Robotic Biofabrication of Living Human Organs

Interview with
Dr. Vladimir Mironov, Director of the World’s First Bioprinting Research Center
February 14, 2007

Vladimir Mironov, MD, PhD, is an Associate Professor in the Department of Cell Biology and Anatomy at the Medical University of South Carolina. He received his MD degree and PhD in developmental biology in Russia and trained in angiogenesis research at the Max Planck Institute in Martinsried, Germany. He is a pioneer of organ printing technology and Director of the Bioprinting Research Center located at MUSC in Charleston, SC. He is a leader of one of the most ambitious tissue engineering projects — the Charleston Bioengineered Kidney Project. The ultimate goal of this project is to engineer living human kidney suitable for clinical implantation. He was a finalist for the prestigious World Technology Award for his development of the concept of organ printing. He is a consultant to several start-up biotech companies and venture capitalists, and a co-founder of two biotech companies. He is an author of more than 100 research papers and several books, and he holds several patents. Together with Canadian artist Tim Fedak, Dr. Mironov just completed educational comic “Adventures in Bioprinting: How to Print a Kidney.” He is an organizer of several workshops on Bioprinting and Vice President of the World Academy for Bioprinting. He is working now on an internet-based, open access introductory course on Robotic Biofabrication, and organizing a National Robotic Biofabrication Engineering Research Center and World Bioprinting Congress.

Q: Vladimir, you just returned from an NIH Kidney Development and Repair Conference where in your invited talk you presented the concept of robotic biofabrication of human kidney. What was reaction of conference participants to your talk?

A: First, I must say that it was a very interesting and inspiring conference. The idea was to create a new emerging field of renal regenerative medicine and bring developmental biologists, molecular biologists, pathologists, nephrologists and tissue engineers together. As one of the conference organizers Prof. Joseph Bonventre put it: “There is an increasing recognition that the sciences of developmental biology and injury and repair are converging to the advantage of both fields”. Basically, we try to understand the determinants of normal and abnormal repair and progressive renal disease, and develop protective and therapeutic strategies. Tissue engineering represents one such therapeutic strategy. Overall reception of my talk was very positive. As one famous developmental biologist from Harvard University said in discussion, we welcome “this bioprinting guy” and what he is doing. The NIH manager strongly suggested that I call him and apply for an NIH grant. I also liked such comments: “I am more convinced in the possibility of tissue engineered kidney now than before your talk. Good luck”. Of course, as always there are a few people (not professional experts in tissue engineering, by the way) for whom even the idea that one can build a complex human organ like the kidney in the next 10-15 years still sounds “ridiculous”. But one never knows what one can or cannot do until one finally tries to do it. Moreover, the building of living human organs suitable for clinical transplantation was the original promise, and still unaccomplished goal, of tissue engineering as a biomedical field. The real question is — are we ready and how much time and investment will it take to build a living human organ?

Q: So, what do think: are we ready?

A: I think it is safe to say that we are practically ready to print organs from an engineering point of view, but we still are facing a lot of challenges, potential pitfalls and unsolved problems from the biological point of view, especially in questions related to cell sources. But we believe, in parallel development, there is much to be learned with model (“clinically irrelevant”) cells.

Q: When you are talking about cell source and “clinically relevant” cells, do you mean human stem cells?

A: Yes, human stem cells are a big issue. It looks like three potential human stem cell sources are emerging. First, the still politically controversial human embryonic stem cells. Second, resident stem or progenitor cells which could be isolated from some organs. Finally, circulated bone marrow derived adult stem cells. Although there is published evidence that indicate all three potential cell sources are reasonable candidates for tissue repair, only further intensive and focused research can answer the question — what cell source is most suitable? The contribution of circulated stem cells is probably the most hotly debated subject. It is important to mention that even after identification of reasonable cell source of human stem cells, it will take a lot of effort to figure out reproducible technologies for their direct differentiation into the desirable cell type. There are 14 different cell types in kidney tubules alone. There are at least five main cell types in kidney glomerulus. Thus, identification, isolation, propagation and directed differentiation of human stem cells is a big challenge. But we assume that with $3 billion from California’s State Proposition 71 stem cell initiative and the recently announced $2 billion New York State stem cell initiative, these problems will be eventually solved. A growing number of excellent and very smart scientists are moving into this field of research, and with appropriate funding, solutions will come. It is just a function of time. In the meantime, we’ll continue to monitor progress in stem cell research, but focus our efforts on designing, developing and testing bioprinting technologies using clinically irrelevant model animal cells or differentiated human renal cells.

Q: Could you explain to our readers what is organ printing or robotic biofabrication?

A: Organ printing is the bioassembly of living 3D human organs using bioprinting technology. It is basically a biomedical application of the well established, rapid prototyping technology. Rapid prototyping is additive manufacturing or layer-by-layer material deposition. The term “organ printing” was first used by the “Nature News” science writer when she described the application of rapid prototyping (3D printing) for designing solid biodegradable scaffolds for tissue engineering on technology developed by Linda Griffiths, Micheal Cima, and Samuel Sacks at MIT. However, it is two step process and is based on the classic concept of solid-scaffold based tissue engineering: first one must print the scaffold and then perform cell seeding using a bioreactor. In our approach we employ a one step process and also we do not use a solid scaffold. Instead we use stimuli-sensitive fluidic hydrogels. The essense of our approach is simultaneous robotic layer-by-layer deposition of living cells and hydrogel. However, in order to be more inclusive, we use the term robotic biofabrication, which is a broad and fuzzy term, but which includes a variety of robotic and diversified rapid prototyping approaches: selective laser scintering, fused deposition modeling, 3D printing, stereolithography, laser engineered net shaping, direct laser writing, centrifugal casting, ink-jet printing and so on… Thus, we operationally define organ printing as computer-aided, layer-by-layer deposition of biologically relevant material with the purpose of engineering functional 3D tissues and organs.

Q: What is the recent progress with ink-jet bioprinting technology?

A: Ink-jet bioprinting technology was invented by our former collaborator Dr. Thomas Boland from Clemson University. He recently got a USA patent on this technology. We published several papers together, but it looks like ink-jet printers do not like cell aggregates. Our recent approach is based on using self-assembling cell aggregates that provide initial desirable high density, in addition to a dispensing device (“bioprinter”) which does not damage cell aggregate organization during the preprinting process. We have used the relatively affordable “Neatco Inc” (Toronto, Canada) robotic device, but the most sophisticated but expensive type of device has been developed by “Sciperio/nScript Inc” (Orlando, FL). We are successfully collaborating with this highly innovative company on a second generation bioprinter. Concerning ink-jet printers, I can say that they are very good for 2D cell patterning, but for 3D tissue printing with initial high cell density, they are probably not the device of first choice. But “never say never” and opportunities for creative innovation in ink-jet printing are unlimited. But recent ink-jet printers are not cell-aggregate friendly which has reduced our initial enthusiasm in their effective use for 3D organ printing. We believe that our strategic shift to cell aggregate friendly dispensing devices was correct. But only time can say who is right. Competition in this field is growing and the field is attracting more and more excellent engineers and researchers. The main beauty of ink-jet bioprinters is that they are compact and affordable. Their use for bioprinting cell-based arrays could be very beneficial.

Q: Why you have selected kidney and not other organs for bioprinting?

A: Paraphrasing President John F. Kennedy’s famous speech at Rice University, we can say: “We chose to do these things not because they are easy, but because they are hard, because that goal will serve to organize and measure the best of our energies and skills, because that challenge is one that we are willing to accept, one we are unwilling to postpone…” The kidney is probably the most complex organ in the human body. I know the kidney very well. I started my scientific career in a student scientific society by studying the ultrastructure of kidney glomerular filter using a transmission electron microscope. I published several papers on kidney glomerular development using scanning electron microscopy. My first lecture as a faculty member in the Department of Histology was about kidney development. I spent two years in Germany by trying to induce fenestrae in endothelial cells using their co-culture with kidney epithelial cells. My poster at my first USA Keystone Symposium on tissue engineering in 1992 was about co-culture of kidney epithelial and endothelial cells. The kidney is a very beautiful organ and I just love it. But the real reason is the urgent clinical need. Every day several dozen patients with chronic end stage renal disease are dying in this country while on the waiting list for kidney transplantation. Something must be done. As President Clinton said “We failed, but at least we have tried”. So we want to try…and not fail. It is definitely very challenging and probably the most ambitious project in the tissue engineering field, but we as a team strongly believe that it is doable and feasible.

Q: What are the main challenges in organ printing technology?

A: We identify 12 milestones in our roadmap for kidney bioprinting. We already accomplished 4 milestones and are working on the fifth. The main challenge is of course finding clinically relevant cell source. The second important challenge is designing a kidney “blueprint” using computer-aided design. We are still working on increasing diversity, optimization and standardization of “bioink“ or self-assembling tissue spheroids. Finding optimal hydrogels for bioprinting is also work in progress. Vascularization and maintenance of viability of the printed construct is a real big challenge. Another challenging part in post-processing is transformation of the printed tissue construct into the living human organ, so-called “accelerated tissue maturation”. Finally, sustainable funding for this project is another challenge. The management of a multidisciplinary team is also not a simple thing. But as one friend told me: “Vladimir, do not worry. It is only upper part of iceberg…’” Thus, we are expecting more hurdles, but we are ready to deal with this and solve any potential problems. We strongly believe that we can and are able to accomplish this project successfully and we have a great committed team of excellent experts, and most important, our University support.

Q: When do you think a first living human organ will be printed?

A: It is a very interesting question. I am a member of the World Future Society. According to predictions published in the journal “Futurist” and some popular scientific journals, it will be around 2030. I am always amazed by people who pretend that they have some sort of technology development chronometer. Development of the artificial heart took 25 years. Tissue engineered products such as skin, blood vessel and bladder took at least 10 years to develop. The kidney is a much more complex organ. I usually answer this question: it is not a function of time but rather a function of investment, or more correctly, carefully managed multidisciplinary efforts in a stable financial supporting environment. In the pharmacological industry it usually takes 10-15 years and $1 billion dollars to bring a new drug to market. I am 52 years old now and I really want to accomplish this project before I go to another world.

Q: How much investment is necessary to develop organ printing technology?

A: Short answer – I do not know and I am afraid that nobody knows exactly. We can only guess. It is interesting that artificial kidney (dialysis apparatus) was developed and clinically tested by Dr. Willem Kolff in NAZI-occupied Netherlands without any substantial financial support. Moreover, paradoxically he somehow benefited from occupation, because NAZI authorities did not allow Dutch companies to work for Dutch citizens and therefore the company that helped Dr. Kolff design the artificial kidney could not send him a bill. But our preliminary estimation is that the entire cost of this project, including building kidney producing plants with GMP standards and clinical trials, will probably be comparable with the cost of developing a new drug. It means around $1 billion dollars. As Prof. Joseph Bonventre (Harvard University) commented during above mentioned NIH conference - “It is an expansive dream”. However, keeping in mind the potential market of $15 billion, it is a reasonable and economically justifiable investment. It is also obvious that this project could not be successfully and completely accomplished in an academic environment. We will need at certain stages of project development strong industrial partners and/or venture capital money for start up companies. By the way, we are already working on this.

Q: How much will a printed kidney cost?

A: This is probably the simplest question. It will cost $250K. Why, because this is the cost of an artificial heart. Also, according to published medical economic studies perfomed by Dr. Schnitzler at Washington University, if recent laws prohibiting selling kidney for transplantation will be somehow changed, then potential kidney vendors can charge $250K. Interestingly, it will be cheaper than dialysis and save healthcare costs for treatment of end-stage kidney diseases. Together with Dr. Andrea Kutinova, a medical economist from New Zealand, we performed independent economic estimations using more recent statistical data and came to a similar conclusion. Thus $250K per kidney is an economically justifiable and affordable price which will save money for Medicare and other health providers. It will also allow to build commercially successful multibillion dollars robotic biofabrication industry and create well paid high tech jobs.

Q: It looks like robotization as well as progress in stem cell biology are essential to the tissue engineering field. What is the potential impact of nanotechnology on tissue engineering?

A: Nanotechnology is already impacting the field and potentially can transform tissue engineering. I can give you at least three, most impressive examples. Electrospinning of synthetic and natural derived polymer allows one to create a most sophisticated nanoscaffold that can biomimick natural extracellular matrix and provide optimal conditions for cell attachment, spreading, growth and differentiation. Second, magnetic iron oxide nanoparticles have been successfully used by Japanese groups for what they call magnetic driven tissue engineering. This approach will allow one to move from “directed tissue self-assembly”, which we are exploring in bioprinting technology ex vivo, to “self-directed tissue self-assembly” which can be used in injectable tissue engineering in vivo. Finally, together with our Dutch collaborators from Utrecht University we demonstrated that relatively low toxic tannic acid-mimicking dendrimers (more correctly dendrons) can be used for stabilization of decellularized natural scaffolds in cardiovascular tissue engineering. This is only the beginning and this list of examples can be extended. Mutually beneficial “marriage” of nanotechnology and tissue engineering is already an ongoing process. One can only expect more exciting things to come in the future.

Q: What are you working on right now?

A: I just finished an educational cartoon book “Adventure in Bioprinting: How to Print a Kidney” together with Canadian artist Tim Fedak. I am working on an internet-based, open access introductory course on bioprinting, and organizating the first certified course on robotic biofabrication. We are applying for a $32 million NSF Robotic Biofabrication Engineering Research Center (ROBERC) grant. We will soon incorporate a new start-up company with the self-explanatory name of Organovo. We are organizing the World Academy for Bioprinting and the first World Bioprinting Congress in Honolulu, Hawaii. Meantime, we are designing new robotic biofabrication tools, planning and doing experiments on building kidney glomeruli and nephron, and bioassembling a vascular tree, and writing papers and grants…just a normal, regular, routine but still very exciting academic life.

Thank for the interview and good luck in your efforts.