– 1
10⁹ hPSC-derived cardiomyoyctes will be required to replace

the considerable number of cells lost in a myocardial infarction

[14]. To this end, bioreactors provide a robust, efficient and scal-

able method to generate an unlimited supply of cells critical in many

clinical indications. Here, we describe the microcarrier-based

expansion of hPSCs in a 3 L bioreactor, resulting in high-fold

expansion of high quality cells within 2 weeks of culture [9]. The

cell inoculum—derived from cells cultured in 2D—are introduced

into the bioreactor with extracellular matrix-coated plastic micro-

carriers. Compared to suspension cultures which use an aggregate-

based method for expansion [7, 15], microcarriers provide a large

surface area on which cells attach and grow [9, 16].

2

Materials

Perform the procedures using aseptic technique inside a Biological

Safety Cabinet unless otherwise noted. Limit the exposure of the

L7™hPSC culture system to light. Store reagents according to

manufacturer’s instructions.

2.1

L7™TFO2 hPSC

Complete Medium

1. Thaw a 10 mL bottle of L7™hPSC medium supplement in

37 ^C. Add the entire contents to 1 L of L7™TFO2 hPSC

basal medium (see Note 1). Invert the bottle twice to mix and

store in 4 ^C until use.

2. 7 L bags of L7™TFO2 hPSC basal medium (see Note 2).

2.2

Passaging

Solutions

1. L7™hPSC passaging solution (see Note 3).

2. F3 hPSC passaging solution (see Note 4).

2.3

Buffers and

Reagents

1. Cell culture grade water.

2. Dulbecco’s Phosphate-Buffered Saline with calcium and mag-

nesium (DPBS^+/+); pH 7–7.6. Store at 15–30 ^C.

3. Dulbecco’s Phosphate-Buffered Saline without calcium and

magnesium (DPBS^/); pH 7–7.6. Store at 15–30 ^C.

4. 250 μg/mL L7™hPSC matrix. Add 4 mL of cell culture grade

water to 1 mg of lyophilized matrix (see Note 5).

5. 10 mM Y-27632 Dihydrochloride (Rho-associated protein

kinase (ROCK) inhibitor). Add 624.4 μL DMSO to 2 mg of

ROCK inhibitor. Mix by pipetting (see Note 6).

2.4

Tubing

1. Assemble the dip tube/perfusion line (see Fig. 1), media feed

line (see Fig. 2) and harvest line extension assembly (see Fig. 3).

Autoclave using the dry cycle (see Note 7).

40

Marites T. Woon et al.