Effective Hypothermic Storage of Human Pluripotent Stem Cell-Derived Cardiomyocytes Compatible With Global Distribution of Cells for Clinical Applications and Toxicology Testing

Abstract

To fully explore the potential of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs), efficient methods for storage and shipment of these cells are required. Here, we evaluated the feasibility to cold store monolayers and aggregates of functional CMs obtained from different PSC lines using a fully defined clinical-compatible preservation formulation and investigated the time frame that hPSC-CMs could be subjected to hypothermic storage. We showed that two-dimensional (2D) monolayers of hPSC-CMs can be efficiently stored at 4°C for 3 days without compromising cell viability. However, cell viability decreased when the cold storage interval was extended to 7 days. We demonstrated that hPSC-CMs are more resistant to prolonged hypothermic storage-induced cell injury in three-dimensional aggregates than in 2D monolayers, showing high cell recoveries (>70%) after 7 days of storage. Importantly, hPSC-CMs maintained their typical (ultra)structure, gene and protein expression profile, electrophysiological profiles, and drug responsiveness. STEM CELLS TRANSLATIONAL MEDICINE 2016;5:658-669 SIGNIFICANCEThe applicability of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) in the clinic/ industry is highly dependent on the development of efficient methods for worldwide shipment of these cells. This study established effective clinically compatible strategies for cold (4°C) storage of hPSC-CMs cultured as two-dimensional (2D) monolayers and three-dimensional (3D) aggregates. Cell recovery of 2D monolayers of hPSC-CMs was found to be dependent on the time of storage, and 3D cell aggregates were more resistant to prolonged cold storage than 2D monolayers. Of note, it was demonstrated that 7 days of cold storage did not affect hPSC-CM ultrastructure, phenotype, or function. This study provides important insights into the cold preservation of PSC-CMs that could be valuable in improving global commercial distribution of hPSC-CMs.

Cryopreservation of Human Pluripotent Stem Cell-Derived Cardiomyocytes: Strategies, Challenges, and Future Directions

“…These results suggest that thawed hPSC-CMs are especially vulnerable to single-cell culture, but may demonstrate improved long-term outcomes in host tissues or under tissue-like conditions. This inference is corroborated by a recent study that demonstrated hPSC-CMs cultured in 3D micro-tissue aggregates also are more resistant to prolonged hypothermic (4°C) storage-induced cell injury compared to their counterparts cultured in 2D monolayers [63]. However, differences in cryopreservation methodology may have also contributed to the observed differences, so these results must be interpreted with caution.…”

“…Therefore, the long-term viability of hPSC-CMs at ultra-low storage temperatures should be evaluated, since this will be an important consideration in future biobanking initiatives. For very short-term storage, cryopreservation may not even be necessary, since recent evidence suggests that hPSC-CMs can be efficiently stored in hypothermic conditions (4°C) for up to 7 days without significantly compromising cell viability [63]. …”

Distinct carbon sources affect structural and functional maturation of cardiomyocytes derived from human pluripotent stem cells

“…Monolayers of human hiPSC-CMs were fixed in 2% (v/v) glutaraldehyde in 0.1 M HEPES buffer (pH 7.4) for 20 min, washed in DPBS and stored in 1% (v/v) glutaraldehyde in DPBS until analyses. Samples were washed, contrasted, dehydrated, infiltrated, embedded, cured, dissected into small blocks and mounted on epon dummy blocks, as previously described 34 . Ultrathin sections of cell monolayers were cut on a Leica UC6 ultramicrotome using a diamond knife.…”

Dynamic expression of SMAD3 is critical in�osteoblast differentiation of PDMCs

“…Human pluripotent stem cells have the potential to assist in the identification of genes associated with �a��alian develo�� genes associated with mammalian develop-s associated with mammalian develop-mammalian development (1,2), and the plasticity of stem cells suggests that they may have a wide range of clinical applications (3,4). Bone tissue engineering has also been explored for clinical purposes (5,6).…”