For differentiation into RPE cells, iPSCs were brought to 95%–100% confluence, maintained for additional 1–2 days and fed with RPE differentiation media (Advanced RPMI 1640, 10% KnockOut serum replacement, 2% B27 supplement, 1% GlutaMAX and 1% Penicillin‐Streptomycin (all ThermoFisher)). Media was partially replaced daily. Once pigmented RPE cell patches appeared (2‐3 weeks), media was partially replaced three times a week, and once established—twice a week. RPE patches were excised with a scalpel, dissociated with TrypLE Select Enzyme (10×) (ThermoFisher), sieved through a 100 μm cell strainer and replated onto Matrigel coated 24‐well plates (125,000 cells/cm2). Once showing pigmentation and hexagonal morphology, RPE cells were expanded by passaging at a 1:2 to 1:3 ratio using TrypLE Select Enzyme (10×). For assays, RPE cells were seeded onto Matrigel coated 0.4 μm PET hanging cell culture inserts (Merck) at a density of 450,000 cells/cm2. Subsequent cell passages were used to generate replicates for the analyses.
Tryple select enzyme 10
Manufactured by Thermo Fisher Scientific
Sourced in United States
TrypLE Select Enzyme (10×) is a cell dissociation reagent used for the detachment of adherent cells from culture surfaces. It is a ready-to-use, 10X concentrated solution that can be diluted with cell culture medium or buffer to the desired working concentration.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Glutamax, by Thermo Fisher Scientific (1 mentions)
Knockout serum replacement (ksr), by Thermo Fisher Scientific (1 mentions)
Advanced rpmi 1640, by Thermo Fisher Scientific (1 mentions)
0.4 μm pet hanging cell culture inserts, by Merck Group (1 mentions)
S2 medium, by Thermo Fisher Scientific (1 mentions)
3 protocols using tryple select enzyme 10
1
Differentiation of iPSCs into RPE cells
2
Influenza Virus Propagation Protocol
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Several influenza A and B viruses (IAV, IBV) were used that are listed in Table 1 . All seed viruses were propagated in adherent MDCK cells (#84121903, ECACC). Infection experiments were carried out in shake flasks, ambr15 vessels, or in DASGIP bioreactors. For infection, either a full or a partial medium exchange was performed. For the latter case, the wv was decreased by half (ambr15) and filled up with infection medium; in the DASGIP system the wv was directly increased to achieve a 1:2 dilution. In all cases, the infection medium contained recombinant trypsin (TrypLE Select Enzyme (10×), #A1217701, ThermoFisher) at a final concentration of 20 USP U/mL. Seed viruses were diluted with phosphate‐buffered saline and added to the cell suspension using a multiplicity of infection (MOI) of 10−3 infectious virions/cell. Virus samples were taken from the supernatant and centrifuged at 3000 × g for 5 min at room temperature to remove cell debris, then aliquoted and stored at −80°C until further analysis.
3
Isolation of Intervertebral Disc Cells
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At least 70 L3 discs were dissected for T1, and at least 50 L3 discs each were dissected for T2 and T3. These discs were collected in an Eppendorf tube containing phosphate-buffered saline (PBS) with 0.04% bovine serum albumin (BSA) on ice. After pipetting out the PBS from briefly centrifuged samples, we added TrypLE Select Enzyme (10×) (ThermoFisher, Waltham, MA, USA, #A1217702). The discs were then incubated in a thermomixer shaken at 500 rpm for 25 min at 37 °C (with the tube being flicked every five minutes). S2 medium (Gibco, Waltham, MA, USA, #21720) supplemented with 10% fetal bovine serum and 2% penicillin/streptomycin were then added to stop the dissociation reaction. Finally, the isolated single cells were washed and resuspended in PBS + 0.04% BSA.
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