2019／03／11

Ver.01

Human Pluripotent Stem Cell Passaging Protocol
This protocol is optimized for BD standard 6-well flat-bottomed tissue culture dishes.
However, the protocol can be adapted to tissue culture vessels of different sizes andconfigurations.

Cell Passaging Steps
1. Pre-warm the required volume of cell culture medium containing 10 μM Y-27632(final concentration) to 37°C.
The typical volume is 2 ml for every well that the cells will be transferred to + 1 ml foreach well that needs to be harvested + extra 1 - 2 ml.
Examples:1. When you pass the cells from 1 well to 6 wells:-> [(2 ml x 6 wells) + 1 ml for harvesting + extra 1 - 2 ml] = 14 - 15 ml medium
2. When you pass the cells from 2 wells to 8 wells:-> [(2 ml x 8 wells) + 2 ml for harvesting + extra 1 - 2 ml] = 19 - 20 ml medium
2. Remove the spent culture medium.
3. Wash the adherent cells gently with 2 ml of D-PBS(-) (Thermo Fisher Scientific) ineach well.
4. Add 600 μl 0.5x TrypLE Select (Thermo Fisher Scientific) diluted in 0.5 mM EDTA(Sigma-Aldrich)/D-PBS(-) to each well.

*See below on how to prepare 0.5 mM

EDTA/D-PBS(-).
5. Incubate the plate(s) in an incubator for 10 minutes at 37°C, 5% CO2.
6. Aspirate the supernatant (TrypLE Select + EDTA) from each well. Wash theadherent cells remaining on the plate gently with 2 ml D-PBS(-).
7. Add 1 ml of the pre-warmed medium (37°C) with the 10 µM Y-27632 to each well.
8. Harvest the cells using a cell scraper, followed by gentle pipetting to generate asingle cell suspension.

Being gentle with the scraper is critical for retaining

high cell viability.
9. Stain the cells with 10 μl of Trypan blue staining solution (Thermo Fisher Scientific).
Determine the number of viable cells either by counting manually with ahaemocytometer or by using an automated cell counter.
10. Adjust the concentration of the cell suspension with the pre-warmed medium
containing 10 µM Y-27632.

The recommended cell density is 2.0 - 5.0 x 104

cells/well in a 6-well plate, depending on the hPSC line used (Miyazaki et al., 2012;2019／03／11

Miyazaki et al., 2017).

Ver.01

The number of cells will increase 100-fold after 6 - 7 days inculture.
11. Add an adequate volume of iMatrix-511. The recommended iMatrix-511
concentration is 0.25 µg/cm2 (4.8 µl of 500 µg/ml iMatrix-511/well).
12. Transfer 2 ml of the cell suspension with the iMatrix-511 to each well.
13. Gently rock the plate back-and-forth and side-to-side to disperse the cells across thesurface.
14. Incubate the cells for 24 hrs before exchanging the plating medium for fresh medium(without Y-27632).

Replace medium on day 3 and day 5, then everyday until thenext passage (usually at day 6 or day 7).

Recommended Volume of Reagents
6 well12 well

24 well

48 well

Approximate Area (cm2)

9.6

4.0

2.0

1.00.25 μg/cm2 iMatrix-511 (μl)

52

10.5

Medium volume (ml)

2

10.5

0.25

TrypLE Select/EDTA/D-PBS(-) (μl)

500

250

12565

*Preparation of 0.5 mM EDTA/D-PBS(-)
1. First, prepare the 0.5 M EDTA/D-PBS(-) stock solution by adding 18.6 g EDTA-2Na(Sigma-Aldrich) to 100 ml D-PBS(-).
dissolved. Adjust the pH to 8.0.

Mix well until EDTA-2Na is completely

Store at room temperature, in the dark, untilneeded.
2. To prepare the 0.5 mM EDTA/D-PBS(-) working solution, use the concentrated
solution in step #1 and make a 1,000-fold dilution by adding D-PBS(-).

Store it at4°C until use. This solution is good for two weeks, any remaining solution after twoweeks should be safely disposed.