Cryopreservation of Human Stem Cells for Clinical Application: A Review

Abstract

Stem cells have been used in a clinical setting for many years. Haematopoietic stem cells have been used for the treatment of both haematological and non-haematological disease; while more recently mesenchymal stem cells derived from bone marrow have been the subject of both laboratory and early clinical studies. Whilst these cells show both multipotency and expansion potential, they nonetheless do not form stable cell lines in culture which is likely to limit the breadth of their application in the field of regenerative medicine. Human embryonic stem cells are pluripotent cells, capable of forming stable cell lines which retain the capacity to differentiate into cells from all three germ layers. This makes them of special significance in both regenerative medicine and toxicology. Induced pluripotent stem (iPS) cells may also provide a similar breadth of utility without some of the confounding ethical issues surrounding embryonic stem cells. An essential pre-requisite to the commercial and clinical application of stem cells are suitable cryopreservation protocols for long-term storage. Whilst effective methods for cryopreservation and storage have been developed for haematopoietic and mesenchymal stem cells, embryonic cells and iPS cells have proved more refractory. This paper reviews the current state of cryopreservation as it pertains to stem cells and in particular the embryonic and iPS cell.

Cryopreservation of Human Pluripotent Stem Cells: Are We Going in the Right Direction?

“…Using mixtures of cryoprotectants helps to reduce the intrinsic toxicity of each, and the method published by Reubinoff et al utilized 20% DMSO, 20% ethylene glycol and 0.5 mol/l sucrose (Reubinoff et al, 2001). However, no studies have been reported so far to determine the permeability of the cells (or colony fragments) to either cryoprotectant, or the intrinsic toxicity of these components to hESCs (Hunt, 2011).…”

“…In contrast, vitrification overcomes the problems associated with ice crystallization in a different manner. Here cryoprotectants are used in high concentrations preventing ice formation entirely (Baust et al, 2009;Hunt, 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).…”

“…Osmotic imbalances can injure cells and programmed cell death may be induced upon thawing (Mathew et al 2002). Further to this, dimethyl sulfoxide (DMSO), the most frequently used CPA, is considered toxic to cells at ambient temperatures (Hunt 2011) and may induce cell differentiation (Santos et al 2003). Patients infused with DMSO-preserved cells can also experience adverse reactions (Cox et al 2012).…”

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

“…It is well known that DMSO is potentially cytotoxic, and transplantation of DMSO-preserved human BM cells has been shown to cause severe adverse reactions. 20 Moreover, the use of human serum albumin, cerebrospinal fluid, fetal bovine serum, and bovine serum albumin as cryoprotectants is also problematic, due to the risk of contamination with human or animal viruses and the potential of infection with prions and other unidentified pathogens. 21 In addition, these are potentially dangerous due to the possible induction of an allergic response.…”