Posts Tagged ‘3D bioprinting’

Use of 3D Bioprinting for Development of Toxicity Prediction Models

Curator: Stephen J. Williams, PhD

SOT FDA Colloquium on 3D Bioprinted Tissue Models: Tuesday, April 9, 2019

The Society of Toxicology (SOT) and the U.S. Food and Drug Administration (FDA) will hold a workshop on “Alternative Methods for Predictive Safety Testing: 3D Bioprinted Tissue Models” on Tuesday, April 9, at the FDA Center for Food Safety and Applied Nutrition in College Park, Maryland. This workshop is the latest in the series, “SOT FDA Colloquia on Emerging Toxicological Science: Challenges in Food and Ingredient Safety.”

Human 3D bioprinted tissues represent a valuable in vitro approach for chemical, personal care product, cosmetic, and preclinical toxicity/safety testing. Bioprinting of skin, liver, and kidney is already appearing in toxicity testing applications for chemical exposures and disease modeling. The use of 3D bioprinted tissues and organs may provide future alternative approaches for testing that may more closely resemble and simulate intact human tissues to more accurately predict human responses to chemical and drug exposures.

A synopsis of the schedule and related works from the speakers is given below:


8:40 AM–9:20 AM Overview and Challenges of Bioprinting
Sharon Presnell, Amnion Foundation, Winston-Salem, NC
9:20 AM–10:00 AM Putting 3D Bioprinting to the Use of Tissue Model Fabrication
Y. Shrike Zhang, Brigham and Women’s Hospital, Harvard Medical School and Harvard-MIT Division of Health Sciences and Technology, Boston, MA
10:00 AM–10:20 AM Break
10:20 AM–11:00 AM Uses of Bioprinted Liver Tissue in Drug Development
Jean-Louis Klein, GlaxoSmithKline, Collegeville, PA
11:00 AM–11:40 AM Biofabrication of 3D Tissue Models for Disease Modeling and Chemical Screening
Marc Ferrer, National Center for Advancing Translational Sciences, NIH, Rockville, MD

Sharon Presnell, Ph.D. President, Amnion Foundation

Dr. Sharon Presnell was most recently the Chief Scientific Officer at Organovo, Inc., and the President of their wholly-owned subsidiary, Samsara Sciences. She received a Ph.D. in Cell & Molecular Pathology from the Medical College of Virginia and completed her undergraduate degree in biology at NC State. In addition to her most recent roles, Presnell has served as the director of cell biology R&D at Becton Dickinson’s corporate research center in RTP, and as the SVP of R&D at Tengion. Her roles have always involved the commercial and clinical translation of basic research and early development in the cell biology space. She serves on the board of the Coulter Foundation at the University of Virginia and is a member of the College of Life Sciences Foundation Board at NC State. In January 2019, Dr. Presnell will begin a new role as President of the Amnion Foundation, a non-profit organization in Winston-Salem.

A few of her relevant publications:

Bioprinted liver provides early insight into the role of Kupffer cells in TGF-β1 and methotrexate-induced fibrogenesis

Integrating Kupffer cells into a 3D bioprinted model of human liver recapitulates fibrotic responses of certain toxicants in a time and context dependent manner.  This work establishes that the presence of Kupffer cells or macrophages are important mediators in fibrotic responses to certain hepatotoxins and both should be incorporated into bioprinted human liver models for toxicology testing.

Bioprinted 3D Primary Liver Tissues Allow Assessment of Organ-Level Response to Clinical Drug Induced Toxicity In Vitro

Abstract: Modeling clinically relevant tissue responses using cell models poses a significant challenge for drug development, in particular for drug induced liver injury (DILI). This is mainly because existing liver models lack longevity and tissue-level complexity which limits their utility in predictive toxicology. In this study, we established and characterized novel bioprinted human liver tissue mimetics comprised of patient-derived hepatocytes and non-parenchymal cells in a defined architecture. Scaffold-free assembly of different cell types in an in vivo-relevant architecture allowed for histologic analysis that revealed distinct intercellular hepatocyte junctions, CD31+ endothelial networks, and desmin positive, smooth muscle actin negative quiescent stellates. Unlike what was seen in 2D hepatocyte cultures, the tissues maintained levels of ATP, Albumin as well as expression and drug-induced enzyme activity of Cytochrome P450s over 4 weeks in culture. To assess the ability of the 3D liver cultures to model tissue-level DILI, dose responses of Trovafloxacin, a drug whose hepatotoxic potential could not be assessed by standard pre-clinical models, were compared to the structurally related non-toxic drug Levofloxacin. Trovafloxacin induced significant, dose-dependent toxicity at clinically relevant doses (≤ 4uM). Interestingly, Trovafloxacin toxicity was observed without lipopolysaccharide stimulation and in the absence of resident macrophages in contrast to earlier reports. Together, these results demonstrate that 3D bioprinted liver tissues can both effectively model DILI and distinguish between highly related compounds with differential profile. Thus, the combination of patient-derived primary cells with bioprinting technology here for the first time demonstrates superior performance in terms of mimicking human drug response in a known target organ at the tissue level.

A great interview with Dr. Presnell and the 3D Models 2017 Symposium is located here:

Please click here for Web based and PDF version of interview

Some highlights of the interview include

  • Exciting advances in field showing we can model complex tissue-level disease-state phenotypes that develop in response to chronic long term injury or exposure
  • Sees the field developing a means to converge both the biology and physiology of tissues, namely modeling the connectivity between tissues such as fluid flow
  • Future work will need to be dedicated to develop comprehensive analytics for 3D tissue analysis. As she states “we are very conditioned to get information in a simple way from biochemical readouts in two dimension, monocellular systems”  however how we address the complexity of various cellular responses in a 3D multicellular environment will be pertinent.
  • Additional challenges include the scalability of such systems and making such system accessible in a larger way
  1. Shrike Zhang, Brigham and Women’s Hospital, Harvard Medical School and Harvard-MIT Division of Health Sciences and Technology

Dr. Zhang currently holds an Assistant Professor position at Harvard Medical School and is an Associate Bioengineer at Brigham and Women’s Hospital. His research interests include organ-on-a-chip, 3D bioprinting, biomaterials, regenerative engineering, biomedical imaging, biosensing, nanomedicine, and developmental biology. His scientific contributions have been recognized by >40 international, national, and regional awards. He has been invited to deliver >70 lectures worldwide, and has served as reviewer for >400 manuscripts for >30 journals. He is serving as Editor-in-Chief for Microphysiological Systems, and Associate Editor for Bio-Design and Manufacturing. He is also on Editorial Board of BioprintingHeliyonBMC Materials, and Essays in Biochemistry, and on Advisory Panel of Nanotechnology.

Some relevant references from Dr. Zhang

Multi-tissue interactions in an integrated three-tissue organ-on-a-chip platform.

Skardal A, Murphy SV, Devarasetty M, Mead I, Kang HW, Seol YJ, Shrike Zhang Y, Shin SR, Zhao L, Aleman J, Hall AR, Shupe TD, Kleensang A, Dokmeci MR, Jin Lee S, Jackson JD, Yoo JJ, Hartung T, Khademhosseini A, Soker S, Bishop CE, Atala A.

Sci Rep. 2017 Aug 18;7(1):8837. doi: 10.1038/s41598-017-08879-x.


Reconstruction of Large-scale Defects with a Novel Hybrid Scaffold Made from Poly(L-lactic acid)/Nanohydroxyapatite/Alendronate-loaded Chitosan Microsphere: in vitro and in vivo Studies.

Wu H, Lei P, Liu G, Shrike Zhang Y, Yang J, Zhang L, Xie J, Niu W, Liu H, Ruan J, Hu Y, Zhang C.

Sci Rep. 2017 Mar 23;7(1):359. doi: 10.1038/s41598-017-00506-z.



A liver-on-a-chip platform with bioprinted hepatic spheroids.

Bhise NS, Manoharan V, Massa S, Tamayol A, Ghaderi M, Miscuglio M, Lang Q, Shrike Zhang Y, Shin SR, Calzone G, Annabi N, Shupe TD, Bishop CE, Atala A, Dokmeci MR, Khademhosseini A.

Biofabrication. 2016 Jan 12;8(1):014101. doi: 10.1088/1758-5090/8/1/014101.


Marc Ferrer, National Center for Advancing Translational Sciences, NIH

Marc Ferrer is a team leader in the NCATS Chemical Genomics Center, which was part of the National Human Genome Research Institute when Ferrer began working there in 2010. He has extensive experience in drug discovery, both in the pharmaceutical industry and academic research. Before joining NIH, he was director of assay development and screening at Merck Research Laboratories. For 10 years at Merck, Ferrer led the development of assays for high-throughput screening of small molecules and small interfering RNA (siRNA) to support programs for lead and target identification across all disease areas.

At NCATS, Ferrer leads the implementation of probe development programs, discovery of drug combinations and development of innovative assay paradigms for more effective drug discovery. He advises collaborators on strategies for discovering small molecule therapeutics, including assays for screening and lead identification and optimization. Ferrer has experience implementing high-throughput screens for a broad range of disease areas with a wide array of assay technologies. He has led and managed highly productive teams by setting clear research strategies and goals and by establishing effective collaborations between scientists from diverse disciplines within industry, academia and technology providers.

Ferrer has a Ph.D. in biological chemistry from the University of Minnesota, Twin Cities, and completed postdoctoral training at Harvard University’s Department of Molecular and Cellular Biology. He received a B.Sc. degree in organic chemistry from the University of Barcelona in Spain.


Some relevant references for Dr. Ferrer

Fully 3D Bioprinted Skin Equivalent Constructs with Validated Morphology and Barrier Function.

Derr K, Zou J, Luo K, Song MJ, Sittampalam GS, Zhou C, Michael S, Ferrer M, Derr P.

Tissue Eng Part C Methods. 2019 Apr 22. doi: 10.1089/ten.TEC.2018.0318. [Epub ahead of print]


Determination of the Elasticity Modulus of 3D-Printed Octet-Truss Structures for Use in Porous Prosthesis Implants.

Bagheri A, Buj-Corral I, Ferrer M, Pastor MM, Roure F.

Materials (Basel). 2018 Nov 29;11(12). pii: E2420. doi: 10.3390/ma11122420.


Mutation Profiles in Glioblastoma 3D Oncospheres Modulate Drug Efficacy.

Wilson KM, Mathews-Griner LA, Williamson T, Guha R, Chen L, Shinn P, McKnight C, Michael S, Klumpp-Thomas C, Binder ZA, Ferrer M, Gallia GL, Thomas CJ, Riggins GJ.

SLAS Technol. 2019 Feb;24(1):28-40. doi: 10.1177/2472630318803749. Epub 2018 Oct 5.


A high-throughput imaging and nuclear segmentation analysis protocol for cleared 3D culture models.

Boutin ME, Voss TC, Titus SA, Cruz-Gutierrez K, Michael S, Ferrer M.

Sci Rep. 2018 Jul 24;8(1):11135. doi: 10.1038/s41598-018-29169-0.

A High-Throughput Screening Model of the Tumor Microenvironment for Ovarian Cancer Cell Growth.

Lal-Nag M, McGee L, Guha R, Lengyel E, Kenny HA, Ferrer M.

SLAS Discov. 2017 Jun;22(5):494-506. doi: 10.1177/2472555216687082. Epub 2017 Jan 31.


Exploring Drug Dosing Regimens In Vitro Using Real-Time 3D Spheroid Tumor Growth Assays.

Lal-Nag M, McGee L, Titus SA, Brimacombe K, Michael S, Sittampalam G, Ferrer M.

SLAS Discov. 2017 Jun;22(5):537-546. doi: 10.1177/2472555217698818. Epub 2017 Mar 15.


RNAi High-Throughput Screening of Single- and Multi-Cell-Type Tumor Spheroids: A Comprehensive Analysis in Two and Three Dimensions.

Fu J, Fernandez D, Ferrer M, Titus SA, Buehler E, Lal-Nag MA.

SLAS Discov. 2017 Jun;22(5):525-536. doi: 10.1177/2472555217696796. Epub 2017 Mar 9.


Other Articles on 3D Bioprinting on this Open Access Journal include:

Global Technology Conferences on 3D BioPrinting 2015 – 2016

3D Medical BioPrinting Technology Reporting by Irina Robu, PhD – a forthcoming Article in “Medical 3D BioPrinting – The Revolution in Medicine, Technologies for Patient-centered Medicine: From R&D in Biologics to New Medical Devices”

Bio-Inks and 3D BioPrinting

New Scaffold-Free 3D Bioprinting Method Available to Researchers

Gene Editing for Gene Therapies with 3D BioPrinting



Read Full Post »

New 3D-printed Device could Help Treat Spinal Cord Injuries

Reporter: Irina Robu, PhD

Every ten minutes, a person is added to the national transplant waiting list in the US alone, where on average 20 people die each day while waiting for a transplant. The shortage of organ donors is not just confined to the US and scientists are turning to technology for help against this worldwide issue.

Bioprinting sounds innovative, but it has a potential to be the next big thing in healthcare and the hope is that printing and transplanting an organ will take a few hours without any risk of rejection from the body. These printed organs are created from the very cells of the body they will re-enter, matching the exact size, specifications and requirements of each individual patient. The artificial creation of human skin, tissue and internal organs sounds like something from the distant future, nevertheless much of it is happening right now in research facilities around the globe and providing new options for treatment.

Medical researchers and engineers at University of Minnesota created a groundbreaking 3-D printed device that could help patients with long term spinal injuries regain some function. A 3-D printed silicone guide, serves as a
platform for specialized cells that are then 3-D printed on top of it. The guide would be surgically implanted into the injured area of the spinal cord where it would serve as a “bridge” between living nerve cells above and below the area of injury.

According to Dr. Ann Parr “This is a very exciting first step in developing a treatment to help people with spinal cord injuries.” The expectation is that this would help patients alleviate pain as well as regain some functions like control of muscles, bowel and bladder. In the current experiments developed at University of Minnesota, years, researchers start with any kind of cell from an adult, such as a skin cell or blood cell which then use to reprogram the cells into neuronal stem cells. The engineers print these cells onto a silicone guide using an exclusive 3-D-printing technology in which the same 3-D printer is used to print both the guide and the cells. The guide keeps the cells alive and allows them to change into neurons. The team developed a prototype guide that would be surgically implanted into the damaged part of the spinal cord and help connect living cells on each side of the injury.

Despite all of these complexities, the hardest part of the entire procedure is being able to keep about 75% of cells during the 3-D printing process. But even with the latest technology, developing the prototype guides wasn’t easy. But although the research is very exciting, we need to be careful to offset expectations against reality. While the research still needs more work, there is no doubt that the future of healthcare and medicine will be very different thanks to this research.



Read Full Post »

First 3D Printed Tibia Replacement

Reporter: Irina Robu, PhD

Current advances have allowed 3D printing of biocompatible materials, cells and supporting components into complex 3D functional living tissues. 3D bioprinting has already been used for the generation and transplantation of several tissues, including multilayered skin, bone, vascular grafts, tracheal splints, heart tissue and cartilaginous structures. Thanks to 3D printing, an Australian man got to keep his leg. The man, Reuben Lichter nearly lost his leg above the knee due to a bacterial infection. Doctors told him that he had osteomyelitis which infected his entire bone. Lichter’s bacterial disease of osteomyelitis affects 2 in every 10,000 people in the United States. He had two choices: an experimental procedure using the 3D printed bone or lose his leg. For Lichter, the choice was easy.

Michael Wagels who served as the lead surgeon performed the world’s first-ever transplant surgery using a 3D printed bone. The scaffold was initially modeled at Queensland University of Technology. Biomedical engineers designed the scaffold to promote bone growth around it and then slowly dissolve over time. To have the body successfully grow around the scaffold, the team introduced tissue and blood vessels from both of Lichter’s legs to the scaffold. The surgery itself happened over five operations at Brisbane’s Princess Alexandra Hospital.

However, the next major challenge for biomedical engineers is how to successfully 3D print organs.


Read Full Post »

3-D Printed Ovaries Produce Healthy Offspring

Reporter: Irina Robu, PhD


Each year about 120,000 organs are transplanted from one human being to another and most of the time is a living volunteer. But lack of suitable donors, predominantly means the supply of such organs is inadequate. Countless people consequently die waiting for a transplant which has led researchers to study the question of how to build organs from scratch.

One promising approach is to print them, but “bioprinting” remains largely experimental. Nevertheless, bioprinted tissue is before now being sold for drug testing, and the first transplantable tissues are anticipated to be ready for use in a few years’ time. The first 3D printed organ includes bioprosthetic ovaries which are constructed of 3D printed scaffolds that have immature eggs and have been successful in boosting hormone production and restoring fertility was developed by Teresa K. Woodruff, a reproductive scientist and director of the Women’s Health Research Institute at Feinberg School of Medicine, at Northwestern University, in Illinois.

What sets apart these bioprosthetic ovaries is the architecture of the scaffold. The material is made of gelatin made from broken-down collagen that is safe to humans which is self-supporting and can lead to building multiple layers.

The 3-D printed “scaffold” or “skeleton” is implanted into a female and its pores can be used to optimize how follicles, or immature eggs, get wedged within the scaffold. The scaffold supports the survival of the mouse’s immature egg cells and the cells that produce hormones to boost production. The open construction permits room for the egg cells to mature and ovulate, blood vessels to form within the implant enabling the hormones to circulate and trigger lactation after giving birth. The purpose of this scaffold is to recapitulate how an ovary would function.
The scientists’ only objective for developing the bioprosthetic ovaries was to help reestablish fertility and hormone production in women who have suffered adult cancer treatments and now have bigger risks of infertility and hormone-based developmental issues.



Printed human body parts could soon be available for transplant


3D printed ovaries produce healthy offspring giving hope to infertile women


Brave new world: 3D-printed ovaries produce healthy offspring


3-D-printed scaffolds restore ovary function in infertile mice


Our Grandkids May Be Born From 3D-Printed Ovaries


Read Full Post »

BioPrinting Basics

Curator: Larry H. Bernstein, MD, FCAP



The ABCs of 3D Bioprinting of Living Tissues, Organs   5/06/2016 

(Credit: Ozbolat Lab/Penn State University)
(Credit: Ozbolat Lab/Penn State University)

Although first originated in 2003, the world of bioprinting is still very new and ambiguous. Nevertheless, as the need for organ donation continues to increase worldwide, and organ and tissue shortages prevail, a handful of scientists have started utilizing this cutting-edge science and technology for various areas of regenerative medicine to possibly fill that organ-shortage void.

Among these scientists is Ibrahim Tarik Ozbolat, an associate professor of Engineering Science and Mechanics Department and the Huck Institutes of the Life Sciences at Penn State University, who’s been studying bioprinting and tissue engineering for years.

While Ozbolat is not the first to originate 3D bioprinting research, he’s the first one at Penn State University to spearhead the studies at Ozbolat Lab, Leading Bioprinting Research.

“Tissue engineering is a big need. Regenerative medicine, biofabrication of tissues and organs that can replace the damage or diseases is important,” Ozbolat told R&D Magazine after his seminar presentation at Interphex last week in New York City, titled 3D Bioprinting of Living Tissues & Organs.”

3D bioprinting is the process of creating cell patterns in a confined space using 3D-printing technologies, where cell function and viability are preserved within the printed construct.

Recent progress has allowed 3D printing of biocompatible materials, cells and supporting components into complex 3D functional living tissues. The technology is being applied to regenerative medicine to address the need for tissues and organs suitable for transplantation. Compared with non-biological printing, 3D bioprinting involves additional complexities, such as the choice of materials, cell types, growth and differentiation factors, and technical challenges related to the sensitivities of living cells and the construction of tissues. Addressing these complexities requires the integration of technologies from the fields of engineering, biomaterials science, cell biology, physics and medicine, according to

“If we’re able to make organs on demand, that will be highly beneficial to society,” said Ozbolat. “We have the capability to pattern cells, locate them and then make the same thing that exists in the body.”

3D bioprinting of tissues and organs

Sean V Murphy & Anthony Atala
Nature Biotechnology 32,773–785(2014)       doi:10.1038/nbt.2958


Additive manufacturing, otherwise known as three-dimensional (3D) printing, is driving major innovations in many areas, such as engineering, manufacturing, art, education and medicine. Recent advances have enabled 3D printing of biocompatible materials, cells and supporting components into complex 3D functional living tissues. 3D bioprinting is being applied to regenerative medicine to address the need for tissues and organs suitable for transplantation. Compared with non-biological printing, 3D bioprinting involves additional complexities, such as the choice of materials, cell types, growth and differentiation factors, and technical challenges related to the sensitivities of living cells and the construction of tissues. Addressing these complexities requires the integration of technologies from the fields of engineering, biomaterials science, cell biology, physics and medicine. 3D bioprinting has already been used for the generation and transplantation of several tissues, including multilayered skin, bone, vascular grafts, tracheal splints, heart tissue and cartilaginous structures. Other applications include developing high-throughput 3D-bioprinted tissue models for research, drug discovery and toxicology.


Future Technologies : Bioprinting

3D printing is increasingly permitting the direct digital manufacture (DDM) of a wide variety of plastic and metal items. While this in itself may trigger a manufacturing revolution, far more startling is the recent development of bioprinters. These artificially construct living tissue by outputting layer-upon-layer of living cells. Currently all bioprinters are experimental. However, in the future, bioprinters could revolutionize medical practice as yet another element of the New Industrial Convergence.

Bioprinters may be constructed in various configurations. However, all bioprinters output cells from a bioprint head that moves left and right, back and forth, and up and down, in order to place the cells exactly where required. Over a period of several hours, this permits an organic object to be built up in a great many very thin layers.

In addition to outputting cells, most bioprinters also output a dissolvable gel to support and protect cells during printing. A possible design for a future bioprinter appears below and in the sidebar, here shown in the final stages of printing out a replacement human heart. Note that you can access larger bioprinter images on the Future Visions page. You may also like to watch my bioprinting video.



Bioprinting Pioneers

Several experimental bioprinters have already been built. For example, in 2002 Professor Makoto Nakamura realized that the droplets of ink in a standard inkjet printer are about the same size as human cells. He therefore decided to adapt the technology, and by 2008 had created a working bioprinter that can print out biotubing similar to a blood vessel. In time, Professor Nakamura hopes to be able to print entire replacement human organs ready for transplant. You can learn more about this groundbreaking work here or read this message from Professor Nakamura. The movie below shows in real-time the biofabrication of a section of biotubing using his modified inkjet technology.


Another bioprinting pioneer is Organovo. This company was set up by a research group lead by Professor Gabor Forgacs from the University of Missouri, and in March 2008 managed to bioprint functional blood vessels and cardiac tissue using cells obtained from a chicken. Their work relied on a prototype bioprinter with three print heads. The first two of these output cardiac and endothelial cells, while the third dispensed a collagen scaffold — now termed ‘bio-paper’ — to support the cells during printing.

Since 2008, Organovo has worked with a company called Invetech to create a commercial bioprinter called the NovoGen MMX. This is loaded with bioink spheroids that each contain an aggregate of tens of thousands of cells. To create its output, the NovoGen first lays down a single layer of a water-based bio-paper made from collagen, gelatin or other hydrogels. Bioink spheroids are then injected into this water-based material. As illustrated below, more layers are subsequently added to build up the final object. Amazingly, Nature then takes over and the bioink spheroids slowly fuse together. As this occurs, the biopaper dissolves away or is otherwise removed, thereby leaving a final bioprinted body part or tissue.


bioprinting stages

As Organovo have demonstrated, using their bioink printing process it is not necessary to print all of the details of an organ with a bioprinter, as once the relevant cells are placed in roughly the right place Nature completes the job. This point is powerfully illustrated by the fact that the cells contained in a bioink spheroid are capable of rearranging themselves after printing. For example, experimental blood vessels have been bioprinted using bioink spheroids comprised of an aggregate mix of endothelial, smooth muscle and fibroblast cells. Once placed in position by the bioprint head, and with no technological intervention, the endothelial cells migrate to the inside of the bioprinted blood vessel, the smooth muscle cells move to the middle, and the fibroblasts migrate to the outside.

In more complex bioprinted materials, intricate capillaries and other internal structures also naturally form after printing has taken place. The process may sound almost magical. However, as Professor Forgacs explains, it is no different to the cells in an embryo knowing how to configure into complicated organs. Nature has been evolving this amazing capability for millions of years. Once in the right places, appropriate cell types somehow just know what to do.

In December 2010, Organovo create the first blood vessels to be bioprinted using cells cultured from a single person. The company has also successfully implanted bioprinted nerve grafts into rats, and anticipates human trials of bioprinted tissues by 2015. However, it also expects that the first commercial application of its bioprinters will be to produce simple human tissue structures for toxicology tests. These will enable medical researchers to test drugs on bioprinted models of the liver and other organs, thereby reducing the need for animal tests.

In time, and once human trials are complete, Organovo hopes that its bioprinters will be used to produce blood vessel grafts for use in heart bypass surgery. The intention is then to develop a wider range of tissue-on-demand and organs-on-demand technologies. To this end, researchers are now working on tiny mechanical devices that can artificially exercise and hence strengthen bioprinted muscle tissue before it is implanted into a patient.

Organovo anticipates that its first artificial human organ will be a kidney. This is because, in functional terms, kidneys are one of the more straight-forward parts of the body. The first bioprinted kidney may in fact not even need to look just like its natural counterpart or duplicate all of its features. Rather, it will simply have to be capable of cleaning waste products from the blood. You can read more about the work of Organovoand Professor Forgac’s in this article from Nature.

Regenerative Scaffolds and Bones

A further research team with the long-term goal of producing human organs-on-demand has created the Envisiontec Bioplotter. Like Organovo’s NovoGen MMX, this outputs bio-ink ’tissue spheroids’ and supportive scaffold materials including fibrin and collagen hydrogels. But in addition, the Envisontech can also print a wider range of biomaterials. These include biodegradable polymers and ceramics that may be used to support and help form artificial organs, and which may even be used as bioprinting substitutes for bone.

Talking of bone, a team lead by Jeremy Mao at the Tissue Engineering and Regenerative Medicine Lab at Columbia University is working on the application of bioprinting in dental and bone repairs. Already, a bioprinted, mesh-like 3D scaffold in the shape of an incisor has been implanted into the jaw bone of a rat. This featured tiny, interconnecting microchannels that contained ‘stem cell-recruiting substances’. In just nine weeks after implantation, these triggered the growth of fresh periodontal ligaments and newly formed alveolar bone. In time, this research may enable people to be fitted with living, bioprinted teeth, or else scaffolds that will cause the body to grow new teeth all by itself. You can read more about this development in this article from The Engineer.

In another experient, Mao’s team implanted bioprinted scaffolds in the place of the hip bones of several rabbits. Again these were infused with growth factors. As reported inThe Lancet, over a four month period the rabbits all grew new and fully-functional joints around the mesh. Some even began to walk and otherwise place weight on their new joints only a few weeks after surgery. Sometime next decade, human patients may therefore be fitted with bioprinted scaffolds that will trigger the grown of replacement hip and other bones. In a similar development, a team from Washington State University have also recently reported on four years of work using 3D printers to create a bone-like material that may in the future be used to repair injuries to human bones.

In Situ Bioprinting

The aforementioned research progress will in time permit organs to be bioprinted in a lab from a culture of a patient’s own cells. Such developments could therefore spark a medical revolution. Nevertheless, others are already trying to go further by developing techniques that will enable cells to be printed directly onto or into the human body in situ. Sometime next decade, doctors may therefore be able to scan wounds and spray on layers of cells to very rapidly heal them.

Already a team of bioprinting researchers lead by Anthony Alata at the Wake Forrest School of Medicine have developed a skin printer. In initial experiments they have taken 3D scans of test injuries inflicted on some mice and have used the data to control a bioprint head that has sprayed skin cells, a coagulant and collagen onto the wounds. The results are also very promising, with the wounds healing in just two or three weeks compared to about five or six weeks in a control group. Funding for the skin-printing project is coming in part from the US military who are keen to develop in situ bioprinting to help heal wounds on the battlefield. At present the work is still in a pre-clinical phase with Alata progressing his research usig pigs. However, trials of with human burn victims could be a little as five years away.

The potential to use bioprinters to repair our bodies in situ is pretty mind blowing. In perhaps no more than a few decades it may be possible for robotic surgical arms tipped with bioprint heads to enter the body, repair damage at the cellular level, and then also repair their point of entry on their way out. Patients would still need to rest and recuperate for a few days as bioprinted materials fully fused into mature living tissue. However, most patients could potentially recover from very major surgery in less than a week.

Cosmetic Applications …

Bioprinting Implications …

More information on bioprinting can be found in my books 3D Printing: Second Editionand The Next Big Thing. There is also a bioprinting section in my 3D Printing Directory. Oh, and there is also a great infographic about bioprinting here. Enjoy!


How to print out a blood vessel

New work moves closer to the age of organs on demand.

Blood vessels can now be ‘printed out’ by machine. Could bigger structures be in the future?SUSUMU NISHINAGA / SCIENCE PHOTO LIBRARY

Read Full Post »

Mid Atlantic LRIG 22nd Annual Technology Showcase: Agenda on 3D Bioprinting on Wednesday, May 11, 2016 at Holiday Inn, 195 Davidson Avenue, Somerset, NJ

Reporter: Stephen J. Williams, Ph.D.


Symposium Speakers and Topics:

Human Organoids
Hatem E. Sabaawy-Director, Production GMP Facility for Cell and Gene Therapy, RBHS-Robert Wood Johnson Medical School, Rutgers Cancer Institute of New Jersey

Intestinal Organoids for Drug Discovery
Richard Visconti-Associate Principal Scientist, Cellular Pharmacology, Merck Research Laboratories, Kenilworth,  New Jersey

3D Bioprinting
Elizabeth Wu-President, WuZenTech, Edison, New Jersey

Building  Your Brand  Through LinkedIn
Stan Robinson, Jr., LinkedIn Consultant, Helping Professionals with Social Selling, Personal Branding

Register at EventBrite here: 

To sign up to be an LRIG member or update your profile, please visit us at
Hoping to see you on May 11th.
Reserve your spot today!


Read Full Post »

3D DNA Images  Nanoscale design of printed vascular tissue

Curator: Larry H. Bernstein, MD, FCAP


New Detailed 3D DNA Images Could Aid Nanoscale Designs

This video shows techniques that scientists used to produce 3-D reconstructions of shape fluctuations in double-helix DNA segments attached to gold nanoparticles [Lei Zhang, Dongsheng Lei, Jessica M. Smith, Meng Zhang, Huimin Tong, Xing Zhang, Zhuoyang Lu, Jiankang Liu, A. Paul Alivisatos, and Gang “Gary” Ren]


Flexible double-helix DNA segments connected to gold nanoparticles are revealed from the 3-D density maps (purple and yellow) reconstructed from individual samples using a Berkeley Lab-developed technique called individual-particle electron tomography or IPET. Projections of the structures are shown in the background grid.[Berkeley Lab]


The general chemical structure of the DNA helix was described by James Watson and Francis Crick in 1953. Over the years that followed, scientists intensely studied the molecular structure of DNA to understand its behaviorin vivo and to exploit its unique properties for nanotechnology purposes.

Now, an international team of scientists working at the Department of Energy’s Lawrence Berkeley National Laboratory (Berkeley Lab) has captured the first high-resolution 3D images from individual double-helix DNA segments attached at either end to gold nanoparticles. The images detail the flexible structure of the DNA segments, which appear as nanoscale jump ropes.

Using a cutting-edge electron microscopy (EM) technique, called individual-particle electron tomography (IPET), the researchers were able to visualize the shapes of the coiled DNA strands, which were sandwiched between polygon-shaped gold nanoparticles, and reconstruct high-resolution 3D images. The EM technique was coupled with a protein-staining process and sophisticated software that provided structural details to the scale of approximately 2 nanometers (two billionths of a meter).

“We had no idea about what the double-strand DNA would look like between the nanogold particles,” noted senior study author Gang Ren, Ph.D., staff scientist in the Molecular Foundry at Berkeley Lab. “This is the first time for directly visualizing an individual double-strand DNA segment in 3-D.”

The findings from this study were published recently in Nature Communications in an article entitled “Three-Dimensional Structural Dynamics and Fluctuations of DNA-Nanogold Conjugates by Individual-Particle Electron Tomography.”

Dr. Ren and his colleagues hope their unique imaging technique will aid in the use of DNA segments as building blocks for molecular devices that function as nanoscale drug-delivery systems, markers for biological research, and components for computer memory and electronic devices. Additionally, the research team speculates that the new method could also lead to images of important disease-relevant proteins that have proven elusive for other imaging techniques and of the assembly process that forms DNA from separate, individual strands.

The Berkeley Lab scientists flash froze samples to preserve their structure for study with cryo-EM imaging. The distance between the two gold particles in individual samples varied from 20 to 30 nanometers based on different shapes observed in the DNA segments. They then collected a series of tilted images of the stained objects and reconstructed 14 electron-density maps that detailed the structure of individual samples using the IPET technique. They gathered a dozen confirmations for the samples and found the DNA shape variations were consistent with those measured in the flash-frozen cryo-EM samples.

While the 3D reconstructions show the basic nanoscale structure of the samples, the investigators are looking at the next steps, which will be to work on improving the resolution to the subnanometer scale.

“Even in this current state we begin to see 3D structures at 1- to 2-nanometer resolution,” Dr. Ren explained. “Through better instrumentation and improved computational algorithms, it would be promising to push the resolution to that visualizing a single DNA helix within an individual protein.”

In future studies, Dr. Ren noted that researchers could attempt to improve the imaging resolution for complex structures that incorporate more DNA segments as a sort of “DNA origami”—with the hope of building and better characterizing nanoscale molecular devices using DNA segments that can, for example, store and deliver drugs to targeted areas of the body.

“DNA is easy to program, synthesize, and replicate, so it can be used as a special material to quickly self-assemble into nanostructures and to guide the operation of molecular-scale devices,” Dr. Ren stated. “Our current study is just a proof of concept for imaging these kinds of molecular devices’ structures.”


Three-dimensional structural dynamics and fluctuations of DNA-nanogold conjugates by individual-particle electron tomography

Lei ZhangDongsheng LeiJessica M. Smith, …., Jiankang LiuA. Paul Alivisatos & Gang Ren
Nature Communications7,Article number:11083

DNA base pairing has been used for many years to direct the arrangement of inorganic nanocrystals into small groupings and arrays with tailored optical and electrical properties. The control of DNA-mediated assembly depends crucially on a better understanding of three-dimensional structure of DNA-nanocrystal-hybridized building blocks. Existing techniques do not allow for structural determination of these flexible and heterogeneous samples. Here we report cryo-electron microscopy and negative-staining electron tomography approaches to image, and three-dimensionally reconstruct a single DNA-nanogold conjugate, an 84-bp double-stranded DNA with two 5-nm nanogold particles for potential substrates in plasmon-coupling experiments. By individual-particle electron tomography reconstruction, we obtain 14 density maps at ~2-nm resolution. Using these maps as constraints, we derive 14 conformations of dsDNA by molecular dynamics simulations. The conformational variation is consistent with that from liquid solution, suggesting that individual-particle electron tomography could be an expected approach to study DNA-assembling and flexible protein structure and dynamics.

Organic–inorganic-hybridized nanocrystals are a valuable class of new materials that are suitable for addressing many emerging challenges in biological and material sciences1, 2. Nanogold and quantum dot conjugates have been used extensively as biomolecular markers3, 4, whereas DNA base pairing has directed the self-assembly of discrete groupings and arrays of organic and inorganic nanocrystals in the formation of a network solid for electronic devices and memory components5. Discretely hybridized gold nanoparticles conjugated to DNA were developed as a molecular ruler to detect sub-nanometre distance changes via plasmon-coupling-mediated variations in dark-field light scattering3, 6. For many of these applications, it is desirable to obtain nanocrystals functionalized with discrete numbers of DNA strands7, 8. In all of these circumstances, the soft components can fluctuate, and the range of these structural deviations have not previously been determined with a degree of rigour that could help influence the future design and use of these assemblies.

Conformational flexibility and dynamics of the DNA-nanogold conjugates limit the structural determination by X-ray crystallography, nuclear magnetic resonance spectroscopy and single-particle cryo-electron microscopic (cryo-EM) reconstruction because they do not crystallize, are not sufficiently small for nuclear magnetic resonance studies and cannot be classified into a limited number of classes for single-particle EM reconstruction. In addition, three-dimensional (3D) structure averaged from tens of thousands of different macromolecular particles obtained without prior knowledge of the macromolecular structural flexibility could result in an absence of flexible domains upon using the single-particle reconstruction method, for example, two ankyrin repeated regions of TRPV1 were absent in its atomic resolution 3D density map9.

A fundamental experimental solution to reveal the structure of a flexible macromolecule should be based on the determination of each individual macromolecule’s structure10. Electron tomography (ET) provides high-resolution images of a single object from a series of tilted viewing angles11. ET has been applied to reveal the 3D structure of a cell section and an individual bacterium at nanometre-scale resolution12. However, reconstruction from an individual macromolecule at an intermediate resolution (1–3nm) remains challenging due to small molecular weight and low image contrast. Although, the first 3D map of an individual macromolecule, a fatty acid synthetase molecule, was reconstructed from negative-staining (NS) ET by Hoppe et al.13, serious doubts have been raised regarding the validity of this structure14, as this molecule received a radiation dose hundreds of times greater than the reported damage threshold15. Recently, we investigated the possibility based on simulated and real experimental NS and cryo-ET images10. We showed that a single-protein 3D structure at an intermediate resolution (1–3nm) is potentially achieved using our proposed individual-particle ET (IPET) method10, 16, 17, 18. IPET, an iterative refinement process using automatically generated dynamic filters and soft masks, requires no pre-given initial model, class averaging or lattice, but can tolerate small tilt errors and large-scale image distortion via decreasing the reconstruction image size to reduce the negative effects on 3D reconstruction. IPET allows us to obtain a ‘snapshot’ single-molecule 3D structure of flexible proteins at an intermediate resolution, and can be even used to reveal the macromolecular dynamics and fluctuation17.

Here we use IPET, cryo-EM and our previously reported optimized NS (OpNS)19, 20 techniques to investigate the morphology and 3D structure of hybridized DNA-nanogold conjugates. These conjugates were self-assembled from a mixture of two monoconjugates, each consisting of 84-bp single-stranded DNA and a 5-nm nanogold particle. The dimers were separated by anion-exchange high-performance liquid chromatography (HPLC) and agarose gel electrophoresis as potential substrates in plasmon-coupling experiments. By OpNS-ET imaging and IPET 3D reconstruction, we reconstruct a total of 14 density maps at a resolution of ~2nm from 14 individual double-stranded DNA (dsDNA)-nanogold conjugates. Using these maps as constraints, we derive 14 conformations of dsDNA by projecting a standard flexible dsDNA model onto the observed maps using molecular dynamics (MD) simulations. The variation of the conformations was largely consistent with that from liquid solution, and suggests that the IPET approach provides a most complete experimental determination of flexibility and fluctuation range of these directed nanocrystal assemblies to date. The general features revealed by this experiment can be expected to occur in a broad range of DNA-assembled nanostructures and flexible proteins.


Although the direct imaging of dsDNA has been previously reported using heavy metal shadowing32, 33 and NS methods34, 35, 36, to the best of our knowledge, the 3D structure of an individual dsDNA strand has not previously been achieved. It has been thought that individual dsDNA would be destroyed under the high energy of the electron beam before a 3D reconstruction, or even a 2D image, is able to be achieved. Our NS tilt images showing fibre-shaped dsDNA bridging two conjugated nanogold particles demonstrated that the dsDNA can in fact be directly visualized using EM, which is consistent with the recently reported single-molecule DNA sequencing technique via TEM36. The resolutions of our density maps ranged from ~14 to ~23Å, demonstrating that an intermediate-resolution 3D structure can be obtained for each individual macromolecule. This capability is consistent with our earlier report of a ~20-Å resolution 3D reconstruction of an individual IgG1 antibody using the same approach16, 17.

Notably, a total dose of ~2,000eÅ−2 used in our ET data acquisition is significantly above the limitation conventionally used in cryo-EM (~80–100eÅ−2), which can be suspected to have certain artefact from radiation damage. In cryo-EM, the radiation damage could cause sample bubbling, deformation and knockout effects; in NS, only the knockout phenomena is often observed, in which the protein is surrounded by heavy atoms that were kicked out by electron beam. Since the sample was coated with heavy metal atoms and were dried in air, the bubbling and deformation phenomena were not usually observed. The heavy metal atoms that coat the surface of the biomolecule can provide a much higher electron scattering than from a biomolecule only inside lighter atoms. The scattering is sufficiently high to provide enough image contrast at our 120-kV high tension; thus, a further increase to the scattering ability by reducing the high tension to 80kV may not be necessary for this NS sample. In addition, the heavy atoms can provide more radiation resistance and allow the sample to be imaged under a higher dose condition. The exact dose limitation for NS is still unknown. The radiation damage related artefact in NS samples is knockout, which could reduce the image contrast and lower the tilt image alignment accuracy and 3D reconstruction resolution. In our study, a total dose of 2,000eÅ−2 did not cause any obvious knockout phenomena, but provides a sufficiently high contrast for the otherwise barely visible DNA conformations in each tilt series. The direct confirmation of visible DNA in each tilt image is essentially important to us to validate each 3D reconstruction, especially considering this relatively new approach.

Our 3D reconstruction algorithm used an ab initio real-space reference-projection match iterative algorithm to correct the centres of each tilt images, in which the equal tilt angle step for 3D reconstruction of a low contrast and asymmetric macromolecule was used. This method is different from recently reported Fourier-based iterative algorithm, termed equally sloped tomography, in which the pseudo-polar fast Fourier transform, the oversampling method and internal lattice of a targeted nanoparticle are used to achieve 3D reconstruction at atomic resolution37.

It is generally challenging to achieve visualization and 3D reconstruction on an individual, small and asymmetric macromolecule by other conventional methods; our method demonstrated its capability for 3D reconstruction of 52kDa 84-bp dsDNA through these studies: IgG1 antibody 3D structural fluctuation17, peptide-induced conformational changes on flexible IgG1 antibody5, floppy liposome surface binding with 53-kDa proteins38, all of which suggest that this method could be used to serve the community as a novel tool for studying flexible macromolecular structures, dynamics and fluctuations of proteins, and for catching the intermediate 3D structure of protein assembling.

DNA-based self-assembling materials have been developed for use in materials science and biomedical research, such as DNA origami designed for targeted drug delivery. The structure, design and control require feedback from the 3D structure, which could validate the design hypothesis, optimize the synthesis protocol and improve the reproducible capability, while even providing insight into the mechanism of DNA-mediated assembly.


Printing Vascular Tissue

from Wyss Institute for Biologically Inspired Engineering at Harvard University

Printing vessel vasculature is essential for sustaining functional living tissues. Until now, bioengineers have had difficulty building thick tissues, lacking a method to embed vascular networks. A 3D bioprinting method invented at the Wyss Institute and Harvard SEAS embeds a grid of vasculature into thick tissue laden with human stem cells and connective matrix. Printed within a custom-made housing, this method can be used to create tissue of any shape. Once printed, an inlet and outlet own opposite ends are perfused with fluids, nutrients, and cell growth factors, which control stem cell differentiation and sustain cell functions. By flowing growth factors through the vasculature, stem cells can be differentiated into a variety of tissue cell types. This vascularized 3D printing process could open new doors to tissue replacement and engineering. Footage credit: David KA.S. Gladman, E. Matsumoto, L.K. Sanders, and J.A. Lewis / Wyss Institute at Harvard University For more information, please visit:

Scaling up tissue engineering

In this video, the Wyss Institute and Harvard SEAS team uses a customizable 3D bioprinting method to build a thick vascularized tissue structure comprising human stem cells, collective matrix, and blood vessel endothelial cells. Their work sets the stage for advancement of tissue replacement and tissue engineering techniques. Credit: Lewis Lab, Wyss Institute at Harvard University


Bioprinting technique creates thick 3D tissues composed of human stem cells and embedded vasculature, with potential applications in drug testing and regenerative medicine

(CAMBRIDGE, Massachusetts)  — A team at the Wyss Institute for Biologically Inspired Engineering at Harvard University and the Harvard John A. Paulson School for Engineering and Applied Sciences (SEAS) has invented a method for 3D bioprinting thick vascularized tissue constructs composed of human stem cells, extracellular matrix, and circulatory channels lined with endothelial blood vessel cells. The resulting network of vasculature contained within these deep tissues enables fluids, nutrients and cell growth factors to be controllably perfused uniformly throughout the tissue. The advance is reported March 7 in the journal Proceedings of the National Academy of Sciences.

“This latest work extends the capabilities of our multi-material bioprinting platform to thick human tissues, bringing us one step closer to creating architectures for tissue repair and regeneration,” says Wyss Core Faculty member Jennifer A. Lewis, Sc.D., senior author on the study, who is also the Hansörg Wyss Professor of Biologically Inspired Engineering at SEAS.

Three-dimensional bioprinting of thick vascularized tissues

David B. Koleskya,1Kimberly A. Homana,1Mark A. Skylar-Scotta,1, and Jennifer A. Lewisa,2     aSchool of Engineering and Applied Sciences, Wyss Institute for Biologically Inspired Engineering, Harvard University, Cambridge, MA 02138
PNAS  2016; 113(12): 3179–3184


Current tissue manufacturing methods fail to recapitulate the geometry, complexity, and longevity of human tissues. We report a multimaterial 3D bioprinting method that enables the creation of thick human tissues (>1 cm) replete with an engineered extracellular matrix, embedded vasculature, and multiple cell types. These 3D vascularized tissues can be actively perfused with growth factors for long durations (>6 wk) to promote differentiation of human mesenchymal stem cells toward an osteogenic lineage in situ. The ability to construct and perfuse 3D tissues that integrate parenchyma, stroma, and endothelium is a foundational step toward creating human tissues for ex vivo and in vivo applications.

The advancement of tissue and, ultimately, organ engineering requires the ability to pattern human tissues composed of cells, extracellular matrix, and vasculature with controlled microenvironments that can be sustained over prolonged time periods. To date, bioprinting methods have yielded thin tissues that only survive for short durations. To improve their physiological relevance, we report a method for bioprinting 3D cell-laden, vascularized tissues that exceed 1 cm in thickness and can be perfused on chip for long time periods (>6 wk). Specifically, we integrate parenchyma, stroma, and endothelium into a single thick tissue by coprinting multiple inks composed of human mesenchymal stem cells (hMSCs) and human neonatal dermal fibroblasts (hNDFs) within a customized extracellular matrix alongside embedded vasculature, which is subsequently lined with human umbilical vein endothelial cells (HUVECs). These thick vascularized tissues are actively perfused with growth factors to differentiate hMSCs toward an osteogenic lineage in situ. This longitudinal study of emergent biological phenomena in complex microenvironments represents a foundational step in human tissue generation.

The ability to manufacture human tissues that replicate the essential spatial (1), mechanochemical (2, 3), and temporal aspects of biological tissues (4) would enable myriad applications, including 3D cell culture (5), drug screening (6, 7), disease modeling (8), and tissue repair and regeneration (9, 10). Three-dimensional bioprinting is an emerging approach for creating complex tissue architectures (10, 11), including those with embedded vasculature (1215), that may address the unmet needs of tissue manufacturing. Recently, Miller et al. (15) reported an elegant method for creating vascularized tissues, in which a sacrificial carbohydrate glass is printed at elevated temperature (>100 °C), protectively coated, and then removed, before introducing a homogeneous cell-laden matrix. Kolesky et al. (14) developed an alternate approach, in which multiple cell-laden, fugitive (vasculature), and extracellular matrix (ECM) inks are coprinted under ambient conditions. However, in both cases, the inability to directly perfuse these vascularized tissues limited their thickness (1–2 mm) and culture times (<14 d). Here, we report a route for creating thick vascularized tissues (≥1 cm) within 3D perfusion chips that provides unprecedented control over tissue composition, architecture, and microenvironment over several weeks (>6 wk). This longitudinal study of emergent biological phenomena in complex microenvironments represents a foundational step in human tissue generation.

Central to the fabrication of thick vascularized tissues is the design of biological, fugitive, and elastomeric inks for multimaterial 3D bioprinting. To satisfy the concomitant requirements of processability, heterogeneous integration, biocompatibility, and long-term stability, we first developed printable cell-laden inks and castable ECM based on a gelatin and fibrinogen blend (16). Specifically, these materials form a gelatin–fibrin matrix cross-linked by a dual-enzymatic, thrombin and transglutaminase (TG), strategy (Fig. 1and SI Appendix, Fig. S1). The cell-laden inks must facilitate printing of self-supporting filamentary features under ambient conditions as well as subsequent infilling of the printed tissue architectures by casting without dissolving or distorting the patterned construct (Fig. 1A). The thermally reversible gelation of the gelatin–fibrinogen network enables its use in both printing and casting, where gel and fluid states are required, respectively (SI Appendix, Fig. S2). Thrombin is used to rapidly polymerize fibrinogen (17), whereas TG is a slow-acting Ca2+-dependent enzymatic cross-linker that imparts the mechanical and thermal stability (18) needed for long-term perfusion. Notably, the cell-laden ink does not contain either enzyme to prevent polymerization during printing. However, the castable matrix contains both thrombin and TG, which diffuse into adjacent printed filaments, forming a continuous, interpenetrating polymer network, in which the native fibrillar structure of fibrin is preserved (SI Appendix, Fig. S3). Importantly, our approach allows arbitrarily thick tissues to be fabricated, because the matrix does not require UV curing (19), which has a low penetration depth in tissue (20) and can be readily expanded to other biomaterials, including fibrin and hyaluronic acid (SI Appendix, Fig. S4).


Fig. 1.

Fig. 1.

Three-dimensional vascularized tissue fabrication. (A) Schematic illustration of the tissue manufacturing process. (i) Fugitive (vascular) ink, which contains pluronic and thrombin, and cell-laden inks, which contain gelatin, fibrinogen, and cells, are printed within a 3D perfusion chip. (ii) ECM material, which contains gelatin, fibrinogen, cells, thrombin, and TG, is then cast over the printed inks. After casting, thrombin induces fibrinogen cleavage and rapid polymerization into fibrin in both the cast matrix, and through diffusion, in the printed cell ink. Similarly, TG diffuses from the molten casting matrix and slowly cross-links the gelatin and fibrin. (iii) Upon cooling, the fugitive ink liquefies and is evacuated, leaving behind a pervasive vascular network, which is (iv) endothelialized and perfused via an external pump. (B) HUVECs growing on top of the matrix in 2D, (C) HNDFs growing inside the matrix in 3D, and (D) hMSCs growing on top of the matrix in 2D. (Scale bar: 50 µm.) (E and F) Images of printed hMSC-laden ink prepared using gelatin preprocessed at 95 °C before ink formation (E) as printed and (F) after 3 d in the 3D printed filament where actin (green) and nuclei (blue) are stained. (G) Gelatin preprocessing temperature affects the plateau modulus and cell viability after printing. Higher temperatures lead to lower modulus and higher HNDF viability postprinting. (H) Photographs of interpenetrated sacrificial (red) and cell inks (green) as printed on chip. (Scale bar: 2 mm.) (I) Top-down bright-field image of sacrificial and cell inks. (Scale bar: 50 µm.). (J–L) Photograph of a printed tissue construct housed within a perfusion chamber (J) and corresponding cross-sections (K and L). (Scale bars: 5 mm.)

To construct thick, vascularized tissues within 3D perfusion chips, we coprinted cell-laden, fugitive, and silicone inks (Fig. 1 H and I). First, the silicone ink is printed on a glass substrate and cured to create customized perfusion chips (Movie S1 and SI Appendix, Fig. S1). Next, the cell-laden and fugitive inks are printed on chip, and then encapsulated with the castable ECM (Fig. 1 J–L and Movie S2). The fugitive ink, which defines the embedded vascular network, is composed of a triblock copolymer [i.e., polyethylene oxide (PEO)–polypropylene oxide (PPO)–PEO]. This ink can be removed from the fabricated tissue upon cooling to roughly 4 °C, where it undergoes a gel-to-fluid transition (14, 23). This process yields a pervasive network of interconnected channels, which are then lined with HUVECs. The resulting vascularized tissues are perfused via their embedded vasculature on chip over long time periods using an external pump (Movie S3) that generates smooth flow over a wide range of flow rates (24).

Movie S3.

Fluorescent microscopy video of different perfusion rates through the embedded vasculature within the printed 3D tissue microenvironments.

Fig. 2.

Three-dimensional vascularized tissues remain stable during long-term perfusion. (A) Schematic depicting a single HUVEC-lined vascular channel supporting a fibroblast cell-laden matrix and housed within a 3D perfusion chip. (B and C) Confocal microscopy image of the vascular network after 42 d, CD-31 (red), vWF (blue), and VE-Cadherin (magenta). (Scale bars: 100 µm.) (D) Long-term perfusion of HUVEC-lined (red) vascular network supporting HNDF-laden (green) matrix shown by top-down (Left) and cross-sectional confocal microscopy at 45 d (Right). (Scale bar: 100 µm.) (E) Quantification of barrier properties imparted by endothelial lining of channels, demonstrated by reduced diffusional permeability of FITC-dextran. (F) GFP-HNDF distribution within the 3D matrix shown by fluorescent intensity as a function of distance from vasculature.

Movie S4.

Confocal microscopy video of cross-section through vascularized tissue after 45 d of perfusion.

To explore emergent phenomena in complex microenvironments, we created a heterogeneous tissue architecture (>1 cm thick and 10 cm3 in volume) by printing a hMSC-laden ink into a 3D lattice geometry along with intervening in- and out-of-plane (vertical) features composed of fugitive ink, which ultimately transform into a branched vascular network lined with HUVECs. After printing, the remaining interstitial space is infilled with an HNDF-laden ECM (Fig. 3A) to form a connective tissue that both supports and binds to the printed stem cell-laden and vascular features. In this example, fibroblasts serve as model cells that surround the heterogeneously patterned stem cells and vascular network. These model cells could be replaced with either support cells (e.g., immune cells or pericytes) or tissue-specific cells (e.g., hepatocytes, neurons, or islets) in future embodiments. The embedded vascular network is designed with a single inlet and outlet that provides an interface between the printed tissue and the perfusion chip. This network is symmetrically branched to ensure uniform perfusion throughout the tissue, including deep within its core. In addition to providing transport of nutrients, oxygen, and waste materials, the perfused vasculature is used to deliver specific differentiation factors to the tissue in a more uniform manner than bulk delivery methods, in which cells at the core of the tissue are starved of factors (25). This versatile platform (Fig. 3A) is used to precisely control growth and differentiation of the printed hMSCs. Moreover, both the printed cellular architecture and embedded vascular network are visible macroscopically with this thick tissue (Fig. 3B).

Fig. 3.

Fig. 3.

Osteogenic differentiation of thick vascularized tissue. (A) Schematic depicting the geometry of the printed heterogeneous tissue within the customized perfusion chip, wherein the branched vascular architecture pervades hMSCs that are printed into a 3D lattice architecture, and HNDFs are cast within an ECM that fills the interstitial space. (B) Photographs of a printed tissue construct within and removed from the customized perfusion chip. (C) Comparative cross-sections of avascular tissue (Left) and vascularized tissue (Right) after 30 d of osteogenic media perfusion with alizarin red stain showing location of calcium phosphate. (Scale bar: 5 mm.) (D) Confocal microscopy image through a cross-section of 1-cm-thick vascularized osteogenic tissue construct after 30 d of active perfusion and in situ differentiation. (Scale bar: 1.5 mm.) (E) Osteocalcin intensity across the thick tissue sample inside the red lines shown in C. (F) High-resolution image showing osteocalcin (purple) localized within hMSCs, and they appear to take on symmetric osteoblast-like morphologies. (Scale bar: 100 µm.) After 30 d (Gand H), thick tissue constructs are stained for collagen-I (yellow), which appears to be localized near hMSCs. (Scale bars: 200 µm.) (I) Alizarin red is used to stain calcium phosphate deposition, and fast blue is used to stain AP, indicating tissue maturation and differentiation over time. (Scale bar: 200 µm.)

In summary, thick, vascularized human tissues with programmable cellular heterogeneity that are capable of long-term (>6-wk) perfusion on chip have been fabricated by multimaterial 3D bioprinting. The ability to recapitulate physiologically relevant, 3D tissue microenvironments enables the exploration of emergent biological phenomena, as demonstrated by observations of in situ development of hMSCs within tissues containing a pervasive, perfusable, endothelialized vascular network. Our 3D tissue manufacturing platform opens new avenues for fabricating and investigating human tissues for both ex vivo and in vivo applications.


Steve Dufourny Hello Mr Bernstein, it is relevant.I have a model with quantum sphères and spherical volumes correlated with my theory of spherisation in theoretical physics(in a very simple resume quant sphères…..spherisation encodings…….Cosmol sphères…….Universal sphères with a central BH)more two équations about matter and energy and sphères.The quantizations and properties can be computed.The adn, amino acids ,protiens INE ASE are the keys.I search the main gravitationalcodes.It is more far than our standard model in logic.Regards


Read Full Post »

Older Posts »