Artificial organs and tissue engineering

Yulia Kondratenko, "Biomolecule" 

We will continue the special project on the problems of aging with a story about the most outstanding and famous researchers who initiated the work on the creation of artificial organs. Most of them are still working on ambitious new projects.

Linda Griffith and Charles Vacanti

Linda Griffith is a professor of bioengineering and mechanical engineering at the Massachusetts Institute of Technology. In 2006, she received the MacArthur Scholarship, also known as the "grant for geniuses". Co-author of a pioneering work on growing cartilage in the shape of a human ear. At the moment, he is developing technologies for the cultivation of 3D cell cultures, and also participates in the "Man on a Chip" project.

Charles Vacanti is a professor at Harvard Medical School. Co-author of pioneering works on growing cartilage in the shape of a human ear, as well as the first anatomically shaped artificial bone (for a patient with a thumb injury). I am convinced of the existence of a way to switch specialized cells to stem cells that does not use genetic modifications. His conviction was not shaken even by the scandal with his former graduate student, Haruko Obokata, who fabricated the results of an experiment to obtain stem cells. Charles Vacanti argued until the last moment that Haruko Obokata's protocols should work. In September last year, after the falsification of data by a Japanese researcher was proved, he went on a one-year sabbatical. Apparently, after his graduation, Charles Vacanti plans to continue searching for a simple way to obtain stem cells.

In the late 1990s, a creepy picture of a mouse with a human ear on its back was distributed on the Internet (Fig. 1). The picture was sent mainly by e-mail, and the signatures to it were lost over time. Many people did not believe that the picture was real, and others began to actively protest against genetic engineering, as a result of which, according to these people, an ugly mouse was born. The picture was real. The human ear on the mouse's back was grown, of course, without the use of genetic modifications (it was already clear at that time that organs were formed by the complex interaction of multifunctional genes, and no "human ear gene" could exist). And the work for which the unfortunate mouse was obtained was one of the pioneers in the field of engineering of artificial human organs [1].

Figure 1. A famous photograph from a work made in the 90s. The animal, contrary to the assumptions of many frightened people, was not subjected to genetic modifications, but only served as an environment in which the synthetic base of the ear was populated with cells deposited on it. Bioreactors more suitable for incubating an artificial organ simply did not exist at that time. Figure from [1].

The ear, in truth, was human only in shape, and its constituent cells were taken from a calf. Nevertheless, the authors of the work, including Linda Griffith and Charles Vacanti, took the first step towards creating such frighteningly complex structures as human organs. There are so few donor organs, and there are so many problems with them (both immunological and psychological) that timidity before creating artificial parts of the human body was simply necessary to overcome.

 The strategy used by Linda Griffith and Charles Vacanti is still popular in the bioengineering of artificial organs with a complex structure. First, a frame is obtained from a degradable polymer, and then it is populated with cells that gradually corrode the frame, divide and master the vacated space. In a less "pure" version of the same method, organ bases obtained from other animals or donors are used, their cells are destroyed, and the resulting matrix is populated with recipient cells. Such an organ cannot be considered completely artificial, and yet, it is better than a donor one, since it does not contain its cells and does not cause rejection by the immune system. This variant of the method is used when the frame is difficult to obtain artificially due to its complex structure or composition and when this frame should be part of the resulting organ, and not corrode in the process of colonization by cells.

The settlement of the frame should take place in conditions as close as possible to the conditions inside the body – with the correct temperature and the flow of nutrient solutions through its parts. Now special reactors are used for this, which have to be adjusted to the shape of a certain organ. And in the first works of the 90s, mice and rats were used as bioreactors, to which the bases of organs populated with cells were simply implanted under the skin. Such animals looked scary, but the goal – the first artificial cartilage in the shape of a human ear – was achieved.

Linda Griffith continued her work in the field of artificial tissue engineering. Now, under her leadership, a three-dimensional culture of liver cells is maintained in a special bioreactor. Such a culture is far from an artificial liver – it does not resemble it in structure, but nevertheless it is suitable for drug research and hepatocyte metabolism in conditions close to natural. The researcher is engaged in the development of organs on chips, which were invented in 2010 by Donald Ingber (we will talk about him later).

Charles Vacanti became interested in the other side of the issue of growing artificial organs – stem cell research. The fact is that the cells needed to grow a new organ are not always convenient (if at all possible) to take from a donor. Therefore, before learning how to grow complex structures from suitable cells, it is first wiser to learn how to obtain these suitable cells. Charles Vacanti was interested in converting cells that are easy to take from a donor (for example, from the surface of the skin) into cells of the required type. To do this, it was necessary to learn how to turn specialized cells into stem cells – that is, capable of acquiring any specialization. And, of course, it is important for bioengineers that the method of reprogramming cells is not too complicated, otherwise the benefits of its use will come to naught. Charles Vacanti was convinced that the body should have a way to switch cells to the stem state* if necessary – such an ability seemed to him too profitable.

 * – Perhaps the solution lies in IPSC – induced pluripotent stem cells, which can be obtained from cells of various specializations. Read about the problems of obtaining them and the risks of using them in the articles "In search of cells for IPSC – step by step to the medicine of the future" and "IPSC Fuse" [2, 3].

Yoshiki Sasai (drawing from the website blogs.nature.com ) is an outstanding bioengineer, a pioneer in the field of obtaining mini–organoids by reproducing the first stages of human embryonic development. He reproduced the initial stages of the development of the cerebral cortex, as well as the ocular glass and the pituitary gland of the embryo. In his laboratory, a young researcher Haruko Obokata was searching for a simple method of turning specialized cells into stem cells. Haruko Obokata fabricated the data on the success of her research. Tired of the attention of the press and accusations of the scientific community of insufficient control over the progress of work under his leadership, Yoshiki Sasai hanged himself on the railing of the stairs of his institute in August 2014 [4].

All living organisms go through a long and difficult path of development before they acquire a final, often very complex structure. If we want to get a copy of an artificial organ, it is worth remembering exactly how this organ is formed in nature. Reproduction of the embryonic development of an organ is a very promising path for bioengineers. Yoshiki Sasai became famous for his work in this area. In 2008, the results of work on reproducing the first stages of the development of no less than the human brain were published [5]. And in 2011, Japanese researchers led by Sasai received the beginnings of the pituitary gland [6] and eye glasses (Fig. 2) [7].

Figure 2. Reproduction of the development of eye glasses (colored green) 
in cell culture. Figure from [7].

Only mini-organoids can be grown "in a test tube" (more precisely, on a Petri dish), because the further stages of their development require a complex three-dimensional environment, which, in turn, must also develop with the growth of the organ. Nevertheless, the selection of conditions that stimulate cells to repeat at least the first stages of organ development already provides a lot of useful data for embryology. In addition, the formation of pathology can be traced on mini-organoids grown from cells with genetic mutations. And of course, mini-organoids are suitable for testing drugs and especially for studying their effect on the early stages of the body's development.

Unfortunately for Yoshiki Sasai, there were works on other topics under his leadership. In early 2014, an article was published in the journal Nature, the first author of which was Haruko Obokata, and the last was Yoshiki Sasai. The article described a surprisingly simple method of reprogramming specialized cells into stem cells – using a short incubation in a solution of citric acid. Stem cells obtained in this way were called STAP (stimulus-triggered acquisition of pluripotency). STAP cells could cause a real revolution in regenerative medicine - with such a simple method, as described by Japanese scientists, stem cells could be obtained in huge quantities. Unfortunately, no other researchers, except Haruko Obokata, were able to obtain STAP cells. Japanese scientists were bombarded with questions from disappointed colleagues and the press, and Haruko Obokata had to repeat the experiments in his own laboratory to prove that the method could work. She didn't succeed. During the investigation under the auspices of the RIKEN Institute, it turned out that Haruko Obokata had manipulated the data of the scandalous publication, and the head of the study, Yoshiki Sasai, did not know about it. In August 2014, the scientist, who was hard going through the scandal around the study, committed suicide. Haruko Obokata did not challenge the decision of the expert commission on the manipulation of the results.

Interestingly, during the scandal, Charles Vacanti (the former head of Haruko Obokata) actively defended Japanese scientists. In the end, he had to admit that the article was withdrawn reasonably, but despite this, he did not give up his favorite idea about the possibility of obtaining stem cells from specialized without laborious genetic modifications. In September last year, Charles Vacanti went on a one-year sabbatical, which has just ended by now.

It is unknown whether a simple way to obtain stem cells will one day be found. Anyway, another area of Yoshiki Sasai's research – the production of organoids – turned out to be very fruitful. In the following years, scientists from different groups managed to obtain mini-organoids of the intestine, stomach and kidneys [8-11]. The latest achievement in this field – organoids of the heart – belongs to the famous specialist in the creation of artificial organs Anthony Atala [12].

Anthony Atala (drawing from the website wakehealth.edu ) – Director of the Wake Forrest Institute of Regenerative Medicine. I learned how to get an artificial bladder, urethra and vagina from patients' own cells. Now there are dozens of people living all over the world with such artificial organs created under the leadership of Anthony Atala. Now the famous bioengineer is working on creating an artificial penis that would suit accident victims and men with congenital pathologies of the reproductive system.

Anthony Atala is the director of the whole Institute of Regenerative Medicine. Under the guidance of a scientist in this field, many remarkable works have been done, more and more complex. Anthony Atala is mainly engaged in the creation of artificial organs of the genitourinary system. He started with the simplest – the bladder [13]. In fact, the bladder is just a bag of cells, and operations in which bladders are made from intestinal tissues have been carried out for quite a long time. Of course, these organs have very different functions – the intestinal walls absorb nutrients, and the bladder simply serves as a reservoir for urine before its excretion. Therefore, of course, I wanted to learn how to get this simple organ from a more suitable material. Anthony Atala used the already mentioned method for this – growing cells on a special anatomically shaped frame. Such artificial bladders were implanted in several boys with pathologies of this organ in 1999. After 5 years of observation, Anthony Atala and colleagues reported that the artificial organs took root well and did not cause complications in the recipients [14]. After that, the scientist moved on to a more complex task – the creation of artificial vaginas. Unlike bladders, these organs have never been artificially obtained. At the same time, the device of the vagina is also not very complicated – it is a tube of cells. In 2005-2009, four girls with rare pathologies in which the reproductive system develops incorrectly were implanted with such artificial vaginas. In 2014, the scientist reported on the success of all operations, thanks to which the grown-up patients were able to live a normal sexual life [15]. At the same time, scientists under the leadership of Anthony Atala learned how to obtain another organ of a tubular structure – the urethra (urethra) [16]. Such artificial organs were implanted in five boys, and the operations were also successful and did not cause complications.

The next step was the most complex organ of the genitourinary system – the penis. Modern surgery already allows patients who have lost a penis due to accidents to sew on a donor organ. The first such operation was carried out back in 2006. However, two weeks after this complicated operation, the patient asked to remove the donor penis [17]. This decision seems strange only at first glance. The penis refers to organs that are donated only posthumously, and getting used to living with the penis of a deceased person is clearly more difficult than living with a donor kidney. For example, the recipient also refused the world's first transplanted arm shortly after the operation [18]. So the engineering of external organs is, in a sense, even more urgent than the engineering of vital parts of the body. After all, while surgeons are provided only with donor organs as material, many complex operations will take place in vain. In addition, in addition to psychological problems, problems of immunological compatibility also arise with donor organs – patients often have to take drugs that suppress the activity of the immune system so that it does not begin to attack a foreign part of the body.

It is much more difficult to construct a penis than just a bubble or a tube of cells, because for the functioning of this organ, the correct structure in its entire volume is necessary. It is absolutely necessary to reproduce the spongy tissue of the cavernous bodies, which swell during erection, as well as the structure of the vessels through which blood flows to this tissue. And, of course, you need to place the urethra in it, which should not be squeezed when the cavernous bodies swell. It is very difficult to reproduce such a structure from scratch, so Anthony Atala uses the collagen bases of donor organs, which are purified from cells using enzymes, to obtain artificial penises. Then it is populated with human cells, to which the organ can later be transplanted without problems (so far such operations have not been performed). According to Anthony Atala, no matter how severe the injury to the penis is, due to the fact that this organ continues inside the pelvis, it is always possible to take cells from a person to grow a new one [19].

Human artificial penises are still in development – in order for them to be transplanted to recipients, they must pass many complex tests. But there are already successful results for rabbits – animals with penises obtained by the Anthony Atala method successfully mate and procreate. However, it was not so easy to switch from rabbits to humans – to get a larger organ, it is not enough to simply proportionally increase the number of cells, incubation time and other parameters. In addition, with an increase in the volume of the organ, the requirements for its internal structure also become higher – after all, each cell of a living organism should be located from the nearest capillary at a distance of no more than 200 micrometers (which is approximately equal to the thickness of a human hair). Therefore, it is always more difficult to grow a large volumetric organ than a flat one (like a fragment of skin), a tubular one (like an artificial urethra) or a sac-shaped one (like a bladder).

Anthony Atala's interests are not limited to the genitourinary system. In his laboratory, work is underway to obtain artificial tissues of the liver, heart and lungs. In 2011, during the TED conference, the famous scientist excited the public by demonstrating a prototype of an artificial kidney obtained by 3D printing [20]. The key word that many did not pay attention to was "prototype" – the artificial kidney had the right shape, and also proved that with the help of 3D printing, you can get something at least superficially similar to the desired object. But the structure of the prototype kidney did not even come close to the complexity of the present organ, which is absolutely necessary for the kidney to perform its function. This organ should consist of the thinnest tubules, entangled with vessels, in order to secrete only unnecessary substances with urine, and return everything useful to the blood. Bioengineers have not yet managed to approach such complexity, and, of course, it was impossible to achieve it in 2011. However, it seems that it is the bioprinting method that will eventually allow scientists to obtain exactly the biological structures that he needs. Another famous bioengineer, Gabor Forgach, has developed and is actively developing this method.

Gabor Forgach (drawing from the website organprint.missouri.edu )

– famous bioengineer and entrepreneur from science. Under his leadership, the first commercial 3D bioprinter was created, on which samples of many tissues have already been printed. Together with his son Andras, he founded the Modern Meadow company, which produces artificial leather and artificial meat for eating.

In 1996, Gabor Forgach drew attention to a fact that has long been known to scientists – cells formed during the division of the embryo can move along it, but once they get to the final destination, they stick together with other cells. This led him to the idea that cells can be used as elementary units for construction – if you choose the right conditions, then the cells stacked in the desired structures will stick together themselves. However, the idea that a special printer could be used for such stacking of cells did not occur to him.

Thomas Boland was the first to think of printing biological objects [21]. He modified a conventional printer in such a way that it became possible to print biological materials on it, for example, proteins or bacteria. The device was not suitable for 3D printing. The idea, however, turned out to be sound, and eventually led to the development of bioprinters capable of printing complex three-dimensional structures.

It took Forgach a long time to develop his idea of self-bonding cells into a technology for producing three-dimensional artificial tissues. It took several years to develop a printer capable of applying this technology. The device had to become quite accurate and delicate in relation to sensitive cellular "ink". Forgacha managed to create such a device called Organovo only in 2009 [22]. In 2010, a human vessel was printed on this first bioprinter, and, which was important for Forgach from the very beginning, without any additional frames. Thanks to this, there is confidence that the organ will contain absolutely nothing that causes immunological rejection in the recipient (if the organ is grown from its own cells).

To make the cells an analogue of printer ink, they are placed in a special gel that does not allow the cells to stick together ahead of time. The printer prints, as a rule, not single cells, but their spherical clusters – spheroids (although the method allows you to use individual cells for printing, which is necessary for some structures), the idea of which also belongs to Gabor Forgach [23]. Each printed layer of cells is separated by a layer of gel, and the finished organ is sent to mature in an incubator. At the same time, the gel used for printing dissolves, and its vascular network develops inside the organ – the thinnest capillaries grow from the vessels. This is very convenient for bioengineers, because they don't know how to get such small vessels yet. In addition, if the organ is transplanted to the recipient, the vascular network of the host will necessarily penetrate into the new part of the body. However, this practice is more suitable for animals than for humans – in his case, it is too dangerous to rely on the fact that the necessary vessels grow into the organ themselves. In addition, it is absolutely impossible to hope that the vessels themselves will grow as needed in the case of organs with a complex structure, such as the kidneys already discussed. So it remains to hope for an increase in the accuracy of 3D printing in the future.

3D bioprinting continues to develop all over the world: in 2010, for the first time, it was possible to print a fragment of skin [24], and in 2014 – a heart valve (Fig. 3, a heart valve printed on a 3D bioprinter. Figure from [25].) [25] and a fragment of liver tissue* [26]).

* – How 3D printing is developing in Russia is described in the article "Organs from the laboratory" [28].

The latest achievement of bioprinting at the moment is a fragment of human nervous tissue with precisely positioned neurons, obtained this year under the guidance of Australian bioengineer Gordon Wells (the very case when it is necessary to print tissue with individual cells, not spheroids) [29].

Gabor Forgach not only initiated 3D printing of human organs for sick people or survivors of an accident. He was also the first to realize that artificial tissues and organs can be useful to all people without exception. Some animal products – such as meat and leather – are so good that it is difficult to create a full-fledged replacement for them. But now, thanks to bioengineering, they can be obtained in an ethical way – without killing animals. Gabor Forgach was the first to think that we already know enough to grow an artificial steak or a piece of leather. Getting them is much easier than many artificial organs that scientists are struggling to develop, and the need for meat and skin is much higher than in human organs. Also, the transition to meat and leather of artificial origin would favorably affect the ecological situation – after all, bioreactors do not trample huge pastures and do not emit such an amount of methane into the atmosphere, which can significantly enhance the greenhouse effect.

Therefore, Forgach's second company, which he founded together with his son Andras – Modern Meadow – grows meat and skin in laboratory conditions [30]. An important aspect of the company's activities is the optimization of methods, since now artificial copies of animal products are expensive. Another problem is that the public is distrustful of lab-grown products. According to a survey conducted in 2014, only 20% of Americans are ready to try meat obtained by laboratory methods [31]. Therefore, Forgach himself tries to prove to people that his products are safe, including by his own example. For example, in 2011, at the TedMed conference, Forgach personally cooked and then ate meat grown in the laboratory [32]. In addition, the bioengineer assures that his laboratories are open to potential customers, and everyone can see how a sausage is made, while "slaughterhouses never invite visitors to watch their work" [31].

Gabor Forgach realized that biotechnologies lack proper technology – many of the methods used in attempts to reproduce the most complex structure of organs were old-fashioned in nature. Biology remains not a very precise science, but when creating artificial organs for living people, according to Forgach, it is unacceptable to expect that the correct structure will somehow form itself. 3D bioprinters follow the trends of the times and realize dreams of precise control over what seems completely chaotic and mysterious – life. And only one direction of bioengineering is perhaps even more technologically advanced and futuristic – organs on chips.

Donald Ingber (drawing from the website newyorker.com )

Donald Ingber is a biologist, famous for his engineering view of living objects, thanks to which the scientist made several discoveries in the field of cell biology (for example, about the influence of mechanical influences on the activity of genes). The author of the idea of an "organ on a chip" – the simplest cellular system located on a standard-sized plate and reproducing the basic functions of the simulated organ. He has created many organs on chips, and is now working on combining ten such organs into a "man on a chip".

Before the beginning of the two thousandth, Donald Ingber studied the biology of cancer – the parameters that affect the development of tumors and metastasis of cancer cells. At the same time, the scientist looked at the living cell as an engineer. The scientist's approach to cell biology research was influenced, oddly enough, by one unusual sculpture that Donald Ingber saw in the mid-70s. The sculpture was designed according to the principle of tensegrity. Such structures consist of strong beams that do not touch each other thanks to a system of stretched cables. The entire structure is supported by precisely balanced tensioning of flexible elements. Donald Ingber suggested that the structure of a living cell can be maintained thanks to the same principles. Indeed, he was able to show, for example, that mechanical influences applied to the cell surface can affect the shape of its nucleus and even the expression of genes. A deep understanding of how mechanical forces affect the structure and function of cells helped the scientist to advance in the study of cancer biology [33].

Probably, this desire to introduce cell research into a more understandable, "mechanical" plane, eventually led Donald Ingber to the idea of organs on chips. An organ on a chip is a plate no bigger than a credit card. In the plate there are cells populated with cells of certain types. The cells are connected by channels simulating blood flow or exchange of tissue fluid between groups of organ cells. Of course, such a device does not reflect the shape of a natural organ, but it models the very essence of its work in the most compact and controlled form. The vital activity of cells in an organ on a chip must be maintained by placing the chip in a special reactor that drives nutrient solutions through the channels of the chip at the correct pressure and maintains a certain temperature and the content of dissolved gases in these liquids.

The most important advantage of organs on chips corresponds to technological trends: it is modularity – the ability to make different combinations of such devices. Chips depicting various organs can be connected to each other to study the effect of these organs on each other, to simulate the movement of pathogenic microbes through various body systems, or to study what happens to drug molecules when it enters the body.

Donald Ingber and his colleagues developed the first device of this type – a lightweight one on a chip – in 2010 [34]. The channels of this device are divided into two parts by a porous membrane, on one side of which there is a layer of lung cells, and on the other – a layer of vessel wall cells. In the part of the channels where the vessel cells were located, blood circulates, and the one where the lung cells are located is filled with air. Special openings lead to both parts of the channels – you can add drugs or, for example, pathogens to simulate their entry into the lung from the air or with blood flow.

Since then, it has been possible to reproduce the work of the kidney [35], liver [36], as well as the intestine with microbiome and peristalsis on chips (Fig. 4) [37]. The development of a chip reflecting the blood-brain barrier device turned out to be particularly interesting for clinical research [38]. The developers have reproduced both the tight contacts between the cells of the brain vessels, and the location of glial cells – features due to which many molecules from the blood cannot easily penetrate into the brain. When testing drug prototypes, it is very useful to find out whether they are able to penetrate the blood-brain barrier, and if so, with what effectiveness. In addition, it was possible to reproduce the device of the hematopoietic niche of the bone marrow on the chip, which is extremely useful for studies of diseases in which the normal development of blood cells is disrupted [39].

4

Figure 4. "Intestines on a chip". a. Device diagram.
A flexible porous membrane lined with intestinal epithelial cells is located horizontally in the center of the microchannel, on the sides of which there are vacuum chambers. b. Photo of the "intestine on a chip" consisting of a transparent PDMS elastomer (polydimethylsiloxane elastomer).
In the direction of the arrows, red and blue liquids are pumped into the lower and upper compartments of the microchannel, respectively, to visualize them. Figure from [37].

Naturally, researchers are striving to create a "man on a chip" – that is, a system of all vital organs in which it will be possible to study the transport of substances and microbes in the body, as well as the influence of organs on each other, in the most accurate way. In addition to Donald Ingber, Linda Griffith is also involved in this ambitious project.

Literature

1. Vacanti C.A., Cimaa L.G., Ratkowskia D., Uptona J., Vacanti J.P. (1991). Tissue engineered growth of new cartilage in the shape of a human ear using synthetic polymers seeded with chondrocytes. MRS Online Proc. Libr. 252, 367;

2. Biomolecule: "In search of cells for IPSC – step by step to the medicine of the future";

3. biomolecule: "IPSK fuse";

4. Baklanov A. (2014). A scientist committed suicide because of a scientific scandal. Website "Snob";

5. Eiraku M., Watanabe K., Matsuo-Takasaki M., Kawada M., Yonemura S., Matsumura M. et al. (2008). Self-organized formation of polarized cortical tissues from ESCs and its active manipulation by extrinsic signals. Cell Stem Cell. 3, 519–532;

6. Suga H., Kadoshima T., Minaguchi M., Ohgushi M., Soen M., Nakano T. et al. (2011). Self-formation of functional adenohypophysis in three-dimensional culture. Nature. 480, 57–62;

7. Eiraku M., Takata N., Ishibashi H., Kawada M., Sakakura E., Okuda S. et al. (2011). Self-organizing optic-cup morphogenesis in three-dimensional culture. Nature. 472, 51–56;

8. Sato T., Vries R.G., Snippert H.J., van de Wetering M., Barker N., Stange D.E. et al. (2009). Single Lgr5 stem cells build crypt–villus structures in vitro without a mesenchymal niche. Nature. 459, 262–265;

9. McCracken K., Catá E.M., Crawford C.M., Sinagoga K.L., Schumacher M., Rockich B.E. et al. (2014). Modelling human development and disease in pluripotent stem-cell-derived gastric organoids. Nature. 516, 400–404;

10. Takasato M., Er P.X., Becroft M., Vanslambrouck J.M., Stanley E.G., Elefanty A.G., Little M.H. (2014). Directing human embryonic stem cell differentiation towards a renal lineage generates a self-organizing kidney. Nat. Cell Biol. 16, 118–126;

11. biomolecule: "A pea-sized stomach + human intestines grown in a mouse";

12. Bargmann J. (2015). Exclusive: watch this video of 3D-printed heart cells beating. Popular Mechanics Website;

13. Atala A. (1999). Tissue engineering for bladder substitution. World J. Urol. 18, 364–370;

14. Wake Forest physician reports first human recipients of laboratory-grown organs. (2006). Wake Forest Baptist Medical Center Website;

15. Raya-Rivera A., Esquiliano D., Fierro-Pastrana R., López-Bayghen E., Valencia P., Ordorica-Flores R. et al. (2014). Tissue-engineered autologous vaginal organs in patients: a pilot cohort study. Lancet. 384, 329–336;

16. De Filippo R., Yoo J.J., Atala A. (2002). Urethral replacement using cell seeded tubularized collagen matrices. J. Urol. 168, 1792–1793;

17. Sample I. (2006). Man rejects first penis transplant. The Guardian website;

18. Transplanted hand amputated. (2001). The New York Times website;

19. Mohammadi D. (2014). The lab-grown penis: approaching a medical milestone. The Guardian website;

20. Atala A. (2011). Printing a human kidney. Video on the TED website;

21. Wilson W.C. Jr. and Boland T. (2003). Cell and organ printing 1: Protein and cell printers. Anat. Rec. A Discov. Mol. Cell Evol. Biol. 272, 491–496;

22. History. Organovo website;

23. Jakab K., Neagu A., Mironov V., Markwald R.R., Forgacs G. (2004). Engineering biological structures of prescribed shape using self-assembling multicellular systems. Proc. Natl. Acad. Sci. USA. 101, 2864–2869;

24. Binder K., Zhao W., Aboushwareb T., Dice D., Atala A., Yoo J.J. (2010). In situ bioprinting of the skin for burns. JACS. 211, s76;

25. Duan B. et al. (2014). Three-dimensional printed trileaflet valve conduits using biological hydrogels and human valve interstitial cells. Acta Biomater. 10, 1836–1846;

26. Renard M. (2014). Organovo announces commercial release of the exVive3D™ human liver tissue. Organovo website;

27. L’Oreal to start 3D-printing skin. (2015). BBC News Website;

28. biomolecule: "Organs from the laboratory";

29. Lozano R., Stevens L., Thompson B.C., Gilmore K.J., Gorkin R. 3rd, Stewart E.M. et al. (2015). 3D printing of layered brain-like structures using peptide modified gellan gum substrates. Biomaterials. 67, 264–273;

30. Forgacs A. (2013). Leather and meat without killing animals. Video on the TED website;

31. Gunther M. (2014). Lunch from a lab? A Brooklyn company builds a better burger. The Guardian website;

32. Gabor Forgacs: In vitro meat – it’s what’s for dinner! (2012). Video on the TEDMED website;

33. Tensegrity. Scholarpedia website;

34. Huh D., Matthews B.D., Mammoto A., Montoya-Zavala M., Hsin H.Y., Ingber D.E. (2010). Reconstituting organ-level lung functions on a chip. Science. 328, 1662–1668;

35. Janq K., Mehr A.P., Hamilton G.A., McPartlin L.A., Chung S., Suh K.Y., Ingber D.E. (2013). Human kidney proximal tubule-on-a-chip for drug transport and nephrotoxicity assessment. Integr. Biol. (Camb). 5, 1119–1129;

36. Nakao Y., Kimura H., Sakai Y., Fujii T. (2011). Bile canaliculi formation by aligning rat primary hepatocytes in a microfluidic device. Biomicrofluidics. 5, 022212;

37. Kim H., Huh D., Hamilton G., Ingber D.E. (2012). Human gut-on-a-chip inhabited by microbial flora that experiences intestinal peristalsis-like motions and flow. Lab. Chip. 12, 2165–2174;

38. Alcendor D., Block F.E. 3rd, Cliffel D.E., Daniels J.S., Ellacott K.L., Goodwin C.R. et al. (2013). Neurovascular unit on a chip: implications for translational applications. Stem Cell Res. Ther. 4, S18;

39. Torisawa Y., Spina C.S., Mammoto T., Mammoto A., Weaver J.C., Tat T. et al. (2014). Bone marrow-on-a-chip replicates hematopoietic niche physiology in vitro. Nat. Methods. 11, 663–669.

Portal "Eternal youth" http://vechnayamolodost.ru 15.02.2015