Growing Stem Cells in 3D Microgels for Regenerative Medicine

Binil Starly, Ph.D., an associate professor in the industrial and systems engineering program at North Carolina State University and co-director of the functional tissue engineering group at the Comparative Medicine Institute, talked about his recent work culturing stem cells in 3D microgels using a low shear stress rotating wheel-type bioreactor. The study detailed the encapsulation and harvesting of stem cells and offered microgels as a viable option for growing millions to billions of the therapeutic stem cells required for regenerative medicine. His current work continues to address the challenges around accurately predicting cell growth within the bioreactor and the ability to harvest the cells safely and efficiently from the gel. He spoke with writer Tanuja Koppal.

Q: What inspired this work? Were there any gaps in manufacturing that this study helped to fill?

Starly: Regenerative medicine manufacturing uses machines to produce living products, and it all begins with the raw material, which in this case is the living cell or tissue. Any organ in the human body is packed with millions of cells, and when trying to re-create, print, or fabricate this living tissue, a significant amount of cells (>10M cells/ml) needs to be packed into the machine initially. The key challenge here is how to get this large amount of living cells produced for regenerative medicine or cell therapy. Traditionally these cells have been grown using flat-plate culture, but this would be economically infeasible for large-scale production in cell therapy/regenerative medicine. Bioreactors that enable the growing of cells in the third dimension, in an automated or semi-automated manner, are found to be more suitable. The current bioreactor technology in the biopharmaceutical industry is mostly suspension-based. However, most of the cells required for regenerative medicine, such as stem cells, are adherent and need a surface to attach and grow on. Microbeads made from glass or polystyrene are commonly used for this purpose, where cells attach to these surfaces and grow for a few weeks. The challenge then is trying to get the cells off these surfaces safely and efficiently for retrieval.

Instead of using microbeads, we decided to encapsulate and grow human adipose-derived stem cells (hASC) within 3D microgels to maximize the number of cells grown within a certain volume. With microbeads you have only the surface area of the bead, whereas with microgels you have the internal volume available to grow the cells and hence improve productivity. After three to four weeks, the microgels are extracted from the bioreactor, and the cells are safely removed. This technology can be used to grow any type of adherent cells as long as the microgel does not negatively interact with the cells. The gel also provides a protective layering for cells that are sensitive to shear stress in a bioreactor.

Q. What are some of the challenges you ran into and were they overcome?

Starly: The biggest challenge was, and still is, trying to figure out how many cells exist within each microgel and when is the right time to retrieve them. In conventional manufacturing, all the raw materials are well-controlled and behave in a predictable fashion. However, in regenerative medicine each patient is different and their cells behave differently, too, in terms of their growth rate and metabolic function. Predicting the yield of cell production is a challenge, and in this study, we used the amount of glucose consumed within the reactor to evaluate cell viability. When the cells are growing and metabolically active, the levels of glucose in the bioreactor continue to drop, while the amount of lactate, a byproduct of metabolism, remains steady or increases slightly. We performed offline sensing by regularly sampling the media from the bioreactor, without disturbing the cells, to measure the amounts of glucose and lactate. Using calibration curves, we then tried to determine the amount of cells in the bioreactor. Since stem cells vary and the rate at which they grow are different, the standard calibration curves don’t necessarily work as well. Hence, the cells have to be routinely monitored.

The second challenge was how to safely and efficiently remove the cells from within the microgel. We did manage to get nearly an 80 percent yield after 21 days, but this would have to be much better for large-scale manufacturing. We do need to make better biomaterials so that the microgels can be de-cross-linked or separated from the cells rather easily and efficiently without any remnants of the gel in the cells.

Q. Where do you see this technology being applied?

Starly: This microgel technology can be used for autologous cell therapy, such as with therapeutic stromal cells, where cells are retrieved from the patient, modified, and put back into the same patient. It can also be used for allogenic cell therapy, where cells derived from one patient are grown and distributed to many other patients, in which case you need to grow a lot more cells. It can also be used for engineering cells from animals, grown outside the animal’s body and made into engineered meat for use as an alternate food source. For instance, in engineered beef production, the amount of animal cells needed to be grown outside the body will push our current technology limits to make it economically feasible. Bioengineers and process engineers have to work together to optimize and automate some of the conditions for growing these cells and making them cost-efficient.

Q. What are your plans in terms of improving the technology and increasing its use?           

Starly: Predicting the cellular yields accurately is going to have to be addressed for use in large-scale production. Offline sensing, by monitoring chemical entities produced by cells in the media, is not ideal in a production-scale setup. Development of new in-line sensors and process control modules that forewarn the operators about any problems or when to stop the production run is critical, if regenerative medicine were to get to the production stage. Custom-designed 3D surfaces can also improve the quality and quantity of cells produced. In the context of growing large number of cells for regenerative medicine, cell therapy, and engineered food, the process has to be made economical. Manufacturing costs can be brought down by automation, but automation is not possible unless the processes are standardized and qualified by the regulatory agencies. Manufacturing institutes such as the National Institute for Innovation in Manufacturing Biopharmaceuticals and Advanced Regenerative Manufacturing Institute have been set up for industry and academia to work together to further develop these methodologies for biomanufacturing.

Tanuja Koppal, Ph.D., is a freelance writer based in Randolph, NJ.         

REFERENCE:

Modeling Human Mesenchymal Stem Cell Expansion in Vertical Wheel Bioreactors Using Lactate Production Rate in Regenerative Medicine Biomanufacturing. Read the full paper in the ASME Digital Collection.