The Importance of Culturing Human Embryonic Stem Cells in Animal-Free Conditions

Human embryonic stem cells (hES) due to their ability to differentiate into all cell types offer great promise for clinical cell based therapies. The majority of hES cell lines available to date have been directly or indirectly exposed to animal material. This exposure can increase the risk of graft vs. host disease and the transfer of animal pathogens. The elimination of all animal material for both the derivation of and long term culture of hES cells is essential for future hES cell based therapies. Finding alternatives to replace animal components is a major focus of hES cell research. There are two primary areas of this research, feeder-free cell culture systems and serum-free media. Feeder-Free Cell Culture Systems The first hES cells were derived and cultured on mouse embryonic fibroblast (mEF) feeder cells (Thomson et al., Science, 1998). Mouse EF feeder cells deliver an unknown collection of factors that allow hES cells to be preserved in an undifferentiated state. A human foreskin feeder has been reported to maintain hES cells in an undifferentiated state, thus supporting the development of animal-free feeder conditions (Amit et al., Reprod. Bio., 2003). Although, the presence of these human feeder cells poses a risk of transferring human viruses to hES cells. Therefore, to develop hES cells for clinical applications, a feeder-free culture system, which includes the critical factors secreted by feeders or other activators of the same signaling pathways, needed to be identified. Xu et al. Nat. Methods, 2005, reported that the bone morphogenetic protein antagonist, Noggin, could synergize with basic fibroblast growth factor to sustain undifferentiated proliferation of hES cells under feeder-free cell culture conditions. The hES cells were cultured on Matrigel-coated plates. Matrigel is the trade name for a gelatinous extract taken from mouse tumor cells that contains extracellular matrices (ECMs). The Matrigel coating provides the scaffolding to which the hES cells first attach and then grow in undifferentiated colonies. Unfortunately, Matrigel is not entirely animal-free. A novel culture using “poly-D-lysine,” a chemically synthesized ECM, has been developed combining a single synthetic matrix, defined medium, and the Rock inhibitor allowed for hES cell self-renewal independent of animal-derived matrices, tight cell contacts, or fibroblastic niche-forming cells (Harb et al., Plos One, 2008). Serum-Free Media Human serum can be used to replace Matrigel (Stojkovic et al., Stem Cells, 2005). However, human serum contains undefined components. Knockout serum replacement (KO) is frequently used in hES cell culture, although it still contains the animal derived product bovine serum albumin and hence is not completely animal-free (Price et al., Internat. Patent Applic. WO98/30679, 1998). Ludwig al., Nat. Biotech., 2006 report on a feeder independent hES cell culture that includes protein components solely derived from recombinant sources or purified from human material. Although, Matrigel and KO serum replacement was still used in this system.

Albumin has been defined as the essential component of serum for stimulating hES cell self-renewal (Garcia-Gonzalo and Belmonte, Plos One, 2008, Xu et al., 2005). Lu et al., PNAS, 2006 report a hES media derived free of animal components except for albumin and fibronectin that supported hES cell expansion without differentiation of the cells.

Animal-free recombinant human serum albumin and fibronectin have been developed. Testing of these animal-free recombinant components in hES cell culture systems may prove beneficial for cellular expansion and the maintenance of undifferentiated cells and would provide for optimal animal-free hES cell culture conditions.

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