Derivation, Maintenance and Downstream Processing of Cardiomyocytes from Human Pluripotent Stem Cells (hPSCs)

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 STEMdiff™ Cardiomyocyte System in a simple workflow as shown in the schematic below:

Cardiomyocyte Differentiation Protocol
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.

Replated Monolayer of hPSC-CMs
Figure A. Replated Monolayer of hPSC-CMs


Replated Single hPSC-CMs
Figure B. Replated Single hPSC-CMs

hPSC-CMs can be dissociated into single cell suspensions using the STEMdiff™ Cardiomyocyte Dissociation Kit (Catalog # 05025) to maintain high levels of viability. The harvested hPSC-CMs can be cryopreserved using the STEMdiff™ Cardiomyocyte Freezing Medium (Catalog #05030). Cryopreserved hPSC-CMs should be thawed into the STEMdiff™ Cardiomyocyte Support Medium (Catalog #05027) first to reduce stress and maintain viability and functional capacities for replating and downstream applications. After 24 hours in Support Medium, transition cells into STEMdiff™ Cardiomyocyte Maintenance Kit (Catalog #05020) 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 (STEMdiff™ Cardiomyocyte Dissociation Kit (Catalog # 05025)) hPSC-CMs can be plated onto the glass coverslips using STEMdiff™ Cardiomyocyte Support Medium (Catalog #05027). After 24 hours in Support Medium, transition cells into STEMdiff™ Cardiomyocyte Maintenance Kit (Catalog #05020) for prolonged maintenance or downstream applications, such as optical mapping, immunocytochemistry, and patch clamp electrophysiology.