described in Subheading 3.2.3. cTnT expression should be

above 80% [3] for successfully differentiated cardiomyocytes

(see Note 17).

10. Take another 2 mL of sample from the spinner culture and

transfer a six-well ULA plate, and then determine the percent-

age of aggregate beating under microscopy (see Note 17).

11. Dissociate rest of cells from the spinner culture as described in

Subheading 3.3.4 (see Note 18). Cryopreserve the cells in

CryoStor^® CS10 Cell Freezing Medium until further charac-

terization, e.g., patch-clamp recording, followed by the

instruction of the manufacturer’s Product Information Sheet

Document #10000000383 version 01.

4

Notes

1. MCs can take up to a few minutes to fully settle. Once settled,

keep track of the amount of supernatant that is removed.

Replace with the same volume of DPBS(
) so the total volume

does not change during the washing process. Some MC loss

during wash is normal however should be minimized.

2. Agitation speed has room for adjustment. Start the speed at

25 rpm and ensure MCs are properly suspended and not

settling in the middle of the spinner flask (see Fig. 5). If MCs

remain improperly suspended, increase the agitation speed up

to 30 rpm.

3. Cells

should

be

detached

easily

after

treatment

with

TrypeLE™-Express, if many cells remain adherent after pipet-

ting, the plate can be incubated in 37 ^C for another 1 or 2 min.

Gentle pipetting is sufficient to remove cells from the culture

surface; rigorous pipetting will result in more cell death.

Fig. 5 Illustration of proper suspension of microcarriers

Integrated Cardiomyocyte Differentiation in Microcarrier Culture

77