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C u l t u r eDate of Issue: May 2019

FCeM® 3D CULTURE STARTER KITPROTOCOL
ver4.3For research use only. Not for use in clinical and diagnostic procedures.

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C u l t u r eTable of Contents
I. Description ............................................................................................................... 2
II. Materials ................................................................................................................. 3
III. Equipment ............................................................................................................. 3
IV. 3D culture medium preparation ................................................................................ 3
V. Procedure ................................................................................................................ 4
0: Protocol outline ..................................................................................................... 4
1: Start of suspension sphere culture from conventional adherent culture ..................... 42: Adding medium at day 1 and 4 .............................................................................. 5
3: Collecting and passaging at day 7 .......................................................................... 6<3-1: Preparation of collecting medium and cell suspension> ...................................... 6<3-2: Trapping and collecting cell spheres> ................................................................ 7<3-3: Splitting cell spheres> ...................................................................................... 8
VI. Appendix ................................................................................................................ 9
Troubleshooting ........................................................................................................ 9<About Starter Kit> .................................................................................................. 9<About 3D cell culture> .......................................................................................... 10For research use only. Not for use in clinical and diagnostic procedures.

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C u l t u r eI. Description
This product is a cell culture kit for ES/iPS cell with FCeM® 3D culture medium. This single use kit makes
it possible to collect and passage cell spheres with simple operation. This product is suitable for daily
ES/iPS cell culture and mass production study.
【Caution】
This product (“Product”) is designed for research and development use only – Do not use it for otherpurposes.

Wear appropriate protective eyewear, clothing, and gloves when handling the Product.

Nissan Chemical Corporation shall not be liable for any damages as the result of (i) misuse, fault ornegligence of or by users or purchasers of the Product, (ii) use of the Product in a manner for which it
was not designed, or (iii) improper storage and handling of the Product.

This protocol is designed for 253G1 human induced pluripotent stem (hiPS) cells grown in mTeSR1
medium with Vitronectin (Thermo, #A14700) coated dish in 2D culture and suspension culture (3D cellculture) of hiPS cells in mTeSR1 based 3D culture medium. If using different cell lines or growth media,
the culture schedule and protocol below may need to be modified.

【Components】

1

Sphere collecting module

1 piece, sterile

2

Sphere splitting module

1 piece, sterile

3

Syringe cap

1 piece, sterile

4

Filling nozzle

1 piece, sterile5

50 mL syringe

2 pieces, sterile

For research use only. Not for use in clinical and diagnostic procedures.

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C u l t u r eII. Materials
・FCeM®-series Preparation Kit for ES/iPS cells (Nissan Chemical, #382-07991 or #385-07981)
・mTeSR1 (STEMCELL Technologies, #ST-85850)
・D-MEM/F-12
・D-PBS (-)
・Dissociation Solution for human ES/iPS cells (Reprocell, #RCHETP002) orCTK solution (0.05 % collagenase IV (Gibco), 0.25 % trypsin (Gibco), and 20 % KSR) or Dispase・10 mM Y-27632 in PBS
・TrypLE™ Select Enzyme (1X), no phenol red (Thermo, #12563011) or
0.25w/v% Trypsin-1mmol/L EDTA･4Na Solution with Phenol Red (FUJIFILM Wako Pure ChemicalCorporation, #209-16941)

III. Equipment
・FCeM® 3D Culture Starter Kit
・50 mL Conical Centrifuge Tube
・15 mL Conical Centrifuge Tube
・1.5 mL Micro tube
・Pipettes and pipette tips
・Serological pipettes
・Incubator（37℃, 5% CO2 condition）
・Centrifuge
・Inverted microscope
・25 - 37℃ water bath・4℃ refrigerator
・NucleoCounter (chemometec, #NC-200) or Cell counter (BioRad, #TC-20) or Hemocytometer・Stopwatch

IV. 3D culture medium preparation
The procedure to mix FP001 Solution with mTeSR1 can be found in the “Instructions for Use” document
included in the FCeM® Preparation Kit for Stem Cells (Nissan Chemical, #382-07991).
The recommended FP001 final concentration is 0.01 - 0.016 wt %.

For research use only. Not for use in clinical and diagnostic procedures.

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C u l t u r eV. Procedure
0: Protocol outline

1: Start of suspension sphere culture from conventional adherent culture*The regent volume of following instructions is for cells from one well of a 6-well plate.
1.

Warm mTeSR1 medium and 3D culture medium to 25 - 30℃ before use.

2.

Remove the existing media from the cells and wash the cells with 2 mL D-PBS (-).

3.

Add 1 mL Dissociation solution (e.g. Dissociation Solution for human ES/iPS cells or CTK solution orDispase).

4.

Incubate at 37℃ in an incubator for 3 - 5 minutes until the cells begin to slough off the well.

5.

Remove the Dissociation solution and add 1 mL mTeSR1 medium.

6.

Gently pipette over the well until the cells become detached and transfer the cell suspension to a
new 15 mL centrifuge tube.
Note: Not breaking up cell colonies into single cells. Appropriate crump size is from 50 to 100 μm.

7.

Add 2 mL mTeSR1 medium to the well and transfer the remaining cells to the 15 mL centrifugetube.

8.

Count cells (operation of Cell counting).

For research use only. Not for use in clinical and diagnostic procedures.

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C u l t u r e

Cell counting
A1. Transfer 500 μL cell suspension into 1.5 mL Micro tube.
A2. Centrifuge at 100 x g for 3 minutes and remove supernatant.
A3. Add 250 L Trypsin solution and suspend the cell pellet.
A4. Incubate for 2 minutes in water bath at 37℃.
A5. Re-suspend the cell pellet firmly at 10 times.
A6. Incubate for 2 minutes in water bath at 37 ℃ again.
A7. Re-suspend the cell pellet firmly at 10 times to breaking up cell colonies into single cells.
A8. Add 250 μL mTeSR1 and re-suspend the cells to neutralize trypsin.
A9. Count the cells using Cell counter.
9.

Transfer 2 – 3 x 106 cells to a new 50 mL centrifuge tube.

10. Centrifuge at 100 x g for 3 minutes and remove supernatant.
11. Tapping the 50 mL centrifuge tube to disperse cell pellet and re-suspend the cell pellet with 10 mL
3D culture medium containing 10 μM Y-27632.
12. After closing the centrifuge tube cap, open the cap slightly to keep air flow.
13. Put the centrifuge tube into incubator (37℃, 5% CO2).
Note: The centrifuge tube cap must not be fully closed.

2: Adding medium at day 1 and 4
1. Warm 20 mL 3D culture medium to 25 - 30℃ before use.
2. Adding the medium to cell suspension in 50 mL centrifuge tube.
3. Gently pipette the cell suspension to make it uniform.
4. After closing the centrifuge tube cap, open the cap slightly to keep air flow.
5. Put the centrifuge tube into incubator (37℃, 5% CO2).
Note: The centrifuge tube cap must not be fully closed.

For research use only. Not for use in clinical and diagnostic procedures.

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3: Collecting and passaging at day 7<3-1: Preparation of collecting medium and cell suspension>
1. Warm mTeSR1 medium, 3D culture medium and D-MEM/F-12 to 25 - 30℃ before use.
2. Dispense 50 mL 3D culture medium to a new 50 mL centrifuge tube.

3. Unseal two syringes and put one syringe “syringe A” on centrifuge tube rack to keep not touched
the tip around. Putting filling nozzle to another syringe “syringe B” tip.
4. Suck up the 3D culture medium (prepared step: 2) into the syringe B with filling nozzle. Direct thesyringe tip up and push air out of the syringe completely.
5. Remove the filling nozzle from the syringe B and put the nozzle to syringe A tip. Put the syringe B oncentrifuge tube rack to keep not touched the tip around.
6. Put syringe cap to syringe B tip and put the syringe B on centrifuge tube rack.

7. Slowly suck up cell suspension with the syringe A.
8. Remove the filling nozzle from syringe A.
For research use only. Not for use in clinical and diagnostic procedures.

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C u l t u r e<3-2: Trapping and collecting cell spheres>

9. Connect the syringe A to the sphere collecting module firmly.
Note: Sphere collection module is symmetrical structure.
Direct the sphere collecting module up and slowly transfer cell suspension to the module to fill themodule with cell suspension.
10. Transfer cell suspension to sphere collecting module slowly; 10 mL cell suspension is transferred atover 10 seconds. (e.g. if you collect 50 mL cell suspension, it takes about 1 minute to transfer it tosphere collecting module.) Receive waste with 50 mL centrifuge tube.

11. Connect the syringe A to the sphere collecting module with air released.
Transfer 3D culture medium to sphere collecting module slowly; 10 mL 3D culture medium istransferred at over 10 seconds, and massage sphere collecting module with fingers softly at 10times every 10 mL transferring.
12. After all 3D culture medium transferred, pull the syringe B plunger to collect residual liquid in thesphere collecting module.
13. Remove the sphere collecting module from syringe B.
For research use only. Not for use in clinical and diagnostic procedures.

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C u l t u r e<3-3: Splitting cell spheres>

14. Put sphere splitting module to syringe B tip filled with cell suspension firmly.
15. Prepare a new 50 mL centrifuge tube and place tip of sphere splitting module to inner side of the 50
mL centrifuge tube lightly.
Transfer cell suspension 10 mL at about 20 seconds with pulling plunger 1 mL every 10 mL
transferring.
16. Count cells (operation of Cell counting).
Cell counting
A1. After gently pipetting the cell suspension, transfer 3 mL cell suspension
to a new 15 mL centrifuge tube.
A2. Add 12 mL D-MEM/F-12 to the 15 mL centrifuge tube and pipette at 10 times.
A3. Centrifuge at 300 x g for 3 minutes and remove supernatant carefully.
A4. Add 250 μL Trypsin solution and suspend the cell pellet.
A5. Incubate for 2 minutes in water bath at 37℃.
A6. Re-suspend the cell pellet firmly at 10 times.
A7. Incubate for 2 minutes in water bath at 37℃ again.
A8. Re-suspend the cell pellet firmly at 10 times to breaking up cell colonies into single cells.
Add 250 μL mTeSR1 and re-suspend the cells to neutralize trypsin.
A9. Count the cells using Cell counter.
17. Adjust seeding density to 2 - 3 x 105 cells/mL by adding 3D culture medium and dispense the cellsuspension to a new 50 mL centrifuge tube.
18. Add Y-27632 to the cell suspension at 10 μM final concentration and gently pipette the cellsuspension to make it uniform.
19. After closing the centrifuge tube cap, open the cap slightly to keep air flow.
20. Put the centrifuge tube into incubator (37℃, 5% CO2).
Note: The centrifuge tube cap must not be fully closed.
For research use only. Not for use in clinical and diagnostic procedures.

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C u l t u r e

VI. Appendix
Troubleshooting<About Starter Kit>
ProblemPossible reason

Solution

Cell recovery rate with sphere

Cell spheres are too small.

Culture cell spheres until thecollecting module is not good.

*Sphere collecting module

diameter size become about 200

separates cell spheres and 3Dμm.

culture medium by sphere size.
Time to transfer cell spheres toTransfer cell suspension tosphere collecting module is too

sphere collecting module more

short.

slowly; 10 mL cell suspension istransferred at over 20 seconds.

Cell spheres remain in sphere

Transfer 3D culture medium tocollecting module.

sphere collecting module more
slowly; 10 mL cell suspension istransferred at over 20 seconds.
Massage sphere collecting
module with fingers softly at
more 10 times every 10 mL
transferring.
Transfer fluids to spherecollecting module without
containing air.

Splitting efficiency with sphere

Splitting velocity is too high.

splitting module is not good.

Transfer cell suspension 10 mL
at over 20 seconds.

Cells are stuck in sphere splitting

Transfer cell suspension withmodule.

pulling plunger 1 mL every 5 mLtransferring.

Cell density is too high.

Dilute cell suspension at under3 x 105 cells/mL.

For research use only. Not for use in clinical and diagnostic procedures.

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C u l t u r e<About 3D cell culture>
ProblemSolution

After 3D culture, cell spheres

Dilute cell suspension over 5 times by D-MEM/F-12.

don’t precipitate byRe-suspend cell suspension more 10 times after dilution.

centrifugation.
Cell spheres gathered up side in

Gently mix the tube by pipetting.

the centrifuge tube.

Warm 3D culture medium at 37℃ before use.

Cells do not growth in 3D

The cells may not be used to the new 3D environment.

condition.

Please contact us.

If you have any questions or encounter any problems or need help with any aspect related to these
instructions, please contact us.

For research use only. Not for use in clinical and diagnostic procedures.

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ADVANCED MATERIALS & PLANNING DEPERTMENT
2-5-1 NIHONBASHI, CHUO-KU, TOKYO 103-6119, JAPANTEL: +81-3-4463-8370

FAX:+81-3-4463-8371

E-mail: fcem@nissanchem.co.jp

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