Tryple

Murine prostate organoids were derived as previously described [26 (link)]. Briefly, murine prostates were harvested from Probasin‐Cre4; Pten^loxP/loxP; Trp53^loxP/loxP genotype mice. Prostates were then digested with Collagenase Type II (Gibco) at 37 °C for 2 h followed by exposure to TrypLE (Gibco) at 37 °C for single‐cell suspension. Y‐27632 (10 μm) was used as a supplement to inhibit anoikis. Epithelial cells isolated from the prostate (35 μL drops) were embedded in basement membrane extracts (Matrigel, Corning, NY, USA) and organoid medium including advanced DMEM/F12 (Thermo Fisher Scientific) supplemented with B27 (Gibco), 10 mm Hepes (Gibco), 10 μm GlutaMAX (Gibco), 1.25 mm N‐Acteyl Cysteine (Millipore Sigma, Burlington, MA, USA), Pen/Strep (Gibco), 50 ng·mL⁻¹ epithelial growth factor, 5% (v/v) R‐spondin 1, 10% (v/v) Noggin, 1 nm DHT (Millipore Sigma), 200 nm A83‐001 (Tocris Bioscience, Bristol, UK). Prostate organoids were trypsinized using TrypLE (Gibco) and passaged weekly.

hiPSC-CMs were dissociated as follows: Briefly, the hiPSC-CMs were washed three times with 0.5 mM EDTA in DPBS. Then, TrypLE (Gibco) was added to the cells and incubated at 37 °C for 10 min before vigorously pipetting to remove cells from the culture plate. RPMI 1640 (Life Technologies) supplemented with B27 (Thermo Fisher Scientific) was added to the cell suspension to neutralize the protease in TrypLE. The cell suspension was centrifuged at 200 g for 10 min before being resuspended in RPMI 1640supplemented with B27, 10%v v⁻¹ fetal bovine serum (Gibco) and 10 µM Y27632 (Biorbyt). 1 ml of this solution was added per 1 million cells. The cells were then disaggregated using a 21 G needle and passed through a 100 µm cell strainer (Falcon). In parallel, the patterned hydrogel was precut into 11 mm diameter circles and placed into a 48 well plate; excess liquid was removed using a sterile gauze. Single cells were seeded to the hydrogel at a density of 50 000 cells per cm² before removing excess solution.

The human umbilical cords were collected in 10 min after the baby’s birth and the informed consents were obtained. The umbilical cords were stored in DMEM/High Glucose (Gibco, USA) with antibiotics (500 units/mL of penicillin and 500 μg/mL of streptomycin) on ice and delivered to the lab. They were minced into 1–3 mm³ fragments immediately and incubated with indicated dissociation reagents according to the instructions. The dissociation reagents include TrypLE (Gibco), 0.05% Trypsin-EDTA (Gibco), Accutase (STEMCELL Technologies), GCD (Gentle Cell Dissociation Reagent, STEMCELL Technologies), SR (ReLeSR™, STEMCELL Technologies), CDB (Cell Dissociation Buffer, enzyme-free, Gibco), ACF (Animal Component-Free Cell Dissociation Kit, STEMCELL Technologies), Collagenase B (STEMCELL Technologies, diluted in DMEM/High Glucose), and DNase I (Sigma), Dispase II (Sigma). Then the isolated single cells of human MSCs were expanded in the NBVbe medium (DMEM/High Glucose plus NB, 50 μg/mL Vc, 10 ng/mL bFGF, and 10 ng/mL EGF). The human MSCs were expanded with TrypLE (Thermo Scientific).

Capacitation of naive hESCs was performed as previously described (Rostovskaya et al., 2019 (link)). Approximately 0.5 × 10⁶ TrypLE-dissociated naive hESCs were seeded in 5i/L/A or alternative naive conditions on one well of a Geltrex (Thermo Fisher, A1413201) coated 6-well plate. After 48 hours, naive media were switched to capacitation media (N2B27 supplemented with 2 μM XAV939). The cells were fed fresh media every 1-2 days and passaged at 70%–80% confluency (about 4-5 days) using TrypLE (GIBCO, 12604054). 10 μM of Y-27632 was added for 24 hr following passaging. Cells were analyzed for the expression of naive and primed-specific genes by qRT-PCR after 10 days. Capacitation was performed in 5% CO₂ and 5% O₂ at 37°C.

Mock and MeV-infected HAE (MOI = 5) were prepared for FACS at 3, 7, and 14 days post-infection. HAE were dissociated by incubation with TrypLE (Gibco) for 30 minutes at 37°C and 5% CO₂. Dissociated cells were collected and centrifuged at 200 x g for 5 minutes. The TrypLE was aspirated, the cells were resuspended in DMEM/F-12 media (Gibco) and kept on ice (~4°C). FACS was performed on a BD FACSAria Fusion (BD Biosciences, San Jose, CA) by the University of Iowa Flow Cytometry Core.

nhPBTEs were seeded in 6.5-mm Transwells (Corning Transwells) and maintained in PC-Ex expansion medium. Upon reaching confluence, the medium was removed from the apical surface, and the cells were fed basolaterally with Pneuma-Cult ALI (air-liquid interface) differentiation medium (Stemcell Technologies) for 5 to 8 weeks to allow differentiation of cells at the air-liquid interface. For the assay, the apical surface was washed with 1× DPBS to remove the existing mucus produced by these cells. Bacterial inoculum was prepared from a log phase culture of NTHi, added to the cells at an MOI of 100 and incubated at 37°C and 5% CO₂ for 1 h. After the incubation period, the supernatant was removed, and the apical surface was washed three times with 1× DPBS to remove nonadherent bacteria. 10× TrypLE (Gibco) was added to the apical surface, and 1× TrypLE was added to the basolateral surface to dissociate the adherent bacteria. Samples were collected in 1× DPBS and plated on chocolate agar. The percentage of adherence was determined as described above. The experiment was carried out at least three times with a minimum of three biological replicates each time.