Enhanced Hypothermic Storage of Neonatal Cardiomyocytes

Abstract

Successful hypothermic storage of primary cells is now recognized as a rate-limiting step in the development of clinical applications in regenerative medicine and in research-related settings. Heart transplantation and stem cell cardiomyoplasty require hypothermic storage for successful harvest/isolation and transport to the recipient. These processes are time constrained, thereby necessitating the need to recover fully functional bioproducts following prolonged hypothermia. Despite numerous attempts, extension of the cold storage interval for myocytes while maintaining viability and function has not been realized. In this study, investigation into several strategic approaches to hypothermic preservation was evaluated in the neonatal rat ventricular cardiomyocyte model (NRVCM). Samples were assessed for survival by a panel of indicators including cellular membrane integrity, metabolic activity and spontaneous contractile function. Cultured NRVCM were held at 4°C for 24–72 h in either standard culture media, ViaSpan® (University of Wisconsin /UW) or HypoThermosol® (HTS) variants (HTS-Base, HTS-DCC or HTS-FRS). Samples stored in HTS-DCC and HTS-FRS yielded overall survival rates 10–30% greater than HTS-Base (p < 0.05), 20 to >70% greater than ViaSpan®(p < 0.05), and at least 50% greater than cells stored in serum supplemented Dulbecco's modified Eagle medium (DMEM) cell culture media (p < 0.01). Upon return to normothermic conditions, NRVCM stored in HTS-FRS for 24 or 48 h regained spontaneous cellular contraction in vitro and 90–95% metabolic activity as compared to 37°C controls. Extension of the storage interval to 72 h resulted in a significant drop in viability; however, cells stored in HTS-FRS maintained adherence and contractile activity. Western blot analysis revealed that extended/suboptimal storage conditions elicited an alteration in the ratio of the mitochondrial proteins Bcl-XL:Bcl-XS toward pro-death signaling, whereas cells that demonstrated nearcontrol levels of metabolic activity and function exhibited pro-survival ratios within the first 8 h following removal from hypothermic storage. These data demonstrate successful preservation of primary NRVCM cultures for 48 h in selected cold-storage solutions that provide protection from the cellular and molecular stresses associated with hypothermic storage. These results provide important basic steps toward increasing the cold-storage window for sensitive biologic products.