Tryple select enzyme

Approval to use the cells for culture and in vitro research was obtained from the patient and the local ethical committee prior to the study. The tumor tissue-derived cells were grown for 4 months in RPMI-1640 culture media supplemented with penicillin/streptomycin (100 units/100 mg), l-glutamine (2 mmol/L), fetal bovine serum (5%, Biowest), and 1× ITS-G (Insulin-Transferrin-Selenium, Gibco). The resulting cell line, designated MISB10, grew continuously when the culture medium was changed twice per week. Confluent cultures were dissociated for 2 min at +37°C using TrypLE Select enzyme (Gibco), and split into new cultures with ratios of 1:2 or 1:3. Cryopreservation was done in CellVation (MP Biomedicals) cryopreservation media. The MISB10 cell line will be available for non-profit research purposes through the corresponding author on reasonable request and in accordance to a specified MTA.

Cell surface expression of CD44 and CD24 was analyzed, based on previous reports [67 (link)]. Briefly, drug-treated cells were harvested with TrypLE Select Enzyme (Gibco) and simultaneously stained with anti-CD44-APC (clone G44-26, BD Pharmigen) and anti-CD24-PE (clone ML5, BD Pharmigen) in PBS that contained 10% FBS. Isotype controls were used to establish the negative fluorescence signal. Samples were analyzed on a FACS Aria III (BD Biosciences) or Attune NxT (Life Technologies), and the data were analyzed with FlowJo, version 8.7 (Tree Star Inc.).

MDA-MB-231 cells (American Type Culture Collection) were cultured in Dulbecco’s Modified Eagle’s Media (Hyclone) supplied with 10% fetal bovine serum (Gibco), 1 mM sodium pyruvate (Gibco), penicillin (100 U/ml), and streptomycin (100 mg/ml; Hyclone). Cells were transfected with Lipofectamine 2000 reagent (Invitrogen) or jetPRIME (Polyplus-transfection) according to the manufacturer’s instructions. HEK293T cells were cultured in RPMI 1640 medium (Hyclone) supplied with 10% fetal bovine serum, 10 mM Hepes, penicillin (100 U/ml), and streptomycin (100 mg/ml; Hyclone). Cells were transfected using Mirus (TransIT-LT1) according to the manufacturer’s instructions. All cells were grown at 37°C with 5% CO₂. For all cell lines, TrypLE Select Enzyme (Gibco) was used for passaging. Cells within passages 3 to 15 were used for all the experiments.

Based on glucose and lactate measurements of supernatants with a blood gas analyzer (ABL 835 FLEX, Radiometer), feeding rates were adjusted and growth rates predicted. Feeding rate was doubled upon predefined measures of lactate.
ASCs were harvested by loading and circulating 180 ml 1x TrypLE Select enzyme (Gibco, Life Technologies) into the system for 20 min, and the cell suspension was collected in the harvest bag of the closed quantum system.

Cultured hPSC colonies were first washed with Dulbecco’s phosphate-buffered Saline (D-PBS; Invitrogen, # 14190144) and incubated with 0.5× TrypLE select enzyme (Gibco, # 50-591-419) and 0.5 mol I-EDTA solution (Nacalai USA, pH 8.0, # 13567-84) at 37 °C, 5% CO₂ for 10 min. hPSCs were quenched with E8 medium containing 10 μM Y27632 and gently pipetted to dissociate to single cells. Single cell suspension was centrifuged at 300 g for 5 min before resuspending resultant cell pellets gently in E8 medium containing 10 μM Y27632. Cell counts of all hPSCs were done in parallel using 4-chip disposable hemocytometers (Bulldog Products). For hPGCLC induction screen in 96-well plates, Ibidi USA μ-plate 96 well black, ibiTreat–tissue culture treated polymer coverslip, sterilized plates (Ibidi, Fisher Scientific, # 89626) were used, where each well was pre-coated with 1% Geltrex for at least 1.5 h at 37 °C and 5% CO₂. Single-cell suspension of each hPSC line was plated in each well at 35,000 cells cm⁻² in technical triplicate. In total, 2.5 h after initial cell seeding, culture medium (E8 medium containing 10 μM Y27632) was changed to fresh E8 medium containing 4% Geltrex and 10 μM Y27632. Culture medium was replenished daily thereafter. Y27632 was removed from culture medium at 24 h after initial cell seeding.

The tissue samples were washed twice in Hanks’ balanced salt solution (HBSS), incubated in 2.5 mg/mL Dispase II (Gibco Cat. No. 17105-041) for 1.5 h at 37 °C and scraped with a scalpel blade to remove the epithelial and endothelial tissue layers. A trephine blade (2 mm diameter) was then used to obtain several punch biopsies of limbal stroma. The biopsies were attached to the bottom of tissue culture dishes using 30 µL of type I collagen gel (1 mg/mL) and subsequently submerged in stromal cell growth medium consisting of Dulbecco’s modified Eagle’s medium (DMEM) with high glucose content (Life Technologies Cat. No. 10313-021), 10% (v/v) foetal bovine serum (FBS), 2 mM L-glutamine (Life Technologies Cat. No. 25030-081) and 1% penicillin/streptomycin solution (Life Technologies Cat. No. 15140-122). The resulting primary culture was subsequently harvested by rinsing with Versene (Gibco Cat. No. 15040066) followed by incubation for 5–10 min in TrypLE select enzyme (Gibco Cat. No. 12563011). Cultured were expanded to passage p2 before re-suspension in 90% FBS/10% dimethyl sulfoxide (DMSO) and storage, at 2 × 10⁶/mL in liquid nitrogen.

Human ESC line hPSC-1 was derived from surplus, blastocyst stage human embryo as described previously^{83 (link)}. Human iPSC line hPSC-2 was generated at Prof. Katriina Aalto-Setälä’s laboratory at University of Tampere from dermal fibroblasts (Table 3). For this study, hPSCs were cultured in xeno- and serum-free Essential 8™ Flex Medium (E8 flex, Thermo Fisher Scientific), supplemented with 50 U/ml Penicillin-Streptomycin (Gibco, Thermo Fisher Scientific) on Corning® CellBIND® -well plates coated with 0.55 µg/cm² human recombinant laminin-521 (LN-521, Biolamina, Sweden). Single cell passaging with TrypLE™ Select Enzyme (Gibco, Thermo Fisher Scientific) was carried out twice a week using split ratio of 40 000-50 000 cells/cm² as described by Hongisto et al.^{84 (link)}.Table 3

Details of the hPSC and AT-MSC cell lines used in this study.

Cell line ID	Sex/Karyotype	Passage	Source/Details	BMI	AGE
hPSC-1	46XX	p28 + 9FF	Blastocyst	—	—
hPSC-2	46XX	p26 + 9FF	Skin/Sendai	—	—
AT-MSC-1	XX	P5	Thigh	23.6	53
AT-MSC-2	XX	P4	Abdominal flanks	22.7	32
AT-MSC-3	XX	P1	Thigh	31.2	50