The human embryonic stem cell line, KhES-1, was originally established by Kyoto University and is routinely maintained, authenticated and tested for contamination in our laboratory (Suemori et al., 2006 (link)). All cells used in this research are below passage number 50. KhES-1 was cultured under feeder-free conditions using StemFit AK02N (RCAK02N, TAKARA BIO) with daily medium change. Cells were routinely passaged every week. On passage day, cells were washed in PBS twice and digested using 50% CTS TrypLE Select Enzyme (Thermo Fisher Scientific, A1285901) at 37°C for 5 min. CTS TrypLE Select Enzyme was then replaced with 2 ml PBS. Cells were collected by gently pipetting and centrifuging at 600 g for 5 min. ROCK Inhibitor Y-27632 (72302, STEMCELL Technologies) (10 µM) was added during passage to improve survival and removed the next day. Laminin 511 E8 fragment (T304, TAKARA BIO) (2.5 µg/ml) was added into the medium during passage as the cell culture matrix. The use of the KhES-1 cell line is approved and performed in accordance with the human embryonic stem cell (ESC) guidelines of the Japanese government.
Stemfit ak02n
Manufactured by Takara Bio
Sourced in Japan
StemFit AK02N is a cell culture medium designed for the maintenance and expansion of human induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs). It provides a defined, serum-free, and xeno-free formulation to support the growth and self-renewal of these cell types.
Automatically generated - may contain errors
Lab products found in correlation
Cts tryple select enzyme, by Thermo Fisher Scientific (1 mentions)
Rock inhibitor y 27632, by STEMCELL (1 mentions)
Imatrix 511, by Takara Bio (1 mentions)
Hygromycin b, by Fujifilm (1 mentions)
Laminin 511, by Nippi (1 mentions)
Hat medium, by Merck Group (1 mentions)
Dulbecco s modified eagle s medium, by Fujifilm (1 mentions)
3 protocols using stemfit ak02n
1
Feeder-free Culture of KhES-1 Cells
2
Generation of Human Induced Pluripotent Stem Cells
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For generation of human iPSCs (hiPSCs), human peripheral blood mononuclear cells (Cellular Technology) were cultured. After transduction of the plasmid mixture (pCXLE-hOCT3/4-shp53-F, pCXLE-hUL, pCXWB-EBNA1, and pCXLE-hSK encoding short hairpin RNA for DNMT3A), the cells were seeded on a 6-well plate coated with iMatrix-511 (Takara) and cultured until hiPSC colony formation. The selected hiPSCs were expanded in StemFit AK02N (Takara).
3
Generation and Selection of HAC/MAC Containing Cell Lines
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This study was approved by the Institutional Animal Care and Use 45 (link) obtained from the ATCC (ATCC ® CCL-121™) were cultured in Dulbecco's modified Eagle's medium (FUJIFILM Wako) with 10% FBS and 1% penicillin/streptomycin. The human immortalised mesenchymal stem cell (hiMSC) 37 (link) line was kindly provided by Dr. J. Toguchida and was cultured in Dulbecco's modified Eagle's medium (FUJIFILM Wako) with 10% FBS and 1% penicillin/streptomycin. HEK293 clones that contained each HAC/MAC were selected with 200 µg/mL hygromycin B (FUJIFILM Wako). HT1080 cell and hiMSC clones that contained HAC2 were selected with 8 and 4 µg/mL blasticidin S, respectively. HT1080 clones that contained MAC2 were selected with 200 µg/mL hygromycin B. For drug selection after transfection using the SIM system, HACs/MACs expressed the HPRT gene for hypoxanthineaminopterin-thymidine (HAT) resistance following gene insertion. HEK293 and HT1080 clones were selected after transfection in HAT medium (Sigma-Aldrich). hiPSC cell line 201B7 (HPS0063) was provided by the RIKEN BRC and cultured in StemFit AK02N (Takara Bio, Kusatsu, Japan) with Laminin-511 (Nippi, Adachiku, Japan). hiPSCs that contained MAC6 with a neomycin resistance gene were selected with 90 µg/mL G418.
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