Cell Preservation in Reparative and Regenerative Medicine: Evolution of Individualized Solution Composition

Abstract

The expanding complexity of biologics banked for therapeutic applications necessitates the development of improved preservation technologies for support of the emerging fields of reparative and regenerative medicine. Currently, a number of media or "solutions" are utilized for the preservation of biologics. Given the diversity of cell systems utilized in the regenerative medicine arena, we hypothesized that the development of unique (individualized) preservation solutions designed to meet the distinct molecular biological requirements of individual systems would provide for enhanced and extended preservation. To evaluate this hypothesis, coronary artery smooth muscle cells (CASMCs), coronary artery endothelial cells (CAECs), hepatic cells (C3A), and skeletal muscle cells (SKMCs) were hypothermically preserved for 2 to 7 days at 4 degrees C in either cell culture medium, University of Wisconsin Solution (UW or ViaSpan), or HypoThermosol (HTS) variants. Cells were then assayed for viability, using the alamarBlue assay as well as calcein-AM, subsequent to their return to normothermic (37 degrees C) temperatures for up to 5 days. CASMC viability was best maintained when preserved in HTS plus Trolox/EDTA, CAEC viability was highest when preserved in HTS plus Trolox, SKMCs stored in HTS plus Trolox/RGD demonstrated enhanced viability, and C3A cells were best preserved in HTS plus FK041. The data suggest that solution compositions that address the differences in cell death mechanisms limiting preservation efficacy can result in targeted improvement matched to specific cell types. These observations support the custom solution hypothesis of cell and tissue preservation.

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

“…This is similar to tissue ischemia and cell death upon reperfusion. Extreme temperature shifts may also induce apoptosis and necrosis (Mathew et al 2004). Viability evaluation, either immediately after hypothermic exposure or following rewarming to normothermic conditions, may give varied results as cells may be programmed for apoptosis upon their return to 37°C or following continuous culture (Rauen et al 2000).…”

“…These cold storage media aim to support cell metabolism and inhibit post-storage necrosis and activation of apoptosis in response to cold temperatures (Hope et al 2011). HTS-FRS has been particularly successful for the storage of cells at 4°C (Mathew et al 2002;Mathew et al 2004). Comprised of ionic components such as Na + and Cl -, pH buffers, energy substrates and osmotic and oncotic stabilizers, the ionic makeup of HTS aims to balance cellular ion concentrations which are altered upon hypothermia and nutrient deprivation (Mathew et al 2004).…”

“…HTS-FRS has been particularly successful for the storage of cells at 4°C (Mathew et al 2002;Mathew et al 2004). Comprised of ionic components such as Na + and Cl -, pH buffers, energy substrates and osmotic and oncotic stabilizers, the ionic makeup of HTS aims to balance cellular ion concentrations which are altered upon hypothermia and nutrient deprivation (Mathew et al 2004). HEPES buffer manages pH fluctuations and provides osmotic support (Taylor 1982;Taylor & Pignat 1982).…”

“…It is of extreme importance that preservation methods are tailored to different cellular systems as each cell type has distinct metabolic, structural and physiological properties (Mathew et al 2004). The success or failure of any cold temperature preservation protocol depends upon the rate of cooling, temperature and fluctuations, medium composition and method and rate of rewarming (Heidemann et al 2011).…”

“…It would be ideal if a platform preservation method could be developed but current research indicates tailoring is needed. For example, viability in coronary artery smooth muscle cells was best maintained in HTS supplemented with Trolox for 7 days at 4°C, whereas skeletal muscle cells required the addition of an RGD peptide to maintain viability (Mathew et al 2004). Stem cells are less robust than most fully differentiated cell types and preservation protocols need to consider maintenance of their potency characteristics.…”