Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) are used for disease modelling, drug discovery, and toxicology screening. Current published differentiation protocols lead to variable efficiencies in the generation of hPSC-CMs across different hPSC lines. Most hPS cell lines require optimization of the initial plating density. Furthermore, once established in culture, processing of hPSC-CMs for downstream assays is cumbersome. Inefficient dissociation of hPSC-CMs and their subsequent cryopreservation/thawing can significantly reduce viability, and hence deprive their utility in downstream applications.

The above challenges can be overcome by using in a simple workflow as shown in the schematic below:

Figure 1. Cardiomyocyte Differentiation Protocol

Starting with an initial seeding density of 3.5 -8 x 10e5 cells/well of a 12 well plate, a confluent monolayer of hPSC-CMs that are of high purity (> 80% cTnT) and yield (> 1x10^6 cTnT+ cardiomyocytes per well of a 12-well plate) can be obtained by day 15 of culture.

hPSC-CMs are terminally differentiated cells and therefore cannot be passaged/expanded indefinitely in culture. However, they can be cryopreserved (as early as day 15 of the protocol above) and thawed and plated onto Corning® Matrigel®-coated culture ware for downstream applications. In general, if the recovery of the hPSC-CM monolayer is required (for example in microelectrode array studies) as shown in Figure A, plate on average 500,000 cells in single well of a 48 well plate (~526315 cells/cm²). However, if single hPSC-CMss are required as shown in Figure B, then plate between 25,000 - 50,000 cells for a single well of 12-well plate (~6580 - 13158 cells/cm²). It is also possible to go as low as 5,000-10,000 cells per well of a 12-well plate (~1315 - 2630 cells/cm²). The lower seeding density can be used for seeding the hPSC-derived cardiomyocytes to run a single cardiomyocyte assay such as patch clamping electrophysiology.

hPSC-CMs can be dissociated into single cell suspensions using the to maintain high levels of viability. The harvested hPSC-CMs can be cryopreserved using the . Cryopreserved hPSC-CMs should be thawed into the first to reduce stress and maintain viability and functional capacities for replating and downstream applications. After 24 hours in Support Medium, transition cells into for prolonged maintenance or downstream applications.

The hPSC-CMs can also be plated on glass coverslips coated with Corning® Matrigel®. Both cryopreserved and freshly dissociated () hPSC-CMs can be plated onto the glass coverslips using . After 24 hours in Support Medium, transition cells into for prolonged maintenance or downstream applications, such as optical mapping, immunocytochemistry, and patch clamp electrophysiology.