Archive for the ‘Cardiovascular and Vascular Systems’ Category

A Rich Tradition of Patient-Focused Care — Richmond University Medical Center, New York’s Leader in Health Care and Medical Education 

Author: Gail S. Thornton, M.A.

Co-Editor: The VOICES of Patients, Hospital CEOs, HealthCare Providers, Caregivers and Families: Personal Experience with Critical Care and Invasive Medical Procedures


Richmond University Medical Center (, an affiliate of The Mount Sinai Hospital and the Icahn School of Medicine, is a 470+ bed health care facility and teaching institution in Staten Island, New York. The hospital is a leader in the areas of acute, medical and surgical care, including emergency care, surgery, minimally invasive laparoscopic and robotic surgery, gastroenterology, cardiology, pediatrics, podiatry, endocrinology, urology, oncology, orthopedics, neonatal intensive care and maternal health. RUMC earned The Joint Commission’s Gold Seal of Approval® for quality and patient safety.

RUMC is a designated Level 1 Trauma Center, a Level 2 Pediatric Trauma Center, a Level 3 Neonatal Intensive Care Unit (NICU), which is the highest level attainable, and a designated Stroke Center, receiving top national recognition from the American Heart Association/American Stroke Association.  Their state-of-the-art Cardiac Catheterization Lab has Percutaneous Coronary Intervention (PCI) capabilities, for elective and emergent procedures in coronary angioplasty that treats obstructive coronary artery disease, including unstable angina, acute myocardial infarction (MI), and multi-vessel coronary artery disease (CAD).

RUMC maintains a Wound Care/Hyperbaric Center and a Sleep Disorder Center on-site at its main campus.  The facility also offers behavioral health services, encompassing both inpatient and outpatient services for children, adolescents and adults, including emergent inpatient and mobile outreach units.  RUMC is the only facility that offers inpatient psychiatric services for adolescents in the community.

In April 2016, RUMC announced its intent to merge with Staten Island Mental Health Society in order to expand its footprint in Staten Island and integrate behavioral health services alongside primary care. As part of New York’s Medicaid reforms, funding is available to incentivize providers to integrate treatment for addiction, mental health issues and developmental disabilities with medical services.

With over 2,500 employees, RUMC is one of the largest employers on Staten Island, New York.


Image SOURCE: Photographs courtesy of Richmond University Medical Center, Staten Island, New York. Interior and exterior photographs of the hospital.


Below is my interview with President and Chief Executive Officer Daniel J. Messina, Ph.D., FACHE, LNHA, which occurred in September, 2016.

What has been your greatest achievement?

Dr. Messina: Professionally, my greatest achievement is my current responsibility – to be President and Chief Executive Officer of one of the greatest hospitals with a strong, solid foundation and rich history. I was born in this hospital and raised on Staten Island, so to me, there is no greater gift than to be part of a transformative organization and have the ability to advance the quality of health care on Staten Island.

My parents taught me the value of responsibility and motivation and instilled in me the drive and tenacity to be the best person I could be – for my employees and for my family. I am a highly competitive person, who is goal-oriented, hands-on and inspired by teamwork. I rarely sit behind my desk as I believe my place is alongside my team in making things happen.

As a personal goal, I recently climbed the 20,000-foot Mount Kilimanjaro in Tanzania. It was the experience of a lifetime. I could not have completed this challenge without the support of the guides and porters who helped me and my group along the way. For me, it was a challenge in proving to myself that I could be out of my comfort zone. My group and I hiked hours and hours each day, dodging rocks and scrambling along rock walls with the goal of reaching the summit. In many ways, it takes a village to climb the mountain, relying on each other in the group to get you to the next level.

In many ways, that is how I see my professional day at the hospital, working with a strong team of dedicated medical staff and employees who are focused on one goal, which is to continue our hard work, continue to improve care and continue to move forward to advance life and health care.

The mission of Richmond University Medical Center, an affiliate of The Mount Sinai Hospital and Mount Sinai School of Medicine, serves the ethnically diverse community of Staten Island, New York, by providing patients with a range of services.

How has your collaboration with the Mount Sinai network helped to expand health care delivery and services for patients of Staten Island, New York?

Dr. Messina: Being able to serve our patients year after year continues to be a top priority, so we are constantly improving upon our rich history of 100 years of exceptional patient-focused care given by our medical and surgical health care professionals as well as innovative technologies and programs created by our award-winning hospital team. We have committed medical specialists, passionate employee staff, exceptional Board of Trustees, supportive elected government officials – all who in their own way contributes to providing the highest level of patient care to the more than 500,000 residents of Staten Island, New York.

As a member of the Mount Sinai Health network, we have found ways to work collaboratively with our academic partner to ensure that our patients’ health care needs not only are fully met but also exceeded. This alliance will facilitate the development of a new, Comprehensive Breast and Women’s Healthcare Center. We have leveraged our Breast and Women’s Health Center with our RUMC general surgeons in conjunction with breast imaging, fellowship-trained physicians from Mount Sinai’s Icahn School of Medicine. The physicians who are granted this renowned fellowship interact with our patients and become an active participant in multidisciplinary breast conferences and resident and medical student education. For patients, this means that they have access to the best minds and latest research, therapies and treatment regimens throughout our network.

What makes Richmond University Medical Center and its specialty areas stand out from other hospitals?

Dr. Messina: We bring the highest level of advanced medicine to our patients. For more than 100 years, we have built a rich history of delivering patient-focused care that is unique. Our organization is recognized as a family organization with strong community spirit and family values. We are proud to be a high-technology/high-touch organization of caring professionals that go above and beyond the standard of health care. Our strengths lie in the areas of acute, medical and surgical care, including emergency care, surgery, minimally invasive laparoscopic and robotic surgery, gastroenterology, cardiology, pediatrics, podiatry, endocrinology, urology, oncology, orthopedics, neonatal intensive care and maternal health.

Each year, we embark upon a comprehensive, robust strategic planning process that involves our senior leadership team, clinical chairs, Board of Trustees as well as our medical and surgical staff and hospital employees that looks out three to five years in the future to determine what is best for the patient. We are each committed in our own way to quality patient care and building an even stronger organization.

Some of our achievements are noteworthy:

  • As a New York City Department of Emergency Services designated Level 1 Trauma Center and Level 2 Pediatric Trauma Center, the only Trauma Center dually verified in New York City, we rely on sophisticated equipment so our medical and surgical specialists are prepared to treat severe conditions within minutes.
  • Our Neonatal Intensive Care Unit (NICU) is a designated Level 3 facility, the highest level attainable. The unit delivers 3,000 babies annually and it was recognized as having the lowest mortality rate in the metropolitan area and a survival rate of 99 percent, that exceeds national benchmarks. Our specialists in our pediatric ambulatory services department treat over 10,000 patients annually and our children’s urgent care area records over 23,000 visits annually.
  • Our state-of-the-art, 38,000-square-foot Emergency Department (ED), which will be replaced by an expanded facility and projected to open in 2018, will provide for more focused care, operational efficiency and flexibility for our staff and patient. We also will be better integrated and connected to the entire hospital campus.

Originally designed to serve 22,000 patients each year, the ED is expected to accommodate an increased volume of patients, which is estimated at 70,000 and give our medical specialists the tools they need to provide the best in care for this volume of patients. In a new patient and family-centered space with 49 treatment positions, the new ED will be connected to the existing hospital, close to surgical services, the radiology department and lab services.

Equally as important, the hospital has been strong in the face of natural disasters, especially Hurricane Sandy which occurred a few years ago, and the new ED is being designed with storm resilient and redundant design to minimize impact from severe weather conditions.

In fact, the New York City Council and the Staten Island Borough President have set aside a combined $13.5 million for this $60+ million project and believe in the transformative impact that it will have on emergency care on Staten Island. These local officials believe that Staten Island residents deserve quality, readily accessible health care.

  • Heroin addiction is an epidemic on Staten Island, so we have a number of programs in place at RUMC’s Silberstein Center to provide outpatient treatment, rehabilitation and clinics, along with group therapy sessions, Alcoholics Anonymous meetings and individual therapy sessions.
  • Our new primary care/walk-in facility in the heart of Staten Island borough is operational and there are no appointments required. Patients can visit with one of three physicians or a nurse practitioner. This off-site facility is not located in the hospital complex and is an expansion of our services outside of the hospital walls.
  • We also maintain a Wound Care Center, Pain Management Center and a Sleep Disorder Center at our facility. In fact, we are the only local facility that offers inpatient psychiatric services for adolescents and we are expanding our capacity to meet the needs of the community.


RUMC has been awarded a top designation jointly by the American Heart Association and the American Stroke Association. What does that mean to the hospital?

Dr. Messina: This designation makes us proud as the recipient of the American Heart Association/American Stroke Association’s Quality Achievement Award for six consecutive years and its first Elite Plus recognition. This means that we have achieved 85 percent or higher adherence in indicators for two or more consecutive 12-month periods to improve quality of patient care and outcomes for stroke patients.

Our cardiac catheterization lab with Percutaneous Coronary Intervention (PCI) capabilities – the newest facility of its kind on Staten Island — now treats semi-urgent and elective coronary procedures.

For patients, this means that we have a commitment to ensure that stroke patients receive the most appropriate treatment according to nationally recognized, research-based guidelines based on the latest scientific evidence. With a stroke, when time is lost, brain is lost, and this award demonstrates our commitment to ensuring patients receive care based on evidenced-based guidelines. We are dedicated to continually improving the quality of stroke care and this recognition helps us achieve that goal.

Studies have shown that hospitals that consistently follow these quality improvement measures can reduce length of stay and 30-day readmission rates and reduce disparities in care. To qualify for the Elite Plus recognition, we met quality measures developed to reduce the time between the patient’s arrival at the hospital and treatment with the clot-buster tissue plasminogen activator, or tPA, the only drug approved by the U.S. Food and Drug Administration to treat ischemic stroke. If given intravenously in the first three hours after the start of stroke symptoms, tPA has been shown to significantly reduce the effects of stroke and lessen the chance of permanent disability. We earned the award by meeting specific quality achievement measures for the diagnosis and treatment of stroke patients at a set level for a designated period.

According to the American Heart Association/American Stroke Association, stroke is the number five cause of death and a leading cause of adult disability in the United States. On average, someone suffers a stroke every 40 seconds; someone dies of a stroke every four minutes; and 795,000 people suffer a new or recurrent stroke each year.

The values of Richmond University Medical Center are summarized in the acronym, WE CARE (Welcoming Energized Compassion Advocacy Respect Excellence). How is this part of your day-to-day life?

Dr. Messina: For more than 100 years, Richmond University Medical Center has

been building a rich history of exceptional patient-focused care for the residents of Staten Island. Each year, we carry that tradition forward by our medically innovative and patient-focused care and services we offer. It is the passion, creativity and caring of everyone who is part of our ‘hospital team’ that moves the organization to new heights.

The chart below summarizes our credo, the values that guide us every day and help us focus on the care and well-being of the people who come through our doors.

We are welcoming and gracious toward each other, and toward all who come to receive our services.

Personnel are energized for quality, creativity, commitment and teamwork.

Compassion is the way we share deep concern and care toward each person.

Advocacy is our activity that promotes the rights and responsibilities of patients, families and staff, in the hospital setting and in the community.

We show respect by recognizing the basic dignity of every person in all our interactions and in the formulation of policies and procedures.

Excellence is our way of demonstrating that we can always be more and always be better.


The Richmond University Medical Center Board is comprised of distinguished leaders of the Staten Island community who are committed to the success of the hospital and to the health of Staten Islanders.

How is this local approach revolutionizing health care for the Staten Island community?

Dr. Messina: The members of our distinguished Board of Trustees, who represent a cross-section of business professionals and community leaders, continue our goal of meeting the needs of our patients and our hospital.

Our Board remains committed to providing solutions for our patients to challenging healthcare issues they face every day and to making a difference in the lives of patients by providing the latest thinking and technology solutions. Our Board Chairperson Kathryn K. Rooney, Esq., and Vice Chairperson Ronald A. Purpora, as well as the other Board members, and even our elected government officials, have a strong connection to Staten Island and we believe it truly ‘takes a village’ to make this organization flourish.

Each year, our Board of Trustees is presented with new opportunities and possibilities for growth and development. That is why their top priority for this past year was approving the construction of a state-of-the-art Emergency Department (ED) as this undertaking will serve both the patients and staff equally. In order to serve the residents of Staten Island properly, the new ED will accommodate an increased number of patients and our medical staff will receive the tools and technology to provide the best in care for our patients.

This past year, we were provided with a $1.5 million gift from the Staten Island Foundation that will go toward the hospital’s capital campaign to construct the new $60 million Emergency Department. We decided to name the RUMC’s Allan Weissglass Pavilion Center for Ambulatory Care, in honor of our long-time community and business leader, who is a founding Board member and Board of Trustees member. Allan Weissglass devoted his time, energy and talent to the success of this hospital over many years.

We are positioning our organization for the future and we continuously build on our strengths, being responsive to the needs of the community. In the past, we saw the patient was the only ‘customer’ of the hospital. Today, that perception is evolving and our ‘customers’ are many.  With the help and support of donors, local foundations, volunteers, staff, and the community, local government officials, we are building a bright future for Richmond University Medical Center.

What is RUMC’s commitment to graduate medical education?

Dr. Messina: Our six Graduate Medical Education (GME) programs in Internal Medicine, Obstetrics and Gynecology, Pediatrics, Psychiatry, and Diagnostic Radiology and Podiatry, signify our commitment to teaching as a cornerstone of our philosophy. Our medical staff are seen as role models for our medical residents and provide quality training, medical education and research capabilities. Our existing medical staff functions as supervising physicians and gives medical residents exposure to specific responsibilities and patient care, as well as scholarly opportunities. One interesting fact is that the doctors we train come back to help treat our patients by using their knowledge and experience to work in our community.

You mentioned that ‘outreach in the community’ as a key factor in the success of the hospital’s mission to enhance the quality of life for residents of Staten Island. What types of activities are under way?

Dr. Messina: Our lifesaving work takes many forms. We are constantly finding new and different ways to engage with our community – to raise awareness and educate on a number of diseases and conditions, and, hopefully move toward better health care. We believe that our patients need to see us outside of a clinical environment, which strengthens our relationship.

For example, over the past year:

  • We sponsored an annual health and wellness expo with the Staten Island Economic Development Corporation that was attended by over 2,000 people to equip the community with knowledge about their health and the local health services available to them.
  • We pioneered an organ donor enrollment day by welcoming 59 visitors and guests who can potentially donate their organs to save lives.
  • We partnered with the New York City Department of Transportation and our own Trauma team to demonstrate and educate the community on car seat safety.
  • Our Dermatologist team took part in the Borough President’s “Back to the Beach” festival by performing skin screenings and distributing sunscreen and information on skin cancer.
  • Our Obstetrics and Gynecology team hosted a baby expo to talk with new mothers and mothers-to-be about services available at the hospital.
  • Our Diabetologist team partnered with the YMCA on a 16-week partnership to curb the diabetes epidemic on Staten Island through information talks and health screenings.
  • We were even present at last year’s Staten Island Yankees home opening baseball game to throw out the first pitch and conduct a blood drive while distributing wellness information.


Since roughly one third of the residents on Staten Island are enrolled in Medicaid or Medicare, what steps are you taking to improve the delivery of treatment for them?

Dr. Messina: We started several initiatives last year that were funded by the federal and state governments to look at the way care is delivered to patients who are enrolled in Medicare and Medicaid. So far, we’ve reduced costs by $3.75 million and realized $1.8 million in shared savings that are re-invested in key hospital programs.

As you know, Medicare and Medicaid are two different government-run programs that were created in 1965 in response to the inability of older and low-income Americans to buy private health insurance. They were part of our government’s social commitment to meeting individual health care needs. Medicare is a federal program that provides health coverage if you are 65 or older or have a severe disability, no matter your income, while Medicaid is a state and federal program that provides health coverage if you have a very low income.

We’ve set up our own Richmond Quality Accountable Care Organization (ACO), that comprises 30 providers serving 7,500 Medicare patients. This innovative program is accountable for the quality, cost and overall care provided to people on Medicare and who are enrolled in the traditional fee-for-service program.  One program that is ongoing is one that we’ve partnered with the Visiting Nurse Service of Staten Island to prevent hospital readmissions and to identify hospitalized patients who would benefit from a higher level of care and home care services.

Another program that is under way for our Medicaid patients is teaching our staff to prevent hospital readmissions by creating an accurate list of medications that a patient takes and comparing that list against physician’s admission, transfer and discharge orders to ensure that the correct medication plan is in place.

We believe that we are transforming the underlying systems with a focus on delivering quality care and hopefully better outcomes for patients.

RUMC recently announced a merger with Staten Island Mental Health Society (SIMHS) to integrate SIMHS’ broad range of behavioral health programs into the hospital’s existing medical and behavioral program throughout Staten Island. What does this merger bring to the community?

Dr. Messina: We believe that the proposed merger between RUMC and the Staten Island Mental Health Society (SIMHS) will provide a strengthened, comprehensive network of behavioral health services across Staten Island.

This partnership will bring together two Staten Island institutions, with a combined 230 years of service to the borough, and create one strong and vibrant organization dedicated to meeting the health needs of the diverse community.

Merging the range of community-based behavioral health services provided by SIMHS with the solid foundation of primary care services provided by RUMC will create a seamless range of behavioral and medical services for our residents. We are in the unique position to transform and enhance the services of these two vital health care providers. The SIMHS will keep its name and become a division of the hospital. The merger is expected to close during calendar year 2017.


Image SOURCE: Photograph of President and Chief Executive Officer Daniel J. Messina, Ph.D., FACHE, LNHA, courtesy of Richmond University Medical Center, Staten Island, New York.

Daniel J. Messina, Ph.D., FACHE, LNHA
President & Chief Executive Officer

Daniel Messina, Ph.D., FACHE, LNHA, became President and Chief Executive Officer of Richmond University Medical Center (RUMC) – an affiliate of The Mount Sinai Hospital and Mount Sinai School of Medicine – in April 2014.

Dr. Messina, a life-long resident of Staten Island, is a seasoned executive with nearly 30 years of healthcare leadership expertise. For the previous 13 years, he served as the System Chief Operating Officer of CentraState Healthcare System in Freehold, New Jersey, where his responsibilities included all System Operations for the Medical Center, Assisted Living Facility, Skilled Nursing and Rehabilitation Center and Continuing Care Retirement Community. While in this role, Dr. Messina developed additional growth strategies that include a new Cancer Center, a Proton Therapy Center, Radio-Surgery, a new Infusion Center and programs in Robotics, Minimally Invasive Surgery, Bariatric and Neurosurgery. Other accomplishments include a new state-of-the-art 26-bed Critical Care Unit, a 49-bed Emergency Department, and the development of an 180,000 sq. ft. Ambulatory Campus and Wellness Center anchored by a 35,000 sq. ft. Medical Fitness Center. Additionally, Dr. Messina developed the Linda E. Cardinale MS Center – one of the largest and most comprehensive MS Centers in the tristate area – leading to a fundraising event that has generated over $2 million.

Dr. Messina received his B.S. in Health Science/Respiratory Therapy from Long Island University Brooklyn, and earned his M.P.A. in Healthcare Administration from LIU Post. He obtained his Ph.D. in Health Sciences and Leadership at Seton Hall University where he currently serves as an adjunct professor in the School of Health and Allied Sciences. He is active in the American College of Health Care Executives, is board certified in healthcare management as an ACHE Fellow, and recently completed a three-year term as Regent for New Jersey.

Dr. Messina serves as trustee on the National Multiple Sclerosis Society, the New Jersey Metro Chapter, and the Alumni Board of Trustees at Seton Hall University. He is a Board member of the VNA Health Group of New Jersey and a member of the Policy Development Committee of the New Jersey Hospital Association. Dr. Messina has been honored by various organizations for his service to the community, including Seton Hall University with the “Many Are One” award, the American College of Healthcare Executives with Senior, Early and Distinguished Service Awards, New Jersey Women Against MS, CentraState Auxiliary, and the Staten Island CYO.

Editor’s note:

We would like to thank William Smith, director of Public Relations, Richmond University Medical Center, for the help and support he provided during this interview.




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Ralph’s Story: An Entertainer at Heart

Patient was diagnosed with heart disease and pulmonary hypertension in January 2016 and had a triple-bypass operation at age 69. Interview was conducted six months post-surgery.

Author: Gail S. Thornton, M.A.

Co-Editor: The VOICES of Patients, HealthCare Providers, Caregivers and Families: Personal Experience with Critical Care and Invasive Medical Procedures


Evergreen, Colorado, an idyllic, peaceful community with an elevation of 8,000 feet west of Denver, offers its residents and visitors a beautiful place for arts and culture, summer and winter sporting activities, and scenic beauty. In fact, Ralph Nichols has lived in the town for more than 20 years.

“This past September [2015] was, particularly, challenging for me, where winter begins quite early for us. It became increasingly painful and difficult to breathe in the freezing temperatures. It seemed that my lungs were inflamed and I couldn’t even stand the cold weather. I thought it might be the beginning of a bad cold, and I wasn’t overly concerned that there was anything terribly wrong.”

At that time, Ralph went to his family physician who performed the usual routine examination with no significant results.

“Many years ago, I developed a mild case of scleroderma, a chronic connective tissue disease. I thought that perhaps my symptoms were the result of some type of inflammation in my body that could be managed with prescription medications.”

Scleroderma is known as an autoimmune disease, which adds an inappropriate amount of collagen to various parts of the body, such as the joints, skin, and later stages, various organs, such as the lungs, in Ralph’s case. Scleroderma can cause the organs to shut down and, eventually, cause death.

“I never let this condition stop me from doing anything as it is life-long condition. It was always something I had to tolerate and work through.”


Image SOURCE: Photographs courtesy of Ralph Nichols and Gabriela Contreras.  Top left: Ralph today. Top right: Ralph recovering one month after surgery. Bottom left and center: Ralph with his medical team. Bottom right: Ralph in rehabilitation center.

Over the brutal Colorado winter, Ralph’s symptoms were getting worse. He had no idea that his life would dramatically change over the next few months. He went to see his family physician again. During this physical examination, Ralph was referred to pulmonary and cardiovascular specialists for a routine electrocardiogram, echocardiogram and stress test in order to further diagnose his symptoms. He had always been relatively healthy and fit and never been seriously ill or hospitalized.

“On the outside, Ralph was the picture of good health,” said his wife, Gabriela. “On the inside, his body was telling him that something was wrong.”

Three months later in December 2015, Ralph met with Dr. Alexandra Smart, a pulmonologist, who ordered a chest x-ray and other diagnostic tests, including a right heart catheterization. At that point, Ralph’s medical team grew. It was then determined that Ralph needed to see other cardiovascular specialists and undergo more tests. In January 2016, he met with Dr. Sameer Mehta, cardiologist at Cardiac & Thoracic Surgery Associates, in Lakewood, Colorado, who reviewed his tests to date, listened to Ralph’s symptoms, and told him he needed both a right and left heart cardiac catheterization.

 “They gave me sedation for the catheterization procedure and went through my neck with a camera to see what was going on with my lungs and heart. We were all singing together on the way to the operating room. During the procedure, my cardiologist found more than he had anticipated.”

The result was not good. Ralph had major blockages in two main arteries that supply blood to his heart muscle compounded by the fact that his lungs were affected by scleroderma.

“The catheterization was alarming. It showed that my arteries were in bad shape. They were both clogged with atherosclerotic plaque; one of them was 99 percent blocked and the other was 85 percent blocked.”

His cardiologist believed that the blockages would not respond to medications quickly or a stent.

“Even though my father had major heart disease and died two years later of cancer at the age of 56, I thought that I would be immune to this particular experience. After all, I was in good health, exercised regularly, lived a reasonable lifestyle and had a great diet.”

 Preparing for Life-Saving and Life-Changing Surgery

Unfortunately, surgery was the next step. Ralph was referred to Dr. Mehta’s colleague, Dr. Patrick D. Rudersdorf, cardiothoracic surgeon at Cardiac & Thoracic Surgery Associates.

“I didn’t leave the hospital that day as expected. Instead, I got a visit from Dr. Rudersdorf and couldn’t believe what he was telling me. My only chance to live was having triple bypass surgery which needed to be done immediately. The doctor met with me that same day to explain the procedure, answer my questions and talk through the details of the rehabilitation period after the surgery.”

Dr. Rudersdorf reassured Ralph that he was doing the right thing and calmed my fears.

“He said that I needed this life-saving surgery because I was at high risk for having a major heart attack. I was shocked, at first, at the thought of the intensity of surgery on my body. It’s a situation that no one likes to be in, but I had to make a decision about alleviating the ongoing pain and pressure in my chest along with shortness of breath due to diseased heart arteries. Coronary bypass surgery was my answer to feeling better — and it essentially gave me my life back.”

Dr. Rudersdorf moved his previously planned morning surgery to another day to accommodate me first thing in the morning. Ralph underwent triple bypass surgery at St. Anthony Hospital in Lakewood, Colorado. The procedure was complex and took eight hours. He was in the hospital for a total of 31 days.

“It was an ordeal that I thought I’d never have to experience. I had no time to call anyone, or time to even contemplate life and death…or even being scared.  My wife Gabriela spent the entire time in the hospital, supported by our dearest friends, Norma Delaney and Garret Annofsky, in addition to keeping family and friends in other parts of the United States and Mexico updated as well. Once the surgery was over, the medical team woke me up and said the procedure was successful, but I was far from being out of the woods.”

Ralph had some complications because of a condition called pulmonary hypertension, a type of high blood pressure that affects the arteries in the lungs and the right side of the heart. According to the Mayo Clinic’s web site, in one form of pulmonary hypertension, tiny arteries in the lungs, called pulmonary arterioles, and capillaries become narrowed, blocked or destroyed. This makes it harder for blood to flow through the lungs, and raises pressure within the lungs’ arteries. As the pressure builds, the heart’s lower right chamber (right ventricle) must work harder to pump blood through the lungs, eventually causing the heart muscle to weaken and fail.

“The pulmonary hypertension limited some of the medications that the doctors would have used during my recovery. It was a tough few days for me in intensive care, hooked up to about 18 monitors. The medical team had to stop and re-start my heart four different times because of atrial fibrillation — finally getting both parts of the heart to dance together in the same rhythm.”

Ralph’s heart was beating abnormally fast and irregular and not functioning the way it should. The doctors restore regular rhythm to the heart by sending an electrical shock to the heart, which is called electrical cardioversion or chemically using antiarrhythmia medications, which is called pharmacologic or chemical cardioversion.

“The doctors shocked my heart first chemically with medications when I was awake. This procedure was the scariest. I was sitting up in bed and felt my heart stop, then the medical team flushed the medication out with saline in order to restart my heart. That procedure was not successful, so that is why the doctors had to shock my heart three more times electrically.

“The reason the doctors stopped my heart was to correct the atrial fibrillation and to get my heart into regular sinus rhythm, which is a wave mode of the heart where everything is synchronized. The doctors did not want me to continue to experience atrial fibrillation because if continued, I would not be able to regain my strength.”

Ralph was finally moved from intensive care to intermediate care after five days and the medical team kept him in intermediate care another 12 days until his heart and lungs got stronger.

“From there, I didn’t go home but instead went to Evergreen Life Center for rehabilitation for two weeks to learn how to walk, climb stairs so that I could access my home on my own, and develop my strength again. The rehab team would let me leave only after making sure I had oxygen in my home.”

After that, Ralph started another phase of his rehabilitation at St. Anthony Cardiac Rehabilitation and Wellness Center. For the next three months, he took part in cardiac rehabilitation three days a week. He passed that with flying colors. Now, he is in another phase of rehabilitation, building his lung capacity two days a week.

Ralph didn’t have the means or even the will to communicate with friends during this tumultuous time, except Gabriela and several close friends who were always at the hospital and rehabilitation center who gave him the strength to continue.

“I finally returned home after many weeks with an enormous feeling of gratitude for each and every one of my friends, as well as the St. Anthony’s hospital team of doctors, nurses, and therapists, who supported me and Gabriela during this exceptional adventure that has certainly changed my life.”

Surely, this experience has been a life-changing experience for Ralph.

 Coronary Artery Bypass Facts

 Coronary artery bypass grafting (CABG, often pronounced “cabbage”) is a surgical treatment for blocked coronary arteries. Coronary arteries supply blood to the heart muscle and when blockages in these arteries form, chest pain, shortness of breath and heart attacks can occur. Catheter procedures performed by interventional cardiologists address the blockages themselves with stents. Coronary bypass surgery performed by cardiac surgeons reroutes the blood around the blockages to supply better blood supply to the heart muscle and is a better treatment option, although more invasive, for certain patients and more durable for most patients.

Life for Ralph Today

Today, Ralph is regaining his strength both in mind and body. He visits the cardiovascular and pulmonary rehabilitation center three times a week for the past few months and walks on their treadmill, lifts weights and pedals the bicycle for one hour, supervised by the therapists. He also sees his medical team for regular check-ups every month, eats healthier with no fat and no salt, and takes a cocktail of medicines daily for his heart and lungs, including amiodarone, furosemide, pitavastatin, and aspirin.

“Almost six months after my surgery, although I am not in the best shape of my life, however, I am in the best spiritual place than ever before. This is a huge milestone for me. I continue to improve my strength, which will make my heart more resilient. There is nothing that I can’t do now, and I am doing everything I can to experience a normal life as far as work and regaining my strength. I find it necessary to move to a warmer climate and lower altitude in order to continue to improve.”

Ralph also is the former lead singer of The Letterman and The Sandpipers, two American easy-listening bands during the 1960-70-80s. He is an entertainer at heart with over 3,000 professional appearances to his credit. He has been performing and recording for over 50 years, traveled the world extensively and performed before members of the Vatican with Pope Pius XII and Royalty with Prince Rainier and Princess Grace Kelly, as well as notables such as Frank and Nancy Sinatra, Tony Bennett, Ronald Reagan, Merv Griffin, Danny Thomas, Shirley Bassey, Rosalind Russell and Bob Hope.

Ralph and his vocal group were dubbed by Billboard Magazine as “the greatest romantic vocal group of all time.” He is also a member of the Vocal Group Hall of Fame, a prestigious honor. He is a true legend as his group has sold more than 20 million recordings, performed live thousands of times, and whose recording of the song “Love” was left by NASA astronauts in a time capsule on the moon.

“I enjoy each and every day and appreciate all that life has to offer.”

Ralph’s next step is to get back to singing and his solo entertainment business, which he holds dear to his heart. That should be a task that he can easily accomplish.


Editor’s note:

We would like to thank Gabriela Contreras, a global communications consultant and patient advocate, for the tremendous help and support that she provided in scheduling time to talk with Ralph Nichols.

Ralph Nichols provided his permission to publish this interview on July 30, 2016.




Other related articles:

Retrieved from

Retrieved from

Other related articles were published in this Open Access Online Scientific Journal include the following: 


People with blood type O have been reported to be protected from coronary heart disease, cancer, and have lower cholesterol levels.


A Patient’s Perspective: On Open Heart Surgery from Diagnosis and Intervention to Recovery

No evidence to change current transfusion practices for adults undergoing complex cardiac surgery: RECESS evaluated 1,098 cardiac surgery patients received red blood cell units stored for short or long periods


ACC/AHA Guidelines for Coronary Artery Bypass Graft Surgery

On Devices and On Algorithms: Arrhythmia after Cardiac SurgeryPrediction and ECG Prediction of Paroxysmal Atrial Fibrillation Onset


Editor’s note:

I wish to encourage the e-Reader of this Interview to consider reading and comparing the experiences of other Open Heart Surgery Patients, voicing their private-life episodes in the ER that are included in this volume.

I also wish to encourage the e-Reader to consider, if interested, reviewing additional e-Books on Cardiovascular Diseases from the same Publisher, Leaders in Pharmaceutical Business Intelligence (LPBI) Group, on

  •  Perspectives on Nitric Oxide in Disease Mechanisms, on Amazon since 6/2/12013

  • Cardiovascular, Volume Two: Cardiovascular Original Research: Cases in Methodology Design for Content Co-Curation, on Amazon since 11/30/2015

  • Cardiovascular Diseases, Volume Three: Etiologies of Cardiovascular Diseases: Epigenetics, Genetics and Genomics, on Amazon since 11/29/2015

  • Cardiovascular Diseases, Volume Four: Regenerative and Translational Medicine: The Therapeutics Promise for Cardiovascular Diseases, on Amazon since 12/26/2015



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What could replace animal testing – ‘Human-on-a-chip’ from Lawrence Livermore National Laboratory

The iCHIP research, Moya said, could have implications for creating new drugs to fight cancer, vaccines or evaluating the efficacy of countermeasures against biowarfare agents.

Lab scientist Heather Enright is leading research into the peripheral nervous system (PNS), which connects the brain to the limbs and organs. The PNS device has arrays of microelectrodes embedded on glass, where primary human dorsal root ganglion (DRG) neurons are seeded. Chemical stimuli such as capsaicin (to study pain response) then flow through a microfluidic cap to stimulate the cells on the platform.

The microelectrodes record electrical signals from the cells, allowing researchers to determine how the cells are responding to the stimuli non-invasively. Microscopic images can be acquired at the same time to monitor changes in intracellular ion concentrations, such as calcium. This platform is the first to demonstrate that long-term culture and chemical interrogation of primary human DRG neurons on microelectrode arrays is possible, presenting researchers with an advantage over current techniques.

Read full article at the SOURCE

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Medical MEMS, BioMEMS and Sensor Applications

Curator and Reporter: Aviva Lev-Ari, PhD, RN


Contents for Chapter 11

Medical MEMS, BioMEMS and Sensors Applications

Curators: Justin D. Pearlman, MD, PhD, FACC, LPBI Group, Danut Dragoi, PhD, LPBI Group and William H. Zurn, Alpha IP


Series E: Patient-centered Medicine

Volume 4:  Medical 3D BioPrinting – The Revolution in Medicine

Editors: Larry H Bernstein, MD FCAP and Aviva Lev-Ari, PhD, RN



Image Source

Image is courtesy of Google Images



Image Source

Stanford Engineering Team Invents Pressure Sensor That Uses Radio Waves | CytoFluidix

Image is courtesy of Google Images


Introduction by Dr. Pearlman


Chapter 1: Blood Glucose Sensors


  • Tiny wireless chip and miniaturized glucose sensor
  • Embedded between two layers of soft contact lens material
  • Accurate glucose monitoring for diabetics
  • Using bodily fluids, i.e. tears
  • Prototypes can generate one reading per second
  • Experimenting with LEDs
  • Early warning for the wearer


Chapter 2: Blood Chemistry Tests – up to 100 Samples


  • Digital tattoo monitors blood below the skin
  • Tattoos are needle-less
    • Sensor-laden transdermal patch
  • Painless for the user Tiny sensors “ink”
  • Can read blood levels of:
    • Sodium, glucose, kidney function
  • Prototypes contain probes
  • Wireless, battery-powered chip
  • Continually test up to a hundred different samples



  • Lateral flow immuno-chromatographic assays
  • Sense the presence of a target analyte in a sample
  • Device connects to the camera on a cell phone
  • Weighs only 65 grams



  • Implantable device for instantaneous blood analysis
  • Wireless data transmission to a doctor
  • Applications include monitoring general health
  • Tailor drug delivery to a patient’s unique needs
  • Includes five sensors and a radio transmitter
  • Powered via inductive coupling from a battery patch
  • Worn outside the body


Chapter 3: Motion Sensors for Head-Impact

3.1       HEAD-IMPACT MONITORING PATCH – STMicro & X2Biosystems

  • Wearable electronic contains MEMS motion sensors
  • Microcontroller, low-power radio transmitter, and power management circuitry
  • Cloud-based system combines athlete concussion history
  • Pre-season neurocognitive function, balance, and coordinate-performance data
  • Creates a baseline for comparison after a suspected injury event


Chapter 4: Drug Delivery & Drug Compliance Monitoring Systems

4.1       Smart Pill delivers Therapeutic Agent Load to target – ELECTRONIC PILL – Phillips

  • Electronic pill to treat gastrointestinal cancer
  • An ingestible pill is swallowed by the patient, finds its way to the tumor, dispenses the drugs and passes harmlessly from the body
  • Smart pill contains reservoir for drug supply, fluid pump for drug delivery, pH sensor (for navigation), thermometer, microprocessor, communication


4.2       Drug Compliance Monitoring Systems

4.2.1    INGESTIBLE BIOMEDICAL SENSOR – Proteus Digital Health

  • Biomedical sensor that monitors medication adherence
  • Embedded into a pill, the sensor is activated by stomach fluid
  • Transmits a signal through the body to a skin patch
  • Indicates whether a patient has ingested material


4.2.2    MICROPUMP DEVICES – Purdue University

  • Device based on skin contact actuation for drug delivery
  • Actuation mechanism only requires body heat
  • Induced actuation can result to a gradient of 100 Pa/oC
  • Sufficient to drive liquid drug through micro-needle arrays
  • Prototypes exhibit low fabrication costs, employment of biocompatible materials and battery-less operation Suitable for single- or multiple-use transdermal drug dispensers



  • Device can deliver a vasoconstrictor agent
  • On demand to injured soldiers to prevent hemorrhagic shock
  • Other applications include medical implants
  • For cancer detection and monitoring
  • Implant can provide physicians and patients
  • Real-time information on the efficacy of treatment


Chapter 5: Remove Monitoring of Food-related Diseases


  • For analyzing food scanned
  • Information to a cloud-based application
  • Examines the results Data is accumulated from many users
  • Used to develop warning algorithms
  • For Allergies, Bacteria


Chapter 6: Skin Protection and Photo-Sensitivity Management


  • Wristband for monitoring UV exposure
  • Allows user to maximize vitamin D production
  • Reducing the risk of sun
  • Over-exposure and skin cancer
  • LED indicators light up as UV exposure accumulates
  • Flashes once the safe UV limit has been reached


6.2       WEARABLE SKIN SENSOR KTH – Chemistry 2011

  • Bio-patch for measuring and collecting vital information through the skin
  • Inexpensive, versatile and comfortable to wear
  • User Data being gathered depends on where it is placed on the body


Chapter 7: Ophthalmic Applications

7.1       INTRAOCULAR PRESSURE SENSOR – Sensimed & ST Microelectronics

  • Smart contact lens called Triggerfish
  • Contact lens can measure, monitor, and control
  • Intra-ocular pressure levels for patients
  • Catch early cases of glaucoma
  • MEMS strain gage pressure sensor
  • Mounted on a flexible substrate MEMS



  • Swept source OCT model for retinal 3D imaging
  • Replaces bulky galvanometer scanners in a handheld OCT probe for primary care physicians
  • Ultrahigh-speed two-axis optical beam steering gimbal-less MEMS mirrors
  • MEMS Actuator with a 2.4 mm bonded mirror and an angular reach of +6°
  • Low power consumption of <100mW including the MEMS actuator driver Retinal 3D Imaging


Chapter 8: Hearing Assist Technologies


  • Eliminates electronics outside the ear
  • Associated with reliability issues and social stigma
  • Accelerometer-based microphone
  • Successfully tested in cadaver ear canals
  • Prototype measures 2.5 x 6.2mm, weighs 25mg


Chapter 9: Lab-on-a-Chip

9.1       ORGAN-ON-A-CHIP – Johns Hopkins University

  • Silicon substrate for living human cells
  • Controlled environment
  • Emulate how cells function inside a living human body
  • Replace controversial and costly animal testing
  • Lab-on-a-chip: a cost effective end to animal testing


Chapter 10: Intra-Cranial Studies: Pressure Measurement, Monitoring and Adaptation

10.1:   CEREBRAL PRESSURE SENSOR – Fraunhofer Institute

  • Sensor to monitor cerebral pressure that can lead to dementia
  • Pressure changes in the brain can be measured and transmitted
  • Reading device outside the patient’s body
  • Operating at very low power, the sensor module
  • Powered wirelessly by the reading device


10.2    WIRELESS, IMPLANTABLE BRAIN SENSOR – National Institute of Biomedical Imaging and Bioengineering

  • Fully implantable within the brain
  • Allow natural studies of brain activity
  • Cord-free control of advanced prosthetics

Wireless charging Prototypes transmitted brain activity data


Chapter 11: Cardiac and Cardiovascular Monitoring System


  • RF-addressed wireless pressure sensor are powered by inductive coupling
  • Do not need batteries MEMS pressure sensor
  • Wireless antenna are inserted near the heart
  • With a catheter, Blood-pressure readings
  • Are sent to a wireless scanner for monitoring Pressure changes
  • Deflect the transducer’s diaphragm
  • Change the LC circuit’s resonant



  • Working prototypes were developed on inexpensive 3D printers
  • The 3D elastic membrane is made of a soft, flexible, silicon material
  • Precisely shaped to match the outer layer of the heart


Chapter 12: microfluidic chips


  • Watertight pump mounted on a disposable skin patch
  • Provides continuous insulin infusion
  • Controlled by a dedicated smart phone device
  • Incorporating a BGM (blood- glucose meter)




Polydimethylsiloxane called PDMS or dimethicone is a polymer widely used for the fabrication and prototyping of microfluidic chips.

It is a mineral-organic polymer (a structure containing carbon and silicon) of the siloxane family (word derived from silicon, oxygen and alkane). Apart from microfluidics, it is used as a food additive (E900), in shampoos, and as an anti-foaming agent in beverages or in lubricating oils.

For the fabrication of microfluidic devices, PDMS (liquid) mixed with a cross-linking agent is poured into a microstructured mold and heated to obtain a elastomeric replica of the mold (PDMS cross-linked).


Why Use PDMS for Microfluidic Device Fabrication?


PDMS was chosen to fabricate microfluidic chips primarily for those reasons:

Human alveolar epithelial and pulmonary microvascular endothelial cells cultured in a PDMS chip to mimick lung functions

  • It is transparent at optical frequencies (240 nM – 1100 nM), which facilitates the observation of contents in micro-channels visually or through a microscope.
  • It has a low autofluorescence [2]
  • It is considered as bio-compatible (with some restrictions).

The PDMS bonds tightly to glass or another PDMS layer with asimple plasma treatment. This allows the production of multilayers PDMS devices and enables to take advantage of technological possibilities offered by glass substrates, such as the use of metal deposition, oxide deposition or surface functionalisation.

PDMS, during cross-linking, can be coated with a controlled thickness on a substrate using a simple spincoat. This allows the fabrication of multilayer devices and the integration of micro valves.

It is deformable, which allows the integration of microfluidic valves using the deformation of PDMS micro-channels, the easy connection of leak-proof fluidic connections and its use to detect very low forces like biomechanics interactions from cells.



  • Ferrite RF radiation Acoustic wave Rectifier
  • Buried in PDMS Implantable miniature pressure sensor
  • Powered by an acoustically actuated cantilever
  • No battery required
  • Acoustic waves in the 200-500 hertz range
  • Cause cantilever to vibrate
  • Scavenging energy to power pressure sensor


Chapter 13: Peropheral Neuropathy Management

13.1    WIRELESS SHOE INSERT – Mobile Health News

  • WIRELESS SHOE INSERT – Mobile Health News
  • Help diabetics manage peripheral nerve damage
  • Insole collects data of where wearers
  • Putting pressure on their feet
  • Transmits wirelessly to a wristwatch-type display
  • Prevent amputations that often stem from diabetic foot ulcers


Chapter 14: Endoscopic Diagnostics Tools


  • For gastrointestinal and urological imaging
  • Alternative to biopsies in cancer detection
  • A laser beam pointed at the mirror is precisely deflected
  • Steered by the scanning mirror to reach a target


Chapter 15: MEMS guided Surgical Tools

15.1    MICROMACHINED SURGICAL TOOLS; SILICON MEMS TWEEZERS – ElectrolQ Used for minimally invasive surgical (MIS)

  • Procedures where diagnosis, monitoring, or treatment of diseases are performed
  • Performing with very small incisions MEMS
  • Based microsurgical tools is a key enabling technology for angioplasty, catheterization, endoscopy, laparoscopy, and neurosurgery


Summary by Dr. Pearlman

  • Multiple projects by Academia & Industry
  • Multiple MEMS devices for measuring body activities.
  • Many patch type devices attached to the skin
  • Devices attached to the eye
  • Smaller is better, lower footprint, lower power





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Third Annual BioPrinting and 3D Printing in the Life Sciences, 21-22 July 2016 at Academia, Singapore General Hospital Campus

Reporter: Aviva Lev-Ari, PhD, RN



Select Biosciences South East Asia are pleased to present Bioprinting and 3D Printing in the Life Sciences, taking place on the 21-22 July 2016 at Academia, the state-of-the-art conference facilities housed within the Singapore General Hospital Campus.

Building on the success of the 2013 and 2014 meetings The International Bioprinting Congress, we have decided to increase the scope of the event for 2016 to include the latest advances within 3D Printing for the Life Science arena.

We are honoured to again be working in partnership with our Conference Chairman, Professor Chua Chee Kai, Executive Director, Singapore Centre for 3D Printing (SC3DP), School of Mechanical and Aerospace Engineering, Nanyang Technological University, Singapore.

We welcome Professor Martin Birchall, from University College London and Assistant Professor Wai Yee Yeong, from Nanyang Technological University as our Keynote Speakers for 2016.

The meeting will include scientific presentations from the leading international experts covering the latest advances developments and techniques within these allied fields, two highly topical panel discussions which will also highlight the views of the international regulatory authorities plus a tour of the facilities at the Centre for 3D Printing, hosted by Professor Chua.

We will provide you with a balanced overview of the industry from the varied perspectives of the leading researchers, solution providers and legislative authorities.

Attending this meeting will give you an excellent opportunity for networking and help you to build long term collaborations within this rapidly developing field.

We hope you can join us.





Join the Third Annual Bioprinting and 3D Printing in the Life Sciences, taking place on the 21-22 July 2016 at Academia, Singapore General Hospital Campus.

Working in partnership with our Conference Chairman, Professor Chua Chee Kai, Executive Director, Singapore Centre for 3D Printing (SC3DP), Nanyang Technological University, Singapore.

We welcome Professor Martin Birchall, from University College London, Assistant Professor Wai Yee Yeong, Programme Director, SC3DP, Nanyang Technological University and Associate Professor Roger Narayan, University of North Carolina at Chapel Hill, as our Keynote Speakers for 2016.

The meeting will include scientific presentations from the following international experts who have already confirmed their participation.

Paulo Jorge Bártolo,

Chair of Advanced Manufacturing Processes & Director of the Manchester Biomanufacturing Centre, University of Manchester

Goh Bee Tin,

Senior Consultant, Department of Oral and Maxillofacial Surgery (OMS), Research Director and Deputy Director, Research and Education , National Dental Centre Singapore

Jerry Fuh,

Professor, National University of Singapore

Michael Golway,

President & CEO, Advanced Solutions, Inc.

Nazia Mehrban,

Post-Doctoral Researcher, University College London

L.P. Tan,

Associate Professor, School of Materials Science and Engineering, Nanyang Technological University

William G Whitford,

Senior Manager, GE Healthcare

Shoufeng Yang,

Associate Professor, University of Southampton

We are still accepting abstract submissions, if you would like to be considered for an oral presentation at this meeting, Submit an abstract for review now!

Oral Presentation Submission Deadline: 31 March 2016

We will address the following subject areas;

3D-Printing Applications in the Life Sciences

4D Bioprinting

Additive Manufacturing Technologies and Substrates

Bio-Ink and Bioprintable Hydrogels

Biofabrication and 3D-Bioprinting Technologies and Tools

Blueprints (Digital Models of Organs in STL Files)

Emerging Trends in Bioprinting

Intellectual Property and Patent Landscape in the Bioprinting Field

Laser Printing

Medical and Non-Medical Applications of Bioprinted Products

New Bioprinters

Organ Printing

Scaffolds and Biomaterials for Tissue Engineering

Technology Platforms for 3D-Printing

The application of Additive Manufacturing and Medical Devices

We hope you can join us for this exciting event, for further details please do not hesitate to contact me.

Best Regards


Linda Eriksson

Conference Manager

Select Biosciences South East Asia Pte. Ltd.

16 Raffles Quay, #33-03 Hong Leong Building,

Singapore 048581

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Biofabrication with Stem Cells

Curator: Larry H. Bernstein, MD, FCAP



Biofabrication  Special Issue:  Dec 2015; 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)

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.



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

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.


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).



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

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

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

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


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

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

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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Progress towards 3D Bioprinting of blood supply: steps towards production of functional blood vessels presage production of  other viable body part replacements

Curator and Writer: Justin Pearlman MD PhD

3D printing of human tissue requires a blood supply to support tissues that are more than a few millimeters thick. In recognition of the importance of achieving bioprinting with functioning vasculature, a Chinese company has committed to building a BioPrinter and BioInk (cell precursors and support) focused on vasculogenesis suitable for human implants. Meanwhile, researchers in the United States and in Australia have succeeded in printing vascular trees, for example, a branching vascular-like structure that supported blood circulation when grafted to a rat. The vascular pathways are printed within surrounding structures as a dissolvable substance (e.g., sugar) subesquently removed. Remaining steps include establishing adequacy of tissue perfusion and sustainable viability of the vessel integrity and function as well as survival of the surrounding tissue. Adult humans maintain abilities to remodel blood vessels (vasculogenesis, arteriogenesis, angiogenesis) so it may suffice to provide printed blood conduits and precursor cells and/or stimulants as a bridge and scaffold to vasculature development plus tissue perfusion in vivo, if they can meet the milestones of adequacy of biosafety, support of blood circulation, tissue perfusion, sustainability, as well as adaptation with promotion of growth and remodelling as needed.


Researchers successfully 3D print blood vessels, a ‘game changer’ for artificial organs

Mopic / Shutterstock

Above: An illustration of the inside of a blood vessel.

Image Credit: Mopic / Shutterstock

Hundreds of thousands of people die annually because the demand for organs far exceeds the donor supply. Artificial organs could save those lives — and scientists just made a huge breakthrough in the field by “bio-printing” artificial vascular networks.

Researchers from the University of Sydney, MIT, Harvard, and Stanford have successfully bio-printed blood vessels, offering 3D-printed organs access to nutrients, oxygen, and waste-disposal routes, according to a study published Monday.

“While recreating little parts of tissues in the lab is something that we have already been able to do, the possibility of printing three-dimensional tissues with functional blood capillaries in the blink of an eye is a game changer,” said Dr. Luiz Bertassoni, the study’s lead author and a University of Sydney researcher.

The vascular network of the human liver.

To 3D print vascular networks, the researchers fabricated fine, interconnected fibers with an advanced bioprinter. Then they coated those fibers with human endothelial cells — these sit between circulating blood and vessel walls in the interior of blood vessels — and subsequently applied a protein-based material. They hardened the whole structure with light, then delicately removed the fibers, leaving behind a complex network of hollow cell material. After a week, those cells organized themselves into stable capillaries.

Cells inside the bioprinted vascular networks survived, differentiated, and proliferated at better rates than cells that received no nutrient supply.

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