An Optimized Culture Medium for Induced Pluripotent Stem Cells

An animal component-free culture medium provides a safe and robust alternative for maintaining induced pluripotent stem cells.

The Scientist Creative Services Team in collaboration with Bio-Techne
Dec 10, 2021

In 2006, Kazutoshi Takahashi and Shinya Yamanaka made a revolutionary breakthrough when they reprogrammed terminally differentiated, lineage-restricted adult somatic cells into a pluripotent state via the expression of transcription factors Oct4, Sox2, Klf4, and c-Myc.1 Now, researchers can differentiate reprogrammed induced pluripotent stem cells (iPSCs) into any cell type and use them for numerous applications, including disease research, drug development, and personalized regenerative stem cell therapy.2

Growing iPSCs in vitro requires a culture medium, growth factors, and an extracellular matrix. The culture medium quality profoundly impacts cell growth, viability, and differentiated cell type. Stem cell researchers desire stable and consistent culture media to support the long-term expansion of iPSCs and to achieve large cell quantities for downstream differentiation protocols.    

Researchers typically culture iPSCs in medium containing animal-derived serum and growth factors. However, serum composition and performance are variable, which affects cell culture quality leading to smaller colony sizes, excessive differentiation, and changes in cell adhesion properties. Further, contaminating animal pathogens are a threat to cells destined for therapeutic use. In addition to improving consistency and safety, removing serum and other undefined animal components, such as growth factors, from the culture medium could simplify purification and downstream processing. Combining purified growth factors and crucial serum components engineered in bacterial systems with a synthetic iPSC culture medium is the common trick manufacturers use to replace animal components.2      

Another critical but potentially problematic component of iPSC cultures are feeder cells, which provide a surface for iPSCs to grow. Murine fibroblasts are commonly used as feeder cells, but they can introduce animal pathogens into cultures. Moreover, feeder cells often secrete proteins that cause progenitor cells to proliferate and differentiate. iPSCs must maintain their pluripotency, and spontaneous differentiation is a major hurdle in establishing stable, long-term pluripotent cultures. To decrease safety concerns and achieve minimal spontaneous differentiation, researchers must irradiate or chemically inactivate feeder cells prior to culturing iPSCs on them. Due to these concerns, there is an incentive for stem cell researchers to adopt serum-free, animal-free culture media.3,4

ExCellerate™ iPSC Expansion Medium is a serum- and feeder-free culture medium devoid of any animal components, providing a stable and reproducible growth environment for iPSCs without the need for additional growth factors and supplements. The absence of animal components reduces batch-to-batch variability and offers consistent performance in growth and viability across different cell lines.

Human iPSC (hiPSC) cell lines of different origins cultured in ExCellerate medium show characteristics of healthy cells, such as densely packed cell colonies that are smooth and round. Even after more than 45 passages, hiPSCs grown on this expansion medium maintain robust expression of Oct4 and Sox2 pluripotency markers. Furthermore, hiPSCs display normal karyotypes even after more than 30 passages, which highlights the genetic stability of these cultures. Lastly, iPSCs maintained on ExCellerate medium can differentiate into numerous cell types, such as cardiomyocytes, hepatocytes, and neurons, and with improved safety profiles, they are suitable for clinical applications such as transplantation.

References

  1. K. Takahashi, S. Yamanaka, “Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors,” Cell, 126(4):663-76, 2006.
  2. M. Doss, A. Sachinidis, “Current challenges of iPSC-based disease modeling and therapeutic implications,” Cells, 8(5):403, 2019.
  3. S. Llames et al., “Feeder layer cell actions and applications,” Tissue Eng Part B Rev, 21(4):345-53, 2015.
  4. S. Salt, “Technology Digest: the importance of serum- and animal component-free media for stem cell-based therapeutics,” https://www.regmednet.com/technology-digest-the-importance-of-serum-and-animal-component-free-media-for-stem-cell-based-therapeutics/, accessed on November 18, 2021.