3D Printer Makes the Tiniest Human Organs Ever Made


Through the use of 3-D printers, mini human organs can be made in all kinds of shapes and sizes. A new experiment by tissue engineers from Wake Forest University has made tiny beating hearts that beat in sync, and another pulsing heart that fused with a spherical, liver.

These printed, mini-organs were by Anthony Atala and his team at the Wake Forest Institute for Regenerative Medicine in Winston-Salem, North Carolina. They represent the first step in developing an entire human body on a chip. The mini-hearts were made by reprogramming human skin cells into heart cells, which were then clumped together in a cell culture. A 3-D printer was then used to give them the desired shape and size, which in this case was a sphere of tissue with a diameter of 0.25 millimeters.

The development of these miniature organs was motivated by a desire to make model systems that mimic the function of life-size organs. Eventually, such a system could create mini-organs that could be linked up to form an entire organ system that could be used to test new treatments or probe the effects of chemicals and viruses.

The production of these mini-organs could potentially serve an alternative to animal testing, which is usually rather costly and doesn’t always produce results that are applicable to humans.

Further work on these mini-organs could also discover ways to expand these organs and make them life-size that they can be used for organ transplants.

3-D Printing to Make Replacement Body Parts


Advances in three-dimensional (3D) printing have produced a swell of interest in artificial organs that are designed to replace, or even enhance, human tissues.

3-D printed organs

At the Inside 3D Printing conference in New York on April 15–17, 2015, researchers from academia and industry are gathering to discuss the growing interest in using three-dimensional (3D) printing to make replacement body parts. Although surgeons are already using 3D-printed metal and plastic implants to replace bones, researchers are looking ahead to printing organs using cells as “ink.”  All the structures shown here were all 3D printed at Wake Forest Baptist Medical Center in Winston-Salem, North Carolina, and include a rudimentary proto-kidney (top left), complete with living cells.

Printed organs, such as a prototype outer ear that was developed by researchers at Princeton University in New Jersey and Johns Hopkins University in Baltimore, Maryland, will be featured at the conference.  This ear is printed from a range of materials: a hydrogel to form an ear-shaped scaffold, cells that will grow to form cartilage, and silver nanoparticles to form an antenna (see M.S. Mannoor et al. Nano Lett. 13, 2634−2639; 2013. This is just one example of the increasing versatility of 3D printing.

This New York meeting, which is being advertised as the largest event in the industry, will provide exposure for a whole world of devices and novelties. But it will also feature serious discussions on the emerging market for printed body parts.

The dream of bioprinting is to print organs that can be used for transplant. For example, at the Wake Forest Baptist Medical Center in Winston-Salem, North Carolina, researchers are developing a 3D-printed kidney. The project is in its early stages and the kidney is far from functional and some doubt that researchers will ever be able to print such a complex organ. Perhaps a more achievable near-term goal might be to print sheets of kidney tissue that could be grafted onto existing kidneys.

Printed replacement for skull

Printed structures made of hard metal or polymers are already on the market for people in need of an artificial hip, finger bone or facial reconstruction. This skull implant (grey) was made by Oxford Performance Materials of South Windsor, Connecticut, and was approved by US regulators in 2013. It is made of a polymer meant to encourage bone growth, to aid integration of the implant into the surrounding skeleton. The company also sells implants for facial reconstruction and for replacing small bones in the feet and hands.

3-D printed lung tree

One of the key advantages of using 3D printing for surgical implants is the opportunity to model the implant to fit the patient. This airway splint (shown on the right branch of the model trachea) was designed by researchers at the University of Michigan in Ann Arbor to fit an infant with a damaged airway. The splint was made out of a material that is gradually absorbed by the body as the airway heals. The research team benefited from the concentration of 3D-printing expertise that has built up in Michigan because of the US automobile industry, which uses the technology for printing prototypes and design samples.

The business of 3-D printing also includes titanium replacement hip joints, which can be tailored to fit individual people, and made-to-order polymer bones to reconstruct damaged skulls and fingers. Printed body parts brought in US $537 million last year, up about 30% on the previous year, says Terry Wohlers, president of Wohlers Associates, a business consultancy firm in Fort Collins, Colorado, that specializes in 3D printing.

3-D printed prosthetics

3D printing can also be used to generate cheap — and creative — prostheses.  A prosthetic hand can cost thousands of dollars, which is a burdensome expense when fitting it to a growing child.  Jon Schull founded a company called e-NABLE that provides free printed prosthetics to those in need, harnessing the efforts of hundreds of volunteers who own consumer-grade 3D printers. “When people get tired of printing Star Wars figurines, they give us a call,” he says.  The cost of materials for a printed prosthesis is about US $35.

3-D animal prosthesis

Also, 3-D printed prostheses are not just for humans.  For example, a duck named Buttercup was born with its left foot turned backwards.  The Feathered Angels Waterfowl Sanctuary in Arlington, Tennessee, arranged for the fowl to receive a new foot, complete with a bendable ankle.  Also in the an eagle, a box turtle and a handful of dogs also have been fitted with 3-D printed prostheses.

Scientists are looking ahead to radical emerging technologies that use live cells as ‘ink’, assembling them layer-by-layer into rudimentary tissues, says Jennifer Lewis, a bioengineer at Harvard University in Cambridge, Massachusetts. Bioprinting firm Organovo of San Diego, California, already sells such tissues to researchers aiming to test experimental drugs for toxicity to liver cells. The company’s next step will be to provide printed tissue patches to repair damaged livers in humans, says Organovo’s chief executive, Keith Murphy.

Lewis hesitates to say that 3D printing will ever yield whole organs to relieve the shortage of kidneys and livers available for transplant. “I would love for that to be true,” she says. “But these are highly complicated architectures.”

Artificial Organs that Fit in Your Hand


New technologies are now available that allow scientists to make mock human organs that can fit in the palm of your hand. These organs-on-a-chip can help test drugs and provide excellent model systems for organ function when they are healthy and when they are diseased.

Think of it: a gut-on-a-chip being developed at the Johns Hopkins School of Medicine can help determine if your heart medicine is actually causing your upset stomach or your diet. This type of technology is a high-tech approach to dealing with a scourge of the low-tech world.

“I’m interested in solving a worldwide problem of diarrheal diseases,” says Dr. Mark Donowitz, who runs this lab and studies diarrheal diseases. According to Donowitz, 800,000 children a year die from diseases like cholera, rotavirus and certain strains of E. coli.

“We’ve failed so far to find drugs to treat diarrhea using cell culture models and mouse intestine,” Donowitz says. Unfortunately. mouse digestive systems don’t react the way human s do to these germs. Therefore, they aren’t very helpful for studying diseases of the gut. Therefore, Donowitz’s team is building his gut-on-a-chip technology in what he hopes will be a superior technique for studying these these diseases.

Postdoctoral researcher Jennifer Foulke-Abel holds the gut-on-a-chip inside the lab at Johns Hopkins School of Medicine.
Postdoctoral researcher Jennifer Foulke-Abel holds the gut-on-a-chip inside the lab at Johns Hopkins School of Medicine.

When you hold one of these devices in the palm of your hand, it is little more than a thin sheet of glass, topped with a plastic microscope slide with a tiny cavity inside. Half a dozen spaghetti-size tubes extend from the device.

“The reason there are so many tubes is we have a vacuum chamber that will cause the membrane to stretch, the way the intestine stretches as it moves food along,” Fouke-Abel explains.

Cells isolated from a human intestine are placed into a tiny chamber around that membrane, and the cells divide, grow and organize themselves into a small version of part of a human gut. The device, when operating, might hold 50,000 gut cells.

The first step of this research is to determine if the cells in the chip react the same way to diseases as cells in the human gut.

“And in all three of the diseases I mentioned, we’ve been able to take that first step,” Donowitz says. “So we know that these appear to be really good models of the human disease.”

To date, the guts-on-a-chip produce digestive enzymes, hormones and mucus, but they don’t yet incorporate other parts of the human intestine, such as blood vessels or nerve cells.

“They all have to be incorporated if you want to move from a simple to a more complex system, which I think you need to do if you are going to reproduce intestinal biology,” Donowitz says.

However, Donowitz’s laboratory is moving in that direction. Once it has built a complete system, they will use it to test potential drugs for the diseases being studied. “We think this could be a real step forward in terms of reducing waste-of-time drug development,” Donowitz says.

While the Donowitz lab at Johns Hopkins is working to develop the gut, other labs scattered around the country are working on other organ systems.

“There’s going to be a brain-on-a-chip, liver, heart and so on,” says Danilo Tagle, who coordinates this overall effort at the National Center for Advancing Translational Sciences, which is part of the National Institutes of Health. The grant structure for this study section will fund the development of 10 organ systems in all.

“The goal is actually to tie them in all together,” Tagle says. To this end, the mini-organs on a chip will collectively work together, much like an entire human being on a chip.

Tagle’s hope is that scientists can build many of these systems, each one based on the cells from an individual person. This would create an array of cell-based stand-ins for research or even diagnoses.

“And so you can identify which part of the population might be more responsive to particular drugs, or identify a subset of the population that might be more vulnerable to the harmful effects of a particular drug,” Tagle says.

According to Tagle, this $75 million, five-year project took off thanks to pioneering work at the Wyss Institute for Biologically Inspired Engineering at Harvard. The research has been so promising, Wyss spun off a private company to pursue it.

“It’s called Emulate,” says Donald Ingber, founding director of the Wyss Institute. “It’s just getting its feet on the ground. We have almost 20 people out of the Wyss Institute who are moving out with it.”

Ingber says it would be too much to expect this technology to replace mice in medical research anytime soon. But he is hoping that this will speed up drug development and make it less expensive, “because if we can identify things that are more likely to work in humans, that’s going to have major impact.”

And there are so many avenues to pursue, he says, there’s plenty of room for both industry and academics to work on building and improving these organs-on-a-chip.

Radio Interview About my New Book


I was interviewed by the campus radio station (89.3 The Message) about my recently published book, The Stem Cell Epistles,

Stem Cell Epistles

It has been archived here. Enjoy.

Embryonic Stem Cells Used to Make Laboratory-Created Thymus


Medical researchesr from UC San Francisco have used embryonic stem cells to construct a functioning mouse thymus in the laboratory. When implanted into a living mouse, this laboratory-made thymus can successfully foster the development of T cells, which the body needs to fight infections and prevent autoimmune reactions.

This achievement marks a significant step toward developing new treatments for autoimmune disorders such as type 1 diabetes and other autoimmune diseases, such as systemic lupus erythematosis and ulcerative colitis.

This research team was led by immunologist Mark Anderson and stem cell researcher Matthias Hebrok. They used a unique combination of growth factors to push the embryonic stem cells into a particular developmental trajectory. After a period of trial and error, they eventually found a formula that produced functional thymus tissue.

In our bodies, the thymus lies just over the top of our heart, and it serves to instruct T lymphocytes (a type of white blood cell) what to attack and what to leave alone. Because T cells serve a vital role in the immune response, the thymus serves a vital function.

thymus

Typically, each T cell attacks a foreign substance that it identifies by binding the foreign substance to its cell surface receptor. This T cell-specific receptor is made in each T cell by a set of genes that are randomly shuffled, and therefore, each T cell has a unique cell receptor that can bind particular foreign molecules. Thus each T cell recognizes and attacks a different foreign substance.

With in the thymus, T cells that attack the body’s own proteins are eliminated. Thymic cells express major proteins from elsewhere in the body. The T cells that enter the thymus first undergo “Positive Selection” in which the T cell comes in contact with self-expressed proteins that are found in almost every cell of the body and are used to tell “you” from something that is not from “you.” In order to destroy cells that do not bear these self-expressed proteins, they must be able to properly identify them. If T cells that enter the thymus cannot properly recognize those self-expressed proteins (known as MHC or major histocompatibility complex proteins for those who are interested), the thymus destroys them. Second, T cells undergo “Negative Selection” in which if the T cell receptor binds to self MHC proteins, that T cell is destroyed to avoid autoimmunity.

The thymus tissue grown in the laboratory in this experiment was able to nurture the growth and development of T cells. It could act as a model system to study patients with fatal diseases from which there are no effective treatments, according the Mark Anderson.

As an example, DiGeorge Syndrome is caused by a small deletion of a small portion of chromosome 22 and infants born with DiGeorge Syndrome are born without a thymus and they usually die during infancy.

Other applications include manipulating the immune system to accept transplanted tissues such as implanted stem cells or organs from donors that are not a match to the recipient.

Anderson said, “The thymus is an environment in which T cells mature and where they also are instructed on the difference between self and nonself.” Some T cells are prepared by the thymus to attack foreign invaders and that includes transplanted tissue. Other T cells that would potentially attack our own tissues are eliminated by the thymus.

Laboratory-induced thymus tissue could be used to retrain the immune system in autoimmune diseases so that the T cells responsible for the autoimmune response eventually ignore the native tissues they are attacking.

Hebrok warns that he and his team have not perfectly replicated a thymus. Only about 15% of the cells are successfully directed to become thymus tissue with the protocols used in this study. Nevertheless, Anderson asserted, “We now have developed a tool that allows us to modulate the immune system in a manner that we never had before.”

Georgetown Team Discovers New Type of Stem Cell


A research group a Georgetown Lombardi Comprehensive Cancer Center has developed a new and powerful stem cell in the laboratory that grows in sheets and has many characteristics desirable for regenerative medicine.

The senior author of this paper, Richard Schlegel, M.D., Ph.D., chairman of the department of pathology at Georgetown Lombardi, a part of Georgetown University Medical Center, said of these new stem cells: “These seem to be exactly the kind of cells that we need to make regenerative medicine a reality.”

The results of his lab’s research has been published in the November 19 online early edition of the Proceedings of the National Academy of Sciences (PNAS). In this publication, they report that their new stem-like cells do not express the same genes as embryonic stem cells and induced pluripotent stem cells (iPSCs). Thus, they do not produce tumors when injected into laboratory animals. Also, these cells are stable, since they differentiate into the cell types desired by researchers.

This publication is a continuation of a study published in December 2011, when Schlegel and his colleagues invented a laboratory technique that could maintain both normal and cancer cells alive indefinitely. Previously such a technique did not exist and it was simply not possible to keep such cells alive in the laboratory indefinitely.

Schlegel and others showed that if they added two different substances to their cells in culture – fibroblast feeder cells and a chemical that inhibits the Rho kinase – they could push the cells to assume a kind of stem-like state. While in this stem cell-like state, the cells would stay alive indefinitely. Once the feeder cells and the inhibitor were withdrawn, the cells reverted back to their original state. In this paper, Schlegel and his team called these laboratory-derived cells “conditionally reprogrammed cells” or CRCs. See Liu X et al. Am J Pathol. 2012 Feb;180(2):599-607.

Could CRCs be used for personalized medicine? A follow-up study suggested that they could. Published in the New England Journal of Medicine in September 2012, they found a patient who had a 20-year history of recurrent respiratory “papillomatosis” (a type of tumor) that had invaded the lung tissue in both lungs. The tumor was difficult to treat and slow-growing, but it stubbornly resisted treatment. Schlegel and his team made CRCs from this patient’s normal and tumorous lung tissue. By utilizing this technique, they discovered that the tumor cells were infected the same virus that causes warts; the human papillomavirus. They then used these cultured tumor CRCs to determine which cancer drug would work the best. They identified a drug called vorinostat as the best candidate, and 3 months after starting treatment, the tumors stopped growing and the prognosis looked substantially better for this patient (see Yuan H, et al. N Engl J Med. 2012 Sep 27;367(13):1220-7).

Of this paper, Schlegel said, “Our first clinical application utilizing this technique represents a powerful example of individualized medicine. It will take an army of researchers and solid science to figure out if this technique will be the advance we need to usher in a new era of personalized medicine.”

The present study is study was published in PNAS compared CRCs to embryonic stem cells and iPSCs. Both embryonic stem cells and iPSCs have been investigated for use in regenerative medicine, but both cells have the drawback to potentially producing tumors when injected into mice and “it is difficult to control what kind of cells these cells differentiate into,” Schlegel says. “You may want them to be a lung cell, but they could form a skin cell instead.”

In contrast, if lung cells are treated to make lung-specific CRCs, they can be expanded in culture to make a huge quantity of lung-specific cells, but when these conditions are withdrawn, the lung-specific CRCs will revert to mature lung cells. This transformation is rather rapid, since the cells become CRCs within three days of adding the inhibitor and the feeder cells. Once the cells lose their stem-like properties and potentially can be safely implanted into tissue.

A comparison of gene expression patterns from CRCs and embryonic stem cells (ESCs) or iPSCs showed that CRCs do not overexpress the same critical genes that embryonic stem cells and iPSCs express. “Because they don’t express those genes, they don’t form tumors and they are lineage committed, unlike the other cells,” Schlegel says. “That shows us that CRCs are a different kind of stem-like cell.”

In this study, Schlegel’s team used cervical cells and made CRCs from them. However, then they placed the cervical cell-derived CRCs on a three-dimensional platform, they grew into a canal-like structure that looked startlingly like a cervix. A very similar result was seen with cells extracted from the trachea. When the trachea-derived CRCs were grown on a 3-D platform, they begin to look like a trachea.

If and when use of CRCs are perfected for the clinic, which will require considerably more work, they have the potential to be used in a wide variety of novel ways. “Perhaps they could be used more broadly for chemosensitivity, as we demonstrated in the NEJM study, for regenerative medicine to replace organ tissue that is damaged, for diabetes — we could remove remaining islet ells in the pancreas, expand them, and implant them back into the pancreas —and to treat the many storage diseases caused by lack of liver enzymes. In those cases, we can take liver cells out, expand them and insert normal genes in them, and put them back in patients,” Schlegel says.

Schlegel added: “The potential of these cells are vast, and exciting research to help define their ability is ongoing.”

Engineered Tissues for Transplantation


Xenotransplantation refers to the transplantation of organs from non-human animals into human patients. Such a procedure can increase the availability of organs for transplantation, but proteins and sugars on the surfaces of animal cells that are not found in human bodies can elicit an immune response against these xenotransplanted organs and tissues. For example, the human immune system recognizes a sugar molecule that coats the surface of pig blood vessels but is absent from human tissues called alpha-1,3-galactose (α-gal). In 2003, David Cooper, who runs the transplantation program at the University of Cape Town Medical School, engineered pigs without the α-1,3-galactosyltransferase gene that produces the α-gal residues. However, there were other problems with pig organs as well.

Tissue engineered organs are grown from a patient’s own cells. Such organs should help increase the availability of organs and avoid the problems of immune rejection that plague the field of xenotransplantation. “Cartilage, skin, and bone are already on the market. Blood vessels are in clinical trials. The progress has been really gratifying,” says Laura Niklason of Yale University.

Such engineered tissues consist of either flat planes or hollow tubes and are relatively simple to produce. Also, they consist of a small number of cell types. However, solid organs, such as the lungs, heart, liver, and kidneys, pose a greater challenge, since they are bigger and contain dozens of cell types. In addition, they have a complex architecture and an extensive network of the most essential component, which are the blood vessels. “Every cell needs to eat and breathe, and each one needs to be close to a source of nutrition and oxygen,” says Joseph Vacanti, who is in charge of the liver transplantation program at Boston Children’s Hospital in Massachusetts. Still, Vacanti is optimistic that it should be possible to produce even these complex organs through tissue engineering. “People differ about whether it’ll be achieved in 5 or 100 years, but most people in the field believe that it’s a realistic goal,” he says.

In 2008, Harald Ott of Massachusetts General Hospital and Doris Taylor of the University of Minnesota dramatically demonstrated the potential of organ engineering by growing a beating heart in the laboratory. These know first-hand, the need for organs for transplantation, since as physician-scientists, they often see patients who badly need transplants, but have no available organs for transplantation. To make engineered hearts, they began by using detergents to strip the cells from the hearts of dead rats. This left behind an extracellular matrix (a white, ghostly, heart-shaped frame of connective proteins such as collagen and laminin). Ott and Taylor used this matrix as a scaffold, and they seeded it with cells from newborn rats and incubated it in a bioreactor, which is a vat that provides cells with the right nutrients, and simulates blood flow. Four days later, the muscles of the newly formed heart began contracting, and after eight days, it started to beat.

This technique is extremely labor-intensive and is known as whole organ decellularization. Think of it as knocking down a house’s walls to reveal its frame, and then replastering it anew with different materials. Because the frame is of the same structure as the original organ and retains the complicated three-dimensional architecture of the organ which includes the branching network of blood vessels. Additionally, it also preserves the armamentarium of complex sugars and growth factors that covers the matrix and provides signaling signposts for growing cells. These signals will nudge the cells into the proper shapes and structures. “The matrix really is smart,” says Taylor. “If we put human cells on human heart matrix, they organize in remarkable ways. We can spend the next 20 years trying to understand what’s in a natural matrix and recreate that, or we can take advantage of the fact that nature’s put it together perfectly.”

Ott and Taylor’s groundbreaking feat of tissue engineering has since been duplicated for several other organs, including livers, lungs, and kidneys. Rodent versions of all have been grown in labs, and some have been successfully transplanted into animals. Recellularized organs have even found their way into human patients. Between 2008 and 2011, Paolo Macchiarini from the Karolinska Institute in Sweden fitted nine people with new tracheas. These tracheas were built from their own cells grown on decellularized scaffolds. Most of these operations were successful (although three of the scaffolds partially collapsed for unknown reasons after implantation). Decellularization has one big drawback: it still depends on having an existing organ, either from a donor or an animal. These disadvantages led Macchiarini to devise a different approach. In March 2011, he transplanted the first trachea built on an artificial, synthetic polymer scaffold. His patient was an Eritrean man named Andemariam Teklesenbet Beyene, who had advanced tracheal cancer and had been given 6 months to live. “He’s now doing well. He’s employed, and his family have [sic] come over from Eritrea. He has no need for immunosuppression and doesn’t take any drugs at all,” says Macchiarini. A few months later, he treated a second patient—an American named Christopher Lyles—in the same way, although Lyles later died for reasons unrelated to the transplantation.

Macchiarini now has gained approval from the US Food and Drug Administration to perform these transplants in the United States on a compassionate basis, for those patients who have no other options. “The final organ will never ever be as beautifully perfect as a natural organ,” says Macchiarini, “but the difference is that you don’t need a donation. It can be offered to a patient in need within days or weeks.” By contrast, even if a donor is found, a simple trachea can take a few months to regrow using a decellularized scaffold.

Other scientists have enjoyed similar success with other organs. In 1999, Anthony Atala of the Wake Forest Institute for Regenerative Medicine successfully grew bladders using artificial scaffolds. He subsequently transplanted them into seven children afflicted with spina bifida. By 2006, all the children had gained better urinary control. Atala has just completed Phase II trials of his artificial bladders.

Vacanti thinks that artificial scaffolds are the future of organ engineering, and the only way in which organs for transplantation could be mass-produced. “You should be able to make them on demand, with low-cost materials and manufacturing technologies,” he says. Such mass production is relatively simple for organs such as tracheas or bladders, since these are simply hollow tubes or sacs. Such tissue engineering is much more difficult for the lung or liver, which have much more complicated structures. However, Vacanti thinks it will be possible to simulate their architecture with computer models, and then fabricate them with modern printing technology, which uses inkjet technology to squirt stem cells unto three-dimension scaffolds that fit the size of the organ of interest. “They obey very ordered rules, so you can reduce it down to a series of algorithms, which can help you design them,” says Vacanti. However, Taylor says that even if the architecture is correct, the scaffold would still need to contain the right surface molecules to guide the growth of any added cells. “It seems a bit of an overkill when nature has already done the work for us,” she says.

Whether the scaffold used by tissue engineers are natural or artificial, clinicians need to seed it with patient’s cells. For bladders or tracheas, enough cells can be collected from the patient by means of a small biopsy. Unfortunately, this will not work if the organ is diseased, or if it is a complex structure composed of multiple tissue types, or, as in the heart, if its cells do not normally divide normally. In such cases, clinicians will need either stem cells, which can divide and differentiate into any cell type, or progenitor cells that are restricted to specific organs. Since 2006, one source of stem cells has been adult tissues, which scientists can now reprogram back into a stem-cell like state using just a handful of genes. Induced pluripotent stem cells or iPSCs, could then be coaxed to develop into a tissue of choice. “For me, the cells have always been the most difficult part,” says Vacanti, “and I’d say the iPSCs are the ideal solution.”