Cell culture. Human CMs were differentiated from HUES9 and HES3 human embryonic stem cells (hESCs) using established protocols (36). The hESCs were expanded in Essential 8 (E8) medium (Life Technologies, catalog no. A1517001) (37) on Geltrex-coated six-well plates (12 μg/cm2; Life Technologies, catalog no. A1413301) with an initial seeding density of 125,000 cells per well for HUES9 and 100,000 cells per well for HES3 cells. The cells were passaged every 4 days to prevent over-confluence. For CM differentiation, hESCs were seeded at a density of 16,000 cells/cm2 for HUES9 and 12,000 cells/cm2 for HES3 cells in E8 medium with 2 μM ROCK inhibitor, thiazovivin (Selleck Chemicals, catalog no. S1459); the medium was changed daily. On the third day after seeding, hESCs were differentiated into CMs. Cells were washed with 1× PBS and incubated with RPMI 1640 medium (Thermo Fisher, catalog no. 21870076) supplemented with B27 minus insulin (Thermo Fisher, catalog no. A1895601) and 1% (v/v) l-glutamine (Thermo Fisher, catalog no. 25030081) plus 6 μM CHIR99021 (LC Laboratories, catalog no. C-6556), a glycogen synthase kinase-3 inhibitor (LC Laboratories, catalog no. S1459), for 2 days. On day 2 of differentiation, cells were washed with 1× PBS and incubated with RPMI/B27 medium and 2 μM Wnt-C59, a Wnt pathway inhibitor (Selleck Chemicals, catalog no. S7037). On days 4 and 6 of differentiation, the medium was changed to RPMI/B27. On days 8 and 10, the medium was changed to CDM3 medium (37) consisting of RPMI 1640 medium supplemented with 1% (v/v) l-glutamine, human albumin (500 μg/ml) (Sigma, catalog no. A9731), and l-ascorbic acid 2-phosphate sesquimagnesium salt hydrate >95% (213 μg/ml) (Sigma, catalog no. A8960). On day 12, spontaneously beating cells were passaged for CM purification. CMs were purified using lactate-supplemented medium, which achieves 95 to 98% purification of CMs, as previously demonstrated (38). Briefly, beating CMs were washed with 1× PBS and detached from the surface with TrypLE express (Thermo Fisher, catalog no. 12604013) for 15 min at 37°C. Detached cells were pipetted into Dulbecco’s modified Eagle’s medium (DMEM)/F12 (Thermo Fisher, catalog no. 11320033) and centrifuged at 200g for 7 min to pellet the cells. CMs were seeded on Matrigel-coated plates (12 μg/cm2) (Corning, catalog no. 356231) with RPMI 1640 lacking glucose (Thermo Fisher, catalog no. 11879020) and supplemented with human albumin (500 μg/ml), l-ascorbic acid-2-phosphate (213 μg/ml), and sodium lactate (7.1 mM) (Sigma, catalog no. L4263). CMs were purified for 5 days and then switched back to CDM3 for at least 2 days before passaging.

Spheroid formation. The spheroids were formed following a previously established protocol (39). Briefly, sterile solution (10 g/liter) of agarose (Invitrogen, catalog no. 16500) was prepared in 0.9% (w/v) NaCl (Sigma, catalog no. S5886) in DI water. The agarose spherical microwells were obtained by casting the agarose into a mold (3D Petri Dish, Microtissues Inc.). The agarose microwells were equilibrated twice in CDM3 medium for 30 min at 37°C. After purification, adherent CM cultures were lifted with TrypLE for 15 min at 37°C. Cells were pipetted into DMEM/F12 and centrifuged at 200g for 7 min. CMs were then seeded at a density of 1.2 × 106 cells/mold for trough-shaped spheroids and 0.65 × 106 cells/mold for round spheroids in 190 μl of CDM3 medium with 2 μM ROCK inhibitor, thiazovivin. The CM spheroids were formed in microwells while incubating at 37°C and 5% CO2 for 1 day, after which the medium was exchanged to CDM3. The cell medium was changed every other day with a fresh CDM3 medium. After 3 to 5 days, when spheroids compacted and began to spontaneously beat, they were harvested by inverting the micromolds and centrifuging for 5 min at 500 rpm. Spheroids 1 and 2 were formed from HUES9 hESCs, while spheroid 3 and spheroids used for biocompatibility studies were formed from HES3 hESCs.

Note: The content above has been extracted from a research article, so it may not display correctly.



Q&A
Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.



We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.