The hiPSC cell line SB Ad3.1 was obtained from the StemBANCC consortium via the Human Biomaterials Resource Centre, University of Birmingham [39 (link)]. Cells were grown in mTeSR1 basal medium supplemented with mTeSR1 5× supplement (Stemcell Technologies, UK), 100 U/ml penicillin and 100 μg/ml streptomycin on cell culture plates coated with DMEM/F12 (Sigma-Aldrich, UK) supplemented with hESC-qualified Matrigel, diluted according to the manufacturer’s instructions and lot number (Corning, UK). All cell lines were maintained at 37°C and 5% CO2 and routinely tested negative for mycoplasma.
Mtesr1 5 supplement
Manufactured by STEMCELL
Sourced in United Kingdom
MTeSR1 5× supplement is a concentrated medium supplement used for the maintenance and expansion of human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) in defined, serum-free conditions.
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2 protocols using mtesr1 5 supplement
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Culturing hiPSC Line SB Ad3.1
2
Characterization of iPSC Cell Line
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The iPSC cell line used in the experiments described herein was obtained from Joseph R. Mazzulli laboratory (Northwestern University, Feinberg School of Medicine, Chicago, Il). This iPSC cell line was produced from primary human fibroblasts by retroviral expression of the reprogramming factors POU5F1, SOX2, MYC/cMYC, and KLF4 (described in [103 (link)]). This cell line has been characterized through the expression of pluripotency markers (i.e. POU5F1, PODXL, SOX2, NANOG) [104 (link)], genomic integrity through G-banding karyotype analysis (described in [105 (link)]), teratoma analysis (described in [106 ]), and RT-PCR analysis of pluripotency markers (described in [106 ]). iPSCs (passage 50–60) were cultured on 6-well plates coated with either Matrigel (VMR, 47743–715) or Vitronectin XF (Stem Cell Technologies, 07180). Medium consisted of mTeSR1 basal medium containing 10% mTeSR1 5× supplement (Stem Cell Technologies, 05825) and was changed every other day. iPSCs were groomed by the manual removal of differentiated cells, passaged en bloc weekly by the manual dissection of iPSC colonies into ~2 mm2 chunks of cells and placement into a Matrigel- or Vitronectin XF-coated 6-well plate. Prior to floor plate induction, iPSCs were grown to 80–90% confluence. iPSC cultures were monitored daily with an in-hood EVOS Core XL cell imaging system (Thermo Fisher Scientific).
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