Transwell polyester membrane insert

For in vitro studies, human RPE cells (LONZA) were used in the fourth passage, seeded at a density of 50000 cells/cm² on Transwell^® polyester membrane inserts with 6.5 mm diameter and 0.4 μm pore size (Corning^®) and maintained in RtEBM^® Basal Medium (LONZA), supplemented with L-Glutamine, FGF-B, and Gentamicin and Amphotericin-B. During the first 3 days, 2.5% fetal bovine serum (FBS) was also included. The medium was replaced every 3–4 days. To study the Scribble protein expression during RPE cell development and maturation, samples at three different time points of culture were collected: 7, 14, and 21 days in culture (DIC). Subsequently, to study the expression of this protein in pathological conditions, once cells reached 21 DIC, they were incubated with the different types of blood serum obtained from patients with ophthalmological problems.

Indirect co-cultures were set up for high resolution melting (HRM) methylation as follows: fibroblasts (NF, TF) were cultivated in 6-well plates (Corning Incorporated Life Sciences, Acton, MA, USA) at a density of 2.5×10⁴ cells/well in DMEM-low glucose. C tumor cells were placed in Transwell® polyester membrane inserts (Corning) at a density of 5×10⁴ cells/insert in RPMI-1640. Forty-eight hours after seeding C cells containing inserts were transferred into wells containing the fibroblast culture. This was followed by addition of 1:1 (V/V) mixture of DMEM-low glucose and RPMI-1640 to the indirect co-cultures and to the control cells growing alone. Ninety-six hours later cell layers were collected for methylation analysis. NF(+C) and TF(+C) indicate fibroblasts isolated from the bottom of the wells, whereas C(+NF) and C(+TF) designate tumor cells isolated from the bottom of the filter compartments of indirect co-culturing plates.

The anti-inflammatory capacity experiments were performed in an in vitro intestinal barrier model since it represents the most suitable condition to study the gut maintenance and defense to inflammatory mediators and the modulation of intestinal epithelial permeability. Caco-2 cellswere seeded onto collagen coated Transwell^® polyester membrane inserts (Corning 3640) and maintained at 37 °C in a 5% CO₂ atmosphere for 21 days, in order to reach the proper differentiation in a polarized epithelial cell monolayer. The basolateral and the apical sides of the insert represent the circulatory and luminal poles of the intestinal epithelium, respectively. Medium was replaced both in apical and basolateral chambers every 3 days, and Transendothelial Electrical Resistance (TEER) was measured every 7 days by means of EVOM EndOhm-12 chamber (World Precision Instruments, Sarasota, FL, USA), to check the formation of a reliable intestinal barrier.
TEER was evaluated as a parameter of barrier functionality after inflammation in cells pretreated or not with cinnamon digested extract. TEER measures were performed before and after the 24 h-inflammatory treatment for every insert, and the ΔTEER [Δ (Ω·cm²)] between the two measures was calculated and analyzed within the groups.

Trans Epithellal Electric Resistance (TEER) is a common indicator to measure the integrity of cell monolayers. TEER is positively correlated with cell monolayers integrity and usually measured by TEER detector. In this study, Caco-2 cells were seeded on Transwell polyester membrane inserts (12 wells, 0.4 μm pores, Corning, NY, USA) and grown until they formed confluent monolayers. TEER was measured utilizing the Millicell-Electrical Resistance System (Merck KGaA, Darmstadt, Germany). TEER values are expressed as Ω*cm² [42 (link)].

The nontransformed porcine intestinal epithelial cell line IPEC-J2, originally isolated from jejunal epithelia of a neonatal unsuckled piglet [17 (link)], was a kind gift of Dr. Jody Gookin, Department of Clinical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, NC, USA. IPEC-J2 cells were maintained on six-well Transwell polyester membrane inserts (Corning Inc., Corning, NY, USA) as it was described previously [18 (link)]. Transepithelial electrical resistance (TEER) measurement of monolayers was performed on alternate days after seeding, from day 5 to 21 of culture, using an EVOM Epithelial Tissue Volt/Ohmmeter (World Precision Instruments, Berlin, Germany).