EFFECTIVE HYPOTHERMIC STORAGE OF HUMAN PLURIPOTENT STEM CELL-DERIVED CARDIOMYOCYTES COMPATIBLE WITH GLOBAL DISTRIBUTION OF CELLS FOR CLINICAL APPLICATIONS AND TOXICOLOGY TESTING Cláudia Correia 1,2 , Alexey Koshkin 1,2 , Madalena Carido 1,2 , Nuno Espinha 1,2 , Pedro Lima 3 , Margarida Serra 1,2 , Paula M Alves 1,2 1 iBET, Oeiras, Portugal; 2 ITQB-UNL, Oeiras, Portugal; 3 Nova Medical School, Lisboa, Portugal AIM E STABLISHMENT OF EFFECTIVE CLINICALLY COMPATIBLE STRATEGIES FOR COLD (4ᴼ C) STORAGE OF hPSC-CMS AS 2D MONOLAYERS AND 3D AGGREGATES The production of cardiomyocytes (CMs) from human pluripotent stem cells (hPSC) holds great promise for patient-specific cardiotoxicity drug testing, disease modeling and cardiac regeneration [1]. The applicability of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) in the clinic and industry is highly dependent on the development of efficient methods for worldwide shipment of these cells. 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 [2]. S TRATEGY B ACKGROUND 2D MONOLAYERS 3D AGGREGATES CELL CHARACTERIZATION HYPOTHERMIC STORAGE STORAGE SOLUTIONS STORAGE TIME MORPHOLOGY STRUCTURE ULTRASTRUCTURE 2D MONOLAYERS RESULTS Legend: Nucleus (Nu); myofibrils (MF); Z-bands (Z); neighbouring CMs (CM1, CM2); intercalated disks (ID) hPSC-CMS WERE STORED FOR 3 (S3), 5 (S5) AND 7 (S7) DAYS AT 4°C IN HYPO THERMOSOL TM [2] S7 - Day 1 S7 - Day 7 FDA / PI NUCVIEW / MITOVIEW S7 - Day 1 S7 - Day 7 CELL VIABILITY NECROSIS - FDA (LIVE CELLS) / PI (DEAD CELLS) APOPTOSIS - MITOVIEW (LIVE CELLS)/NUCVIEW (APOPTOTIC CELLS, ACTIVE CASPASE-3) METABOLIC ACTIVITY RECOVERY PRESTOBLUE ASSAY HYPOTHERMIC S TORAGE OF hPSC-CMS MF Z Z MF MF CM2 CM1 TRANSMISSION ELECTRON MICROSCOPY (TEM) METABOLOMICS CULTURE S YSTEM CELL RECOVERY TRYPAN BLUE ASSAY 3D A GGREGATES CELL VIABILITY NECROSIS - FDA (LIVE CELLS) / PI (DEAD CELLS) METABOLIC ACTIVITY RECOVERY PRESTOBLUE ASSAY SCANNING ELECTRON MICROSCOPY (SEM) TRANSMISSION ELECTRON MICROSCOPY (TEM) S7 - Day 7 Legend: Nucleus (Nu); myofibrils (MF); Z-bands (Z); mitochondria (M) MONOLAYERS OF hPSC-CMS CAN BE EFFICIENTLY COLD STORED FOR 3 DAYS WITHOUT COMPROMISING CELL RECOVERY , METABOLIC ACTIVITY AND ULTRASTRUCTURE ꭗ CELL VIABILITY DECREASED WHEN THE COLD STORAGE INTERVAL WAS EXTENDED TO 7 DAYS hPSC-CMS ARE MORE RESISTANT TO PROLONGED HYPOTHERMIC STORAGE-INDUCED CELL INJURY IN 3D AGGREGATES THAN IN 2D MONOLAYERS, SHOWING HIGH CELL RECOVERIES (>70%) AFTER 7 DAYS OF STORAGE AGGREGATE STRUCTURE/SIZE AND CELL ULTRASTRUCTURE WERE MAINTAINED AFTER 7 DAYS OF STORAGE 1 2 3 0 2 0 4 0 6 0 8 0 1 0 0 S 7 C o n t r o l B e f o r e S t o r a g e S I R P A V C A M c T N T 0 1 2 3 4 0.0 0.2 0.4 0.6 0.8 1.0 1.2 S3 S5 S7 Time post-storage (day) Frequency (in relation to non-stored cells) hPSC-CM CHARACTERIZATION FLOW CYTOMETRY BEATING FREQUENCY IMMUNOFLUORESCENCE MICROSCOPY Visit us @ www.ibet.pt Animal Cell Technology Unit, iBET & ITQB-UNL , Portugal Scale-up and Manufacturing of Cell-based Therapies V, January 2017, San Diego, CA, USA ACKNOWLEDGEMENTS: The authors acknowledge the financial support received from FCT, Portugal (PTDC/BIO/72755/2006), from the European Commission (Hyperlab:223011) and from the FP7 EU project CARE-MI (HEALTH-2009_242038). CC acknowledges the funding from FCT (SFRH/BD/51573/2011). REFERENCES: [1] Oh, Y., Wei, H., Ma, D., Sun, X. & Liew, R. Clinical applications of patient-specific induced pluripotent stem cells in cardiovascular medicine. Heart 98, 443–9 (2012). [2] Correia, C., Koshkin, A., Carido, M. et al. Effective Hypothermic Storage of Human Pluripotent Stem Cell-Derived Cardiomyocytes Compatible With Global Distribution of Cells for Clinical Applications and Toxicology Testing. Stem Cells Trans Med. 5;111(3):658-69 (2016). ELECTROPHYSIOLOGY ANALYSIS DRUG RESPONSIVENESS Normalized values Normalized values CALCIUM TRANSIENTS AFTER 7 DAYS OF STORAGE, HPSC-CMS MAINTAINED THEIR TYPICAL PROTEIN EXPRESSION PROFILE, SARCOMERIC STRUCTURE, BEATING FREQUENCY , ELECTROPHYSIOLOGICAL PROFILES AND DRUG RESPONSIVENESS CONCLUSION DEVELOPMENT OF EFFICIENT STRATEGIES FOR HYPOTHERMIC STORAGE OF 2D MONOLAYERS AND 3D AGGREGATES OF HPSC-CMS 2D MONOLAYER OF HPSC-CMS CAN BE EFFICIENTLY STORED DURING 3 DAYS (80% OF CELL RECOVERY) 3D AGGREGATES OF HPSC-CM PROVIDE BETTER CELL RECOVERY COMPARING TO 2D MONOLAYER AFTER 7 DAYS OF STORAGE 7 DAYS OF HYPOTHERMIC STORAGE DID NOT AFFECT THE PHENOTYPE AND FUNCTION OF HUMAN PSC-CMS STORED EITHER AS MONOLAYERS OR AGGREGATES STEP FORWARD TOWARDS THE GLOBAL COMMERCIAL DISTRIBUTION OF hPSC-CMS