12 mm transwell with 0.4μm pore polycarbonate membrane insert

A cytotoxicity test
was performed using mouse embryonic fibroblasts (NIH-3T3). All cells
were used at passages 4–6. The 3T3 cells were seeded at a density
of 1 × 10⁴ cells per well on uncoated 24-well polycarbonate
culture plates (Sigma-Aldrich) coupled with 12 mm Transwell with 0.4
μm Pore Polycarbonate Membrane Insert (Corning) for 24 h under
5% CO₂ in a humidified incubator at 37 °C. After 24
h, the materials were placed inside the inserts and were thereby in
indirect contact with the cells.
The cytotoxic effect of the
coated material heterotelechelic copolymers 3 with cells was assessed
in vitro using trypan blue (0.4%) (Thermo Fischer Scientific) dye
exclusion. After 24 and 48 h, cells were dissociated into a single-cell
suspension using trypsin-EDTA (0.05%) and phenol red (Thermo Fischer
Scientific). The cell suspension was added to 1:1 (vol/vol) of FBS
and centrifuged at 300g. The supernatant was then
discarded and the pellet was resuspended in 1 mL of DPBS with 10%
BSA. 10 μL of the cell suspension was mixed 1:1 with the trypan
blue solution and the percentage of live cells was quantified using
a TC20 automated cell counter (Bio-Rad).

Human primary esophageal epithelial cells were purchased from Cell Biologics (Chicago, IL, USA; Cat. # H-6046) cultivated in complete human epithelial cell medium provided by the vendor and used until passage seven. For the purpose of indirect co-culture one confluent flask (75 mm²) of H-6046 cells was seeded on three separate 12-well transwell plates with inserts (Corning, 12 mm transwell with 0.4 μm pore polycarbonate membrane insert). The inserts were precoated with 0.1% gelatin (15 min, 37 °C). Once cells grew to confluence on the transwell inserts (~48h) the media was replaced with starvation media (human epithelial cell media without supplements and FBS). After 4-12 h of starvation, 1 million isolated PBMC from healthy volunteers were resuspended in starvation media and added in the bottom compartment of the transwell plate. Control PBMC were added to bottom compartments, where no epithelial cells were grown. After 72 h, PBMC were collected from bottom compartments and processed for flow cytometry. For the purpose of direct co-culture, epithelial cells were seeded in 6-well plates. Once confluent, epithelial cells were starved as described previously and allogenic PBMC were added directly to cells for the duration of 72 h. After that, PBMC were collected and processed for flow cytometry.