Proliferation Control Medium for Basic Cultureof Human Pluripotent Stem Cells
Xyltech BOF-01
User's Guide
Ver. 4.4

Oct, 2017
BOURBON Biomedical Advanced Research Laboratories, Inc.

Protocol: Human induced pluripotent stem (iPS) cell culture proliferation
control using Xyltech BOF-01

1. Product features
Xyltech BOF-01 is a maintenance medium for human pluripotent stem cells (hPSCs)
grown on a feeder layer of cells.
Xyltech BOF-01 (BOF-01) suppresses the proliferation of hPSCs, maintaining them in an
undifferentiated state. BOF-01 can be used by completely replacing the basal culturemedium (e.g. DMEM/F12) of the hPSCs. The cells can be maintained with this medium
for about 3 days (up to 72 hours, without changing the medium) under normal culture
conditions (37°C, 5% CO2).
After suppression of cell proliferation with BOF-01 medium, cell growth can be resumed
by changing back to normal growth medium.
This product does not contain glucose.
2. Precautions
This product, BOF-01, is a basal medium for hPSCs grown on feeder cells. It cannot beused under feeder free culture conditions.
This product is for research purposes only, and is not intended for use in the treatment ordiagnosis of humans or animals.
3. Preparation method
Replace the basal cell culture medium with BOF-01. An example of the composition ofnormal human ES/iPS medium and BOF-01 medium is described in section 4-1.
References are listed in section 7.
4. Method for cell culture: Example of human ES/iPS medium and BOF-01 medium
4-1. Cells and Reagents・Human iPS cells (grown on feeder cells, 60mm culture dish)・Feeder Cells: Mitomycin C (MMC) treated mouse embryonic fibroblast (MEF) (60mmculture dish)・Xyltech BOF-01
・DMEM/F12
・KnockOut Serum Replacement (KSR)
・Non-Essential Amino Acid Solution (NEAA)
・L-Glutamine
・2-Mercaptoethanol
・Penicillin/Streptomycin
・DPBS without calcium and magnesium (DPBS--)・Dissociation solution for human ES/iPS Cells (Collagenase Type IV, Trypsin, KSR: CTKsolution)

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Normal Growth Medium(Human ES/iPS Medium)
DMEM/F1220% KSR
0.1 mM NEAA
2 mM L-Glutamine
0.1 mM 2-Mercaptoethanol
50 units/ml Penicillin
50 mg/ml Streptomycin
4 ng/ml bFGF

BOF-01 Medium
BOF-01
20% KSR0.1 mM NEAA
2 mM L-Glutamine
0.1 mM 2-Mercaptoethanol
50 units/ml Penicillin
50 mg/ml Streptomycin
4 ng/ml bFGF

4-2. Proliferation control of human iPS cells with BOF-01
1. Prepare Xlytech BOF-01 medium, (see composition in example above) andprewarm in a 37°C water bath.
2. After 1 to 3 days passage, select human iPS cells which are relatively small incolony size (up to 50 cells per colony), remove the culture medium and replacewith 5 ml of BOF-01 medium.*
3. Maintain the cells in a 5% CO2 incubator for up to 3 days. There is no need tochange the BOF-01 medium over this time period.
4. After up to 3 days of proliferation-controlled culture with BOF-01 medium, replace
the medium with human ES/iPS normal growth medium and continue to culture
the cells overnight.
5. The next day, check the density and morphology of cell colonies with a phase
contrast microscope before proceeding with cell culture and experiments.
* We recommend using relatively small sized colonies to maintain the culture forup to 3 days. If the cell density is high (over 60-70% confluency), it isrecommended to replace BOF-01 medium with fresh BOF-01 medium earlier
than 3 days to avoid the potential increase of floating cells.
4-3. Passaging and proliferation control of human iPS cells (weekend-free culturemethod)
1. Warm BOF-01 medium, DPBS-- and dissociation solution, in a 37°C water bath.
2. Remove culture medium from confluent human iPS cells.
3. Add 2 ml of DPBS-- to wash the cells, then remove the DPBS--.
4. Add 0.5 ml of dissociation solution and incubate at 37°C for 2-5 minutes.
5. Remove the dissociation solution, add 2 ml of DPBS-- to wash the cells thenremove the DPBS--.
6. Add 2 to 4 ml of BOF-01 medium to the cells, pipette slowly, several times, thendislodge the iPS cell colonies from the feeder layer, and make clumps of 20-30cells.
7. Remove the culture supernatant from a 60mm culture dish containing a newfeeder layer of MMC treated MEF cells and add the clumps of iPS made in step 6
by diluting them in BOF-01 medium (for example make a 5 fold dilution by adding
1 ml of cell clump suspension to 4 ml of BOF-01 medium) and seeding them overthe feeder cells. Gently shake the dish to seed the cell clumps uniformly.
8. Maintain the cells without changing the BOF-1 medium for (up to) 3 days in a37°C, 5% CO2 incubator.
39. After 3 days, remove BOF-01 medium from the human iPS cells, then add humanES/iPS medium.
10. On the following day, observe the cells with a phase contrast microscope, markcolonies that tend to differentiate, and remove by suction. After changing back tohuman ES/iPS medium, change medium every day.
Cells are typicallysubcultured between a 1:3 (3 fold dilution) and 1:6 (6 fold dilution) ratio, and every
4 to 5 days under normal growth conditions.

Below is an example of the schedule for the weekend-free culture method.

Cell passaging with
BOF-01 medium

Cell passaging

Week 1MONTUE

Back tonormal growth medium

Week 2MONWED

THU

FRI

Cell maintenance withproliferation suppression(No medium change)

SATSUN

Maintain cell culture, experiments, repeat cell passaging schedule with
BOF-01 medium on FridayTUE

WEDTHUFRISATSUN*Cultured human iPS cells with normal growth medium (human ES/iPS medium)
are passaged on Friday afternoon using BOF-01 medium. Cells can be incontinuous culture without replacing the BOF-01 medium during the weekend, thenchanging back to the normal growth medium on Monday morning.
* The timing of passage needs to be adjusted to the cell’s condition and growth rate.
5. Additional information
Human iPS cells may change the morphology of colonies while cultured in BOF-01
medium. The original morphology can be resumed after changing back to normal
growth medium (Phase images show example of an 201B7 cells).

Before changing toBOF-01 medium

Cells maintained withBOF-01 medium

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24 hours after changing backto human ES/iPS medium6. References
1) Curr Protoc Stem Cell Biol. 2009 Jun; Chapter 4: Unit 4A.2.
2) Protocol of Kyoto University iPS Laboratory (CiRA); Revision Ver.2, March 5,
2009“Generation of Human Induced Pluripotent Stem Cells”
https://www.cira.kyoto-u.ac.jp/e/research/img/protocol/hipsprotocolv2_090304.pdf
7. Product inquiries
BOURBON Biomedical Advanced Research Laboratories, Inc
1 Ekimae, Kashiwazaki Niigata, 945-8611, Japan
TEL: 0257-23-2769 E-mail: support@bourbon-barl.co.jp* The protocols shown here are based on our experimental experience. Adjustments tothe seeding density, and passage timing may be required according to the cell line of yourusage. This product (Xyltech BOF–01) is intended for research purposes only, and is not
permitted for use in the treatment or diagnosis of humans or animals.

Copyright© 2017 BOURBON Biomedical Advanced Research Laboratories, Inc., All rights reserved.

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