Human epithelial kidney (HEK)293 and HEK293T cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Wako, Japan) supplemented with 10% fetal bovine serum (FBS) at 37°C and in an atmosphere of 5% CO2. Chinese hamster ovary (CHO) cells were maintained in Ham’s F-12 (Wako, Japan) with 10% FBS at 37°C in the presence of 5% CO2. The hiPSC line HPS0006, generated from skin tissue [22 (link)], was provided by the RIKEN BRC through the Project for Realization of Regenerative Medicine and the National Bio-Resource Project of the MEXT, Japan. The hiPSCs were maintained on iMatrix-511 (Nippi, Japan)-coated culture dishes in serum-free StemFit AK02N medium (Reprocell, Japan) at 37°C in the presence of 5% CO2.
Stemfit ak02n medium
Manufactured by ReproCELL
Sourced in Japan
StemFit AK02N medium is a cell culture medium designed for the maintenance and expansion of human pluripotent stem cells. It supports the undifferentiated growth of these cells while preserving their pluripotency.
Automatically generated - may contain errors
Lab products found in correlation
Imatrix 511, by Nippi (2 mentions)
Ham s f12, by Fujifilm (1 mentions)
Dulbecco s modified eagle, by Fujifilm (1 mentions)
Synthemax, by Corning (1 mentions)
Cellbind, by Corning (1 mentions)
Mccoy s 5a medium, by Thermo Fisher Scientific (1 mentions)
Hct116, by American Type Culture Collection (1 mentions)
Accutase, by Innovative Cell Technologies (1 mentions)
Imatrix 511 silk, by Nippi (1 mentions)
Hank s balanced salt solution, by Thermo Fisher Scientific (1 mentions)
Essential 8 medium, by Thermo Fisher Scientific (1 mentions)
Pbs edta, by Thermo Fisher Scientific (1 mentions)
Essential 8 flex medium, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640 b 27, by Thermo Fisher Scientific (1 mentions)
Nhcf 5, by Lonza (1 mentions)
7 protocols using stemfit ak02n medium
1
Cell Culture Protocols for HEK293, CHO, and hiPSCs
2
Feeder-free Episomal iPSC Generation
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RCS-iPSCs were established by transfection of patient PMNCs with episomal vectors encoding OCT4, SOX2, KLF4, L-MYC, LIN28, and p53 shRNA as previously described34 (link). iPSCs were maintained with feeder-free cultures using StemFit AK02N medium (ReproCELL, Tokyo, Japan) on cell culture plates (CellBIND; Corning, NY, USA) coated with Synthemax (Corning). The medium was changed daily, and passage was performed at 80–90% confluence. iPSCs were routinely monitored for mycoplasma contamination. Three iPSC cell lines (585A1, 585B1, and 648A1), which were generated from PMNCs of 30 s healthy Japanese men using the same method, were used as control iPSCs.
3
Cell Culture Protocols for HCT116 and 201B7 iPSCs
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HCT116 (American Type Culture Collection, CCL-247) cells were maintained in McCoy’s 5A Medium (Life Technologies) supplemented with 10% bovine growth serum (Biowest, S1820). 201B7 human iPS (RIKEN BRC, HPS0063) cells were maintained in StemFit AK02N medium (ReproCELL) in dishes coated with iMatrix-511 (Nippi). Both cell lines were cultured in a humidified incubator with 5% CO2 and 95% air at 37 °C.
4
Maintenance and Differentiation of Human iPSCs
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The human iPSC line Ff-I01s04 was provided by Kyoto University. Accession Number of Ff-I01s04 is ‘CVCL_C1DY’ in Cellosaurus of Expasy (Swiss Bioinfomatics Resource Portal). As we previously reported [14 (link)], all iPSC lines were maintained using the dish coated by iMatrix-511 silk (Nippi, Tokyo, Japan) and StemFit AK02N medium (ReproCELL, Yokohama, Japan). The medium was changed at day 1, 3, 5, and 6; the detachment for passage and differentiation was performed using accutase (Innovative Cell Technologies, San Diego, CA, USA) at day 7. The use of human iPSCs was approved by the ethics committee of The Institute of Medical Science, The University of Tokyo (Approval Code: 2019-4-0716).
5
Isolation of Human Hair Follicle Stem Cells
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Human scalp
hair follicles were obtained from androgenetic alopecia (AGA) patients
after informed consent was obtained. This study was performed in accordance
with protocols approved by the Institutional Ethics Committee of the
YNU Ethical Committee for Medical and Health Research (Authorization
No. Hitoi-2018-16), and Kanagawa Institute of Industrial Science and
Technology (Authorization No. S-2019-01). All of the experimental
procedures were conducted in accordance with the principles of the
Declaration of Helsinki. The hair bulb, which contained DP, was removed
in DMEM, supplemented with 10 mM HEPES, 10% (v/v) FBS, and 1% (v/v)
penicillin-streptomycin. The remaining epithelial components were
treated with 4.8 U/mL dispase II and 100 U/mL collagenase in a mixture
(1:1) of Hanks’ balanced salt solution (Thermo Fisher Scientific,
Inc.) and PBS for 10 min at 37 °C. The collagen sheath was surgically
removed and treated with 0.05% trypsin in PBS for 60 min at 37 °C.
The debris and undissociated tissues were removed by passing through
a 40 μm mesh cell strainer. After centrifugation (180g, 3 min), the human HFSCs were suspended in StemFit AK02N
medium (Reprocell, Japan) and counted.
hair follicles were obtained from androgenetic alopecia (AGA) patients
after informed consent was obtained. This study was performed in accordance
with protocols approved by the Institutional Ethics Committee of the
YNU Ethical Committee for Medical and Health Research (Authorization
No. Hitoi-2018-16), and Kanagawa Institute of Industrial Science and
Technology (Authorization No. S-2019-01). All of the experimental
procedures were conducted in accordance with the principles of the
Declaration of Helsinki. The hair bulb, which contained DP, was removed
in DMEM, supplemented with 10 mM HEPES, 10% (v/v) FBS, and 1% (v/v)
penicillin-streptomycin. The remaining epithelial components were
treated with 4.8 U/mL dispase II and 100 U/mL collagenase in a mixture
(1:1) of Hanks’ balanced salt solution (Thermo Fisher Scientific,
Inc.) and PBS for 10 min at 37 °C. The collagen sheath was surgically
removed and treated with 0.05% trypsin in PBS for 60 min at 37 °C.
The debris and undissociated tissues were removed by passing through
a 40 μm mesh cell strainer. After centrifugation (180g, 3 min), the human HFSCs were suspended in StemFit AK02N
medium (Reprocell, Japan) and counted.
6
Feeder-free Culture of Human iPSCs and ESCs
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The experiments using human iPSCs and ESCs were approved by the Ethics Committee of the Department of Medicine and Graduated School of Medicine, Kyoto University. Informed consent was obtained from all donors from whom iPSCs were derived. The iPSC line 585A135 (link) was cultured in Essential 8 medium (Thermo Fisher Scientific). The ESC line KhES-336 (link) and iPSC line Ff-I0137 (link) were cultured in StemFit AK02N medium (Reprocell). The cells were maintained under feeder-free conditions, passaged every 3–4 days using 0.5 mM EDTA/PBS (Thermo Fisher Scientific) and gentle pipetting, and routinely examined for mycoplasma contamination.
7
Cardiac Organoid Differentiation from hiPSCs
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Human Ventricular Cardiac Fibroblasts (NHCF-V) were purchased from Lonza (CC-2904) and cultured under manufacturer indications. Feeder-free hiPSCs (1390C1) were maintained in StemFit® AK02N medium (Reprocell) on iMatrix-511 (nippi) coated plates. Human heart organoids were differentiated from PSCs by using a published method. Briefly, iPSCs were dissociated with Accumax and resuspended in Essential 8 Flex medium (Gibco) containing 10 μM ROCK inhibitor Y-27632. To generate EBs, 10,000 cells were seeded at a final volume of 100 μl per well in round bottom low-attachment HEMA-coated 96-well plates on day -2. Fresh Essential 8 Flex medium was added the next day. On day 0, Essential 8 Flex medium was removed, and differentiation was performed in RPMI 1640/B-27, minus insulin (Gibco) containing CHIR99021 (4 μM), BMP4 (1.25 ng/ml) and ActivinA (1 ng/ml). On day 1, the medium was replaced with fresh RPMI 1640/B-27, minus insulin. On day 2, the medium was changed to RPMI 1640/B-27, minus insulin containing Wnt-C59 (2 μM). On day 4, the medium was replaced with fresh RPMI 1640/B-27, minus insulin. On day 6, the medium was replaced with fresh RPMI 1640/B-27 (Gibco). On day 7, organoids were treated with 2 μM CHIR99021 in RPMI 1640/B-27 for 1 h. From day 7 onwards, the medium was changed every other day until day 15.
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