Mesenchymal Stromal Cell Culture – Challenges and solutions for isolation, expansion and maintenance

In this podcast and accompanying article, we talked with Dr. Jennifer Chain, Scientific Director of Research and Development, Oklahoma Blood Institute about isolation of mesenchymal stromal cells from cadaveric bone marrow and the differences between live and cadaveric donors.  We also discussed the expansion and maintenance of mesenchymal stromal cells in culture including media design and selection.

I began my interview by asking Dr. Chain to talk about the Oklahoma Blood Institute (OBI) and her work there. She said that the Oklahoma Blood Institute is the sixth largest independent blood bank in the US and their network extends across Oklahoma, Arkansas and parts of Texas with almost a thousand employees. They collect close to three hundred thousand blood products every year and since blood products are regulated as drugs by the FDA, they are actually a not-for-profit pharmaceutical company and manufacture close to twelve hundred drugs every day.

Their primary mission is to collect, test, store and distribute safe blood products to area hospitals where they use those products for transfusion. Recently OBI has been extending their mission for the community by helping support the growing cell therapy and regenerative medicine industries.

I then asked Jennifer to talk about some of the specific projects that they are working on. She explained that they are a contract research lab that works with labs and companies that need access to human cells or they need OBI to help isolate, stimulate and culture cells to support their research projects. She went on to say that she does culture work to support cancer research and other immune studies throughout the country. For example, one project that she is working on is manufacturing a cell-based treatment for dry eye disease for clinical trials. In this instance, she worked with an eye doctor and a scientist to turn their research protocol into a clinical manufacturing protocol. Results show that it has been helping people heal their dry eye disease. Soon they’ll move the manufacturing of that treatment into their clean room space and scale up for a larger clinical trial.

Another project involves Gamma Delta cells. Gamma Delta cells normally live in the tissue and help fight off infections and cancer development in the tissues and organs. Dr. Chain found these cells in a blood source and was able to study them. She found that the population is primed for a cancer response and believes they would make a good adopted T-cell therapy or a platform for CAR-T Therapy. She is gathering more data on the function, as well as optimizing the culture and expansion protocol in order to be fit for clinical use and then would be interested in working with partners.

Last, she told us about OBI’s work to develop cadaveric bone marrow derived mesenchymal stromal cells as a suitable product for research and therapy development.

I followed up by asking why they were working to isolate these cells from cadaveric bone marrow. She explained that there is a shortage of mesenchymal stromal cells (MSCs) that can be used for therapy and there are hundreds of clinical trials that are studying the potential use of them. The two most common sources of MSCs are bone marrow and umbilical cord tissue. Bone marrow donation is associated with risks, it’s painful and a lot of people just don’t want to donate. Umbilical cord tissue, although it seems to be widely available, is actually difficult to obtain in large amounts. The consenting process is long and involved with issues to protect the donor. In addition, it is more difficult to obtain MSCs from umbilical cord. So, there is a real need to find other sources of MSCs that are more sustainable. OBI has access to some of this tissue and if they can show data that cadaveric bone marrow derived MSCs expand and function like expected then they provide a more sustainable source for these cells.

I asked how similar the MSCs are from live versus cadaveric donors. Dr. Chain said that there really is no reason to think that they would be different, but will need proof if the cells will be used to develop therapies. Regulators will also need to see data before any of these therapies can be approved. So far, cadaveric MSCs do express the ISCT surface marker phenotypes, seen as early as the first passage and it lasts through at least passage four. They also have similar metabolic activities and similar proliferative potential as living donor derived.

Next, I asked about the key factors in culturing MSCs. Jennifer said that it is important to have specially defined media optimized for MSC growth. It is good to have access to growth media that’s already optimized, but she does think it’s important to also use a protein supplement like platelet lysate or human serum in your cultures. In looking at comparisons, protein supplementation increases the growth rate in these cells.

I then asked her to describe specific challenges she has seen in culturing these cells. She said that every cell type grows differently and has different needs. You have to troubleshoot until it works. When culturing human primary cells, MSCs and corneal cells, you see that cells from different donors grow differently so you have to adapt from individual to individual. Some don’t grow it all and some grow slowly, so you really have to adapt. Another challenge is contamination – bacterial fungal, and mycoplasma – and it is almost impossible to learn when or how it happened. The greater challenge with mycoplasma is that it is not detectable by eye, but it drastically changes the growth and functional properties of your cells. You really have to practice good technique and you have to test your cultures for things like mycoplasma to make sure that they’re not contaminated.

I further asked Dr. Chain what other factors were important when selecting media for expansion and maintenance of cells. She explained that it is important to see documentation from the supplier that the media is going to support the growth of the cells that you’re studying. Also when scientists publish their data, they need to be able to show that the basic phenotype of their cells is preserved. While suppliers can provide a recommendation, it is important for scientists to also test to determine ideal supplementation for their cultures.

She went on to say that the industry is really moving away from animal derived products like fetal bovine serum and that she thinks it is a good thing. She said that she would discourage using FBS at all if plans are to go toward a clinical trial.

I then asked Dr. Chain how she sees her work evolving, particularly in the area of moving away from FBS. She said that she was really fortunate to have received a Reagent Grant Award from Biological Industries USA. With this award, she was granted $25,000 to use toward products and this included their NutriStem® MSC growth media, human platelet lysate and other products to support OBI’s research. She went on to say that she has been really fortunate to have this resource available and it has really accelerated her research with MSCs. Her short-term goal is to gather enough data to prove that cadaveric MSCs are useful for therapy and to identify ideal donor characteristics and storage conditions. The long-term goal is to find partners for their different projects.

Finally, I asked if she could describe the types of collaborations that OBI is looking for. She said that they are not at the point where they have partners for the cadaveric-derived MSCs yet. They are still trying to gather data to attract partners. They expect these types of partners to be academic labs that are looking to transition from basic research into clinical trials and spin out small companies.

Chain Mentions: We have blood center partners called BioPartners. The group consists of six independent blood centers and the goal is to share protocols and promote standardization of cell therapy product collection. We are hoping to expand this into the larger Blood Centers of America, which is a network of fifty-two independent blood centers.

I closed the interview by asking if there was anything else that she wanted to add for listeners. Dr. Chain encouraged scientists working with MSCs to really know their cells and that the more they know, the better they will understand the clinical data down the road. Also if there are any groups looking for a manufacturing partner, OBI is establishing clean room space to support multiple cell therapy projects and the blood center. OBI has a lot of expertise in developing FDA compliant manufacturing protocols and is looking to grow with the needs of partners. If you’re working with a nonprofit CMO with existing infrastructure like OBI, it could save a lot of money and time on development and might give you a strategic advantage when it’s time to market your therapy.

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