3.3.4

Evaluation the

Pluripotency of hiPSCs in

Spinner Flask Cultures by

Flow Cytometry (Fig. 3)

1. To evaluate the pluripotency of spinner-grown hiPSCs after

6 days of expansion: Apart from taking 1 mL of sample for

cell counting and aggregates imaging, take 2 mL of sample

from the spinner culture, as described in Subheading 3.3.3,

step 1.

2. Allow MC aggregates to settle and gently remove cell culture

medium without distributing the MC aggregates.

3. Wash the MC aggregates two times with DPBS(
).

4. Add 2 mL of warm TrypLE™Express and incubate at 37 ^C for

5–7 min on a 75 rpm shaker for cell dissociation to obtain

single-cell suspension.

5. After incubation, pipet up and down to break clumps and

detach cells from MC, and dilute cell suspension by adding

2 mL of DPBS(
).

6. Separate the single cells from MC by using a 40-μm strainer.

7. Centrifuge the single cells at 300–400  g for 3 min.

8. Wash the cells two times with DPBS(
).

9. Perform flow cytometry as described in Subheading 3.2.3 (see

Fig. 2) with pluripotency markers (see Table 1),

10. The expression of pluripotency markers Oct3/4 and Tra-1-60

should both be above 90% [3].

3.4

Cardiac

Differentiation in

Microcarrier Spinner

Cultures (Fig. 4)

Production of cardiomyocytes in an integrated bioprocess of stem

cell expansion and differentiation in MC spinner cultures. The cell

line with high cardiogenic potential (Subheading 3.2) and high

proliferative potential in MC spinner culture (Subheading 3.3)

will be used.

Fig. 3 Evaluating cell-line proliferation in a microcarrier spinner culture

Integrated Cardiomyocyte Differentiation in Microcarrier Culture

75