Large‐scale compatible methods for the preservation of human embryonic stem cells: Current perspectives

Abstract

Human embryonic stem cells (hESCs) and hESC‐derived cells are of great interest, not only because of their therapeutic potential but also their prospective uses in in vitro drug and toxicity screening. The ability to preserve these cells is critical, allowing for the generation of quality‐controlled stocks of cells, transport of cells between sites, and avoiding the need for expensive and time‐consuming continuous culture. Current methodologies, namely conventional slow freezing and vitrification, can successfully preserve hESCs and their differentiated progeny, retaining the key characteristics of the cells. However, there is a significant gap between the number of cells potentially needed to either treat patients or run a high‐throughput drug screen and how many cells can be preserved using these techniques. Therefore, this review focuses on the scalability of slow freezing and vitrification, identifying key barriers to success and whether they can be overcome. Given the precedent with other mammalian cells in using slow freezing to successfully preserve large quantities of cells and its compatibility with current and emerging culture methods for hESCs, it is likely to become the method of choice for cryopreserving these cells at scale. However, issues other than scale still exist; therefore, alternatives to cryopreservation should also be explored. Here, the potential to lyophilize hESCs for long‐term storage is considered as one such alternative. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011

Low temperature cell pausing: an alternative short-term preservation method for use in cell therapies including stem cell applications

“…Whilst conventional slow freezing generally yields over 90% viability post-thaw (Kleeberger et al 1999;Kotobuki et al 2005), for certain cell types such as human embryonic stem cells (hESC) grown as colonies, poor survival has been reported (Li et al 2010;Xu et al 2010). Vitrification is an alternative approach for storage of hESC (Li et al 2010), yet is disadvantaged due to higher concentrations of cryoprotective agents (CPAs), smaller storage volumes (typically 1-20µl per cryostraw) (Unger et al 2008;Coopman 2011;Hunt 2011) and the risk of contamination from open cryostraws (Mirabet et al 2012). Diminished functionality of cryopreserved human hepatocytes has also been documented; with cells losing their adherence capabilities and exhibiting an altered metabolic profile post-thaw (Terry et al 2006).…”

“…Furthermore, the use of animal derived components in cryopreservation, such as fetal bovine serum as a vehicle solution, risks contamination with adventitious agents (Merten 1999). Despite some drawbacks, cryopreservation at -80°C or -20°C is commonly used to store and transfer cells between the manufacturing site and medical facility (Coopman 2011;Harel 2013). Research to improve cryopreservation protocols, using safer, naturally derived CPAs such as trehalose, and xeno-free serum alternatives, is in progress for application to therapeutically relevant cells.…”

“…The exact number of cells needed is dependent on disease type and prevalence but ranges from 10 6 to 10 9 per patient (Mason & Dunnill 2009). Cryopreservation is limited in its ability to preserve cells at scale with most cryovials and cryobags storing between 1-20 x 10 6 cells per ml (Coopman 2011;Heidemann et al 2010). Being devoid of specialist storage products and apparatus, cell pausing at ambient temperatures generates opportunity for larger scale storage and transport.…”

Cryopreservation and Cell Banking for Autologous Mesenchymal Stem Cell-Based Therapies

“…25,26 In addition to the above areas, efforts to optimize the cryopreservation process have focused on the use of more environmentally-friendly cryopreservation agents, lowering the overall cost of reagents, manual handling and storage, and using slow-rate cooling methods. 27,28 These efforts have resulted in good, reproducible yields, maintenance of similar phenotypes and cell surface markers, cells remaining untransformed, no microbiological contaminations, and comparable growth rates to non-cryogenically treated cells.…”

“…In addition, significant advances might be made, perhaps in lyophilization of cells or even bone marrow, which could provide additional benefits such as significantly longer viability, simpler long-term storage, and less costly shipping. 39 Finally, this future is not likely to happen until several autologous products are launched and can optimize the business model by reinvesting profits from the product. Time will tell, but early data are encouraging that cell therapy products may become a mainstream medical treatment in the near future.…”