2. Cytodex 1 microcarriers (Cytiva). For preparation of Cytodex

1 stock, refer to Cytiva’s related documents “Instructions for

use - 18111979 AE” (see Note 1).

3

Methods

All procedures are carried out in a Biosafety cabinet Class II, Type

A2 (BSC), unless otherwise stated. In addition, all cell incubations

and culturing are done in a 5% CO2 incubator at 37 ^C unless

otherwise stated. All spinner cultures are agitated at 25–30 rpm

(see Note 2) unless otherwise stated.

3.1

hiPSC Culture

1. hiPSCs are grown as 70–90% confluent monolayers on Gel-

trex^®-coated 60-mm dishes in mTeSR™1, as described in the

manufacturer’s

Product

Information

Sheet,

Document

#10000003789-PIS_03. Passaging is performed at a ratio of

1:10 every 6–7 days by ReLeSR™, as described in the manu-

facturer’s Document #28207 Version 1_4_0.

3.2

Screening for

High Cardiac

Differentiation Potency

hiPSC Lines (Fig. 1)

The optimal concentration of CHIR may vary between cell lines

based on their sensitivity to the treatment. Therefore, an initial test

of treating cells at various concentrations (0–14 μM) [3, 7] is

needed.

3.2.1

hiPSC Seeding for

CM Differentiation

1. When hiPSCs (60-mm dish) reach 70–80% confluence, aspirate

the spent mTeSR™1 and wash cells once with 4 mL of DPBS

(
).

2. Dissociate the cells into single-cell suspension by TrypeLE™-

Express, as described in the manufacturer’s user guide Pub.

No. MAN0007321 Rev. 2.0 (see Note 3).

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

cell counter, as described in the manufacturer’s Application

Note No. 0258 (Rev 1.2) or other cell counting methods

(e.g., Trypan blue staining with hemocytometer).

Fig. 1 Screening for high cardiac differentiation potency hiPSC lines

Integrated Cardiomyocyte Differentiation in Microcarrier Culture

71