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

Trypan blue staining with hemocytometer).

3. Seed 1  10⁷ cells in 5 mL mTeSR™1 with 10 μM Y-27632

into the 125-mL Spinner Flask containing Geltrex™-coated

MC prepared in Subheading 3.3.1. Now total volume of

medium in the flask is 30 mL.

4. Place the flask in static condition at 37^C in CO2 incubation for

4–6 h to allow initial cell attachment.

5. After 6 h of static incubation, top up with mTeSR™1 (contain-

ing 10 μM Y-27632) into the flask to a total volume of 50 mL.

6. Place the spinner back to the magnetic stirrer in the CO2

incubator, and the culture is then stirred for 6 days [9, 10]

(see Note 13).

7. Change 80% of medium daily: Take out the spinner flask and

put in static for 5 min to allow the MC aggregates to settle

down by gravity. Then remove 80% (40 mL) of the spent

medium by a 25-mL pipette carefully without disturbing the

MC aggregates. Subsequently replenish 80% (40 mL) of fresh

mTeSR™1 into the flask. Place the spinner back to the mag-

netic stirrer in the CO2 incubator and continue culture at

stirring of 25–30 rpm.

3.3.3

Cell Counting and

Imaging during Cultivation

1. Take 1 mL of sample from the spinner culture daily for cell

counting and imaging: Transfer the spinner flask from the

incubator to a BSC, placing the flask on a stir plate set at

25–30 rpm. With the culture in the stir mode, remove the

cap from one sidearm of the flask. Take out about 1 mL of

the culture using 10-mL serological pipette. Then close the cap

and place the spinner flask back to the magnetic stirrer in the

CO2 incubator and continue the culture at stirring of

25–30 rpm.

2. For cell counting, take out 2 100 μL aliquots (using blunt

200-μL tips) from the 1 mL sample and transfer to 1.5-mL

tubes (see Note 14). Count the cell number as aggregates,

using NucleoCounter^® automated cell counter, as described

in the manufacturer’s Application Note No. 0262 (Rev 1.1) or

other cell counting methods (see Note 15).

3. For cell imaging, take out around 500 μL of aliquot (using

blunt 1000-mL tips) from the 1 mL sample and transfer to a

well of 24-well plate. Visualizing the cells under phase-contrast

microscopy. At least 10 pictures should be taken in order to

measure the aggregate size using image analysis software, e.g.,

Image-J (see Note 16).

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Valerie Ho et al.