2.3

Reagents and

Consumables for

Microcarrier Cultures

1. Cytodex^® 1 (Cytiva).

2. Costar^® ultra-low attachment (ULA) six-well plates (Corning).

3. Corning^® 125-mL Disposable Spinner Flask with 70-mm top

cap and two angled sidearms, sterile.

4. Sigmacote^® (Sigma-Aldrich).

5. Duran^® 500-mL laboratory glass bottles, with blue PP screw

cap, for preparation of microcarriers.

2.4

Media and Small

Molecules for CM

Differentiation

1. RPMI-1640 with glucose and L-glutamine (Gibco #11875085).

2. B27 without insulin (Gibco #A1895601).

3. L-Ascorbic acid 2-phosphate (Sigma-Aldrich # A8960).

4. CHIR99021 (CHIR) (Tocris).

5. Endo IWR1 (IWR) (Tocris).

2.5

Antibodies and

Reagents

See Table 1.

2.6

Preparation of

Extracellular Matrices

for hPSCs Cultures

1. To

prepare

GelTrex™,

refer

to

Gibco’s

user

guide

#MAN0007332 Rev. 3.0.

2. Briefly, thaw a 5-mL vial of Geltrex™LDEV (lactose dehydro-

genase elevating virus) free reduced growth factor basement

membrane matrix overnight at 4 ^C.

3. Add 50 mL of cold DMEM/F12 medium to a 50-mL conical

tube. Dilute 500 μL of Geltrex™into the cold DMEM/F12

Table 1

Antibodies and reagents for cardiomyocyte characterization

Pluripotency markers

Oct3/4

R&D Systems

#MAB1759

Tra-1-60

Biolegend

#330602

Cardiac specific markers

Hepatocyte nuclear factor 4 alpha (HNF4a)

Cell signaling

#3113S

Mesenchymal fibroblast marker CD44

Cell signaling

#3570

Cardiac transcription factor, Homeobox protein

NKX-2.5 (NKX2-5)

Cell signaling

#8792

Cardiac troponin T (cTnT)

ThermoScientific

#MA5-12960

Myosin regulatory light chain 2, atrial isoform (MLC2a)

Synaptic systems

#311011

Secondary antibodies

FITC-conjugated goat anti-mouse IgG

ThermoScientific

#A11029

FITC-conjugated goat anti-rabbit IgG

ThermoScientific

#A11034

Reagents

Para-formaldehyde

1% BSA

Triton X-100

Integrated Cardiomyocyte Differentiation in Microcarrier Culture

69