4. Resuspend cells in 100 μL of 1% BSA/Triton X-100 buffer

containing cardiac-specific primary antibodies (see Table 1).

Incubate for 30 min at room temperature.

5. Centrifuge the stained cell suspension at 300–400  g for

3 min, then discard the supernatant.

6. Wash once with 1% BSA/PBS buffer.

7. Resuspend in 100 μL of secondary antibody (1:500) (see

Table 1) in 1% BSA/PBS buffer. Incubate for 30 min at room

temperature in dark.

8. Centrifuge the stained cell suspension at 300–400  g for

3 min. Discard the supernatant.

9. Resuspend cells in 200 μL of 1% BSA/PBS buffer. Keep cells on

ice in dark until ready to analyze by flow cytometry.

10. The hiPSC line(s) with high cardiac differentiation potency

(i.e., high expression of NKX2-5, cTnT, and MLC2a, but

low in HNF4a and CD44) will be selected for testing of

expansion in microcarrier culture (see Note 9).

3.3

Testing of Cell

Expansion in

Microcarrier Cultures

3.3.1

Preparation of

GelTrex-Coated

Microcarriers

1. To prepare Geltrex™-coated microcarriers (Cytodex^® 1):

Transfer 33.7 mL of cold DMEM/F12, 5 mL of Cytodex^®

1 (MC) suspension (50 mg of Cytodex^® 1) and 1.3 mL of

Geltrex™[8–10] into the 50-mL conical tube (see Note 10).

2. Place the centrifuge tube horizontally onto bench rocker at

4 ^C for overnight gentle agitation.

3. After agitation, store in 4 ^C until ready to use.

4. Pre-warm Geltrex™-coated MC (step 3) in 37 ^C water bath

for

about

20–30

min.

Spin

down

in

a

centrifuge

at

300–400  g for 5 min.

5. Carefully aspirate supernatant from conical tube (see Note 11).

6. Resuspend the coated MC with 25 mL of mTeSR™1 supple-

mented with 10 μM Y-27632.

7. Transfer all 25 mL of resuspended MC into a Corning^®

125-mL Disposable Spinner Flask and put the flask on a Corn-

ing^® Four Position Magnetic Stirrer placed in a 37^C CO2

incubator.

3.3.2

Seeding Cells into

Spinner Flask

1. Dissociate the selected hiPSCs (with high cardiac differentia-

tion potency chosen in Subheading 3.2) into single-cell sus-

pension

by

TrypeLE™-Express,

as

described

in

the

manufacturer’s user guide Pub. No. MAN0007321 Rev. 2.0.

Ensure there are no cell clumps (see Note 12).

2. Count the live cell number using NucleoCounter^® automated

cell counter, as described in the manufacturer’s Application

Integrated Cardiomyocyte Differentiation in Microcarrier Culture

73