Tryple select enzyme 10x

Cre^{ERT2 wt/ki}; Gt(ROSA)26Sor < tm1Ytchn > /J^wt/ki and Cre^{ERT2 wt/ki}; Tet1/2/3^fl/fl; Gt(ROSA)26Sor < tm1Ytchn > /J^wt/ki embryos were produced by timed matings of Cre^{ERT2 wt/wt}; Gt(ROSA)26Sor < tm1Ytchn > /J^wt/wt with wildtype (WT) C57BL/6 J and Tet1^fl/fl; Tet2^fl/fl; Tet3^fl/fl mice. Mouse embryonic stem cells (mESC) were derived from E3.75 blastocysts flushed out from the uterine horn of time-mated females. Blastocysts were collected and hatched on mitomycin C mitotically inactivated MEFs plated 1 day earlier. After attachment, SRES media was replaced every second day until day 6 after attachment of the blastocyst to the feeder MEFs layer. On day 6, the expanded blastocysts were dissociated using TrypLE™ Select Enzyme (10X) (Gibco, A1217702) and dissociated cells were plated on mitotically inactivated MEFs. Cells were grown and SRES media + 2i inhibitors was changed every second day until compact cell colonies with typical ESC colony morphology formed. mESC were passaged 2–3 times before being cryopreserved. All mESC lines were tested routinely and found to be free of any mycoplasma contamination.

Epidermis was mechanically fragmented into small pieces using scissors and incubated for 15 min in a stirring 5 mL of TrypLE Select Enzyme 10X (Gibco^TM) at 37 °C (8 cycles). After each cycle, digested solution containing the cells and pieces of tissue was filtered using a Cell Strainer of 100 µm (Fisherbrand^®). The filtered cell suspension was neutralized using 10 mL of keratinocyte medium and kept while remaining tissue was digested again. After the 8th cycle, the cell suspension was centrifuged (10 min/24 °C/300× g) [11 (link)]. Cells were counted and seeded at 130,000 cells/cm² on a feeder layer of irradiated human fibroblasts (8000 cells/cm²) and cultured in keratinocyte medium. This primary culture was named passage 0 (P0). The estimated time and cost (considering only the price of the enzymes) for the epithelial cell isolation were 5 h and USD 81.00, respectively.

SARS-CoV-2 variants (BA.2, and BA.5) were obtained from saliva samples of COVID-19 patients in Thailand and propagated using A549-ACE2-TM2 cells as previously described (17 (link)). The full-length viral spike gene was then amplified using RT-PCR (Clontech, Mountain View, USA), and the sequences were confirmed through Sanger sequencing. Prior to use in infection experiments, cell culture supernatants were processed to remove cell debris via centrifugation. Titration of viral stocks was performed on A549-ACE2-TM2 cells that were cultured in Opti-MEM supplemented with recombinant trypsin (10 µl/ml) in TrypLE™ Select Enzyme (10X) (Thermo Scientific).

This study was approved by the Ethics Committee of the Graduate School of Medicine, Tohoku University (Permission number: 2019‐1‐430). The amniotic membrane was collected with the consent of a pregnant woman undergoing a scheduled cesarean section at the Department of Obstetrics, Tohoku University Hospital. The amniotic membrane was collected from the placenta, which was removed after delivery by cesarean section. The isolation of AE cells was performed according to the protocol of Gramignoli et al.¹⁷ TrypLE Select Enzyme (10X) (Thermo Fisher Scientific, Waltham, MA, USA) was added to 1 g of amniotic membrane and incubated at 35 rpm for 30 min at 37°C to isolate AE cells. The AE cells that had undergone the filtration process were placed in the cell preservation solution NutriFreez D10 (Biological Industries, Cromwell, CT, USA) and stored frozen at –80°C or lower.
Cryopreserved AE cells were thawed at 37°C for 1 min and washed with Dulbecco's phosphate‐buffered saline (PBS). Two milliliters of normal saline were then added per 1.0 × 10⁷ of AE cells.

For the RPE differentiation experiments, hPSCs on matrigel in 6-well plates were grown to high density, ensuring that the cells formed a confluent monolayer. Then the medium was changed to Advanced RPMI 1640 Medium (AdRPMI) (Thermo Fisher Scientific; Waltham, United States) with B-27® Supplement (50X) (B27) (Thermo Fisher Scientific), 10% Knock-Out Serum Replacement (Thermo Fisher Scientific), 1% 100 X GlutaMAX (Thermo Fisher Scientific), and 1% Penicillin-Streptomycin Solution (Thermo Fisher Scientific). RPE differentiation medium was replaced every day for the first 3 weeks and twice a week thereafter. Differentiation continued for 3–4 months. When pigmented areas reached 3–5 mm in size, they were mechanically dissected under a stereomicroscope using a 10 µl pipette tip, dissociated with TrypLE™ Select Enzyme (10X) (Thermo Fisher Scientific), filtered through a 40 µm cell strainer (Thermo Fisher Scientific) and re-plated for enrichment and maturation at a density of 4.5 × 10⁵ cells per cm² on plates or 0.33 cm² PET hanging cell culture inserts (Merck Millipore; Billerica, United States) pre-coated with growth factor reduced matrigel (Corning; Corning, United States).

iPSCs were grown to confluence and maintained for two additional days before proceeding with spontaneous differentiation using RPE media (Advanced RPMI (ThermoFisher), 10% knockout serum replacement (ThermoFisher), 2% B27 supplement (ThermoFisher), 1% GlutaMAX (ThermoFisher), 1% Penicillin‐Streptomycin (ThermoFisher). The media was then partially replaced every 2 to 3 days until pigmented cell patches appeared. The pigmented patches were surgically excised, disassociated using TrypLE Select Enzyme (10X) (ThermoFisher) and seeded on Matrigel‐coated plates in RPE media.