Which stem cell culture media is right for you?

Learn more about what to consider when selecting a cell culture media for human pluripotent stem cells.

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Nov 23, 2017
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Human pluripotent stem cells (hPSC) represent a promising cell source for the development of regenerative cell therapies and several studies have shown their ability to differentiate into disease-relevant cell types such as dopaminergic neurons or cardiomyocytes. However, successful application of these cells as therapeutic agents will depend on their effective and efficient expansion and culture. Use of xenogenic media or those with high concentrations of growth factors can lead to a culture of hPSCs with heterogeneous differentiation. Culture media can be divided into the following subsets based on the level of definition1:

  • Serum-containing media (commonly 10-20% FBS)
  • Reduced-serum media (commonly 1-5% FBS)
  • Serum-free media (synonymous with defined media)
  • Xeno-free media (contains proteins/peptides, but only from human origin, no non-human proteins)
  • Chemically-defined media (with only recombinant proteins and/or hormones), also known as Animal Component Free (ACF) media

In this editorial, learn more about the features of cell culture media to be considered when selecting the optimal medium for your application.

Xenogenic and serum-free

Xenogenic media, such as fetal bovine serum (FBS) containing media, means that the components in the media come from a species different to that of the human cells being cultured. Xenogenic media and xenogenic additives (like serum) provide a range of macromolecules, proteins, attachment factors, nutrients, hormones and growth factors.

The first human stem cells were cultured using techniques borrowed from mouse ES cells, with media containing FBS. However, it became evident that the rich, undefined components in the serum easily triggered the sensitive cells to spontaneously differentiate2. The variable nature of xenogenic media means that culturing reliable and homogenous stem cell populations can be challenging, can lead to contamination, lot-to-lot variations and poor reproducibility.

Utilizing a xeno- or serum-free media may still contain undefined human-derived products such as hormones, growth factors and various proteins but it can be utilized in cell production during the translation of a cell therapy from research to the clinic.

Feeder-free

In a feeder-dependent culture, human pluripotent stem cells (hPSCs) are cultured on a layer of inactive murine or human fibroblast cells to support the growth of the hPSCs. Introduction of animal-derived cells is problematic and although human feeder cells can be utilized, they can be highly variable and difficult to define.

Feeder-free culture of hPSCs allows a more efficient and controllable system. Without feeder cells, the culture is entirely dependent on the quality of the medium for growth and proliferation of healthy stem cells. The first medium designed for feeder-free culture of hPSCs was mTeSR1®. The combination of mTeSR1 medium and Matrigel® substrate creates a very rich environment to support hPSCs. However, mTeSR1 medium contains animal-derived components and relies on very high amounts of bFGF to support undifferentiated hPSCs.

Chemically defined

A more streamlined medium for feeder-free cell culture is NutriStem® hPSC Medium. This product is completely chemically defined, xeno-free and contains very low levels of growth factors and other proteins, including bFGF. The low protein composition avoids potential bias, inhibition or other effects on subsequent differentiation of the cells.

A chemically defined media means that the quantity and quality of all the components are known. Using a completely chemically defined medium allows your hPSCs to grow in a controlled and consistent manner, making them ideal to be administered as a cellular therapy without lot-to-lot variability. Eliminating all non-human components, thus creating a completely xeno-free environment, reduces health risks for downstream applications and reduces potential immunological reactions from stem cells or their derivatives.

In a recent RegMedNet webinar, speaker Dr Qasim Rafiq commented: “I think we should try and define as much of our process as possible but that may not be possible in all instances and processes. I think that’s one of the major barriers for serum-free, xeno-free cell culture media supplements is that it can be very difficult for certain processes to adapt effectively [but] there are many alternatives to FBS available”.3

Utilizing a high quality media that has been optimized for specific cells and your application can overcome this potential challenge.

NutriStem® hPSC Medium mentioned above is a defined, xeno-free, serum-free medium optimized for the growth and expansion of hPSCs, removing the need for high levels of growth factors and cytokines.

Figure 1: Human Embryonic Stem Cells (H1, passage 6) were seeded in 96-well plates (Matrigel coated) in BI's NutriStem® hPSC XF and competitor's media. Stem cell media were changed every 24 hours.

In addition to the NutriStem® hPSC mentioned above, BI has developed a new defined, xeno-free, serum-free medium. NutriStem® V9 XF, as well as NutriStem® hPSC, is specially designed to support the growth of hPSCs. NutriStem® V9 utilizes vitronectin and enzyme-free passage using EDTA and contains components that maintain hPSCs in the long-term. Long-term cultures are shown to have superior proliferation rates and maintenance of pluripotency when NutriStem® V9 XF is implemented.

Figure 2: Nucleocounts performed on equal volume of cell suspension Chemometech during long-term expansion of H1 cultured in NutriStem® V9 XF medium and E8 under a weekend-free feeding regime using 0.5μg/cm2 Vitronectin ACF. NutriStem® V9 XF medium shows superior proliferation rate in long-term culture (over 20 passages).

Preserving your hPSCs

Effective cryopreservation of hPSCs without compromising their physiology and function is vital if their potential in therapeutics, as well as disease and drug modelling, is to be realized. Utilizing a suitable freezing medium such as CryoStem® ensures high viability and recovery after thawing and preserves markers of pluripotency. If a serum-free and xeno-free environment has been implemented, it is important to maintain the same conditions during cryopreservation.

Conclusion

In conclusion, it is important to consider the culture medium used to get the best out of your hPSCs. Considerations when selecting a culture medium include the application of your hPSCs, the scope for translation and the number and quality of cells required. If you are unsure, enlisting the expertise of cell culture media manufacturers such as Biological Industries will ensure that the method you select to culture your hPSCs will be efficient and effective for your application.

References

  1. Jayme DW, Smith SR. Media formulation options and manufacturing process controls to safeguard against introduction of animal origin contaminants in animal cell culture. Cytotechnology. 33(1-3):27-36. (2000)
  2. Biological Industries. An Introduction to the Evolution of Human Pluripotent Stem Cell Culture Systems. RegMedNet (2017)
  3. RegMedNet. Q&A transcription: Why are we still using FBS in our processes? RegMedNet (2017)

Writing assistance from RegMedNet was used in the production of this article.


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Biological Industries

Biological Industries (BI) provides stem cell researchers with high-quality cell culture reagents including NutriStem® – a serum-free; xeno-free stem cell media for the growth and expansion of hPSCs and MSCs. In addition, our CryoStem™ Freezing Media and MSCgo™ Differentiation Kits are used by researchers all over the world. BI also offers contract and custom media formulations, products for mycoplasma detection and elimination, cell culture contamination prevention, and human cytogenetics products. BI’s facilities are cGMP compliant and ISO-certified, with the highest levels of quality management systems in place.

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