i90~210¹ Sizej
Code No. 197­17571 (100 mL)
193­17573 (100 mL~4)
StemSureö hPSC Medium ¢
StemSureö hPSC Medium ¢ is appropriate for maintaining
human pluripotent stem cells (hPSCs), human ES cells and
human iPS cells, in a feeder­free, serum­free and animal­free
environment. This medium is low in protein, and does not contain
albumin.
This product is for laboratory research use only.
Procedures :
ƒPreparation of complete StemSureö hPSC Medium„
1. Thaw StemSureö hPSC Medium ¢ at 2­10 ú for several
hours or overnight.
NOTE : Do not thaw the frozen medium at 37 ú. After
thawing, store this medium at 2­10 ú in the dark and use
within a week. Avoid repeating freeze­thaw.
2. Add 35­100 ng/mL basic fibroblast growth factor (bFGF)
(Code No. 064­05381, 068­05384) (final concentration) to the
thawed StemSureö hPSC Medium ¢ , and gently mix well to
prepare the complete StemSureö hPSC Medium (sshPSC
medium).
NOTE : The concentration of 35 ng/mL is recommended
as a final concentration of bFGF. When you transfer
hPSCs cultured in other feeder­free medium to the
sshPSC medium, the same concentration of bFGF used
might be suitable for the sshPSC medium also.
3. Before use, warm the sshPSC medium to room temperature.
NOTE : Do not warm the sshPSC medium in a water
bath.
ƒTransferring of hPSCs from the culture on feeder
layers to the feeder­free culture„
NOTE : When hPSCs cannot be directly transferred from
feeder­dependent culture with mouse embryonic fibroblasts
(MEFs) to feeder­free culture which uses the sshPSC
medium, a preculture in MEF­conditioned medium [Xu,
C., et al.: Nat. Biotechnol., 19, 971, (2001)], prior to using
the sshPSC medium might be effective to transfer
smoothly.
Preparation of Matrigelö­coated plates
1. Thaw Matrigelö at 4 ú. To avoid gelation, do not store at
room temperature.
2. Dilute Matrigelö in cold DMEM/Ham's F­12 and gently mix
well by pipetting (300 ÊL of Matrigelö in 25 mL of
D­MEM/Ham's F­12).
3. Coat 6 well­plate with 1.4 mL of the diluted Matrigelö per
well (9.5 cm2).
4. Incubate at room temperature for at least 1 hour before use.
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Passaging
1. Aspirate the culture medium of a feeder­dependent cultured
well.
2. Add 0.4 mL of CTK solution [Suemori, H., et al.: Biochem.
Biophys. Res. Commun ., 345, 926, (2006). ] per well of 6­well
plate.
3. Incubate at 37 ú with 5% CO2 for 5 minutes.
4. Aspirate the CTK solution, and add 2 mL per well of the
sshPSC medium and suspend the colonies in small clumps of
about 100 cells by pipetting with a P1000 micropipette.
5. Transfer the cell suspension to a 15mL tube, and add 6 mL of
the sshPSC medium and gently mix by inverting the tube
several times.
6. Spin at 1,000 rpm (about 170 ~ g) at room temperature for 3
minutes.
7. Remove the supernatant, and add 2 mL per well of ROCKi {
medium¦ and resuspend the pellet.
NOTE : Do not break up the clumps to a smaller number
of cells, especially single cells.
¦ ROCKi { medium : sshPSC medium with 10 Êmol/L
Y­27632, which is an inhibitor of p160­Rho­associated
coiled kinase (ROCK).
8. Aspirate the coating solution from the Matrigelö­coated
wells.
9. Transfer 2 mL of the cell suspension to the well.
NOTE : Do not plate the cells at low­density. This will
lead to the decrease in the viability of the cells and the
increase in the differentiation of the plated cells.
NOTE : After the transfer to the feeder­free condition,
MEFs will remain present for the first 1­3 passages. To
eliminate MEFs from the culture, the cell suspension is
plated to a non­coated or a gelatin­coated well, and incu­
bate at 37 ú with 5% CO2 for about 1 hour. Residual
MEFs, not hPSC clumps, adhere to the well. The suspen­
sion containing hPSC clumps is harvested from the well,
and then plated to a Matrigelö coated­well.
10. Incubate at 37 ú with 5% CO2.
11. Replace the medium daily with 2 mL of sshPSC medium per
well.
12. Passage the cells at 5 to 7 days.
ƒPassaging in sshPSC medium„
1. Aspirate the culture medium, and rinse the cells once with
D­PBS(„).
2. Remove D­PBS(„), and add 1 mL of StemProö Accutase,
TrypLETM Select or TrypLETM Express per well.
NOTE : Do not use trypsin, Dispaseö and CTK solution.
3. Incubate at 37 ú with 5% CO2 for 5 minutes.
4. Add 2 mL of the ROCKi { medium, and suspend the colonies
to single cells by using a P1000 micropipette.
5. Transfer the cell suspension to a 15 mL tube.
6. Spin at 1,000 rpm (about 170 ~ g) at room temperature for 3
minutes.
7. Aspirate the supernatant and suspend the pellet with 2 mL of
the ROCKi { medium.
8. Count the viable cell numbers.
9. Aspirate the coating solution from the Matrigelö­coated
wells (see Preparation of Matrigelö­coated plates).
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10. Add 2 mL of the ROCKi { medium per well, and plate 1 ~ 105
cells per well.
NOTE : Do not plate the cells at low­density. This will
lead to the decrease in the viability of the cells and the
increase in the differentiation of the plated cells.
NOTE : In early passages, when the cells do not adapt
to the feeder­free culture, 2­3 ~ 105 cells per well should
be plated. The optimal split ratio is different in hPSC
clones. The optimal plating cell number should be
adjusted carefully for reproducible culture. In general,
the cells should reach 80­90% confluent of the well at 4 or
5 days after plating.
11. Incubate at 37 ú with 5% CO2.
12. The next day (at day 1), replace the medium with 2 mL of the
sshPSC medium per well, not the ROCKi { Medium.
13. Replace the medium daily with 2 mL of the sshPSC medium
per well. The replacement of medium is unnecessary on day 2.
14. Passage the cells when cells grow to cover approximately
80­90% of the well, usually every 4­6 days.
Storage : Store at | 20 ú in the dark.
Avoid repeating freeze­thaw. After thawing, store at
2­10 ú in the dark and use within a week.
Package :
Code No.
Packaging
197­17571
100 mL
193­17573
100 mL ~ 4
Related Reagents
ED­MEM/Ham's F­12 with L­Glutamine, Phenol Red,
HEPES and Sodium Pyruvate (Code No.042­30555)
EFibroblast Growth Factor (basic), Human, recombinant,
Animal­derived­free (Code No. 064­05381, 068­05384)
EY­27632 (Code No. 257­00511, 253­00513, 251­00514)
ED­PBS(„) (Code No. 045­29795)
EMatrigelö hESC­Qualified Matrix (Code No. 643­55461,
Corning No. 354277)
E6 Well Clear TC­Treated Multiple Well Plates (Code No.
649­01111, Corning No. 3516)
R[h No. 197­17571 (100 mL)
193­17573 (100 mL~4)
StemSureö hPSC|n¢
StemSureö hPSC |n¢ÍA³tB[_[|{ðºÅqg
½\«²×Eiqg iPS ×E¨æÑqg ES ×EjÌ۝|{É
gpū鳌´A®¨Rˆ¬ªsÜÌtÌ|nÅ·B{iÍA
á^“pN¿|nÅ èAA‹u~“ðÜñŢܹñB
{iͤ†pŽòÅ·B
[€
ì]
ƒ®S|ǹõ„
1. {ið 2 ` 10 úŔžÔ©çêÓ©¯ÄäÁ­èZð·éB
F 37 úÅZðµÈ¢Å­¾³¢BZðãA 2 ` 10 úÌ
ÊÅÛ¶µA 1 TÔÈàɲgp­¾³¢B€‹Zð
ÍJèԳȢŭ¾³¢B
2. ZðãÌ{iɖî«üÛè×E¬·öqi bFGF ji Code
No. 064­05381, 068­05384 jðIZx 35 ` 100 ng/mL ÆÈé
æ¤YÁµäÁ­èÆæ­¬ºA®S|nð²»·éiÈ
ºA sshPSC |nÆ¢¤jB
F bFGF ÌZxÍA 35 ng/mL ÅYÁ·é±Æð„§µ
Ü·ªAùɳtB[_[|{É黵½×Eð{iÅ
|{·éê‡ÍA²gp|nƯ¶ZxÌ bFGF ðYÁ·
éûªAKµÄ¢éꇪ èÜ·B
3. sshPSC |nðgpOɺ·Éß·B
F·…ÍgpµÈ¢Å­¾³¢B
ƒI“tB[_[|{©ç³tB[_[|{ÖÌÚs„
F }EXٙüÛè×Ei MEF jðp¢½I“tB[_[
|{†Ì×EðA sshPSC |nÖ¼ÚÚsÅ«È¢ê‡ÍA
MEF é»|n [ Xu, C., et al. : Nat. Biotechnol, 19(10), 971,
(2001). ] Å|{ãA sshPSC |nÉÚsµÄ­¾³¢B
Matrigelö R[gvŒ[g̀õ
Wako Pure Chemical Industries, Ltd.
1­2, Doshomachi 3­Chome, Chuo­Ku, Osaka 540­8605, Japan
Telephone : {81­6­6203­3741
Facsimile : {81­6­6201­5964
http://www.wako­chem.co.jp
Wako Chemicals USA, Inc.
Wako Chemicals GmbH
1600 Bellwood Road
Richmond, VA 23237
U.S.A.
Telephone : {1­804­271­7677
Facsimile : {1­804­271­7791
http://www.wakousa.com
Fuggerstrasse 12
D­41468 Neuss
Germany
Telephone : {49­2131­311­0
Facsimile : {49­2131­311100
http://www.wako­chemicals.de
| 3/6 |
1. Matrigelö ð 4 úÅZð·éBQ‹»ðð¯é½ßº·ÅZ
ðµÈ¢Å­¾³¢B
2. Matrigelö ðâpµ½ D­MEM/Ham's F­12 ÅóßµAsyb
eB“OÅæ­¬ºéi Matrigelö 300 ÊL ð D­MEM/Ham's
F­12 25 mL Åóß·éjB
3. óßµ½ Matrigelö ntð 6 EF‹vŒ[gÉ 1.4 mL/well
i 9.5 cm2 jYÁ·éB
4. º·Å 1 žÔÈãC“L…x[g·éB
p
ã
1. I“tB[_[|{†ÌEF‹©ç|n𜎷éB
2. CTK nt [ Suemori, H., et al . : Biochem. Biophys. Res.
Commun ., 345, 926, (2006). ] ð 6 EF‹vŒ[gÉ 0.4
mL/well ÅYÁ·éB
| 4/6 |
3. 37 úA 5% CO2 C“L…x[^[Å 5 ªÔÃu·éB
4. CTK nt𜎷éB sshPSC |n 2 mL ðÁ¦A P1000 }
CNsybgÅN‰“vª 100 ÂöxÌ×EÉÈéæ¤É
sybeB“O·éB
5. ×Eœ÷tð 15 mL `…[uÉÚ·B sshPSC |n 6 mL ð
Á¦A”ñ]|¬a·éB
6. 1,000 rpm iñ 170 ~ g jÅ 3 ªÔAº·Å“S·éB
7. ã´ðœŽµA ROCKi {|n¦ ð 2 mL/well ÅYÁµAÄ
œ÷·éB
FN‰“vðªUµ·¬È¢æ¤ÉÓµÄ­¾³¢B
¦ ROCKi {|nF sshPSC |nÉ Y­27632 i p160­Rho ‹
‡Li[[jQÜE ROCKi jðIZx 10 Êmol/L ÆÈé
æ¤ÉYÁµ½|nB
8. Matrigelö ÅR[gÏÝÌEF‹©çR[eB“Ontð
œŽ·éB
9. ×Eœ÷tð 2 mL/well Ådí·éB
F×E¶…¦ªáºµ½èAª»×EªÁ·é½ßA
áZxÅ×EðdíµÈ¢Å­¾³¢B
F MEF ͳtB[_[|{ÚsãA 1 ` 3 pãÍc¶
µÜ·B MEF 𜎷é½ßÉÍA×Eœ÷tðR[g
µÄ¢È¢EF‹Ü½Í[‰`“R[gµ½EF‹Éd
íµA 37 úA 5 ÷ CO2 C“L…x[^[Åñ 1 žÔÃu
µÄ­¾³¢Bc¶ MEF ÍEF‹Éڅ·é½ßAã´
ðñû·é±ÆÉæèA hPSC N‰“vðÜÞ×Eœ÷t
ðñûÅ«Ü·Bñûµ½×Eœ÷tð Matrigelö ÅR[
gÏÝÌEF‹ÉdíµÄ­¾³¢B
10. 37 úA 5 ÷ CO2 C“L…x[^[ÅÃu·éB
11. sshPSC |nðp¢Ä 2 mL/well ňú|nð··éB
12. 5 ` 7 úÚÅpã·éB
ƒsshPSC|nÉæépã„
1. |n𜎵A×Eð D­PBS ( „ )Åêxôò·éB
2. D­PBS(„) 𜎵AStemProö Accutase ATrypLETM Select
é¢Í TrypLETM Express ð 1 mL/well YÁ·éB
FgŠvV“A Dispaseö A CTK ntÍgpµÈ¢Å­
¾³¢B
3. 37 úA 5 ÷ CO2 C“L…x[^[Å 5 ªÔÃu·éB
4. ROCKi {|n 2 mL/well ðYÁµAP1000 }CNsybg
ðp¢ÄARj[ðV“O‹Z‹ÖªU·éB
5. ×EªUtð 15 mL `…[uÉÚ·B
6. 1,000 rpm iñ 170 ~ g jÅ 3 ªÔAº·Å“S·éB
7. ã´ðœŽµA ROCKi {|n 2 mL Å×EyŒbgðœ÷
·éB
8. ¶×E”ðvª·éB
9. Matrigelö ÅR[gÏÝÌEF‹©çR[eB“Ontðœ
Ž·éi Matrigelö R[gvŒ[g̀õðQÆjB
10. ROCKi {|nð 2 mL/well ÅYÁµA 1 ~ 105 cells/well Æ
Èéæ¤É×Eðdí·éB
F×E¶…¦ªáºµ½èAª»×EªÁ·é½ßA
áZxÅ×EðdíµÈ¢Å­¾³¢B
F³tB[_[pã|{‰úÌ×EÍÀèµÄ¢È¢
½ßA×E”ð 2 ` 3 ~ 105 cells/well Ådíµ½ûªDÜ
µ¢ê‡ª èÜ·BÅKÈ×EdíZxÍ×EN[
“ÉæÁÄÙÈèÜ·B×EdíZxÍ|{ 4 ` 5 úÚÅ
80 ` 90 ÷ÌR“t‹G“gÆÈéæ¤É²ßµÄ­¾³
¢B
11. 37 úA 5 ÷ CO2 C“L…x[^[ÅÃu·éB
12. ‚úi|{ 1 úÚjA sshPSC |nðp¢Ä 2 mL/well Å
|nð··éBsshPSC |nÖÌ Y­27632 ÌYÁÍsvÅ·B
13. sshPSC |nðp¢Ä 2 mL/well ňú|nð··éB|n
ð·Í|{ 2 úÚÌÝsvÅ·B
14. 80 ` 90 ÷ÌR“t‹G“góÔÅpã·éi|{ 4 ` 6 ú
Új
B
[Û
Ç]
Ê| 20 úÛÇB
€‹ZðÍJèԳȢŭ¾³¢BZðãÍÊŠ2 ` 10 ú
ÅÛǵA 1 TÔÈàɲgp­¾³¢B
[ï
•]
Code No.
197­17571
ï •
100 mL
193­17573
100 mL ~ 4
[ÖA»i]
ED­MEM/Ham's F­12 i L­ O‹^~“AtFm[‹ŒbhA
HEPES As‹r“_igŠE€ÜLji Code No. 042­
30555)
EüÛè×E¬·öqi–î«jAqgAg·¦ÌA®¨Rˆ
¨tŠ[i bFGF ji Code No. 064­05381, 068­05384 j
EY­27632 i Code No. 257­00511, 253­00513, 251­00514 j
ED­PBS ( „ )i Code No. 045­29795 j
EMatrigelö hESC­Qualified Matrix i Code No. 643­55461,
Corning No. 354277 j
E}‹`v‹EF‹vŒ[g 6 EF‹½êi Code No. 649­
01111, Corning No. 3516 j
STUVW XYZ[\
1405K01
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