Optimization of serum free media for the propagation of primary and stem cells in animal and human serum component free conditions

Introduction

Optimization of serum free media can be a complex and daunting challenge for scientists working to bring a cellular therapy to market. When considering raw material sourcing, it is important to limit animal or human serum derived components as these are major sources of variability and potential viral contamination risks. Inclusion of recombinant versions represents a viable alternative to circumvent issues associated with serum-derived proteins. However, utilization of these components can sometimes bring new hurdles to light. Here, I discuss serum free media design and formulation for an array of primary and stem cell systems. Using recombinant animal free proteins, I can provide guidance to convert any media to be completely void of any human serum or animal serum-derived components. Further, through this guidance, I can share some tricks to the trade that I’ve learned along the way to maximize the performance of your cell system. This Ask the Expert session is sponsored by InVitria and is hosted by Randall Alfano. Mr. Alfano, Cell Culture Scientist, joined InVitria in 2012. He currently develops animal free proteins and supplements for various cell systems including stem cells. He has over 6 years’ experience in recombinant protein expression and purification as well as medium development for CHO-based biomanufacturing and stem cells. Randall was awarded his Ph.D. in 2009 from the Texas A&M College of Medicine Health Science Center in Cell Biology.

please take advantage of the opportunity to ask our expert a question and participate in a lively discussion on designing serum free cell culture media.

Question 1

I am currently culturing MSCs with platelet lysate, do you think there is reason to switch to completely animal free or is avoiding serum enough?

Human platelet isolate has been considered to be an alternative to serum in the expansion of MSC. Although platelet isolate is free of bovine components, there is still risk of viral contamination since this is derived from human serum. In addition, this material can vary from lot to lot which warrants the qualification of a lot prior to the incorporation into a manufacturing process.

Question 2

We would like to initiate a large media component screen to optimize our MSC production. The challenge is that for what we want to screen it is going to be very time/labor intense. Can you advise on your experience using automation to simplify this?

Automation of cell culture processes is a great way to simplify media component screening. However, it is quite pricey. If you have the resources to incorporate automation of media development, there are several options. If there are limiting resources, there are specific methods and techniques that one can use to minimize the cost of media development and still screen large numbers of components with relative ease. If you would like more information on these approaches, I would be happy to further discuss.

Question 3

In your opinion if you have determined that you can’t get rid of serum completely is there any advantage (beyond cost) to trying to reduce it?

Total elimination of serum versus serum reduction will be determined by the end application of the cell-based product. For therapeutic applications, it is best to totally eliminate serum for variability and safety reasons. If you find that you are having difficulty in removing serum completely, I would be interested in talking with you further to see if we can solve your issue. Simply reducing serum in a medium is a viable alternative if the end use allows (i.e. R&D). There are a few advantages to having a reduced serum medium versus a serum free medium. Namely, it is generally well known that cells cultured in serum are more durable and can withstand environmental insults (i.e. temperature and pH shifts during handling) slightly better than when cultured in serum free media.

Question 4

I am working on reducing serum in our iPCS culture. What steps would you recommend for weaning and in your experience how low have you gotten in serum concentration?

I would highly recommend a step wise reduction in serum for iPSC over multiple passages. There are a variety of serum free medias currently marketed for iPSCs, so these cells can be propagated in serum free conditions. In addition, we have developed in house animal component free formulations that are capable of expanding iPSC for at least 12 passages while retaining all of the desired phenotypic traits. When combined with a recombinant vitronectin matrix, these cells can be expanded in media that is completely void of any human or animal serum components. If you are interested in formulations such as these, we can further discuss

Question 5

We don’t have an immediate need to eliminate serum in our culture but are seeing some problems with variability. Do you see a reduction in variability issues with serum reduction and how does that compare with eliminating altogether? MSCs

In my experience with serum-based media in expanding MSC, I’ve found that reducing serum does seem to improve variability issues. However, improvement would depend on the extent of serum reduction as well as the composition of the substituted defined formulation. With this said, serum reduction does have some issues. Generally speaking, the problem with serum reduction in MSC is that a defined formulation that is optimized for one lot of serum may have inadequate levels of components when paired with a different lot of serum. Therefore, if MSC performance variability is an issue, I would recommend eliminating serum all together.

Question 6

Do you have any recommendations on a coating matrix that you use for MSCs?

If you are using serum-containing medium, a tissue culture-treated flask should work just fine without the need for any additional treatment for good MSC growth. However, once you withdraw serum, MSC adhesion can become an issue. 1-5 ug/squared centimeter of human serum fibronectin is probably the simplest way to recover MSC adhesion in the absence of serum. There are also a number of xeno-free defined commercially available matrices such as CellStart that also work very well. In our lab, we actually prefer not to coat flasks as this is a bit time consuming and can get expensive. We use the CellBIND surface from Corning and have good luck with cell adhesion and maintenance of multipotency in MSC.

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