients.

“It typically takes about 12 years and $1.8 billion to produce one FDA-approved drug,” said Shaochen Chen, NanoEngineering professor at the UC San Diego Jacobs School of Engineering. “That’s because over 90 percent of drugs don’t pass animal tests or human clinical trials. We’ve made a tool that pharmaceutical companies could use to do pilot studies on their new drugs, and they won’t have to wait until animal or human trials to test a drug’s safety and efficacy on patients. This would let them focus on the most promising drug candidates earlier on in the process.”

The work was published the week of Feb. 8 in the online early edition of Proceedings of the National Academy of Sciences.


Abstract of Deterministically patterned biomimetic human iPSC-derived hepatic model via rapid 3D bioprinting

The functional maturation and preservation of hepatic cells derived from human induced pluripotent stem cells (hiPSCs) are essential to personalized in vitro drug screening and disease study. Major liver functions are tightly linked to the 3D assembly of hepatocytes, with the supporting cell types from both endodermal and mesodermal origins in a hexagonal lobule unit. Although there are many reports on functional 2D cell differentiation, few studies have demonstrated the in vitro maturation of hiPSC-derived hepatic progenitor cells (hiPSC-HPCs) in a 3D environment that depicts the physiologically relevant cell combination and microarchitecture. The application of rapid, digital 3D bioprinting to tissue engineering has allowed 3D patterning of multiple cell types in a predefined biomimetic manner. Here we present a 3D hydrogel-based triculture model that embeds hiPSC-HPCs with human umbilical vein endothelial cells and adipose-derived stem cells in a microscale hexagonal architecture. In comparison with 2D monolayer culture and a 3D HPC-only model, our 3D triculture model shows both phenotypic and functional enhancements in the hiPSC-HPCs over weeks of in vitro culture. Specifically, we find improved morphological organization, higher liver-specific gene expression levels, increased metabolic product secretion, and enhanced cytochrome P450 induction. The application of bioprinting technology in tissue engineering enables the development of a 3D biomimetic liver model that recapitulates the native liver module architecture and could be used for various applications such as early drug screening and disease modeling.

Fernando

I wonder how equivalent are these hepatic cells derived from human induced pluripotent stem cells (hiPSCs) compared with the real hepatic cell populations.
All cells in our organism share the same DNA info, but every tissue is special for what genes are expressed and also because of the specific localization in our body (which would mean different surrounding environment for each tissue). I am not sure about how much of a step forward this is. Induced hepatic cells are known, but this 3-D print does not have liver shape or the different cell sub-types you would find in the liver.

I agree with your observation that having the same DNA information doesn’t account for variability of cell function within an organ. The regulation of expression is in RNA translation, and that is subject to regulatory factors related to noncoding RNAs and to structural factors in protein folding. The result is that chronic diseases that are affected by the synthetic capabilities of the liver are still problematic – toxicology, diabetes, and the inflammatory response, and amino acid metabolism as well. Nevertheless, this is a very significant step for the testing of pharmaceuticals. When we look at the double circulation of the liver, hypoxia is less of an issue than for heart or skeletal muscle, or mesothelial tissues. I call your attention to the outstanding work by Nathan O. Kaplan on the transhydrogenases, and his stipulation that there are significant differences between organs that are anabolic and those that are catabolic in TPNH/DPNH, that has been ignored for over 40 years. Nothing is quite as simple as we would like.

Fernando commented on 3-D printed liver

3-D printed liver Larry H. Bernstein, MD, FCAP, Curator LPBI 3D-printing a new lifelike liver tissue for drug …

I wonder how equivalent are these hepatic cells derived from human induced pluripotent stem cells (hiPSCs) compared with the real hepatic cell populations.
All cells in our organism share the same DNA info, but every tissue is special for what genes are expressed and also because of the specific localization in our body (which would mean different surrounding environment for each tissue). I am not sure about how much of a step forward this is. Induced hepatic cells are known, but this 3-D print does not have liver shape or the different cell sub-types you would find in the liver.

 

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Lifelong Contraceptive Device for Men: Mechanical Switch to Control Fertility on Wish

Reporter and Curator: Dr. Sudipta Saha, Ph.D.

There aren’t many options for long-term birth control for men. The most common kinds of male contraception include

  • condoms,
  • withdrawal / pulling out,
  • outercourse, and
  • vasectomy.

But, other than vasectomy none of the processes are fully secured, comfortable and user friendly. Another solution may be

  • RISUG (Reversible Inhibition of Sperm Under Guidance, or Vasalgel)

which is said to last for ten years and no birth control pill for men is available till date.

VIEW VIDEO

http://www.mdtmag.com/blog/2016/01/implanted-sperm-switch-turns-mens-fertility-and?et_cid=5050638&et_rid=461755519&type=cta

Recently a German inventor, Clemens Bimek, developed a novel, reversible, hormone free, uncomplicated and lifelong contraceptive device for controlling male fertility. His invention is named as Bimek SLV, which is basically a valve that stops the flow of sperm through the vas deferens with the literal flip of a mechanical switch inside the scortum, rendering its user temporarily sterile. Toggled through the skin of the scrotum, the device stays closed for three months to prevent accidental switching. Moreover, the switch can’t open on its own. The tiny valves are less than an inch long and weigh is less than a tenth of an ounce. They are surgically implanted on the vas deferens, the ducts which carry sperm from the testicles, through a simple half-hour operation.

The valves are made of PEEK OPTIMA, a medical-grade polymer that has long been employed as a material for implants. The device is patented back in 2000 and is scheduled to undergo clinical trials at the beginning of this year. The inventor claims that Bimek SLV’s efficacy is similar to that of vasectomy, it does not impact the ability to gain and maintain an erection and ejaculation will be normal devoid of the sperm cells. The valve’s design enables sperm to exit the side of the vas deferens when it’s closed without any semen blockage. Leaked sperm cells will be broken down by the immune system. The switch to stop sperm flow can be kept working for three months or 30 ejaculations. After switching on the sperm flow the inventor suggested consulting urologist to ensure that all the blocked sperms are cleared off the device. The recovery time after switching on the sperm flow is only one day, according to Bimek SLV. However, men are encouraged to wait one week before resuming sexual activities.

Before the patented technology can be brought to market, it must undergo a rigorous series of clinical trials. Bimek and his business partners are currently looking for men interested in testing the device. If the clinical trials are successful then this will be the first invention of its kind that gives men the ability to control their fertility and obviously this method will be preferred over vasectomy.

 

References:

 

https://www.bimek.com/this-is-how-the-bimek-slv-works/

 

http://www.mdtmag.com/blog/2016/01/implanted-sperm-switch-turns-mens-fertility-and?et_cid=5050638&et_rid=461755519&type=cta

 

http://www.telegraph.co.uk/news/worldnews/europe/germany/12083673/German-carpenter-invents-on-off-contraception-switch-for-sperm.html

 

http://www.discovery.com/dscovrd/tech/you-can-now-turn-off-your-sperm-flow-with-the-flip-of-a-switch/

 

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Contribution to Inflammatory Bowel Disease (IBD) of bacterial overgrowth in gut on a chip

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

Contributions of microbiome and mechanical deformation to intestinal bacterial overgrowth and inflammation in a 

human gut-on-a-chip 
Gut-On-a-Chip Holds Clues for Treating Inflammatory Bowel Diseases
Greg Watry
Human intestinal epithelial cells cultured in the Wyss Institute's human gut-on-a-chip form differentiated intestinal villi when cultured in the presence of lifelike fluid flow and rhythmic, peristalsis-like motions. Here the villi are visible using a traditional microscope (left) or a confocal microscope (right); when the same villi are stained with fluorescent antibodies, it clearly reveals the nuclei in the intestinal cells (blue) and their specialized apical membranes when they contact the intestinal lumen (green). Credit: Wyss Institute at Harvard University
Human intestinal epithelial cells cultured in the Wyss Institute’s human gut-on-a-chip form differentiated intestinal villi when cultured in the presence of lifelike fluid flow and rhythmic, peristalsis-like motions. Here the villi are visible using a traditional microscope (left) or a confocal microscope (right); when the same villi are stained with fluorescent antibodies, it clearly reveals the nuclei in the intestinal cells (blue) and their specialized apical membranes when they contact the intestinal lumen (green). Credit: Wyss Institute at Harvard University

Roughly the size of a computer memory stick and made of clear flexible polymer, the human gut-on-a-chip was created by Harvard Univ.’s Wyss Institute in 2012. Three years later, researchers are utilizing the technology in hopes of creating new therapies for inflammatory bowel diseases (IBD).

The Centers for Disease Control and Prevention estimates that between 1 and 1.3 million people suffer from IBD, including such diseases as ulcerative colitis and Crohn’s disease. With origins still mysterious, IBD is currently incurable.

“It has not been possible to study…human intestinal inflammatory diseases, because it is not possible to independently control these parameters in animal studies or in vitro models,” wrote the researchers in Proceedings of the National Academy of the Sciences. “In particular, given the recent recognition of the central role of the intestinal microbiome in human health and disease, including intestinal disorders, it is critical to incorporate commensal microbes into experimental models, however, this has not been possible using conventional culture systems.”

Additionally, static in vitro methods fail to replicate the pathophysiology of human IBD.

But the hollow-channeled microfluidic gut-on-a-chip successfully simulates the human intestine’s physical structure, microenvironment, peristalsis-like motion, and fluid flow.

“With our human gut-on-a-chip, we can not only culture the normal gut microbiome for extended times, but we can also analyze contributions of pathogens, immune cells, and vascular and lymphatic endothelium, as well as model specific diseases to understand the complex pathophysiological responses of the intestinal tract,” said Donald Ingber, founding director of the Wyss Institute.

The device was “used to co-culture multiple commensal microbes in contact with living human intestinal epithelial cells for more than a week in vitro and to analyze how gut microbiome, inflammatory cells, and peristalsis-associated mechanical deformations independently contribute to intestinal bacterial overgrowth and inflammation,” the researchers wrote.

Thus far, use of the device has yielded two interesting observations.

Four proteins—called cytokines—work together to trigger an inflammatory responses that exacerbate the bowel, the researchers found. Potentially, this new discovery could lead to the development of treatments that block the cytokine interaction.

Another observation, the researchers noted, is that “by ceasing peristalsis-like motions while maintaining luminal flow, lack of epithelial deformation was shown to trigger bacterial overgrowth similar to that observed in patients with ileus and inflammatory bowel disease,” according to the researchers.

The researchers believe the micro-device may one day be applicable to precision medicine. Eventually, a custom treatment may arise from scientists using a patient’s gut microbiota and cells on a human gut-on-a-chip.

 

 

Contributions of microbiome and mechanical deformation to intestinal bacterial overgrowth and inflammation in a human gut-on-a-chip
Hyun Jung Kima,1, Hu Lia,2, James J. Collinsa,b,c,d,e,f,3, and Donald E. Ingbera,g,h,
http://www.pnas.org/content/early/2015/12/09/1522193112.full.pdf

A human gut-on-a-chip microdevice was used to coculture multiple commensal microbes in contact with living human intestinal epithelial cells for more than a week in vitro and to analyze how gut microbiome, inflammatory cells, and peristalsis-associated mechanical deformations independently contribute to intestinal bacterial overgrowth and inflammation. This in vitro model replicated results from past animal and human studies, including demonstration that probiotic and antibiotic therapies can suppress villus injury induced by pathogenic bacteria. By ceasing peristalsis-like motions while maintaining luminal flow, lack of epithelial deformation was shown to trigger bacterial overgrowth similar to that observed in patients with ileus and inflammatory bowel disease. Analysis of intestinal inflammation on-chip revealed that immune cells and lipopolysaccharide endotoxin together stimulate epithelial cells to produce four proinflammatory cytokines (IL-8, IL-6, IL-1β, and TNF-α) that are necessary and sufficient to induce villus injury and compromise intestinal barrier function. Thus, this human gut-on-a-chip can be used to analyze contributions of microbiome to intestinal pathophysiology and dissect disease mechanisms in a controlled manner that is not possible using existing in vitro systems or animal models.

 

Significance The main advance of this study is the development of a microengineered model of human intestinal inflammation and bacterial overgrowth that permits analysis of individual contributors to the pathophysiology of intestinal diseases, such as ileus and inflammatory bowel disease, over a period of weeks in vitro. By studying living human intestinal epithelium, with or without vascular and lymphatic endothelium, immune cells, and mechanical deformation, as well as living microbiome and pathogenic microbes, we identified previously unknown contributions of specific cytokines, mechanical motions, and microbiome to intestinal inflammation, bacterial overgrowth, and control of barrier function. We provide proof-of-principle to show that the microfluidic gut-on-a-chip device can be used to create human intestinal disease models and gain new insights into gut pathophysiology.

 

Various types of inflammatory bowel disease (IBD), such as Crohn’s disease and ulcerative colitis, involve chronic inflammation of human intestine with mucosal injury and villus destruction (1), which is believed to be caused by complex interactions between gut microbiome (including commensal and pathogenic microbes) (2), intestinal mucosa, and immune components (3). Suppression of peristalsis also has been strongly associated with intestinal pathology, inflammation (4, 5), and small intestinal bacterial overgrowth (5, 6) in patients with Crohn’s disease (7) and ileus (8). However, it has not been possible to study the relative contributions of these different potential contributing factors to human intestinal inflammatory diseases, because it is not possible to independently control these parameters in animal studies or in vitro models. In particular, given the recent recognition of the central role of the intestinal microbiome in human health and disease, including intestinal disorders (2), it is critical to incorporate commensal microbes into experimental models; however, this has not been possible using conventional culture systems. Most models of human intestinal inflammatory diseases rely either on culturing an intestinal epithelial cell monolayer in static Transwell culture (9) or maintaining intact explanted human intestinal mucosa ex vivo (10) and then adding live microbes and immune cells to the apical (luminal) or basolateral (mucosal) sides of the cultures, respectively. These static in vitro methods, however, do not effectively recapitulate the pathophysiology of human IBD. For example, intestinal epithelial cells cultured in Transwell plates completely fail to undergo villus differentiation, produce mucus, or form the various specialized cell types of normal intestine. Although higher levels of intestinal differentiation can be obtained using recently developed 3D organoid cultures (11), it is not possible to expose these cells to physiological peristalsis-like motions or living microbiome in long-term culture, because bacterial overgrowth occurs rapidly (within ∼1 d) compromising the epithelium (12). This is a major limitation because establishment of stable symbiosis between the epithelium and resident gut microbiome as observed in the normal intestine is crucial for studying inflammatory disease initiation and progression (13), and rhythmical mechanical deformations driven by peristalsis are required to both maintain normal epithelial differentiation (14) and restrain microbial overgrowth in the intestine in vivo (15).

Thus, we set out to develop an experimental model that would overcome these limitations. To do this, we adapted a recently described human gut-on-a-chip microfluidic device that enables human intestinal epithelial cells (Caco-2) to be cultured in the presence of physiologically relevant luminal flow and peristalsislike mechanical deformations, which promotes formation of intestinal villi lined by all four epithelial cell lineages of the small intestine (absorptive, goblet, enteroendocrine, and Paneth) (12, 16). These villi also have enhanced barrier function, drug-metabolizing cytochrome P450 activity, and apical mucus secretion compared with the same cells grown in conventional Transwell cultures, which made it possible to coculture a probiotic gut microbe (Lactobacillus rhamnosus GG) in direct contact with the intestinal epithelium for more than 2 wk (12), in contrast to static Transwell cultures (17) or organoid cultures (11) that lose viability within hours under similar conditions. In the present study, we leveraged this human gut-on-a-chip to develop a disease model of small intestinal bacterial overgrowth (SIBO) and inflammation. We analyzed how probiotic and pathogenic bacteria, lipopolysaccharide (LPS), immune cells, inflammatory cytokines, vascular endothelial cells and mechanical forces contribute individually, and in combination, to intestinal inflammation, villus injury, and compromise of epithelial barrier function. We also explored whether we could replicate the protective effects of clinical probiotic and antibiotic therapies on-chip to demonstrate its potential use as an in vitro tool for drug development, as well as for dissecting fundamental disease mechanisms.

 

Fig. 1. The human gut-on-a-chip microfluidic device and changes in phenotype resulting from different culture conditions on-chip, as measured using genome-wide gene profiling. (A) A photograph of the device. Blue and red dyes fill the upper and lower microchannels, respectively. (B) A schematic of a 3D cross-section of the device showing how repeated suction to side channels (gray arrows) exerts peristalsis-like cyclic mechanical strain and fluid flow (white arrows) generates a shear stress in the perpendicular direction. (C) A DIC micrograph showing intestinal basal crypt (red arrow) and villi (white arrow) formed by human Caco-2 intestinal epithelial cells grown for ∼100 h in the gut-on-achip under medium flow (30 μL/h) and cyclic mechanical stretching (10%, 0.15 Hz). (Scale bar, 50 μm.) (D) A confocal immunofluorescence image showing a horizontal cross-section of intestinal villi similar to those shown in Fig. 1C, stained for F-actin (green) that labels the apical brush border of these polarized intestinal epithelial cells (nuclei in blue). (Scale bar, 50 μm.) (E) Hierarchical clustering analysis of genome-wide transcriptome profiles (Top) of Caco-2 cells cultured in the static Transwell, the gut-on-a-chip (with fluid flow at 30 μL/h and mechanical deformations at 10%, 0.15 Hz) (Gut Chip), or the mechanically active gut-on-a-chip cocultured with the VSL#3 formulation containing eight probiotic gut microbes (Gut Chip + VSL#3) for 72 h compared with normal human small intestinal tissues (Duodenum, Jejunum, and Ileum; microarray data from the published GEO database). The dendrogram was generated based on the averages calculated across all replicates, and all branches in the cluster have the approximately unbiased (AU) P value equal to 100. The y axis next to the dendrogram represents the metric for Euclidean distance between samples. Corresponding pseudocolored GEDI maps analyzing profiles of 650 metagenes between samples described above (Bottom).

 

Fig. 2. Reconstitution of pathological intestinal injury induced by interplay between nonpathogenic or pathogenic enteroinvasive E. coli bacteria or LPS endotoxin with immune cells. (A) DIC images showing that the normal villus morphology of the intestinal epithelium cultured on-chip (Control) is lost within 24 h after EIEC (serotype O124:NM) are added to the apical channel of the chip (+EIEC; red arrows indicate bacterial colonies). (B) Effects of GFP-EC, LPS (15 μg/mL), EIEC, or no addition (Control) on intestinal barrier function (Left). Right shows the TEER profiles in the presence of human PBMCs (+PBMC). GFP-EC, LPS, and EIEC were added to the apical channel (intestinal lumen) at 4, 12, and 35 h, respectively, and PBMCs were subsequently introduced through the lower capillary channel at 44 h after the onset of experiment (0 h) (n = 4). (C) Morphological analysis of intestinal villus damage in response to addition of GFP-EC, LPS, and EIEC in the absence (−PBMC) or the presence of immune components (+PBMC). Schematics (experimental setup), phase contrast images (horizontal view, taken at 57 h after onset), and fluorescence confocal micrographs (vertical cross-sectional views at 83 h after onset) were sequentially displayed. F-actin and nuclei were coded with magenta and blue, respectively. (D) Quantification of intestinal injury evaluated by measuring changes in lesion area (Top; n = 30) and the height of the villi (Bottom; n = 50) in the absence (white) or the presence (gray) of PBMCs. Intestinal villi were grown in the gut-on-a-chip under trickling flow (30 μL/h) with cyclic deformations (10%, 0.15 Hz) during the preculture period for ∼100 h before stimulation (0 h, onset). Asterisks indicate statistical significance compared with the control at the same time point (*P < 0.001, **P < 0.05). (Scale bars, 50 μm.)

 

Recapitulating Organ-Level Intestinal Inflammatory Responses. During inflammation in the intestine, pathophysiological recruitment of circulating immune cells is regulated via activation of the underlying vascular endothelium. To analyze this organ-level inflammatory response in our in vitro model, a monolayer of human microvascular endothelial cells (Fig. 3 C and D and Fig. S6 A and C) or lymphatic endothelial cells (Fig. S6 B and C) was cultured on the opposite (abluminal) side of the porous ECM-coated membrane in the lower microchannel of the device to effectively create a vascular channel (Fig. 3C). To induce intestinal inflammatory responses, LPS (Fig. 3 C and D) or TNF-α (Fig. S6) was flowed through the upper epithelial channel for 24 h, and then PBMCs were added to the vascular channel for 1 h without flow (Fig. 3 C and D). Treatment with both LPS (or TNF-α) and PBMCs resulted in the activation of intercellular adhesion molecule-1 (ICAM-1) expression on the surface of the endothelium (Fig. 3 C and D, Left, and Fig. S6) and a significant increase (P < 0.001) in the number of PBMCs that adhered to the surface of the capillary endothelium compared with controls (Fig. 3D). These results are consistent with our qPCR results, which also showed up-regulation of genes involved in immune cell trafficking (Fig. S5). Neither addition of LPS nor PBMCs alone was sufficient to induce ICAM-1 expression in these cells (Fig. 3D), which parallels the effects of LPS and PBMCs on epithelial production of inflammatory cytokines (Fig. 3A) as well as on villus injury (Fig. 2 B and D).

Evaluating Antiinflammatory Probiotic and Antibiotic Therapeutics On-Chip. To investigate how the gut microbiome modulates these inflammatory reactions, we cocultured the human intestinal villi with the eight strains of probiotic bacteria in the VSL#3 formulation that significantly enhanced intestinal differentiation (Fig. 1E and Fig. S1B). To mimic the in vivo situation, we colonized our microengineered gut on a chip with the commensal microbes (VSL#3) first and then subsequently added immune cells (PBMCs), pathogenic bacteria (EIEC), or both in combination. The VSL#3 microbial cells inoculated into the germ-free lumen of the epithelial channel primarily grew as discrete microcolonies in the spaces between adjacent villi (Fig. 4A and Movie S3) for more than a week in culture (Fig. S7A), and no planktonic growth was detected. These microbes did not overgrow like the EIEC (Fig. 2A and Movie S2), although occasional microcolonies also appeared at different spatial locations in association with the tips of the villi (Fig. S7 B and C). The presence of these living components of the normal gut microbiome significantly enhanced (P < 0.001) intestinal barrier function, producing more than a 50% increase in TEER relative to control cultures (Fig. 4B) without altering villus morphology (Fig. 4C). This result is consistent with clinical studies suggesting that probiotics, including VSL#3, can significantly enhance intestinal barrier function in vivo (18).

To mimic the effects of antibiotic therapies that are sometimes used clinically in patients with intestinal inflammatory disease (29), we identified a dose and combination of antibiotics (100 units per mL penicillin and 100 μg/mL streptomycin) that produced effective killing of both EIEC and VSL#3 microbes in liquid cultures (Fig. S9) and then injected this drug mixture into the epithelial channel of guton-a-chip devices infected with EIEC. When we added PBMCs to these devices 1 h later, intestinal barrier function (Fig. 4B) and villus morphology (Fig. 4C) were largely protected from injury, and there was a significant reduction in lesion area (Fig. 4D). Thus, the gut-on-a-chip was able to mimic suppression of injury responses previously observed clinically using other antibiotics that produce similar bactericidal effects.

Analyzing Mechanical Contributions to Bacterial Overgrowth. Finally, we used the gut-on-a-chip to analyze whether physical changes in peristalsis or villus motility contribute to intestinal pathologies, such as the small intestinal bacterial overgrowth (SIBO) (5, 6) observed in patients with ileus (8) and IBD (7). When the GFPEC bacteria were cultured on the villus epithelium under normal flow (30 μL/h), but in the absence of the physiological cyclic mechanical deformations, the number of colonized bacteria was significantly higher (P < 0.001) compared with gut chips that experienced mechanical deformations (Fig. 5A). Bacterial cell densities more than doubled within 21 h when cultured under conditions without cyclic stretching compared with gut chips that experienced physiological peristalsis-like mechanical motions, even though luminal flow was maintained constant (Fig. 5B). Thus, cessation of epithelial distortion appears to be sufficient to trigger bacterial overgrowth, and motility-induced luminal fluid flow is not the causative factor as assumed previously (7).

 

Discussion One of the critical prerequisites for mimicking the living human intestine in vitro is to establish a stable ecosystem containing physiologically differentiated intestinal epithelium, gut bacteria, and immune cells that can be cultured for many days to weeks. Here we leveraged a mechanically active gut-on-a-chip microfluidic device to develop an in vitro model of human intestinal inflammation that permits stable long-term coculture of commensal microbes of the gut microbiome with intestinal epithelial cells. The synthetic model of the human living intestine we built recapitulated the minimal set of structures and functions necessary to mimic key features of human intestinal pathophysiology during chronic inflammation and bacterial overgrowth including epithelial and vascular inflammatory processes and destruction of intestinal villi.

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Surgical Separation of Conjoined Twins been Computer-Aided with CT and 3D BioPrinting

Reporter: Aviva Lev-Ari, PhD, RN

 

From: “PR Newswire for Journalists” <push_services@prnewswire.com>

Sent: Wednesday, December 02, 2015 4:04 PM

To: info@newmedinc.com

Subject: CT and 3-D Printing Aid Surgical Separation of Conjoined Twins

 

CT and 3-D Printing Aid Surgical Separation of Conjoined Twins

CHICAGO, Dec. 2, 2015 /PRNewswire-USNewswire/ — A combination of detailed CT imaging and 3-D printing technology has been used for the first time in the surgical planning for separation of conjoined twins, according to a study presented today at the annual meeting of the Radiological Society of North America (RSNA).

Conjoined twins, or twins whose bodies are connected, account for approximately one of every 200,000 live births. Survival rates are low and separating them through surgery is extremely difficult because they often share organs and blood vessels.

Specialists at Texas Children’s Hospital in Houston brought a new approach to these challenges when they set out to surgically separate Knatalye Hope and Adeline Faith Mata, conjoined twins from Lubbock, Texas. Knatalye and Adeline were born on April 11, 2014, connected from the chest all the way down to the pelvis.

“This case was unique in the extent of fusion,” said the study’s lead author, Rajesh Krishnamurthy, M.D., chief of radiology research and cardiac imaging at Texas Children’s Hospital. “It was one of the most complex separations ever for conjoined twins.”

To prepare for the separation surgery, Dr. Krishnamurthy and colleagues performed volumetric CT imaging with a 320-detector scanner, administering intravenous contrast separately to both twins to enhance views of vital structures and help plan how to separate them to ensure survival of both children. They used a technique known as target mode prospective EKG gating to freeze the motion of the hearts on the images and get a more detailed view of the cardiovascular anatomy, while keeping the radiation exposure low.

“The CT scans showed that the babies’ hearts were in the same cavity but were not fused,” Dr. Krishnamurthy said. “Also, we detected a plane of separation of the liver that the surgeons would be able to use.”

The team translated the CT imaging results into a color-coded physical 3-D model with skeletal structures and supports made in hard plastic resin, and organs built from a rubber-like material. The livers were printed as separate pieces of the transparent resin, with major blood vessels depicted in white for better visibility. The models were designed so that they could be assembled together or separated during the surgical planning process. The surgical team used the models during the exhaustive preparation process leading up to the surgery.

On February 17, a little more than 10 months after they were born, the Mata twins underwent surgical separation by a team of more than 26 clinicians, including 12 surgeons, six anesthesiologists and eight surgical nurses. The official separation took place approximately 18 hours into the 26-hour surgery.

The 3-D models proved to be an excellent source of information, as there were no major discrepancies between the models and the twins’ actual anatomy.

“The surgeons found the landmarks for the liver, hearts and pelvic organs just as we had described,” Dr. Krishnamurthy said. “The concordance was almost perfect.”

Dr. Krishnamurthy expects the combination of volumetric CT, 3-D modeling, and 3-D printing to become a standard part of preparation for surgical separation of conjoined twins, although barriers remain to its adoption.

“The 3-D printing technology has advanced quite a bit, and the costs are declining. What’s limiting it is a lack of reimbursement for these services,” he said. “The procedure is not currently recognized by insurance companies, so right now hospitals are supporting the costs.”

Besides assisting clinicians prepare for surgery, the 3-D model also served another important function: helping the twins’ parents, Elysse and John Eric Mata, understand the process.

“When I showed the mother the model and explained the procedure, she held my hand and thanked me,” Dr. Krishnamurthy recalled. “They said, ‘For the first time, we understand what is going to happen with our babies.'”

Knatalye Hope returned home in May 2015 and her sister Adeline Faith came home a month later. They are both doing well and have a Facebook page, “Helping Faith & Hope Mata,” with updates on their progress.

Co-authors on the study are Nicholas Dodd, B.S., Darrell Cass, M.D., Amrita Murali and Jayanthi Parthasarathy, B.D.S., M.S., Ph.D.

Note: Copies of RSNA 2015 news releases and electronic images will be available online at RSNA.org/press15 beginning Monday, Nov. 30.

RSNA is an association of more than 54,000 radiologists, radiation oncologists, medical physicists and related scientists, promoting excellence in patient care and health care delivery through education, research and technologic innovation. The Society is based in Oak Brook, Ill. (RSNA.org)

For patient-friendly information on CT, visit RadiologyInfo.org.

SOURCE Radiological Society of North America (RSNA)

Radiological Society of North America (RSNA)

CONTACT: RSNA Newsroom, 1-312-791-6610; Before 11/28/15 or after 12/3/15: RSNA Media Relations, 1-630-590-7762; Linda Brooks, 1-630-590-7738, lbrooks@rsna.org; Maureen Morley, 1-630-590-7754, mmorley@rsna.org

Web Site: http://www.rsna.org

SOURCE

From: “Dr. Katie Katie Siafaca” <info@newmedinc.com>

Reply-To: “Dr. Katie Katie Siafaca” <info@newmedinc.com>

Date: Thursday, December 3, 2015 at 2:00 PM

To: Aviva Lev-Ari <AvivaLev-Ari@alum.berkeley.edu>

Subject: fw: CT and 3-D Printing Aid Surgical Separation of Conjoined Twins

 

 

 

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FDA Guidance On Source Animal, Product, Preclinical and Clinical Issues Concerning the Use of Xenotranspantation Products in Humans – Implications for 3D BioPrinting of Regenerative Tissue

Reporter: Stephen J. Williams, Ph.D.

 

The FDA has submitted Final Guidance on use xeno-transplanted animal tissue, products, and cells into human and their use in medical procedures. Although the draft guidance was to expand on previous guidelines to prevent the introduction, transmission, and spread of communicable diseases, this updated draft may have implications for use of such tissue in the emerging medical 3D printing field.

This document is to provide guidance on the production, testing and evaluation of products intended for use in xenotransplantation. The guidance includes scientific questions that should be addressed by sponsors during protocol development and during the preparation of submissions to the Food and Drug Administration (FDA), e.g., Investigational New Drug Application (IND) and Biologics License Application (BLA). This guidance document finalizes the draft guidance of the same title dated February 2001.

For the purpose of this document, xenotransplantation refers to any procedure that involves the transplantation, implantation, or infusion into a human recipient of either (a) live cells, tissues, or organs from a nonhuman animal source, or (b) human body fluids, cells, tissues or organs that have had ex vivo contact with live nonhuman animal cells, tissues or organs. For the purpose of this document, xenotransplantation products include live cells, tissues or organs used in xenotransplantation. (See Definitions in section I.C.)

This document presents issues that should be considered in addressing the safety of viable materials obtained from animal sources and intended for clinical use in humans. The potential threat to both human and animal welfare from zoonotic or other infectious agents warrants careful characterization of animal sources of cells, tissues, and organs. This document addresses issues such as the characterization of source animals, source animal husbandry practices, characterization of xenotransplantation products, considerations for the xenotransplantation product manufacturing facility, appropriate preclinical models for xenotransplantation protocols, and monitoring of recipients of xenotransplantation products. This document recommends specific practices intended to prevent the introduction and spread of infectious agents of animal origin into the human population. FDA expects that new methods proposed by sponsors to address specific issues will be scientifically rigorous and that sufficient data will be presented to justify their use.

Examples of procedures involving xenotransplantation products include:

  • transplantation of xenogeneic hearts, kidneys, or pancreatic tissue to treat organ failure,
  • implantation of neural cells to ameliorate neurological degenerative diseases,
  • administration of human cells previously cultured ex vivo with live nonhuman animal antigen-presenting or feeder cells, and
  • extracorporeal perfusion of a patient’s blood or blood component perfused through an intact animal organ or isolated cells contained in a device to treat liver failure.

The guidance addresses issues such as:

  1. Clinical Protocol Review
  2. Xenotransplantation Site
  3. Criteria for Patient Selection
  4. Risk/Benefit Assessment
  5. Screening for Infectious Agents
  6. Patient Follow-up
  7. Archiving of Patient Plasma and Tissue Specimens
  8. Health Records and Data Management
  9. Informed Consent
  10. Responsibility of the Sponsor in Informing the Patient of New Scientific Information

A full copy of the PDF can be found below for reference:

fdaguidanceanimalsourcesxenotransplatntation

An example of the need for this guidance in conjunction with 3D printing technology can be understood from the below article (source http://www.geneticliteracyproject.org/2015/09/03/pig-us-xenotransplantation-new-age-chimeric-organs/)

Pig in us: Xenotransplantation and new age of chimeric organs

David Warmflash | September 3, 2015 | Genetic Literacy Project

Imagine stripping out the failing components of an old car — the engine, transmission, exhaust system and all of those parts — leaving just the old body and other structural elements. Replace those old mechanical parts with a brand new electric, hydrogen powered, biofuel, nuclear or whatever kind of engine you want and now you have a brand new car. It has an old frame, but that’s okay. The frame wasn’t causing the problem, and it can live on for years, undamaged.

When challenged to design internal organs, tissue engineers are taking a similar approach, particularly with the most complex organs, like the heart, liver and kidneys. These organs have three dimensional structures that are elaborate, not just at the gross anatomic level, but in microscopic anatomy too. Some day, their complex connective tissue scaffolding, the stroma, might be synthesized from the needed collagen proteins with advanced 3-D printing. But biomedical engineering is not there yet, so right now the best candidate for organ scaffolding comes from one of humanity’s favorite farm animals: the pig.

Chimera alarmists connecting with anti-biotechnology movements might cringe at the thought of building new human organs starting with pig tissue, but if you’re using only the organ scaffolding and building a working organ from there, pig organs may actually be more desirable than those donated by humans.

How big is the anti-chimerite movement?

Unlike anti-GMO and anti-vaccination activists, there really aren’t too many anti-chemerites around. Nevertheless, there is a presence on the web of people who express concern about mixing of humans and non-human animals. Presently, much of their concern is focussed on the growing of human organs inside non-human animals, pigs included. One anti-chemerite has written that it could be a problem for the following reason:

Once a human organ is grown inside a pig, that pig is no longer fully a pig. And without a doubt, that organ will no longer be a fully human organ after it is grown inside the pig. Those receiving those organs will be allowing human-animal hybrid organs to be implanted into them. Most people would be absolutely shocked to learn some of the things that are currently being done in the name of science.

The blog goes on to express alarm about the use of human genes in rice and from there morphs into an off the shelf garden variety anti-GMO tirade, though with an an anti-chemeric current running through it. The concern about making pigs a little bit human and humans a little bit pig becomes a concern about making rice a little bit human. But the concern about fusing tissues and genes of humans and other species does not fit with the trend in modern medicine.

Utilization of pig tissue enters a new age 

pigsinus

A porcine human ear for xenotransplantation. source: The Scientist

For decades, pig, bovine and other non-human tissues have been used in medicine. People are walking around with pig and cow heart valves. Diabetics used to get a lot of insulin from pigs and cows, although today, thanks to genetic engineering, they’re getting human insulin produced by microorganisms modified genetically to make human insulin, which is safer and more effective.

When it comes to building new organs from old ones, however, pig organs could actually be superior for a couple of reasons. For one thing, there’s no availability problem with pigs. Their hearts and other organs also have all of the crucial components of the extracellular matrix that makes up an organ’s scaffolding. But unlike human organs, the pig organs don’t tend to carry or transfer human diseases. That is a major advantage that makes them ideal starting material. Plus there is another advantage: typically, the hearts of human cadavers are damaged, either because heart disease is what killed the human owner or because resuscitation efforts aimed at restarting the heart of a dying person using electrical jolts and powerful drugs.

Rebuilding an old organ into a new one

How then does the process work? Whether starting with a donated human or pig organ, there are several possible methods. But what they all have in common is that only the scaffolding of the original organ is retained. Just like the engine and transmission of the old car, the working tissue is removed, usually using detergents. One promising technique that has been applied to engineer new hearts is being tested by researchers at the University of Pittsburgh. Detergents pumped into the aorta attached to a donated heart (donated by a human cadaver, or pig or cow). The pressure keeps the aortic valve closed, so the detergents to into the coronary arteries and through the myocardial (heart muscle) and endocardial (lining over the muscle inside the heart chambers) tissue, which thus gets dissolved over the course of days. What’s left is just the stroma tissue, forming a scaffold. But that scaffold has signaling factors that enable embryonic stem cells, or specially programed adult pleuripotent cells to become all of the needed cells for a new heart.

Eventually, 3-D printing technology may reach the point when no donated scaffolding is needed, but that’s not the case quite yet, plus with a pig scaffolding all of the needed signaling factors are there and they work just as well as those in a human heart scaffold. All of this can lead to a scenario, possibly very soon, in which organs are made using off-the-self scaffolding from pig organs, ready to produce a custom-made heart using stem or other cells donated by new organ’s recipient.

David Warmflash is an astrobiologist, physician, and science writer. Follow @CosmicEvolution to read what he is saying on Twitter.

And a Great Article in The Scientist by Dr. Ed Yong Entitled

Replacement Parts

To cope with a growing shortage of hearts, livers, and lungs suitable for transplant, some scientists are genetically engineering pigs, while others are growing organs in the lab.

By Ed Yong | August 1, 2012

Source: http://www.the-scientist.com/?articles.view/articleNo/32409/title/Replacement-Parts/

.. where Joseph Vacanti and David Cooper figured that using

“engineered pigs without the a-1,3-galactosyltransferase gene that produces the a-gal residues. In addition, the pigs carry human cell-membrane proteins such as CD55 and CD46 that prevent the host’s complement system from assembling and attacking the foreign cells”

thereby limiting rejection of the xenotransplated tissue.

In addition to issues related to animal virus transmission the issue of optimal scaffolds for organs as well as the advantages which 3D Printing would have in mass production of organs is discussed:

To Vacanti, artificial scaffolds are the future of organ engineering, and the only way in which organs for transplantation could be mass-produced. “You should be able to make them on demand, with low-cost materials and manufacturing technologies,” he says. That is relatively simple for organs like tracheas or bladders, which are just hollow tubes or sacs. Even though it is far more difficult for the lung or liver, which have complicated structures, Vacanti thinks it will be possible to simulate their architecture with computer models, and fabricate them with modern printing technology. (See “3-D Printing,” The Scientist, July 2012.) “They obey very ordered rules, so you can reduce it down to a series of algorithms, which can help you design them,” he says. But Taylor says that even if the architecture is correct, the scaffold would still need to contain the right surface molecules to guide the growth of any added cells. “It seems a bit of an overkill when nature has already done the work for us,” she says.

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New FDA Draft Guidance On Homologous Use of Human Cells, Tissues, and Cellular and Tissue-Based Products – Implications for 3D BioPrinting of Regenerative Tissue

Reporter: Stephen J. Williams, Ph.D.

The FDA recently came out with a Draft Guidance on use of human cells, tissues and cellular and tissue-based products (HCT/P) {defined in 21 CFR 1271.3(d)} and their use in medical procedures. Although the draft guidance was to expand on previous guidelines to prevent the introduction, transmission, and spread of communicable diseases, this updated draft may have implications for use of such tissue in the emerging medical 3D printing field.

A full copy of the PDF can be found here for reference but the following is a summary of points of the guidance.FO508ver – 2015-373 HomologousUseGuidanceFinal102715

In 21 CFR 1271.10, the regulations identify the criteria for regulation solely under section 361 of the PHS Act and 21 CFR Part 1271. An HCT/P is regulated solely under section 361 of the PHS Act and 21 CFR Part 1271 if it meets all of the following criteria (21 CFR 1271.10(a)):

  • The HCT/P is minimally manipulated;
  • The HCT/P is intended for homologous use only, as reflected by the labeling, advertising, or other indications of the manufacturer’s objective intent;
  • The manufacture of the HCT/P does not involve the combination of the cells or tissues with another article, except for water, crystalloids, or a sterilizing, preserving, or storage agent, provided that the addition of water, crystalloids, or the sterilizing, preserving, or storage agent does not raise new clinical safety concerns with respect to the HCT/P; and
  • Either:
  1. The HCT/P does not have a systemic effect and is not dependent upon the metabolic activity of living cells for its primary function; or
  2. The HCT/P has a systemic effect or is dependent upon the metabolic activity of living cells for its primary function, and:
  3. Is for autologous use;
  4. Is for allogeneic use in a first-degree or second-degree blood relative; or
  5. Is for reproductive use.

If an HCT/P does not meet all of the criteria in 21 CFR 1271.10(a), and the establishment that manufactures the HCT/P does not qualify for any of the exceptions in 21 CFR 1271.15, the HCT/P will be regulated as a drug, device, and/or biological product under the Federal Food, Drug and Cosmetic Act (FD&C Act), and/or section 351 of the PHS Act, and applicable regulations, including 21 CFR Part 1271, and pre-market review will be required.

1 Examples of HCT/Ps include, but are not limited to, bone, ligament, skin, dura mater, heart valve, cornea, hematopoietic stem/progenitor cells derived from peripheral and cord blood, manipulated autologous chondrocytes, epithelial cells on a synthetic matrix, and semen or other reproductive tissue. The following articles are not considered HCT/Ps: (1) Vascularized human organs for transplantation; (2) Whole blood or blood components or blood derivative products subject to listing under 21 CFR Parts 607 and 207, respectively; (3) Secreted or extracted human products, such as milk, collagen, and cell factors, except that semen is considered an HCT/P; (4) Minimally manipulated bone marrow for homologous use and not combined with another article (except for water, crystalloids, or a sterilizing, preserving, or storage agent, if the addition of the agent does not raise new clinical safety concerns with respect to the bone marrow); (5) Ancillary products used in the manufacture of HCT/P; (6) Cells, tissues, and organs derived from animals other than humans; (7) In vitro diagnostic products as defined in 21 CFR 809.3(a); and (8) Blood vessels recovered with an organ, as defined in 42 CFR 121.2 that are intended for use in organ transplantation and labeled “For use in organ transplantation only.” (21 CFR 1271.3(d))

Contains Nonbinding Recommendations
Draft – Not for Implementation

Section 1271.10(a)(2) (21 CFR 1271.10(a)(2)) provides that one of the criteria for an HCT/P to be regulated solely under section 361 of the PHS Act is that the “HCT/P is intended for homologous use only, as reflected by the labeling, advertising, or other indications of the manufacturer’s objective intent.” As defined in 21 CFR 1271.3(c), homologous use means the repair, reconstruction, replacement, or supplementation of a recipient’s cells or tissues with an HCT/P that performs the same basic function or functions in the recipient as in the donor. This criterion reflects the Agency’s conclusion that there would be increased safety and effectiveness concerns for HCT/Ps that are intended for a non-homologous use, because there is less basis on which to predict the product’s behavior, whereas HCT/Ps for homologous use can reasonably be expected to function appropriately (assuming all of the other criteria are also met).2 In applying the homologous use criterion, FDA will determine what the intended use of the HCT/P is, as reflected by the the labeling, advertising, and other indications of a manufacturer’s objective intent, and will then apply the homologous use definition.

FDA has received many inquiries from manufacturers about whether their HCT/Ps meet the homologous use criterion in 21 CFR 1271.10(a)(2). Additionally, transplant and healthcare providers often need to know this information about the HCT/Ps that they are considering for use in their patients. This guidance provides examples of different types of HCT/Ps and how the regulation in 21 CFR 1271.10(a)(2) applies to them, and provides general principles that can be applied to HCT/Ps that may be developed in the future. In some of the examples, the HCT/Ps may fail to meet more than one of the four criteria in 21 CFR 1271.10(a).

III. QUESTIONS AND ANSWERS

  1. What is the definition of homologous use?

Homologous use means the repair, reconstruction, replacement, or supplementation of a recipient’s cells or tissues with an HCT/P that performs the same basic function or functions in the recipient as in the donor (21 CFR 1271.3(c)), including when such cells or tissues are for autologous use. We generally consider an HCT/P to be for homologous use when it is used to repair, reconstruct, replace, or supplement:

  • Recipient cells or tissues that are identical (e.g., skin for skin) to the donor cells or tissues, and perform one or more of the same basic functions in the recipient as the cells or tissues performed in the donor; or,
  • Recipient cells that may not be identical to the donor’s cells, or recipient tissues that may not be identical to the donor’s tissues, but that perform one or more of the same basic functions in the recipient as the cells or tissues performed in the donor.3

2 Proposed Approach to Regulation of Cellular and Tissue-Based Products, FDA Docket. No. 97N-0068 (February. 28, 1997) page 19. http://www.fda.gov/downloads/biologicsbloodvaccines/guidancecomplianceregulatoryinformation/guidances/tissue/ ucm062601.pdf.

3“Establishment Registration and Listing for Manufacturers of Human Cellular and Tissue-Based Products” 63 FR 26744 at 26749 (May 14, 1998).

Contains Nonbinding Recommendations
Draft – Not for Implementation

1-1. A heart valve is transplanted to replace a dysfunctional heart valve. This is homologous use because the donor heart valve performs the same basic function in the donor as in the recipient of ensuring unidirectional blood flow within the heart.

1-2. Pericardium is intended to be used as a wound covering for dura mater defects. This is homologous use because the pericardium is intended to repair or reconstruct the dura mater and serve as a covering in the recipient, which is one of the basic functions it performs in the donor.

Generally, if an HCT/P is intended for use as an unproven treatment for a myriad of

diseases or conditions, the HCT/P is likely not intended for homologous use only.4

  1. What does FDA mean by repair, reconstruction, replacement, or supplementation of a recipient’s cells or tissues?

Repair generally means the physical or mechanical restoration of tissues, including by covering or protecting. For example, FDA generally would consider skin removed from a donor and then transplanted to a recipient in order to cover a burn wound to be a homologous use. Reconstruction generally means surgical reassembling or re-forming. For example, reconstruction generally would include the reestablishment of the physical integrity of a damaged aorta.5 Replacement generally means substitution of a missing tissue or cell, for example, the replacement of a damaged or diseased cornea with a healthy cornea or the replacement of donor hematopoietic stem/progenitor cells in a recipient with a disorder affecting the hematopoietic system that is inherited, acquired, or the result of myeloablative treatment. Supplementation generally means to add to, or complete. For example, FDA generally would consider homologous uses to be the implantation of dermal matrix into the facial wrinkles to supplement a recipient’s tissues and the use of bone chips to supplement bony defects. Repair, reconstruction, replacement, and supplementation are not mutually exclusive functions and an HCT/P could perform more than one of these functions for a given intended use.

  1. What does FDA mean by “the same basic function or functions” in the definition of homologous use?

For the purpose of applying the regulatory framework, the same basic function or functions of HCT/Ps are considered to be those basic functions the HCT/P performs in the body of the donor, which, when transplanted, implanted, infused, or transferred, the HCT/P would be expected to perform in the recipient. It is not necessary for the HCT/P in the recipient to perform all of the basic functions it performed in the donor, in order to

4 “Human Cells, Tissues, and Cellular and Tissue-Based Products; Establishment Registration and Listing” 66 FR 5447 at 5458 (January 19, 2001).

5 “Current Good Tissue Practice for Human Cell, Tissue, and Cellular and Tissue-Based Product Establishments; Inspection and Enforcement” 69 FR 68612 at 68643 (November 24, 2004) states, “HCT/Ps with claims for “reconstruction or repair” can be regulated solely under section 361 of the PHS Act, provided the HCT/P meets all the criteria in § 1271.10, including minimal manipulation and homologous use.”

Contains Nonbinding Recommendations
Draft – Not for Implementation

meet the definition of homologous use. However, to meet the definition of homologous use, any of the basic functions that the HCT/P is expected to perform in the recipient must be a basic function that the HCT/P performed in the donor.

A homologous use for a structural tissue would generally be to perform a structural function in the recipient, for example, to physically support or serve as a barrier or conduit, or connect, cover, or cushion.

A homologous use for a cellular or nonstructural tissue would generally be a metabolic or biochemical function in the recipient, such as, hematopoietic, immune, and endocrine functions.

3-1. The basic functions of hematopoietic stem/progenitor cells (HPCs) include to form and to replenish the hematopoietic system. Sources of HPCs include cord blood, peripheral blood, and bone marrow.6

  1. HPCs derived from peripheral blood are intended for transplantation into an individual with a disorder affecting the hematopoietic system that is inherited, acquired, or the result of myeloablative treatment. This is homologous use because the peripheral blood product performs the same basic function of reconstituting the hematopoietic system in the recipient.
  2. HPCs derived from bone marrow are infused into an artery with a balloon catheter for the purpose of limiting ventricular remodeling following acute myocardial infarction. This is not homologous use because limiting ventricular remodeling is not a basic function of bone marrow.
  3. A manufacturer provides HPCs derived from cord blood with a package insert stating that cord blood may be infused intravenously to differentiate into neuronal cells for treatment of cerebral palsy. This is not homologous use because there is insufficient evidence to support that such differentiation is a basic function of these cells in the donor.

3-2. The basic functions of the cornea include protecting the eye by forming its outermost layer and serving as the refracting medium of the eye. A corneal graft is transplanted to restore sight in a patient with corneal blindness. This is homologous use because a corneal graft performs the same basic functions in the donor as in the recipient.

3-3. The basic functions of a vein or artery include serving as a conduit for blood flow throughout the body. A cryopreserved vein or artery is used for arteriovenous access during hemodialysis. This is homologous use because the vein or artery is supplementing the vessel as a conduit for blood flow.

3-4. The basic functions of amniotic membrane include covering, protecting, serving as a selective barrier for the movement of nutrients between the external and in utero

6 Bone marrow meets the definition of an HCT/P only if is it more than minimally manipulated; intended by the manufacturer for a non-homologous use, or combined with certain drugs or devices.

Contains Nonbinding Recommendations
Draft – Not for Implementation

environment, and to retain fluid in utero. Amniotic membrane is used for bone tissue replacement to support bone regeneration following surgery to repair or replace bone defects. This is not a homologous use because bone regeneration is not a basic function of amniotic membrane.

3-5. The basic functions of pericardium include covering, protecting against infection, fixing the heart to the mediastinum, and providing lubrication to allow normal heart movement within chest. Autologous pericardium is used to replace a dysfunctional heart valve in the same patient. This is not homologous use because facilitating unidirectional blood flow is not a basic function of pericardium.

  1. Does my HCT/P have to be used in the same anatomic location to perform the same basic function or functions?

An HCT/P may perform the same basic function or functions even when it is not used in the same anatomic location where it existed in the donor.7 A transplanted HCT/P could replace missing tissue, or repair, reconstruct, or supplement tissue that is missing or damaged, either when placed in the same or different anatomic location, as long as it performs the same basic function(s) in the recipient as in the donor.

4-1. The basic functions of skin include covering, protecting the body from external force, and serving as a water-resistant barrier to pathogens or other damaging agents in the external environment. The dermis is the elastic connective tissue layer of the skin that provides a supportive layer of the integument and protects the body from mechanical stress.

  1. An acellular dermal product is used for supplemental support, protection, reinforcement, or covering for a tendon. This is homologous use because in both anatomic locations, the dermis provides support and protects the soft tissue structure from mechanical stress.
  2. An acellular dermal product is used for tendon replacement or repair. This is not homologous use because serving as a connection between muscle and bone is not a basic function of dermis.

4-2. The basic functions of amniotic membrane include serving as a selective barrier for the movement of nutrients between the external and in utero environment and to retain fluid in utero. An amniotic membrane product is used for wound healing of dermal ulcers and defects. This is not homologous use because wound healing of dermal lesions is not a basic function of amniotic membrane.

4-3. The basic functions of pancreatic islets include regulating glucose homeostasis within the body. Pancreatic islets are transplanted into the liver through the portal vein,

7 “Human Cells, Tissues, and Cellular and Tissue-Based Products; Establishment Registration and Listing” 66 FR 5447 at 5458 (January 19, 2001).

6

Contains Nonbinding Recommendations
Draft – Not for Implementation

for preservation of endocrine function after pancreatectomy. This is homologous use because the regulation of glucose homeostasis is a basic function of pancreatic islets.

  1. What does FDA mean by “intended for homologous use” in 21 CFR 1271.10(a)(2)?

The regulatory criterion in 21 CFR 1271.10(a)(2) states that the HCT/P is intended for homologous use only, as reflected by the labeling, advertising, or other indications of the manufacturer’s objective intent.

Labeling includes the HCT/P label and any written, printed, or graphic materials that supplement, explain, or are textually related to the product, and which are disseminated by or on behalf of its manufacturer.8 Advertising includes information, other than labeling, that originates from the same source as the product and that is intended to supplement, explain, or be textually related to the product (e.g., print advertising, broadcast advertising, electronic advertising (including the Internet), statements of company representatives).9

An HCT/P is intended for homologous use when its labeling, advertising, or other indications of the manufacturer’s objective intent refer to only homologous uses for the HCT/P. When an HCT/P’s labeling, advertising, or other indications of the manufacturer’s objective intent refer to non-homologous uses, the HCT/P would not meet the homologous use criterion in 21 CFR 1271.10(a)(2).

  1. What does FDA mean by “manufacturer’s objective intent” in 21 CFR 1271.10(a)(2)?

A manufacturer’s objective intent is determined by the expressions of the manufacturer or its representatives, or may be shown by the circumstances surrounding the distribution of the article. A manufacturer’s objective intent may, for example, be shown by labeling claims, advertising matter, or oral or written statements by the manufacturer or its representatives. It may be shown by the circumstances that the HCT/P is, with the knowledge of the manufacturer or its representatives, offered for a purpose for which it is neither labeled nor advertised.

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Biofabrication with Stem Cells

Curator: Larry H. Bernstein, MD, FCAP

 

 

Biofabrication  Special Issue:  Dec 2015; 7(4).    http://iopscience.iop.org/1758-5090/7/4

 

Three-dimensional bioprinting of embryonic stem cells directs highly uniform embryoid body formation

Liliang Ouyang1,2,6, Rui Yao1,2,6, Shuangshuang Mao1,2, Xi Chen3, Jie Na3 and Wei Sun1,2,4,5
Biofabrication, Volume 7(4)    http://iopscience.iop.org/article/10.1088/1758-5090/7/4/044101/meta

With the ability to manipulate cells temporarily and spatially into three-dimensional (3D) tissue-like construct, 3D bioprinting technology was used in many studies to facilitate the recreation of complex cell niche and/or to better understand the regulation of stem cell proliferation and differentiation by cellular microenvironment factors. Embryonic stem cells (ESCs) have the capacity to differentiate into any specialized cell type of the animal body, generally via the formation of embryoid body (EB), which mimics the early stages of embryogenesis. In this study, extrusion-based 3D bioprinting technology was utilized for biofabricating ESCs into 3D cell-laden construct. The influence of 3D printing parameters on ESC viability, proliferation, maintenance of pluripotency and the rule of EB formation was systematically studied in this work. Results demonstrated that ESCs were successfully printed with hydrogel into 3D macroporous construct. Upon process optimization, about 90% ESCs remained alive after the process of bioprinting and cell-laden construct formation. ESCs continued proliferating into spheroid EBs in the hydrogel construct, while retaining the protein expression and gene expression of pluripotent markers, like octamer binding transcription factor 4, stage specific embryonic antigen 1 and Nanog. In this novel technology, EBs were formed through cell proliferation instead of aggregation, and the quantity of EBs was tuned by the initial cell density in the 3D bioprinting process. This study introduces the 3D bioprinting of ESCs into a 3D cell-laden hydrogel construct for the first time and showed the production of uniform, pluripotent, high-throughput and size-controllable EBs, which indicated strong potential in ESC large scale expansion, stem cell regulation and fabrication of tissue-like structure and drug screening studies.

With the capability of self-renewal and differentiating into all somatic cell types, embryonic stem cells (ESCs) hold great promise as an in vitro model system for studies in early embryonic development, as well as a robust cell source for applications in diagnostics, therapeutics, and drug screening [1]. Derived from the inner cell mass of a blastocyst, ESCs requires delicate culture condition and trend to cluster together, and in particular, forms three-dimensional (3D) cellular spheroids termed embryoid body (EB) [2]. In order to better understand stem cell niche and regulation of ESC differentiation and reprogramming, in vitro recapitulation of the spatial distribution of cells, cell–cell and cell–matrix interactions, is of paramount importance [35]. Compared with 2D monolayer culture, 3D cell culture is believed to confer a higher degree of clinical and biological relevance to in vitro model [6, 7], since the spatial arrangement of cells and extra-cellular matrix could influence cell differentiation and function both in vivo [8] and in vitro[9]. Therefore, reconstruction of 3D cell microenvironment is critical to directing stem cell fate and generating cell sources for tissue engineering, regenerative medicine and drug screening studies.

By mimicking some of the spatial and temporal aspects of in vivo development, EB is a basic 3D model for ESCs culture and differentiation studies. It was reported that the size and uniformity of EBs could vastly influence stem cell fate [1012]. Various methods have been used to fabricate such cellular spheroid, basically including static suspension, hanging-drop and multiwell culture, most of which doesn’t involve biomaterials. Static suspension method inoculate suspension of ESCs onto non-adhesive plate to allow cells spontaneously aggregate into spheroid. This method is easy to operate, but showed limited control over the EBs size and shape due to the probability that ESCs encounter each other accidentally [13]. Hanging-drop is a common method to produce size-controlled homogeneous EBs, where droplets of ESCs suspension are pipetted onto the lid of a Petri dish and EBs was generate by gravity after overturning the dish [14]. However, manual pipetting is labor intensive and the reproducibility varies with operators. Multiwell culture offers high-throughput solution for EB formation through cell aggregation in uniformly shaped microwell arrays but requires expensive microwell culture plates [10, 15]. Besides, there are few customized microwell culture plates available in the market.

Recent advances in bioprinting technologies facilitated the precise deposition of ESCs in a reproducible manner. Xu et al [16] and Shu et al [17] printed ESCs suspension solution into 2D patterns as hanging-drop approach for EB formation, without the cell-biomaterial interaction. Corr and Xie [18, 19] applied laser direct-write method in bioprinting of mouse ESCs together with gelatin. ESCs maintained the pluripotency while proliferation and formed EB. EB size can be controlled by cell density and colony size. However, these studies just generated 2D cellular array without 3D cell–matrix interactions, and cell–cell interaction happens within one drop but not among different drops. To better recapitulate the characteristics of in vivo cell microenvironment, 3D customized cell/matrix construct with macro-porous structure might be a preferred choice. To our knowledge, there has been no report about bioprinting of ESCs into 3D cell-laden constructs.

The extrusion-based temperature-sensitive 3D bioprinting technology was developed in our lab and has been utilized for bioprinting of hepatocytes [20], adipose tissue-derived stem cells (ADSCs) [21], C2C12 cells [22], hela cells [23] and 293FT cells [24]. Most commonly used biomaterials for this technology are gelatin and alginate. Gelatin, a type of denatured collagen, is widely used as a coating for feeder layer-free mouse ES cell culture. Alginate, extracted from brown algae, is proving to have a wide applicability in tissue engineering and drug delivery and also used in embedding mouse ESCs for EB formation [25]. It has been proved in many studies that encapsulation of ESCs in hydrogels would direct EB formation with the maintenance of pluripotency [2628]. Hence, we hypothesized that the bioprinting of 3D ESC-laden construct would maintain the stem cell pluripotency and address the challenges associated with the current methods for EB formation.

In this study, we investigated the feasibility of applying extrusion-based temperature-sensitive 3D bioprinting technology in bioprinting of ESCs with hydrogels into 3D macro-porous structure, with the maintenance of viability, pluripotency, cell growth and to direct EB formation. Printing process parameters were optimized to obtain a high cell survival rate (90%) after printing process and construct formation. Stem cell pluripotency was examined by the expression of stem cell markers (octamer binding transcription factor 4 (Oct4), stage specific embryonic antigen 1 (SSEA1) and a homeodomain-bearing transcriptional factor (Nanog)) and the ability to form EBs. The regulation of EB formation in the 3D bioprinted construct was systematically compared with commonly used methodology, where EB formation relies on cell aggregating as well as cell proliferation. Results demonstrated that this novel technology generated pluripotent, high-throughput, highly uniform and size controllable EBs under static culture condition without complex equipment. This study established the feasibility of fabricating 3D in vitro tissue-like model using ESCs for the first time, creating engineered microenvironment for pluripotent stem cells with the ability of placing cells and materials spatially in a reproducible manner.

 

Results 

3.1. 3D bioprinting and cell viability optimization

In this study, many process parameters, e.g. nozzle inner diameter, nozzle insulation temperature and chamber temperature were examined to optimize cell viability after 3D construct fabrication. It was demonstrated that larger nozzle diameter resulted in higher cell viability (figure 2(A)). Specially, the cell viability under Nozzle-160 μm (81.59% ± 1.74%) was lower than those under Nozzle-260 μm (88.06% ± 1.98%), Nozzle-410 μm (89.59% ± 0.71%) and Nozzle-510 μm (90.84% ± 1.02%), with significant differences. Nozzle diameter of 260 μm, 410 μm and 510 μm showed no significant differences in terms of cell viability.

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Figure 2. The influence of bioprinting parameters on ESC viability is determined by fluorescence live/dead staining. (A) The influence of printing nozzle inner diameter on ESC viability (Insu-30 °C and Cham-10 °C). (B) The influence of nozzle insulation temperature and chamber temperature on ESC viability. Insu-25 °C means keeping the nozzle insulation temperature at 25 °C. Cham-4 °C means setting the chamber temperature at 4 °C, and so as others. (C) The fluorescent staining images show the live (green) and dead (red) cells at different days during culture period. Scale bar: 100 μm.

Insulation and chamber temperatures were altered to study their influences on cell viability (figure 2(B)). As a positive control, ESCs/hydrogel mixture without bioprinting were stained with fluorescence live/dead reagent, and showed 93.14% ± 1.31% cell viability. When insulation temperature was set at 25 °C (labeled as ‘Insu-25 °C’), cell viability increased with the chamber temperature from 55.52% ± 2.37% under 4 °C (labeled as ‘Cham-4 °C’) to 78.22% ± 2.55% under 10 °C (labeled as ‘Cham-10 °C’) with significant differences. When the insulation temperature was set at 30 °C (labeled as ‘Insu-30 °C’), nearly 90% ESCs remained alive under the chamber temperature of 7 °C and 10 °C (labeled as ‘Cham-7 °C’ and ‘Cham-10 °C’), significantly more than that under Cham-4 °C (72.40% ± 2.46%). To achieve both high ESC viability and a clear construct configuration, the process parameter combination of Nozzle-260 μm, Cham-10 °C and Insu-30 °C was chosen.

After culturing for three days, few cells were found dead, which were isolated from living EBs (figure 2(C)). On day 5 and day 7, a few dead cells were observed on the edge of EBs. About 5% ESCs were stained dead on day 7. As the static culturing continued, 9.69% ± 1.77%, 17.72% ± 2.91% and 40.64% ± 2.06% were found dead on day 8, day 9 and day 10, respectively (supplement 2). So, we chose 7 days as the culture period in the following analysis.

3.2. Construct structural stability and EB formation

A 3D cellular construct with the cross section of 8 mm × 8 mm and height of 1 mm was fabricated under the optimized process parameter. The 3D construct demonstrated macro-porous grid structure in which the hydrogel threads were evenly distributed according to the computer design (figure 3(A)). Both the width of the threads and the gap between the threads were homogeneous, that is 728.2 μm ± 24.9 μm and 424.3 μm ± 17.8 μm, respectively, suggesting 3D cellular construct formation in a highly controlled manner. ESCs were embedded uniformly in the hydrogel matrix threads, developing a specific 3D microenvironment.

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Figure 3. Images of the printed cellular model with grid structure. (A) Full view of the cellular construct. (B) Phase-contrast images demonstrating the cell morphology and distribution of different cell density at day 3, day 5 and day 7. Scale bar: 1 mm.

During the culture period, ESCs tended to grow as spheroid cellular aggregates, also known as EB. The cell density in the 3D hydrogel construct were determined by the initial cell density in the ESC/alginate/gelatin mixture and showed significant influence on the yield and density of EBs formed in the construct (figure 3(B)). It was demonstrated by semi-quantitative analysis of figure 3(B) that, the percentage of area occupied by EBs varied from 52% to 85% when initial cell density changed from 0.5 mln mL−1 to 2.0 mln mL−1 . Most of the EBs were contained in the hydrogel threads in the culturing period. However, when the initial cell density was as high as 2.0 mln ml−1, some of the EBs were observed running off from the threads into the throughout holes.

3.3. Cell proliferation

ESCs formed spheroid EBs in the 3D hydrogel construct and the diameter of the EBs enlarged with culturing time while keeping their spatial location in the hydrogel thread, indicating EB formation by ESC proliferation rather than aggregation (figure 4(A)). Compared with traditional 2D culture, ESCs showed different proliferation rate indicated by the OD value measure by CCK-8 kit (figure 4(B)). The normalized OD value of the 3D in situ group grew faster than that of 2D from day 1 to day 3, while slowing down after day 3 and being much less than that of 2D at day 7. However, 3D harvest group showed a generally faster growth rate than 2D during the one week culturing, with a significant difference. In addition, the diameter of EB was also measured to indicate ESC proliferation rate. When comparing the normalized EB volume with normalized 2D OD value, 3D samples also maintained a significantly faster growth rate than 2D, though the EB volume had huge variance (figure 4(B)).

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Figure 4. EB growing and cell proliferation. (A) Magnified images of the same location in 3D printed cellular construct at different times. (B) ESC proliferation in the 3D construct compared with 2D culture. All the date were normalized to the value of day 1. Scale bar: 200 μm.

Pluripotency markers, i.e. Oct4, SSEA1 and Nanog were analyzed to determine the pluripotency maintenance of ESCs after 7 day culture in the 3D hydrogel construct. Immunofluorescence staining and flow cytometry analysis showed that almost all of the cells within the EB were successfully stained both Oct4 and SSEA1. Because of the limitation of confocal capacity when dealing with large scale aggregates, the central part of the EB was darker than the edge (figure5(A)). Flow cytometry analysis demonstrated that 97.2% and 99.0% cells were positively stained with Oct4 and SSEA1 respectively (figure 5(B)). The qRT-PCR results demonstrated that the gene expression level of Oct4 and Nanog in our 3D samples were close to those in 2D (within the deviation of ±3%), without significant difference, confirming that cells have maintained pluripotency (figure 5(C)).

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Figure 5. ESC pluripotency at day 7 was determined by CLSM, flow cytometry and qRT-PCR. (A) Immunofluorescence images of EBs stained with Oct4, SSEA1 and DAPI. (B) Quantification of 3D dissociated cells marked with Oct4 and SSEA1 by using flow cytometry. (C) Gene expression of Oct4 and Nanog in 3D versus 2D by using qRT-PCR. Scale bar: 50 μm.

EBs were harvested from the 3D hydrogel construct at different time intervals to analyze EB morphology (figure 6(A)). Most of the EBs were separated without fusion. The center part of the EBs was darker than edge part, especially at day 5 and day 7, indicating the 3D sphere structure of EBs. Through analyzing the size of 250 random EBs for each sample, the histogram of EB diameter were obtained, showing a Gauss distribution curve (figure 6(B)). The results demonstrated that the EB size increased significantly from about 50 μm to about 110 μm when the construct was cultured from day 3 to day 7 (figure 6(C)). Cell density had little influence on EB average size. However, increased cell density would result in the reduction of the uniformity of EB size, especially at day 7; the EB diameter of 2.0 mln mL−1 group at day 7 was vastly heterogeneous, with a deviation of 42.30 μm, which was much more than those of other two groups.

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Figure 6. EB formation in different cell density: (A) optical images of released EBs at different days. (B) EB diameter and (C) EB circularity distributions at different days. Summary of the (D) diameter and (E) circularity. 250 EBs were applied for diameter and circularity measurements for each group. Scale bar: 200 μm.

Circularity was measured to assess the quality of EBs (figure 6(D)). For the 0.5 mln mL−1 group, most of the EBs were close to a standard spheroid with the circularity centered in 0.9 for the three time points. As to the other two groups, the circularity at day 3 is similar to that of 0.5 mln mL−1group, while the circularity frequency peaks had a significant decrease at day 5 and day 7. In particular, about 20% EBs had a circularity under 0.8 at day 5 and day 7 for the 2.0 mln mL−1group. In general, the circularity decreased with the increase of culture time and initial cell density in the hydrogel (figure 6(E)).

3.6. Comparison with other EB formation methods

Considering this was a novel methodology of EB formation, we systematically compared the commonly used static suspension and hanging drop methods with the 3D bioprinting method for EB formation. As demonstrated by the phase-contrast images (figure 7(A)), EBs generated by static suspension method showed more uncontrollable morphology rather than round spheroid. The distribution of EB diameter clearly demonstrated that 3D bioprinting technology generated EBs with higher uniformity compared with static suspension technology, especially for the larger EB diameter, i.e. 60 ~ 70 μm and 100 ~ 110 μm regions (figure 7(B)). In particular, the EBs with 30 ~ 50 μm diameter presented vastly irregular shape in suspension technology, which was confirmed by the circularity curve (figure 7(C)). On the other hand, EBs generated by 3D bioprinting technology showed higher circularity regardless of the diameter regions, suggesting more regular shape (figure 7(C)). More characteristic like EB forming motivation, size control method, EB diameter range, uniformity, yield, operation complexity were compared among 3D bioprinting technology, static suspension technology and hanging drop technology, as listed in table 1.

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Figure 7. Comparison of static suspension and 3D bioprinting technology for generating EBs. (A) Phase-contrast images showing the morphology of EBs generated by static suspension technology and 3D bioprinting technology. (B) The EB diameter histograms presented the distribution of EB size with a Gauss distribution fitting. (C) The circularity curves contrasted the EB qualities.

Table 1.  Comparison of three EB forming methods.
Hanging-drop Suspension 3D print
Forming mechanism Aggregation by gravity Self-aggregation Proliferation
Size control Time and cell density Time and cell density Mainly time
Diameter range 50 ~ 500 μm 50 ~ 500 μm 30 ~ 200 μm
Uniformity High Low Medium-high
Yield Low High High
Operation Time-consuming for seeding and medium refresh Complex for medium refresh Time-saving and easy for medium refresh

 

4. Discussion

3D cell culture environment and tissue-like models have drawn great attention because they can be tuned to promote certain levels of cell differentiation and tissue organization, which is difficult in traditional 2D culture systems for their failing to reconstitute the in vivo cellular microenvironment [30, 31]. Various 3D culture systems have been developed to study the cellular behavior affected by spatial and temporal cell–cell and cell–matrix interactions. Among these methods, 3D bioprinting, typically containing jet-, laser- and extrusion-based methods, is a promising technique to manipulate cells/matrix deposition and ultimately generate 3D complex tissues or organs. This technique have been used in printing cells derived from adult, embryonic and even tumor tissues for tissue engineering and drug screening applications. With the capacity to expand unlimitedly in vitro and differentiate into a variety of therapeutic cell types, ESCs have generated great enthusiasm and are being applied in bioprinting studies until recently. As a relatively sensitive cell type, ESCs might suffer greater problems in a printing process compared with other types of cells. Several studies had been conducted to print ESCs, maintaining their viability and pluripotency [1619]. Instead of creating 3D tissue-like constructs, these studies were more likely to generate cellular droplet array with precise control of distribution. Here we described the work of establishing a 3D ESC-laden hydrogel construct using extrusion-based bioprinting technology. The results demonstrated high proliferation rate of pluripotent ESCs in the hydrogel construct, and a versatile technology for generating highly uniform and high throughput EBs.

Cell viability after 3D bioprinting and construct formation was determined when evaluating the limitations of bioprinting ESCs. Cells would be lysed or damaged due to osmotic effects in the solution, heat increase and mechanical stress during printing. In the protocol presented in this work, about 6.86% ± 1.31% cells were dead during the cell/hydrogel solution preparation process before 3D bioprinting (figure 2(B)). We assumed this was caused by cell dissociation process, together with the osmosis and stirring operation of hydrogel materials. In an inkjet printing study, 15% Chinese Hamster Ovary cells were detected dead before printing process [32]. Thermal effects of the ejector reservoir in the inkjet printing process and laser force in laser-based printing would be the cause of cell death, in addition to the impact force when cellular droplets were jetted to a rigid substrate in a very short time. Under a different fabricating strategy, the extrusion-based bioprinter extruded the cell-laden cylinders softly on the substrate and controlled the temperature under 30 °C, without the concerns about the thermal and sharply impacting effects. However, cells would inevitably suffer from shear force when the cell-laden hydrogels were continuously extruded through a limited space in the nozzle. We hypothesized that nozzle size and hydrogel viscosity would influence shear force and hence influence cell viability. The cell viability data of different nozzle sizes, chamber and insulation temperatures supported this hypothesis (figures 3(A) and (B)). In our previous study, more than 90% Hela cells were alive after bioprinting under the parameters of Insu-25 °C/Cham-4 °C and Nozzle-260 μm [23], while the viability of ESCs was only 55.52% ± 2.37% under the same parameter combination. When increasing the insulation and chamber temperature to 30 °C and 10 °C respectively, the viability showed a significant increase to 90%. Taking into the account of cell death before bioprinting, optimized parameters led to only 5% cell death during printing, indicating a broad future applicability of this technique to various cell types ranging from tumor cells to ESCs. Additionally, few dead cells were observed during one-week culture period (figure3(C)). On the other hand, when the culture period was extended to more than 7 days, more and more ESCs suffered from apoptosis and lysis, possibly due to contact inhabitation and insufficient mass transfer to the center of EB with the increasing of EB size. Therefore, 7 days was chose as the experiment time window for this study.

Apart from cell viability, the maintenance of pluripotency is another essential criterion for ESCs regulation and application. The results of immunofluorescence staining and FACS analysis showed a high expression rate (98%) of stem cell pluripotent markers Oct4 and SSEA1 at day 7 (figure 4), indicating that cells remained undifferentiated state during the whole experimental period. Naturally, it can be inferred that the printing process also had little influence on ESC pluripotency.

In the cell-laden hydrogel culture system, both the cell type and matrix material could influence cell growth. Human mesenchymal stem cells remained alive but did not proliferate when encapsulated in alginate [33, 34]. While human ADSCs could proliferated for a short period of time in alginate hydrogel microspheres but showed significantly higher proliferation rate in gelatin/alginate microspheres [35]. As a widely used hydrogel, alginate has the disadvantages of low cell adhesiveness and poor support for cell proliferation [36]. Adding gelatin would improve the cellular adhesive condition and hence favor cell expansion. In this study, the fabricated multilayered constructs offered a 3D microenvironment surrounded by gelatin/alginate materials for ESCs to adhere, self-renew, and cellular spheroid, termed EB, was generated in situ because of cell proliferation. Once EB was formed, the spheroid structure supported expansion of subpopulations with differing proliferation, nutrition and oxygenation status compared with conventional monolayer system. It is reported that the proliferation of mouse ESCs was higher when embedded in fibrin gels versus 2D suspension culture [27]. Similarly, in this study, ESCs in 3D constructs proliferated faster than 2D culture sample when being released from hydrogel to read OD value. This operation was aimed to avoid the influence of interactions between reagent molecular and matrix materials (figure 6 and supplement 3). Additionally, the enlargement of EB diameter, which also reflected ESC proliferation, confirmed this result (figure 6).

Typically stimulated via generation of EBs, ESC differentiation depends on numerous cues throughout the EB environment, including EB size and shape, as well as their uniformities. In general, several characteristics should be concerned for EB formation system, including reproducibility, symmetry, ease of use and scalability [37]. In the traditional EB formation methodology, like suspension and hanging-drop, EBs were created via cell gathering and proliferation. In these methods, it was essential to get a balance between allowing necessary ESC aggregation for EB formation and preventing EB agglomeration for efficient cell growth and differentiation [14]. Static suspension cultures produced a large number of EBs with simple operation, but the size and shape of the resulting EBs were highly uncontrollable and irregular due to the tendency of EBs to agglomerate after initial formation, as shown in figure 7. Hanging-drop method served as a golden tool to generate uniform and reproducible EBs with fully aggregating of cells under gravity and non-agglomeration of EBs in different drops. However, it faced the intrinsic limitation of scalability. The 3D bioprinting method presented in this study addressed some of the problems, producing massively homogeneous EBs with regular shape and controllable shape. In this 3D cell-laden hydrogel system, ESCs were immobilized and restricted to aggregate with each other, and would not agglomerate until they are large enough to connect with each other. When the initial cell density was increased, the average distance between two original EBs was closer and these EBs are more likely to agglomerate with each other while proliferation, which is also one of the concerns when we choose the experiment time period. As a result, the EB uniformity of 2.0 mln mL−1 group was not that good as those of 0.5 mln mL−1 and 1.0 mln mL−1 groups, especially after culturing for one week (figure 6). Without the initial cell aggregating, the size of EBs in our model was mainly determined by the culture time. Also, it would take longer to reach the same scale of EB diameter compared with suspension method, probably due to the physical constrain of the matrix material. For example, it took 5 days and 2 days to get EBs ranging 60 ~ 70 μm for 3D printing and suspension methods, respectively (figure7). Besides, thanks to the interconnected channels design in the 3D construct which allowed mass transfer, EBs could be produced in a large scale by changing the construct volume and cell density. In the six-layer construct with 1.0 million cells per milliliter for example, EBs got a stable yield of about 3000 cm−2, while the EB yield by suspension technology was about 900 cm−2(seeding 0.5 million cells in a 35 mm dish) and no more than 10 EBs [38] could be produced in 1 cm2 area in hanging-drop method, which was also demonstrated by our experiments (supplement 4).

In summary, this study presented the high throughput production of pluripotent, uniform, regular and controllable EBs with the diameter smaller than 150 μm during one week culture. In a gelatin-based laser printing method, EBs with the diameter of about 100 μm were also generated to avoid EB agglomeration in gels [19]. EBs with different size exhibit different gene expression and differentiation fate. Park et al [39] found that 100 μm diameter EBs of mouse ESCs expressed increased ectoderm markers while 500 μm diameter EBs expressed endoderm and mesoderm markers. Furthermore, Messana et al [12] demonstrated that mouse ESCs derived from small EBs (<100 μm) had a greater chondrogenic potential than those from larger EBs. Hwang [10] reported that human endothelial cell differentiation was increased in smaller EBs (150 μm) while cardiogenesis was enhanced in larger EBs (450 μm). However, large EBs might be associated with limited mass transfer and the diffusion of biochemical through EBs is demonstrated to be linked to differentiation of ESCs [40]. While the effect of EB size on differentiation remains to be shown in our model, we hypothesize that EBs with the diameter smaller than 150 μm would mediate specific differentiation trajectory, which will be confirmed in the future work.

Demonstrating the advantages of reproducibility, high throughput, regular shape and controlled size, we believe this is a versatile technology for EB generation. But, this 3D printing system does not serve as an EB formation method solely. The ESC-laden hydrogel 3D construct can be dissolved at a proper time point to harvest massive EBs with desired size for ES cell research. Or, the ESC-laden hydrogel 3D construct can be maintained to perform 3D ESC differentiation studies to explore the regulation of EB size, matrix material and 3D structure on ESC differentiation lineages. Furthermore, this technology hold the potential to serve as a versatile tool for the generation of tissue-like structure and organ/tissue on chip based on controlled ESC differentiation.

5. Conclusion

In this study, we reported successful bioprinting of mouse ESCs with hydrogel into a 3D multilayered construct for the first time. Extrusion-based bioprinting technology was applied. Upon parameter optimization, ESCs demonstrated high viability of 90% after 3D printing and construct formation. Cells continued self-renewal in the construct and exhibited a higher proliferation rate compared with conventional 2D culture. 98% cells expressed the canonical pulripotent markers Oct4 and SSEA1 at day 7, indicating that most of the ESCs remained undifferentiated state after printing and culturing. Large quantities of uniform EBs with regular shape and adjustable size were generated through cell proliferation, while avoiding EBs agglomeration. This work indicated the feasibility of fabricating complex 3D tissue-like model based on pluripotent stem cells for applications in pharmacy, regenerative medicine, stem cell expansion and biology studies.

 

Bioprinting of human pluripotent stem cells and their directed differentiation into hepatocyte-like cells for the generation of mini-livers in 3D

Alan Faulkner-Jones1,2, Catherine Fyfe3, Dirk-Jan Cornelissen1,2, John Gardner3, Jason King3,4,Aidan Courtney3,4 and Wenmiao Shu1,2
http://iopscience.iop.org/article/10.1088/1758-5090/7/4/044102/meta

We report the first investigation into the bioprinting of human induced pluripotent stem cells (hiPSCs), their response to a valve-based printing process as well as their post-printing differentiation into hepatocyte-like cells (HLCs). HLCs differentiated from both hiPSCs and human embryonic stem cells (hESCs) sources were bioprinted and examined for the presence of hepatic markers to further validate the compatibility of the valve-based bioprinting process with fragile cell transfer. Examined cells were positive for nuclear factor 4 alpha and were demonstrated to secrete albumin and have morphology that was also found to be similar to that of hepatocytes. Both hESC and hiPSC lines were tested for post-printing viability and pluripotency and were found to have negligible difference in terms of viability and pluripotency between the printed and non-printed cells. hESC-derived HLCs were 3D printed using alginate hydrogel matrix and tested for viability and albumin secretion during the remaining differentiation and were found to be hepatic in nature. 3D printed with 40-layer of HLC-containing alginate structures reached peak albumin secretion at day 21 of the differentiation protocol. This work demonstrates that the valve-based printing process is gentle enough to print human pluripotent stem cells (hPSCs) (both hESCs and hiPSCs) while either maintaining their pluripotency or directing their differentiation into specific lineages. The ability to bioprint hPSCs will pave the way for producing organs or tissues on demand from patient specific cells which could be used for animal-free drug development and personalized medicine.

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Content from this work may be used under the terms of the Creative Commons Attribution 3.0 licence. Any further distribution of this work must maintain attribution to the author(s) and the title of the work, journal citation and DOI.

 

New drug development can take 10 to 20 years with an estimated average of about 9 to 12 years [1, 2]. In addition, only around 16% of the drugs that begin preclinical testing are approved for human use [3]. Some of this low success rate can be attributed to the different responses that animals and humans have to the drugs being tested; some drugs have to be withdrawn from market due to toxic effects on human organs such as liver and heart, despite being tested safely on animals. A possible solution to this might be the creation of human pluripotent stem cell (hPSC) -derived micro-tissues which could be used with organ-on-a-chip devices [47]. These micro-tissues are expected to produce the same or similar physiological reaction that the entire organ would but on a much smaller scale. This would result in scalable, faster and potentially more reliable drug testing platform, and hopefully an end to animal testing.

hPSCs are the ideal cells to use for this application due to their ability to self-renew indefinitely, which enables large populations of cells to be created easily in vitro, and their pluripotency which means that they can be differentiated into any required adult cell type [813]. Pluripotent stem cells can be divided into embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). Human ESCs (hESCs) were first isolated from early human blastocysts in 1998 [14]. Any tissue construct created from hESCs for implantation in vivo would require the patient to receive immunosuppressive drugs and ethical issues still restrict some applications due to their source. iPSCs have neither of these drawbacks as they can be created from harvested adult cells from the patient requiring treatment and as such any implanted cells derived from these iPSCs should not be rejected by the patient’s immune system but may require immunosuppressive drugs at a greatly reduced dosage. In 2006 Shinya Yamanaka discovered that iPSCs can be derived from somatic cells by retrovirally transducing them with four transcription factors—Oct3/4, Sox2, Klf4 and C-myc [15, 16]. These cells have the same self-renewal and differentiation capabilities as ESCs but with the added advantage that iPSCs can be used for autologous therapies. These unique characteristics make pluripotent stem cells ideal for use in a number of applications such as clinical tissue engineering, novel drug discovery and testing for the pharmaceutical industry [8,9, 17, 18].

In the field of biofabrication, great advances are being made towards fabricating 3D tissue and organs with very fine spatial control of cell deposition. From the very first paper that was published investigating printing of biological cells (or bioprinting), tissue engineering was identified as a major application for this new technology [19]. If more complex structures such as organs and tissues were to be printed, the bioprinter would need the ability to transfer microscopic patterns of viable cells of multiple cell types into well-defined three-dimensional arrays that closely mimic the tissue structure. There has been much progress in the development and establishment of several different bioprinting techniques for 3D live constructs [2022] including those based on laser pulses, inkjets and other more novel approaches. It is an inescapable fact that cells will be subjected to some level of stress during deposition, regardless of the printing technique being used. For example, cells printed by non-contact methods will be affected when they impact on the substrate at some incident velocity, which would result in extreme deceleration and shear stress [2326]. Shear stress is applied to cells pushed through nozzle orifices and capillary tubes [24, 2742] and the actuation is provided via pressure, heat, or high frequency vibration which can also be damaging to the cells [30, 31, 4346]. If cells are exposed to laser energy the radiation can cause genetic damage [29, 4754] and shear forces are applied during cavitation and jet formation [23, 55]. Ultrasonic actuation for cell transfer would subject the cells to stress in the form of heat and vibration [56, 57]. Therefore, it is important to validate the response of printed cells to any particular bioprinting process in terms of their viability and more importantly their biological functions.

We previously reported the results of the first experiments printing hESCs using a valve-based printing approach including their response to the printing process in the form of post-printed viability and pluripotency validation [37]. However, if hPSCs are to be used for producing human tissues on demand for drug testing, their post-printing differentiation must be reproducibly directed to the required lineages for each tissue. Unfortunately homogenous cellular differentiation of hPSCs into some germ layers has proved difficult [12, 13]. Here, we report the first investigation into the bioprinting of human iPSCs, their response to the valve-based printing process as well as their post-printing differentiation into hepatocyte-like cells (HLCs). HLCs that are in the process of differentiating are bioprinted and examined to further validate the compatibility of the valve-based bioprinting process with fragile cell transfer. Finally, 3D hydrogel structures were designed and printed out with encapsulated hESC-derived HLCs and the viability and hepatic characteristics of the cells were investigated.

 

A newer version of our previously reported cell printing platform [37] has been developed. Four nanolitre dispensing systems, each comprising a solenoid valve (VHS Nanolitre Dispense Valve, Lee Products Ltd) with 101.6 μm internal diameter nozzles (Minstac Nozzle, Lee Products Ltd), were attached to static pressure reservoirs for the bio-ink solution to be dispensed from via flexible tubing. The nanolitre dispensing system and bio-ink reservoirs were mounted onto the tool head of an enclosed custom built micrometer-resolution 3-axis XY–Z stage (figure 1). This newer cell printing platform improved on the previous version by reducing the overall size and weight of the machine, allowing it to be mounted inside a standard tissue culture hood during experiments requiring a sterile environment. Other enhancements included the two extra nanolitre dispensing systems, taking the total up to four, a more robust electronics and custom firmware was developed which improved the reliability and speed of the machine and two separate pressure channels were included, allowing for differential bio-ink dispensing conditions. Unless otherwise stated standard printing conditions were used: for 2D, printing was carried out using a pulse time of 8 ms at an inlet pressure of 0.6 bar using a nozzle with an internal diameter of 101.6 μm; for 3D, printing was carried out using a pulse time of 400 μs at an inlet pressure of 1.0 bar for sodium alginate solution and a pulse time of 400 μs at an inlet pressure of 0.5 bar for calcium chloride solution both using nozzles with an internal diameter of 101.6 μm.

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Figure 1. (a) Schematic drawing of the cell printer system; (b) detailed schematic of the micro-solenoid valve; (c) schematic of the combinatorial printing process for alginate hydrogel creation; (d) a 3D printed alginate tube structure approximately 13 mm tall printed with 1.5% w/v Sodium Alginate and 600 mM (6%) Calcium Chloride solutions in Millipore water (scale bar 2 mm).

 

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The process of in vivo liver organogenesis occurs in the developing foregut, when newly specified hepatic cells separate from the endodermal sheet and form a dense 3D structure known as a hepatoblast (liver bud) [74, 75]. It is hypothesized that arranging the hESC–HLCs in 3D during the differentiation process may yield more mature hepatocytes than conventional 2D differentiation. The hESC differentiation protocols are more efficient and robust than hiPSC protocols therefore only hESC-derived HLCs were printed in 3D.

In order for this technique to be useful for tissue engineering applications, structures need to be tall enough to allow cells to interact in a three-dimensional environment. The concentration of alginate solution was set to 1.5% w/v to improve the mechanical strength of the hydrogel and allow it to support further layers. Circular structures with a large number of layers were designed and printed out in the wells of a multi-well plate to allow the structures to be cultured post-printing. These resulting structures were photographed for analysis and are shown in figure 7below.

These structures were printed out in a matter of minutes and are strong enough to support their own weight and the weight of further layers (as seen in figure 1(d)). The structures spread slightly, but by slightly altering the volume ratio, concentrations and surface properties this spreading can be reduced.

Approximately one hour post-printing one of the HLC-laden alginate ring structures was examined using a confocal microscope; the 3D image is shown in figure 8(a). Cell viability was calculated to be 55.5% using the Imaris confocal microscope software. Cell viability declined over the first 24 h which resulted in low cell numbers for hepatic marker testing following the 3D differentiation process, but the viability remained stable for the remainder of the differentiation process. At day 23 of the differentiation process, the cells in the remaining structures were harvested and stained for the presence of hepatic markers. As shown in figure 8(b), cells are positive for albumin which demonstrates their hepatic lineage. The normal time required for 2D differentiation of hPSC-HLCs is 17–24 d. However, based on the results of albumin secretion in the medium, we observed the 3D printed cells have taken longer to reach the maximum albumin secretion than the 2D control as shown in figure 8(c). Interestingly, when analyzing the difference between 20 and 40 layer printed tube structures, we noticed close-to proportional increase in albumin secretion to the number of layers as shown in figure 8(d). This indicates that the permeability of the alginate hydrogel allows nutrition and differentiation reagents to enter the structure and support 3D differentiation and maturation processes of the cells, regardless of the height of the printed structure.

Research is currently underway including investigations to improve the 3D viability and adjusting the differentiation protocol that may facilitate higher albumin secretion. For example, the optimization of hydrogel formation as well as enhanced cell density may improve the differentiation process for hPSCs in 3D [21, 76, 77].

4. Conclusions

To the best of our knowledge, this study is the first to demonstrate that hiPS cells can be bioprinted without adversely affecting their biological functions including viability and pluripotency. Importantly, we verified that our valve-based printing process is gentle enough to not affect the pluripotency of both hESCs and hiPSCs. A number of different hPSC lines were directed to differentiate into HLCs. Cells were printed during the differentiation process and showed no differences in hepatocyte marker expression and similar morphology when compared to a non-printed control. We previously reported the results of an investigation into the response of hESCs to the valve-based printing process. Here we build on that study, performing a deeper investigation to compare the response of hiPSCs and hESCs to the printing process using flow cytometry. The effect of nozzle geometry was investigated and the effects of nozzle length on the post-printing viability of cells were recorded; longer nozzles lower the post-printing viability of the cells. We printed hESC-derived HLCs in a 3D alginate matrix and tested for viability and hepatic markers during the remaining differentiation and they were found to be hepatic in nature. The ability to bioprint hPSCs while either maintaining their pluripotency or directing their differentiation into specific cell types will pave the way for producing organs or tissues on demand from patient specific cells which could be used for animal-free drug development and personalized medicine.

 

 

Large scale industrialized cell expansion: producing the critical raw material for biofabrication processes

Arun Kumar1 and Binil Starly1,2

http://iopscience.iop.org/article/10.1088/1758-5090/7/4/044103

Cellular biomanufacturing technologies are a critical link to the successful application of cell and scaffold based regenerative therapies, organs-on-chip devices, disease models and any products with living cells contained in them. How do we achieve production level quantities of the key ingredient—’the living cells‘ for all biofabrication processes, including bioprinting and biopatterning? We review key cell expansion based bioreactor operating principles and how 3D culture will play an important role in achieving production quantities of billions to even trillions of anchorage dependent cells. Furthermore, we highlight some of the challenges in the field of cellular biomanufacturing that must be addressed to achieve desired cellular yields while adhering to the key pillars of good manufacturing practices—safety, purity, stability, potency and identity. Biofabrication technologies are uniquely positioned to provide improved 3D culture surfaces for the industrialized production of living cells.

Biofabrication of tissue constructs by 3D bioprinting of cell-laden microcarriers

Riccardo Levato1,2, Jetze Visser3, Josep A Planell1, Elisabeth Engel1,2,4, Jos Malda3,5 andMiguel A Mateos-Timoneda2,1

http://iopscience.iop.org/article/10.1088/1758-5082/6/3/035020

Bioprinting allows the fabrication of living constructs with custom-made architectures by spatially controlled deposition of multiple bioinks. This is important for the generation of tissue, such as osteochondral tissue, which displays a zonal composition in the cartilage domain supported by the underlying subchondral bone. Challenges in fabricating functional grafts of clinically relevant size include the incorporation of cues to guide specific cell differentiation and the generation of sufficient cells, which is hard to obtain with conventional cell culture techniques. A novel strategy to address these demands is to combine bioprinting with microcarrier technology. This technology allows for the extensive expansion of cells, while they form multi-cellular aggregates, and their phenotype can be controlled. In this work, living constructs were fabricated via bioprinting of cell-laden microcarriers. Mesenchymal stromal cell (MSC)-laden polylactic acid microcarriers, obtained via static culture or spinner flask expansion, were encapsulated in gelatin methacrylamide-gellan gum bioinks, and the printability of the composite material was studied. This bioprinting approach allowed for the fabrication of constructs with high cell concentration and viability. Microcarrier encapsulation improved the compressive modulus of the hydrogel constructs, facilitated cell adhesion, and supported osteogenic differentiation and bone matrix deposition by MSCs. Bilayered osteochondral models were fabricated using microcarrier-laden bioink for the bone compartment. These findings underscore the potential of this new microcarrier-based biofabrication approach for bone and osteochondral constructs.

Microstereolithography and characterization of poly(propylene fumarate)-based drug-loaded microneedle arrays

Yanfeng Lu1, Satya Nymisha Mantha1, Douglas C Crowder2, Sofia Chinchilla2, Kush N Shah2,3,4,Yang H Yun2, Ryan B Wicker5 and Jae-Won Choi1

http://iopscience.iop.org/article/10.1088/1758-5090/7/4/045001/meta

Drug-loaded microneedle arrays for transdermal delivery of a chemotherapeutic drug were fabricated using multi-material microstereolithography (μSL). These arrays consisted of twenty-five poly(propylene fumarate) (PPF) microneedles, which were precisely orientated on the same polymeric substrate. To control the viscosity and improve the mechanical properties of the PPF, diethyl fumarate (DEF) was mixed with the polymer. Dacarbazine, which is widely used for skin cancer, was uniformly blended into the PPF/DEF solution prior to crosslinking. Each microneedle has a cylindrical base with a height of 700 μm and a conical tip with a height of 300μm. Compression test results and characterization of the elastic moduli of the PPF/DEF (50:50) and PPF/drug mixtures indicated that the failure force was much larger than the theoretical skin insertion force. The release kinetics showed that dacarbazine can be released at a controlled rate for five weeks. The results demonstrated that the PPF-based drug-loaded microneedles are a potential method to treat skin carcinomas. In addition, μSL is an attractive manufacturing technique for biomedical applications, especially for micron-scale manufacturing.

Controlling shape and position of vascular formation in engineered tissues by arbitrary assembly of endothelial cells

Hiroaki Takehara1,4, Katsuhisa Sakaguchi2, Masatoshi Kuroda3, Megumi Muraoka3, Kazuyoshi Itoga1,Teruo Okano1 and Tatsuya Shimizu1
http://iopscience.iop.org/article/10.1088/1758-5090/7/4/045006/meta

 

Cellular self-assembly based on cell-to-cell communication is a well-known tissue organizing process in living bodies. Hence, integrating cellular self-assembly processes into tissue engineering is a promising approach to fabricate well-organized functional tissues. In this research, we investigated the capability of endothelial cells (ECs) to control shape and position of vascular formation using arbitral-assembling techniques in three-dimensional engineered tissues. To quantify the degree of migration of ECs in endothelial network formation, image correlation analysis was conducted. Positive correlation between the original positions of arbitrarily assembled ECs and the positions of formed endothelial networks indicated the potential for controlling shape and position of vascular formations in engineered tissues. To demonstrate the feasibility of controlling vascular formations, engineered tissues with vascular networks in triangle and circle patterns were made. The technique reported here employs cellular self-assembly for tissue engineering and is expected to provide fundamental beneficial methods to supply various functional tissues for drug screening and regenerative medicine.

The influence of printing parameters on cell survival rate and printability in microextrusion-based 3D cell printing technology

Yu Zhao1,2, Yang Li1,2, Shuangshuang Mao1,2, Wei Sun1,2,3,4 and Rui Yao1,2
http://iopscience.iop.org/article/10.1088/1758-5090/7/4/045002/meta

Three-dimensional (3D) cell printing technology has provided a versatile methodology to fabricate cell-laden tissue-like constructs and in vitro tissue/pathological models for tissue engineering, drug testing and screening applications. However, it still remains a challenge to print bioinks with high viscoelasticity to achieve long-term stable structure and maintain high cell survival rate after printing at the same time. In this study, we systematically investigated the influence of 3D cell printing parameters, i.e. composition and concentration of bioink, holding temperature and holding time, on the printability and cell survival rate in microextrusion-based 3D cell printing technology. Rheological measurements were utilized to characterize the viscoelasticity of gelatin-based bioinks. Results demonstrated that the bioink viscoelasticity was increased when increasing the bioink concentration, increasing holding time and decreasing holding temperature below gelation temperature. The decline of cell survival rate after 3D cell printing process was observed when increasing the viscoelasticity of the gelatin-based bioinks. However, different process parameter combinations would result in the similar rheological characteristics and thus showed similar cell survival rate after 3D bioprinting process. On the other hand, bioink viscoelasticity should also reach a certain point to ensure good printability and shape fidelity. At last, we proposed a protocol for 3D bioprinting of temperature-sensitive gelatin-based hydrogel bioinks with both high cell survival rate and good printability. This research would be useful for biofabrication researchers to adjust the 3D bioprinting process parameters quickly and as a referable template for designing new bioinks.

A new method of fabricating a blend scaffold using an indirect three-dimensional printing technique

Jin Woo Jung1,3, Hyungseok Lee1,3, Jung Min Hong1, Jeong Hun Park1, Jung Hee Shim2, Tae Hyun Choi2and Dong-Woo Cho1
http://iopscience.iop.org/article/10.1088/1758-5090/7/4/045003/meta

Due to its simplicity and effectiveness, the physical blending of polymers is considered to be a practical strategy for developing a versatile scaffold having desirable mechanical and biochemical properties. In the present work, an indirect three-dimensional (i3D) printing technique was proposed to fabricate a 3D free-form scaffold using a blend of immiscible materials, such as polycaprolactone (PCL) and gelatin. The i3D printing technique includes 3D printing of a mold and a sacrificial molding process. PCL/chloroform and gelatin/water were physically mixed to prepare the blend solution, which was subsequently injected into the cavity of a 3D printed mold. After solvent removal and gelatin cross-linking, the mold was dissolved to obtain a PCL–gelatin (PG) scaffold, with a specific 3D structure. Scanning electron microscopy and Fourier transform infrared spectroscopy analysis indicated that PCL masses and gelatin fibers in the PG scaffold homogenously coexisted without chemical bonding. Compression tests confirmed that gelatin incorporation into the PCL enhanced its mechanical flexibility and softness, to the point of being suitable for soft-tissue engineering, as opposed to pure PCL. Human adipose-derived stem cells, cultured on a PG scaffold, exhibited enhanced in vitro chondrogenic differentiation and tissue formation, compared with those on a PCL scaffold. The i3D printing technique can be used to blend a variety of materials, facilitating 3D scaffold fabrication for specific tissue regeneration. Furthermore, this convenient and versatile technique may lead to wider application of 3D printing in tissue engineering.

 

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