Part 3
Rudimentary liver grown in vitro
20 June 2012
Japanese scientists have used induced stem cells to create a liver-like tissue in a dish.
Although they have yet to publish their results and much work remains to be done, the achievement could have big clinical implications. If the results bear out, they would also constitute a significant advance in the ability to coax stem cells to self-organize into organs.
Induced pluripotent stem cells could be a useful source of human organs such as livers.
The work was presented by Takanori Takebe, a stem-cell biologist at Yokohama City University in Japan, at the annual meeting of the International Society for Stem Cell Research in Yokohama last week. “It blew my mind,” said George Daley, director of the stem-cell transplantation programme at the Boston Children’s Hospital in Massachusetts, who chaired the session.
“It sounds like a genuine advance,” says Stuart Forbes, who studies liver regeneration at the University of Edinburgh, UK. Forbes, who also works as a consultant for Scotland’s liver-transplantation unit, says that the advance could one day help to avoid the “bleak outcome” currently experienced by the many patients who don’t survive long enough to get a new liver.
But the liver described by Takebe has a long way to go before that. Takebe told how his team grew the organ using induced pluripotent stem cells (iPS), created by reprogramming human skin cells to an embryo-like state. The researchers placed the cells on growth plates in a specially designed medium; after nine days, analysis showed that they contained a biochemical marker of maturing liver cells, called hepatocytes.
At that key point, Takebe added two more types of cell known to help to recreate organ-like function in animals: endothelial cells, which line blood vessels, taken from an umbilical cord; and mesenchymal cells, which can differentiate into bone, cartilage or fat, taken from bone marrow. Two days later, the cells assembled into a 5-millimetre-long, three-dimensional tissue that the researchers labelled a liver bud — an early stage of liver development.
Under development
The tissue lacks bile ducts, and the hepatocytes do not form neat plates as they do in a real liver. In that sense, while it does to some degree recapitulate embryonic growth, it does not match the process as faithfully as the optic cup recently reported by another Japanese researcher. But the tissue does have blood vessels that proved functional when it was transplanted under the skin of a mouse. Genetic tests show that the tissue expresses many of the genes expressed in real liver. And, when transferred to the mouse, the tissue was able to metabolize some drugs that human livers metabolize but mouse livers normally cannot. The team claims that its work is “the first report demonstrating the creation of a human functional organ with vascular networks from pluripotent stem cells”.
Takebe says the success depended on properly timing the addition of the other two cell types. “It took over a year and hundreds of trials,” says Takebe.
The team says that the tissue's three dimensions will give it advantages over simple cell-replacement therapies. It could be used for long-term replacement or short-term graft while the recipient waits for a suitable liver donor, or in cases in which doctors anticipate that the native liver will eventually regain its function. But such applications would require extensive development, including making sure that the tissue contains the proper arrangement of lobules.
It won’t be easy, says Forbes. To treat the commonest reason for liver transplants, chronic liver disease, the cells would have to be stable, potentially for many years, in the patient. But it is not clear whether that would be possible, especially considering that they would be exposed to many toxins and pathogens. Furthermore, the organ would need to stay the right size, without atrophying or developing cancerous growth. “Any deviation from the mature phenotype could be catastrophic for the graft,” says Forbes.
A niche in the market
Other researchers have developed competing technologies using scaffolds to build three-dimensional liver-like structures. Sangeeta Bhatia, a bioengineer at the Massachusetts Institute of Technology in Cambridge, for example, has produced a scaffold-based graft1 that doesn’t try to recapitulate development but has proved to be functional and transplantable in mice. Bhatia is now working on increasing the number of hepatocytes present on the two-centimetre graft, to ensure that it is useful in the clinic. "One billion cells is the next frontier," she says.
In the meantime, Takebe and the rest of the team, led by Hideki Taniguchi, also a stem-cell biologist at Yokohama City University — who are collaborating on the project with researchers at Sekisui Medical, a biotechnology firm based in Tokyo — hope that his liver bud could be useful for toxicity testing in drug screening, for which bile ducts are not needed. Many conventional hepatocyte cells that are transplanted to mice for in vivo testing last for only two or three days, but the drug and its various metabolites might take weeks to metabolize, so toxic effects might not be apparent in such testing. Takebe says his graft has the necessary staying power.
Many researchers are already growing hepatocyte-like cells: Bhatia, for example, has already commercialized a device that uses bioengineered cells for drug testing2. However, Takebe’s liver bud has the advantage of being grown from iPS cells, rather than, for example, the primary human hepatocytes used in Bhatia's graft, which could make it useful in modelling rare diseases or examining the specific genetic backgrounds of the iPS cell donors.
Markus Grompe, who studies liver disease at the Oregon Health and Science University in Portland, says that Takebe's team is "on the right track”. Still, he says, the liver cells need to function much more efficiently than they do at present. On the basis of a cursory inspection of Takebe's data presented at the meeting, Grompe says that the liver bud was producing only a small fraction of the albumin — a plasma protein that is a key marker of liver function — that it should. But Takebe says that since his group generated the data presented at the Yokohama meeting, procedural improvements have already led to higher levels of albumin.
The next step for the team is to try to make the liver bud more liver-like, by including structures such as bile ducts.
http://www.nature.com/news/rudimentary-liver-grown-in-vitro-1.10848
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Miniature human liver grown in mice
03 July 2013
Cells self-organize and grow into functional organs after transplantation.
Transplanting tiny 'liver buds' constructed from human stem cells restores liver function in mice, researchers have found. Although preliminary, the results offer a potential path towards developing treatments for the thousands of patients awaiting liver transplants every year.
The liver buds, approximately 4 mm across, staved off death in mice with liver failure, the researchers report this week in Nature1. The transplanted structures also took on a range of liver functions — secreting liver-specific proteins and producing human-specific metabolites. But perhaps most notably, these buds quickly hooked up with nearby blood vessels and continued to grow after transplantation.
The results are preliminary but promising, says Valerie Gouon-Evans, who studies liver development and regeneration at Mount Sinai Hospital in New York. “This is a very novel thing,” she says. Because the liver buds are supported by the host’s blood system, transplanted cells can continue to proliferate and perform liver functions.
However, she says, the transplanted animals need to be observed for several more months to see whether the cells begin to degenerate or form tumours.
There is a dire scarcity of human livers for transplant. In 2011, 5,805 adult liver transplants were done in the United States. That same year, 2,938 people died waiting for new livers or became too sick to remain on waiting lists.
However, attempts to create complex organs in the laboratory have been challenging. Takanori Takebe, a stem-cell biologist at Yokohama City University in Japan who co-led the study, believes this is the first time that people have made a solid organ using induced pluripotent stem cells, which are created by reprogramming mature skin cells to an embryo-like state.
Testing whether liver buds could help sick patients is years away, says Takebe. Apart from the need for longer-term experiments in animals, it is not yet possible to make liver buds in quantities sufficient for human transplantation.
In the current work, Takebe transplanted buds surgically at sites in the cranium or the abdomen. In future work, Takebe hopes to create liver buds small enough to be delivered intravenously in mice and, eventually, in humans. He also hopes to transplant the buds to the liver itself, where he hopes they will form bile ducts, which are important for proper digestion and were not observed in the latest study.
Self-organizing structures
The researchers make the liver buds from three types of human cells. First, they coax induced pluripotent stem cells into a cell type that expresses liver genes. Then they add endothelial cells (which line blood vessels) from umbilical cord blood, and mesenchymal stem cells, which can make bone, cartilage and fat. These cell types also come together as the liver begins to form in the developing embryo.
“It’s a great day for developmental biology,” says Kenneth Zaret, who studies regenerative medicine and liver development at the University of Pennsylvania in Philadelphia. “By reconstituting cell interactions that we know are important for natural liver progression, they get what appears to be robust, mature tissue.”
The project began with an unexpected phenomenon, says Takebe. Hoping to find ways of to make vascularized liver tissues, he tried culturing multiple cell types together and noticed that they began to self-organize into three-dimensional structures. From there, the process for making liver buds took hundreds of trials to tweak parameters such as the maturity and ratios of cells.
Other organs
This strategy takes a middle path between two common strategies in regenerative medicine. For simple, hollow organs such as the bladder and trachea, researchers seed scaffolds with living cells and then transplant the entire organ into patients. Researchers have also worked to create pure cultures of functional cells in the laboratory, hoping that cells could be infused into patients, where they would establish themselves. But even if the cells work perfectly in the laboratory, says Gouon-Evans, the process of harvesting cells can damage them and destroy their function.
Zaret thinks that the liver buds work might encourage an intermediate approach. “Basically, put the cells in a room together and let them talk to each other and make the organ.”
Self-organizing structures from stem cells have also been observed for other organ systems, such as the optic cup, an early structure in eye development2. And 'mini-guts' have been grown in culture from single human stem cells3.
Takebe believes that the self-organizing approach might also be applicable to other organs, such as lung, pancreas and kidney.
http://www.nature.com/news/miniature-human-liver-grown-in-mice-1.13324
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Russian scientists planning the first 3D-printed organ transplant on mice
April 27, 2015
Russian scientists are planning the first 3D-printed organ transplant on mice. Humans could be next.
Russia's 3D Bioprinting Solutions laboratory, the first facility to successfully print a mouse's thyroid gland, is getting ready to transplant artificial organs to living mice. If successful, the experiment could pave the way for the production of 3D-printed human glands.
Back in March 2015 Moscow-based 3D Bioprinting Solutions lab (founded in 2013) became the first such facility to successfully bioprint a thyroid gland – or, to quote the scientists themselves, a "construct" of the organ. Now the researchers are preparing the transplant of several of these glands into living mice. The results of the experiment will be made public in July 2015 at the Second International Congress on Bioprinting in Singapore. The head researcher Vladimir Mironov told RBTH that he is expecting positive results.
Scientists claim they are ready to start the 3D printing of human thyroid glands. All they need for the first batch are follicular cells, which are responsible for the production and secretion of thyroid hormones.
Researchers of the 3D Printing Solutions Lab, Moscow, Russia
According to the World Health Organization (WHO), 665 million people in the world are affected by thyroid disorders. In Russia, about 140,000 people suffer from various types of thyroid disease and each year 10,000 Russian citizens have to undergo a thyroidectomy, or the surgical removal of the gland.
Thyroid dysfunction caused by cancer cannot be treated with pharmacological therapy. Not even a donor organ transplantation can help in this case, says Andrey Polyakov, the head of the microsurgery department at the Moscow Oncology Research Institute. "The reason for this is that the patients who receive organ transplants have to undergo immunosuppression therapy that can in turn speed up the development of cancer cells," Polyakov explains. According to him, the transplantation of 3D printed organs and tissues can be conducted without immunosuppression.
It should come as no surprise that a 3D printed gland does not fit in the conventional biological hierarchy. The existing system recognizes only molecules, tissues, organs, organ systems and organisms. The object printed at 3D Bioprinting Solutions is therefore unclassifiable.
"A tissue is a group of cells of the same kind," says Mironov. "An organ is a group of tissues. The construct we created is closer to an organ, as it consists of several types of tissues, has blood vessels and can function at the level of an organism."
The scientists chose a thyroid gland as this organ is relatively simple, making it an uncomplicated subject for research work. Besides, it was the first organ transplanted from one human being to another.
Alexander Mitryashkin, engineer, 3D Bioprinting Solutions
The researchers at 3D Bioprinting Solutions took the existing technology of 3D printing currently used to work with diverse materials like plastic, ceramics and metal, and adapted it to work with living cells. The process itself is called 'layer-by-layer production'.
Bioprinting looks like this: at first, the printer sprays a thin layer of gel made of fibrin, a protein involved in the clotting of blood. Embedded in the gel are microscopic spheres consisting of tissue, which subsequently form a three-dimensional structure.
Video
Mironov came up with the idea of bioprinting when he discovered that separate ring moieties in a chicken embryo's heart was able to merge to form a tube. He understood that it was possible to form living tissues out of separate cells and groups of cells.
The original bioprinter created by 3D Printing Solutions consists of three basic elements: a mechanical positioning device, a dispenser and a central processing unit (CPU). Essentially, a bioprinter is a simple robot that can move in three directions. It is equipped with an automated syringe that can dispense either fibrin gel or tissue spheroids.
There are, of course, other companies in the world aiming to commercialize 3D bioprinting technology, such as Organovo in the United States, Cyfuse in Japan and Regenhu in Switzerland. The technology offered by 3D Bioprinting Solutions is unique because aside from the cell-based gel, the Russian lab uses the tissue spheroids mentioned above as "building blocks."
"Last year we filed a patent for our bioprinter design and for the methods of printing we invented," Mironov told RBTH.
The original bioprinter created by 3D Printing Solutions
The printed "organ constructs" will soon be transplanted to mice. The procedure will be no different than a regular organ transplant. The mice used in the experiment have already been subjected to a treatment of radioactive iodine that shut down their thyroid glands, causing hormone deficiency.
Scientists will monitor the mice over the course of a month to determine if their thyroid glands are no longer functioning. "We will review the levels of thyroxine that are supposed to go down significantly because of the suppression of thyroid activity," reports Elena Bulanova of 3D Printing Solutions.
Video
In late April 2015 the researchers will transplant the printed glands to the mice and will observe them to see if the hormone levels are restored. If they are, this will mean the artificial organs work. It will take a month for the grafts to be integrated completely into the bodies of the mice.
The experiment will involve outbred mice of the so-called CD1 strain. "Those mice have minimal variations in morphology and behavior," says Bulanova. "Twelve animals will be used in total; six of them will form the control group, which will not receive the transplant, and the other six will get the grafts."
"We are certain that the gland is functional," says Mironov. "In fact, we are mostly concerned by the perspective of the graft hyperactivity, which can cause hyperthyroidism." According to Mironov, the laboratory conducted all necessary theoretical calculations and morphometric studies before beginning the experiment.
The printed organ constructs are already widely used by pharmaceutical companies for toxicological studies, says Youssef Hesuani, the executive director of 3D Printing Solutions. For instance, California-based Organovo cooperated with the international healthcare company F. Hoffmann-La Roche AG to test an unnamed medication. "We know that while the drug showed no toxicity during the tests involving a monolayer of cells, the experiments on a 3D liver construct provided the opposite results," Hesouani told RBTH.
Which organs do you expect to be printed in the next two or three years?
V.M.: Thyroid glands, blood vessels, skin and hair, as well as cartilage, bone and adipose tissue. Some organs from this list have in fact already been printed.
By your estimates, a printed organ will cost between 200,000 and 250,000 dollars. Does this mean that only the wealthy will be able to afford them?
V.M.: The history of technological progress shows that once a hi-tech product enters mass production by automated means and starts to be widely used on the market, it becomes tens, scratch that, thousands of times cheaper. So there is no doubt that 3D printed organs will become more affordable with time.
Do you expect foreign clients?
V.M.: Yes, our product is capable of entering the global market. In China alone there are 1.5 million people in need of an organ transplant.
Do you think Russia will be able to create an infrastructure for printed organ transplantation?
V.M.: It is possible, yes. But the government will need to cooperate with private businesses. This will require millions of dollars of investment, but will in time allow the healthcare system to save a lot of money on the treatment of patients. Besides, a country that does not invest in the development of such technologies today will later have to buy it from others, which will be much more expensive.
Short history of bioprinting
2013
In 2013 a team led by Takanori Takebe, a stem-cell biologist at Yokohama City University in Japan, successfully transplanted tiny "liver buds" constructed from human stem cells to mice. The scientists have promised to create a fully functional human liver by 2019.
2014
In 2014 Sabine Costagliola, a researcher at the Free University of Brussels, regenerated thyroid tissue using the embryonic stem cells of mice. The tissue was later transplanted to a mouse and started producing thyroxine. Dr. Terry Davies, an endocrinologist from New York City, has recently managed to do the same – regenerate thyroid tissue – with human embryonic stem cells.
2015
Researchers at 3D Printing Solutions are currently waiting for the results of research involving the regeneration of thyroid tissue from induced pluripotent stem cells – i.e. adult cells that have been genetically reprogrammed to an embryonic stem-cell state.
http://rbth.com/longreads/bioprint/index.html
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Russian scientists successfully implant the first 3D-printed thyroid gland
November 2, 2015
A Russian company announced a successful experiment implanting 3D-printed thyroid glands into mice, and the results will be published next week, said Dmitri Fadin, development director at 3D Printing Solutions.
"We had some difficulties during the study, but in the end the thyroid gland turned out to be functional," Mr. Fadin told RBTH.
3D Bioprinting Solutions printed the thyroid gland - or to be exact, the gland's organ construct - in March of this year. At that time, scientific laboratories were saying that they will start printing human thyroid glands if the experiment is successful.
3D Bioprinting Solutions uses existing 3D print technology that makes items from plastic, ceramic and metals, but it had to make adaptations for biological material, that is, for cells. Before transplanting the artificial gland, scientists "carved out" a thyroid in the mice using radioactive iodine.
Vladimir Mironov founded 3D Bioprinting Solutions in 2013. He a tissue engineer, and co-founder of two startups in the U.S., Cardiovascular Tissue Technology, and Cuspis.
http://rbth.com/science_and_tech/20...ant-the-first-3d-printed-thyroid-gland_536205
Russia's revolution in medicine – plans to 3D print a human thyroid
December 18, 2015
Vladimir Mironov, head of 3D Bioprinting Solutions, said his laboratory is ready to start printing a human thyroid gland after a successful experiment on mice. The next organ will be the kidney.
The Moscow-based laboratory, 3D Bioprinting Solutions, announced on Dec. 17 that it has completed a unique experiment to 3D print a mouse's thyroid “organ construct.” The 3D printed thyroid gland was not rejected by the mouse's body, and is functioning.
The company will soon print human organs – first a thyroid gland, and then a kidney. Exact dates have not been disclosed.
“The laboratory’s plans include the publication of an article on the results of the experiment in major scientific journals, and the transition to the next stage of work – the bioprinting of a human thyroid,” said Vladimir Mironov, chief scientific officer at 3D Bioprinting Solutions. “Once this is done, we plan to focus our efforts on developing the bioprinting technology for a kidney. There is much concern today over the lack of human kidneys for transplantation.”
Earlier this year, on March 15, 3D Bioprinting Solutions printed a mouse’s thyroid organ construct on Russia’s first bioprinter, FABION. During the experiment, which lasted several months, the printed constructs were accepted and proved their viability. That experiment’s results were first presented to the international scientific community on Nov. 8 at the International Conference on Biofabrication in Utrecht, Netherlands.
http://rbth.com/science_and_tech/20...cine-plans-to-3d-print-a-human-thyroid_552525
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Chinese Company Releases World's First 3D Blood Vessel Bio-printer
Oct 25, 2015
Revotek, a company based in Chengdu, capital of southwest China's Sichuan Province, released the world's first 3D blood vessel bio-printer on Sunday.
With two nozzles working alternatively, the bio-printer can finish a 10-centimeter blood vessel within two minutes.
"The core of the printer is the BioBrick, in which there are stem cells. Given certain environment and conditions, it (the stem cell) can, according to our requirements, differentiate into the cells we need," said Kang Yujian, chief scientist at Revotek.
The BioBrick refers to a stem cell producing system with biomimetic function. As for 3D blood vessel bio-printing, the major difference setting it apart from other 3D printings is that it has to keep the stem cells active during the process.
"The achievement (of making the 3D blood vessel bio-printer) is not just about printing one blood vessel, but finding the method of sustaining vascular cells and other active substances. The method is useful in blood vessel printing, and in the printings of livers, kidneys and other organs," said Dai Kerong, a academician at the Chinese Academy of Engineering.
Dai added that although the breakthrough has a lot of potentials, there can be a long time before it can be applied to human medical care.
http://news.cctvplus.tv/NewJsp/news.jsp?fileId=323244