Millicell cell culture insert

Cells were cultured in Dulbecco’s modified Engle’s medium (DMEM, Sigma-Aldrich) supplemented with a 10% fetal bovine serum (FBS, Gibco), 1% non-essential amino acids (NEAA, Sigma-Aldrich), and a 5% antibiotic–antimycotic solution (Sigma-Aldrich). Incubation was carried out at 37 °C in a humidified atmosphere of 5% CO₂ in air, and the medium was changed every 2–3 days. After the cells had reached a confluence of 80–90%, trypsinization was performed using 0.1% trypsin and 0.02% EDTA in PBS (Sigma-Aldrich). Caco-2 cells were counted with handheld automated cell counter (Scepter™ 2.0 Cell Counter, Merck Millipore) using appropriate sensors (60 µm Scepter Cell Counter Sensors, Merck Millipore). The Caco-2 cells used in this study were in 45–48 passages. For the transport experiment, Caco-2 cells were seeded in cell culture inserts with polycarbonate membranes (parameters: 30 mm diameter, 0.4 μm pore size, 4.2 cm² grown surface area, Millicell Cell Culture Insert, Millipore) at a cell density of 0.5 × 10⁶/insert, and incubated in six-well culture plates. The medium was changed every second day in the volumes of: 1.5 mL on the apical side (AP) and 2.0 mL on the basolateral side (BL), as previously described by Jarmołowska et al. [93 (link)].

We used the Millicell® Cell Culture insert (Millipore, Billerica, MA, USA), according to the manufacturer's instructions. Briefly, human epidermal keratinocytes and human fibroblasts were cocultured in transwell system. For HEKn, 1 × 10³ cells were seeded in the upper chamber, with 8 μm-pore filters and lower chambers were seeded with fibroblasts at 5 × 10⁴ cells for 24 h. PVE, 11β-HSD inhibitor, and cortisone were added to the upper wells. After 72 h of incubation, cell culture medium and NHFs were harvested, and ELISA and real-time qPCR were performed to measure the levels of collagen.

Organotypic cultures were performed following the protocol previously described [47 (link)]. Sprague–Dawley pups (P5-P7) were purchased from Harlan Laboratories (UK). Pups were decapitated using scissors, and brains were removed and incubated in cold-Hank’s solution for 5 min, and then dissected using the optic chiasm as a landmark to produce a hypothalamic block. The block was placed onto a Mcllwain Tissue Chopper and 400 μm sections containing the PVN were cut then transferred to ice-cold Hank’s solution and incubated for 1 h before being placed onto a Millicell Cell Culture Insert (30 mm, hydrophilic PTFE, 0.4 μm; Millipore PICM03050) in a 6-well tissue culture plate with 1.1 ml of culture medium. The culture sections were incubated at 37 °C, 5 % (v/v) CO₂ for 14 days before performing an experiment. The culture medium was replaced with fresh media every 2 days, and 4 days before the experiment the medium was replaced with serum-free medium. For treatments, culture medium was replaced with serum-free medium containing 10 μM FSK (Sigma) and/or 100nM DEX. At appropriate time points, the membrane was frozen on dry ice, and then the PVN was punched out using a 1 mm diameter micropunch (Fine Scientific Tools). The punched tissue was dispersed into a 1.5 ml tube for RNA extraction using Qiazol Reagent with the Qiagen RNeasy kit as described below.

Caco-2 cells were seeded on a permeable polycarbonate insert (Millicell cell culture insert, 12 mm diameter, 0.4 µm pore size, Millipore Corporation, German) in 12-well tissue culture plates with a density of 1.0×10⁵ cells per insert. When cultured 17∼21 days after seeding, fluorescein permeability and transepithelial electrical resistance (TEER) measurement (Millicell-ERS epithelial volt-ohm meter, Millipore Corporation, German) were used to evaluate the integrity of the Caco-2 cell monolayers. The apparent permeability coefficient (P_app) of fluorescein was less than 5×10⁻⁷ cm/s and the TEER value of the monolayers used in the transport study was more than 600 Ω·cm². Before initiation of transport studies, the cell monolayers were washed with warm HBSS. Then, HBSS containing 24R-epimer (1, 5 and 20 µM) or 24S-epimer (1, 5 and 20 µM) was loaded into either apical or basolateral chambers, in the absence or presence of 10 µM verapamil (P-gp inhibitor) pre-incubated for 1 h. Aliquots from the receiver chamber were collected after incubation for 30, 60, 120 and 180 min at 37°C and added an equal volume of HBSS. The collected solutions were stored at −20°C until analysis. All experiments were conducted in triplicate.