title: 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.
  
  
  CONCLUSIONS
  The 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.
authors.affiliation: Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Oeiras, Portugal iBET, Instituto de Biologia Experimental e Tecnológica, Oeiras, Portugal.
authors.forename: Cláudia
authors.initials: C
authors.lastname: Correia
authors.affiliation: Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Oeiras, Portugal iBET, Instituto de Biologia Experimental e Tecnológica, Oeiras, Portugal.
authors.forename: Alexey
authors.initials: A
authors.lastname: Koshkin
authors.affiliation: Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Oeiras, Portugal iBET, Instituto de Biologia Experimental e Tecnológica, Oeiras, Portugal.
authors.forename: Madalena
authors.initials: M
authors.lastname: Carido
authors.affiliation: Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Oeiras, Portugal iBET, Instituto de Biologia Experimental e Tecnológica, Oeiras, Portugal.
authors.forename: Nuno
authors.initials: N
authors.lastname: Espinha
authors.affiliation: Center for Physiology and Pathophysiology, Institute for Neurophysiology, Medical Faculty, University of Cologne, Cologne, Germany.
authors.forename: Tomo
authors.initials: T
authors.lastname: Šarić
authors.affiliation: Nova Medical School, Universidade Nova de Lisboa, Lisboa, Portugal.
authors.forename: Pedro A
authors.initials: PA
authors.lastname: Lima
authors.affiliation: Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Oeiras, Portugal iBET, Instituto de Biologia Experimental e Tecnológica, Oeiras, Portugal mserra@ibet.pt.
authors.forename: Margarida
authors.initials: M
authors.lastname: Serra
authors.affiliation: Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Oeiras, Portugal iBET, Instituto de Biologia Experimental e Tecnológica, Oeiras, Portugal.
authors.forename: Paula M
authors.initials: PM
authors.lastname: Alves
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D003080:Cold Temperature / Q000009:adverse effects; D015925:Cryopreservation; D003451:Cryoprotective Agents / Q000494:pharmacology*; D005240:Feasibility Studies; D005786:Gene Expression Regulation; D005819:Genetic Markers; D005838:Genotype; D006801:Humans; D051379:Mice; D032383:Myocytes, Cardiac / Q000187:drug effects* / Q000378:metabolism / Q000648:ultrastructure; D010641:Phenotype; D039904:Pluripotent Stem Cells / Q000187:drug effects* / Q000378:metabolism / Q000637:transplantation / Q000648:ultrastructure; D013997:Time Factors; D018675:Toxicity Tests / Q000379:methods*; D014186:Transportation
chemical_list: D003451:Cryoprotective Agents; D005819:Genetic Markers
keywords: Cell therapy; Human pluripotent stem cell-derived cardiomyocytes; Hypothermic storage; Three-dimensional aggregates; Two-dimensional monolayer cultures