biostudies-literatureUnknownProndzynski MNCATS NIH HHSNHLBI NIH HHSIn the last decade human iPSC-derived cardiomyocytes (hiPSC-CMs) proved to be valuable for cardiac disease modeling and cardiac regeneration, yet challenges with scale, quality, inter-batch consistency, and cryopreservation remain, reducing experimental reproducibility and limiting clinical translation. Here, we report a robust cardiac differentiation protocol that uses Wnt modulation and a stirred suspension bioreactor to produce on average 124 million hiPSC-CMs with >90% purity using a variety of hiPSC lines (19 differentiations; 10 iPSC lines). After controlled freeze and thaw, bioreactor-derived CMs (bCMs) showed high viability (>90%), interbatch reproducibility in cellular morphology, function, drug response and ventricular identity, which was further supported by single cell transcriptomes. bCMs on microcontact printed substrates revealed a higher degree of sarcomere maturation and viability during long-term culture compared to monolayer-derived CMs (mCMs). Moreover, functional investigation of bCMs in 3D engineered heart tissues showed earlier and stronger force production during long-term culture, and robust pacing capture up to 4 Hz when compared to mCMs. bCMs derived from this differentiation protocol will expand the applications of hiPSC-CMs by providing a reproducible, scalable, and resource efficient method to generate cardiac cells with well-characterized structural and functional properties superior to standard mCMs.bioRxiv : the preprint server for biology2024.02.24.581789https://www.ebi.ac.uk/biostudies/studies/S-EPMC10925150biostudies-literatureEfficient and reproducible generation of human iPSC-derived cardiomyocytes using a stirred bioreactor.PMC10925150UH3 HL141798UH3 TR003279Trembley MAMilosh JBPu WTBerkson PSweat MEAnyanwu NJProndzynski MBortolin RHCordoves AMBezzerides VJTharani YLiu XShani KMayourian JZhang YWalker DParker KKCotton JLiu FfalseEfficient and reproducible generation of human iPSC-derived cardiomyocytes using a stirred bioreactor.In the last decade human iPSC-derived cardiomyocytes (hiPSC-CMs) proved to be valuable for cardiac disease modeling and cardiac regeneration, yet challenges with scale, quality, inter-batch consistency, and cryopreservation remain, reducing experimental reproducibility and limiting clinical translation. Here, we report a robust cardiac differentiation protocol that uses Wnt modulation and a stirred suspension bioreactor to produce on average 124 million hiPSC-CMs with >90% purity using a variety of hiPSC lines (19 differentiations; 10 iPSC lines). After controlled freeze and thaw, bioreactor-derived CMs (bCMs) showed high viability (>90%), interbatch reproducibility in cellular morphology, function, drug response and ventricular identity, which was further supported by single cell transcriptomes. bCMs on microcontact printed substrates revealed a higher degree of sarcomere maturation and viability during long-term culture compared to monolayer-derived CMs (mCMs). Moreover, functional investigation of bCMs in 3D engineered heart tissues showed earlier and stronger force production during long-term culture, and robust pacing capture up to 4 Hz when compared to mCMs. bCMs derived from this differentiation protocol will expand the applications of hiPSC-CMs by providing a reproducible, scalable, and resource efficient method to generate cardiac cells with well-characterized structural and functional properties superior to standard mCMs.2024-01-01T00:00:00Z2024 Feb2025-04-04T20:18:24.995Z2025-04-04T20:18:24.995ZS-EPMC109251503846426910.1101/2024.02.24.581789