Cryopreservation of human pluripotent stem cell-derived cardiomyocytes is not detrimental to their molecular and functional properties

Abstract

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have emerged as a powerful platform for in vitro modelling of cardiac diseases, safety pharmacology, and drug screening.All these applications require large quantities of well-characterised and standardised batches of hiPSC-CMs. Cryopreservation of hiPSC-CMs without affecting their biochemical or biophysical phenotype is essential for facilitating this, but ideally requires the cells being unchanged by the freeze-thaw procedure. We therefore compared the in vitro functional and molecular characteristics of fresh and cryopreserved hiPSC-CMs generated from two independent hiPSC lines. While the frozen hiPSC-CMs exhibited poorer replating than their freshly-derived counterparts, there was no difference in the proportion of cardiomyocytes retrieved from the mixed population when this was factored in.Interestingly, cryopreserved hiPSC-CMs from one line exhibited longer action potential durations.These results provide evidence that cryopreservation does not compromise the in vitro molecular, physiological and mechanical properties of hiPSC-CMs, though can lead to an enrichment in ventricular myocytes. It also validates this procedure for storing hiPSC-CMs, thereby allowing the same batch of hiPSC-CMs to be used for multiple applications and evaluations. 3 Keywords • Human pluripotent stem cell-derived cardiomyocytes • Cryopreservation • Cardiac electrophysiology • Ventricular cardiomyocytes

Simple Workflow and Comparison of Media for hPSC‐Cardiomyocyte Cryopreservation and Recovery

“…The review by Preininger and colleagues is a helpful navigator (Preininger et al., 2016). Among the publications that do openly report or specifically investigate cryopreservation of hPSC‐CMs, as noted in the Introduction, there are widely varying rates of recovery (Burridge et al., 2015; Chen et al., 2015; van den Brink et al., 2020; Xu et al., 2011), and anecdotally recovery rates tend to be lower. Early reports also cryopreserved hPSC‐CMs that had been differentiated at a lower purity and consistency (Xu et al., 2011); hence, their methods and recovery rates may not be wholly applicable now.…”

“…Few studies address the potential effects of cryopreservation on hPSC‐CMs with detailed analysis, and to our knowledge there are no in‐depth comparisons of cryopreservation media. One report by Brink and colleagues investigates the effect of cryopreservation on the electrophysiology and transcripts of two hPSC lines (van den Brink et al., 2020). Interestingly, they find that following cryopreservation there is mostly no effect on key electrophysiological parameters, although one hPSC line did exhibit slight increases in ventricular, ion channel, and cytoskeletal marker expression compared with non‐cryopreserved hPSC‐CMs (van den Brink et al., 2020).…”

“…The inclusion of KSR in CM Suspension and Freeze media is likely to be beneficial to hPSC‐CMs by bolstering B‐27 in terms of survival factors (e.g., insulin and transferrin), and increasing the amount of protein via bovine serum albumin (BSA), which among other things will help to quench protease activity following dissociation with 10× TrypLE. Slow dropwise resuspension as a way of avoiding osmotic shock to hPSC‐CMs immediately following thawing is noted as critical in several protocols and papers (e.g., Breckwoldt et al., 2017; NCardia, 2018; van den Brink et al., 2020). RevitaCell is a pro‐survival supplement commonly used in hPSC culture to avoid anoikis during single‐cell passaging, containing a Rho‐associated kinase (ROCK) inhibitor and anti‐oxidant factors.…”

“…RevitaCell is a pro‐survival supplement commonly used in hPSC culture to avoid anoikis during single‐cell passaging, containing a Rho‐associated kinase (ROCK) inhibitor and anti‐oxidant factors. The application of RevitaCell or some other pro‐survival supplement for the first day following re‐plating of hPSC‐CMs by us and others (Breckwoldt et al., 2017; van den Brink et al., 2020) is partly based on observations by Laflamme et al., whereby inclusion of a pro‐survival cocktail increased engraftment size of hESC‐CMs in rat hearts (Laflamme et al., 2007). Although we have not rigorously compared hPSC‐CM recovery after re‐plating with or without RevitaCell, we would nonetheless recommend inclusion of some kind of pro‐survival supplement in the medium on the day of thawing.…”

“…Even on a small scale, reliable freezing and thawing of hPSC‐CM batches greatly facilitates project management and experimental reproducibility. However, hPSC‐CMs appear to be less robust towards freeze‐thawing than many other cell types, with reported viable recovery rates varying greatly from 50% to 84% (Burridge, Holmström, & Wu, 2015; Chen et al., 2015; van den Brink et al., 2020; Xu et al., 2011). Over nearly a decade, various options in terms of protocols and reagents for cryopreservation have appeared, and although a few papers provide some detail or investigate the functional effects of cryopreservation on hPSC‐CMs, there is not yet a consensus with regards to workflow or best practice.…”