Protocol for Feeder-Free culturing of human iPS cells

Protocol for Feeder-Free culturing ofhuman iPS cells

Date Created: August 4, 2021
Date of revision: November 30, 2023

Public Interest Incorporated Foundation

CiRA Foundation
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Protocol for Feeder-Free culturing of human iPS cells

Table of Contents
Thawing of iPS cell frozen stock ........................................................... 3
Medium exchange ................................................................................ 5
Passage of iPS cells (from 6-well plate to 6-well plate) ......................... 6
Cryopreservation of iPS cells (from 6-well plate) .................................. 9Additional information ........................................................................ 122

Protocol for Feeder-Free culturing of human iPS cells

Thawing of iPS cell frozen stock

Materials
Cells:

Frozen vial of human iPS cellsReagents:

iMatrix-511 (Laminin-511 E8) (Nippi, 892012)
10 mM Y-27632 (FUJIFILM Wako Pure ChemicalCorporation, 036-24023)
PBS (Nakalai Tesque, 14249-24)
StemFit AK03N (StemFit medium) (Ajinomoto)
Trypan blue solution (Sigma-Aldrich, T8154)

Plasticware:

6 well plates
15/50 mL conical tubes
Plastic PipettesProcedure1.

Preparation of culture medium and reagents

1)

Leave the StemFit AK03N medium at room temperature for 30 minutes
before starting.

2)

Dispense 2 mL of StemFit into a 50 mL tube; Add 2 µL of Y-27632 solution
and mix well (final concentration 10 µM). →" StemFit+Y medium."2.

Coating of 6-well plates

1)

Prepare the required volume of PBS in 50 mL tubes (1.5 mL x number ofwells).

2)

Prepare the required volume of iMatrix-511 solution (9.2 µL x number ofwells) and add to PBS.

3)

Mix well and add 1.5 mL to each well.

4)

Immediately shake the plate to spread the solution throughout, then incubate
for at least 1 hour at 37°C in 5% CO2 incubator.

5)

Remove the plate being coated from the incubator.

6)

Add 0.75 mL of StemFit medium to each well, blend well throughout, andremove.

7)

Add 1.5 mL of StemFit+Y medium to each coated well. Add 2 mL of PBS toeach empty well.

8)

Place all plates at 37°C in 5% CO2 incubator.

3Protocol for Feeder-Free culturing of human iPS cells

3.

Thawing of frozen cells

1)Preheat a water bath (or a melting apparatus) to 37°C.

2)

Add 5 mL of StemFit medium into a 50 mL conical tube.

3)

Remove the frozen vial of iPS cell from the liquid nitrogen storage container
and thaw it in the water bath for about 1 minute until a few ice particles
remain.

4)

Add the thawed cell suspension in step 3) into StemFit medium in the 50 mL
tube prepared in 2).

5)

Centrifuge the cells at 160 × g for 5 min at 23°C and remove the supernatant.

6)

Add 0.3 mL of StemFit+Y medium and suspend cells by pipetting (6 times).

7)

Take 10 µL of the cell suspension, add 10 µL of trypan-blue solution, mix, andcount the cells.

8)

Seed 65,000 viable cells into each well of a coated 6-well plate. Immediately
shake the plate to spread the cells evenly.

9)

Check cells using a microscope.

10)

Culture the plates at 37°C in 5% CO2 incubator.

4Protocol for Feeder-Free culturing of human iPS cells

Medium exchange*The day after cell thawing or cell passage, medium needs to be changed to Y27632 free StemFit medium.

MaterialsCells:

6-well plate seeded with cells

Reagents:

StemFit AK03N (StemFit medium) (Ajinomoto)Equipment:

Plastic Pipettes

Procedure1)

Leave StemFit medium at room temperature for 30 minutes prior to use.

2)

Check the condition of the cells using a microscope (cell viability, presence ofiPS cell-like colonies) and take photographs if necessary.

3)

Remove medium and gently add 3 mL of StemFit medium per well (allow it torun down the walls of the well).

4)

Incubate at 37°C in 5% CO2 incubator.

*Guideline for medium exchange:
 For passaging on day 6 of culture: Change the medium on day 1 (day 1 aftercell seeding), day 4 and day 5.
 For passaging on day 7 of culture: Change the medium on day 1, day 4 andday 6.

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Protocol for Feeder-Free culturing of human iPS cells

Passage of iPS cells (from 6-well plate to 6-well plate)Materials
Cells:

6-well plate seeded with cells*Passage is carried out when the colonies have started to grow and are
not too large. Usually, we passage cells on day 6 or 7 of culture.
Cell status before passage(Scale bar; 200 µm)

Reagents:

iMatrix-511 (Laminin-511 E8) (Nippi, 892012)
10 mM Y-27632 (FUJIFILM Wako Pure ChemicalCorporation, 036-24023)
PBS (Nakalai Tesque, 14249-24)
StemFit AK03N (StemFit medium) (Ajinomoto)
0.5×TrypLE Select solution
Trypan blue solution (Sigma-Aldrich, T8154)

Plasticware:

6 well plates
15/50 mL conical tubes
Plastic Pipettes
Cell scraper6

Protocol for Feeder-Free culturing of human iPS cells

Procedure1.

Preparation of culture media and reagents

1)

Leave the StemFit AK03N medium at room temperature for 30 minutes
before starting.

2)

Dispense 2 mL of StemFit into a 50 mL tube; Add 2 µL of Y-27632 solutionand mix well (final concentration 10 µM). →" StemFit+Y medium."2.

Coating of 6-well plate1)

Prepare the required volume of PBS in 50 mL tubes (1.5 mL x number ofwells).

2)

Prepare the required volume of iMatrix-511 solution (9.2 µL x number ofwells) and add to PBS.

3)

Mix well and add 1.5 mL to each well.

4)

Immediately shake the plate to spread it throughout, then place it in a 37°C,5% CO2 incubator for at least 1 hour.

5)

Remove the plate being coated from the incubator.

6)

Add 0.75 mL of StemFit to each well, spread throughout, and remove.

7)

Add 1.5 mL of StemFit+Y medium to each coated well. Add 2 mL of PBS toeach empty well.

8)

Place the plate at 37°C in 5% CO2 incubator.

3.

Passage

1)

Check the condition of the cells using a microscope (cell viability, presence ofiPS cell-like colonies) and take photographs if necessary.

2)

Remove medium and add 1 mL of PBS. Wash entire cells by shaking the plate,then remove PBS.

3)

Add 1 mL of 0.5×TrypLE Select solution and shake the plate to spread thesolution throughout.

4)

Place 6-well plate in the incubator and leave for up to 10 minutes.

5)

Examine the cells using a microscope to see whether cell-cell adhesions areloosened for almost all cells and whether individual cells can be recognized.

7Protocol for Feeder-Free culturing of human iPS cells

6)

Remove 0.5 x TrypLE Select solution from each well. Gently add 2 mL of PBS,
wash entire cells by shaking the plate, and then remove the PBS.

7)

After adding 1 mL of new StemFit medium, scrape the cells with a cell
scraper* (If multiple clones or strains are to be passed together, scrape cells
in all wellswith cell scrapers before proceeding).

8)

Gently pipet the cells to break them apart (10 times) and collect them in anew tube.

9)

Take 10 µL of cell suspension, and add 10 µL of Trypan-blue solution, mix
and count the cells.

10)Transfer the required number of cells (13000 viable cells per well) to one wellof a coated 6-well plate. Immediately shake the plate to spread the cells
evenly. For seeding with whole cells, add 1/1000 volume of Y-27632 to thecell suspension.

11)

Check the cells in each well using a microscope. Incubate at 37°C in 5% CO2incubator.

12)

The day after passages, the medium is replaced (see "Medium Replacement,page 5").

Note: In our internal validation, the method of adding PBS instead of StemFit and detaching andcollecting cells by pipetting also showed no difference in cell survival and proliferation rates
and expression of undifferentiated markers (OCT3/4).

8Protocol for Feeder-Free culturing of human iPS cells

Cryopreservation of iPS cells

Materials
Cells:

6-well plate seeded with cells

Cell status before cryopreservation(Scale bar; 200 µm)

Reagents:

StemFit AK03N (StemFit medium) (Ajinomoto)
PBS (Nakalai Tesque, 14249-24)
0.5×TrypLE Select solution
Trypan blue solution (Sigma-Aldrich, T8154)
STEM-CELLBANKER (Zenogen Pharma, CB045/047)

Equipment:

BICELL (Japan Freezer, BICELL)□Plasticware:

15/50 mL conical tubes
Plastic Pipettes
Cell scraper
Cryovials (1.0 mL)

1)ProcedureCheck the condition of the cells using a microscope (cell viability, presence ofiPS cell-like colonies) and take photographs if necessary.

2)

Remove medium and add 1 mL of PBS. Wash all cells by shaking the plate,then remove PBS.

9Protocol for Feeder-Free culturing of human iPS cells3)

Add 1 mL of 0.5×TrypLE Select solution and shake the plate to spread thesolution throughout.

4)

Return 6-well plate to the incubator and leave for up to 10 minutes.

5)

Examine the cells using a microscope to see whether cell-cell adhesions
areloosened for almost all cells and whether individual cells can be recognized.

6)

After removing 0.5 x TrypLE Select solution, gently add 2 mL of PBS andwash the entire cell by shaking the plate. Then remove PBS.

7)

Add 1 mL of new StemFit medium and scrape the cells with a cell scraper* (Ifmultiple clones or strains are to be passed together, scrape cells in all wellswith cell scrapers before proceeding).

8)

Gently pipet the cells to break them apart (10 times) and collect them in anew tube.

9)

Transfer the cell suspension to a 50 mL tube (Combine all well suspensions inone tube).

10)

Mix the cell suspension by pipetting (6 times) and roughly measure totalliquid volume with scales on the pipette surface.

11)

Remove 10 µL of cell suspension, and add 10 µL of Trypan-blue solution, mix
and count the cells.

12)

Calculate the number of cryovials to freeze based on total number of viable
cells (the number of cryovialsx 2.4 x 105 cells).

13)

Calculate the number of viable cells required from the total number ofcryovials (the number of cryovials x 2.4 x 105 cells)

14)

Prepare new conical tubes and aliquot cell suspension with required amountof viable cells.

15)

Perform centrifugation (160 x g, 23°C, 5 min). Prepare the required numberof new cryotubes.

16)

After centrifugation, remove the supernatant.

10Protocol for Feeder-Free culturing of human iPS cells

17)

Resuspend the pellet by gentle tapping, add STEM-CELLBANKER to aconcentration of 1.2 x 106 cells/mL and suspend by pipetting (8 times).
Dispense 200 µL of the cell suspension into each cryovials.

18)

After aliquoting, the cryovials are placed in pre-cooled bicelles and frozen at 80°C.

19)Transfer to liquid nitrogen tank within a few days for storage.

Note: In our internal validation, the method of adding PBS instead of StemFit and detaching andcollecting cells by pipetting also showed no difference in cell survival and proliferation rates andexpression of undifferentiated markers (OCT3/4).

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Protocol for Feeder-Free culturing of human iPS cells

Additional information
Preparation of StemFit(AK03N) medium
Reagents:StemFit AK03N A solution 400 mL (stored at 4°C)
StemFit AK03N B solution 100 mL (stored at -30°C)
StemFit AK03N C solution 2 mL (stored at -30°C)

Dissolve liquids B and C at 4°C or room temperature (at least 8 hours, o/n).
Plasticware:

Disposable pipettes

Procedure1)

Mix liquid B by pipetting and add to a bottle of liquid A.

2)

Add 2 mL of liquid C to the bottle of liquid A.

3)

Close the lid of liquid A tightly and mix well.

4)

Dispense the mixture into 50 mL tubes or bottles.

5)Store at 4°C or -80°C.

Preparation of 0.5×TrypLE Select solution (0.5 mM EDTA/PBS final
concentration 0.75 mM)
Reagents:

Cell Therapy Systems TrypLE Select CTS (Thermo FisherScientific, A12859)
0.5 mmol/L-EDTA/PBS solution (Nacalai Tesque, 13567-84)

Plasticware:

Disposable pipettes□

Equipment:

250 mL filter system (0.2 µm filter)

Procedure1)

Dispense 90 mL of TrypLE Select into a 250 mL storage bottle.

2)

Add 90 mL of 0.5 mM EDTA/PBS solution to the storage bottle in 1) and mix bypipetting (about 5 times).

3)

Close the bottle lid and mix well.

*The expiration date should be 6 months at room temperature (if the expiration dateof the original reagent is less than 6 months, follow the expiration date of theoriginal reagent).

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Protocol for Feeder-Free culturing of human iPS cells

Reagent amount and seeded cell number according to culture scale.
Table 1. Amount of laminin (iMatrix-511MG; 0.5 mg/mL) and medium used in thecoatingScale

Cultivation

laminin quantityarea(iMatrix-511)

PBS volume

6-well plate*.

9.2 cm2

9.2 µL

1.5 mL90 mm Dish257 µL

9.3 mL

57 cm

Amount of StemFit+Ymedium
1.5 mL
9 mL*per well
Table 2: Reagent amounts during cell passaging.

Scale

0.5× TrypLE SelectamountAmount of PBS added(After TrypLEtreatment)

StemFit Addition Amount(During cell detachment)

6-well plate*.

1 mL

2 mL

1 mL

90 mm Dish

3 mL

10 mL

5 mL

*per well
Table 3. number of cells seeded during cell passages.
Scale

Number of cells seeded

6-well plate*.

1.3 x 104 cells

90 mm Dish

8×104 cells*per well

Created by:

Kyoto University iPS Cell Research Foundation
Cell Preparation Facility FiT

13Protocol for Feeder-Free culturing of human iPS cells

References:1.

A novel efficient feeder-free culture system for the derivation of human induced pluripotent
stem cells
M. Nakagawa et al, Scientific Reports, 4:3594 (2014) DOI: 10.1038/srep03594

2.

An Efficient Non-viral Method to Generate Integration-Free Human iPS Cells from Cord
Blood and Peripheral Blood Cells
K. Okita et al, Stem Cells, 31(3):458-66 (2013) DOI: 10.1002/stem.1293

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