Transwell polyester membrane cell culture insert

Transwell® polyester membrane cell culture inserts (6.5 mm diameter, 0.4 μm pore size; Corning Life Sciences, Amsterdam, The Netherlands) were used for culturing human airway epithelial and endothelial cells. Airway epithelial cells were cultured on the apical side and endothelial cells on the basal side of the permeable culture insert. After coating both sides of the membrane with collagen, inserts were turned upside‐down and 5 × 10⁴ HUVECs in a volume of 50 μl endothelial medium were seeded on the basal side of the membrane. Endothelial cells were left in a humidified incubator at 37°C, 5% CO₂ for 2 h to adhere. Non‐adherent cells were gently washed off and the inserts replaced into a 24‐well plate with 500 μl endothelial medium in the basolateral compartment. Airway epithelial cells were seeded in the apical compartment at a density of 1.5 × 10⁵ cells in 200 μl epithelial cell medium. Cells were co‐cultured for up to 8 days and media was changed every 2 and 3 days. The formation of the physical barrier was monitored by measuring the ionic permeability by transepithelial resistance (TER) using an EVOM voltohmmeter (World Precision Instruments, Aston, UK). TER measurements were corrected for the resistance of an empty Transwell (170 Ω) and were expressed as Ω × cm².

Corning^® Transwell^® polyester membrane cell culture inserts (Corning, NY) were used to mimic the in vivo state and the tumour microenvironment. Initially, fresh medium was added to the multiple-well plate and the transwell insert; then incubated overnight to enhance cell attachment. Ovarian cancer cell lines (IGROV1 and SKOV3) were then seeded in the transwell insert compartment at the density of 3 × 10⁵ then returned at 37°C in an incubator with humidified atmosphere containing 5% CO₂. The next day, ovarian cancer cell lines were exposed to 42°C for 1 hour, then PBMC were added to the lower compartment immediately following density gradient isolation at 1/10 ratio of IGROV1 or SKOV3 to PBMC. PBMC and ovarian cancer cells were harvested 24 and 48 hours after hyperthermic exposure (42°C for 1 hour).

Co-culture of TILs with NSCLC cells was performed with the trans-well assay [45 (link)]. Freshly isolated TILs (5 × 10⁵ cells) were placed in the lower compartment of Corning® Transwell® polyester membrane cell culture inserts. Same number of NSCLC cells were seeded in the upper compartment and co-cultured with TILs for 4 days. TILs were then collected for analyzing macrophages and Treg cells with flow cytometry.