DOI: 10.6092/unibo/amsacta/6660
title: ConcePTION WP3 task 3.2. porcine Mammary Epithelial Cells (pMECs) in vitro model for epithelial barrier

description: This data set contains data related to the characterization of an appropriate in vitro animal model based on porcine epithelial mammary cells (pMECs). The objective of the research was to verify the accuracy of pMECs as solid translational model for the study of mammary epithelial barrier. The results showed that it was possible to isolate, cultivate, and maintain three pure epithelial cell lines, each from a different animal. The pMECs maintained until P10 showed a typical cobblestone morphology and doubling time data indicated that cells grew with a similar rate (MG2: 43±13h; MG3 47±14h; MG8: 49±16h, as reported in the data file CONCEPTION_WP3_pMECs_DoublingTime_17032021.csv). DNA index was normal for all the three cell populations with a mean value of 0.996±0.047 (see data file CONCEPTION_WP3_pMECs_DNAIndex_17032021.xlsx). Regarding the cell cycle, selected single populations showed the three distinct phases (G0/G1, S and G2/M) that could be recognized in a proliferating cell population (see data file CONCEPTION_WP3_pMECs_CellCycle_17032021.csv). Immunofluorescence analysis of the typical epithelial markers indicated that the isolated pMECs showed a diffuse positivity to pan-cytokeratins antibody with a correct cytoplasmatic localization. All the three pMECs cell lines expressed epithelial markers Epithelial-Cadherin (E-Cad) and Cytokeratin 18 (CK18), confirming their epithelial origin (see data file CONCEPTION_WP3_pMECs_FlowCytometer_17032021.csv). In particular pMECs-MG8 showed a bimodal peak for CK18 indicating that in the cell culture are present two different subpopulations: 27.3% with a dim fluorescence and 72,7% with a bright fluorescence. The barrier function of pMECs was evaluated via TEER and fluorescein sodium transport. For MG2 and MG3 pMECs, a plateau in TEER values (368 ± 48 and 393 ± 56 Ωcm2 respectively) was already reached after 2-3 days, followed by a quick drop in TEER values. Similarly, fluorescein sodium transport reached values around 0.06 % at day 2, and increased again after day 4. MG8 pMECs showed a different pattern in both TEER and fluorescein sodium transport: the initial increase in TEER was slower, a maximum of 829 Ωcm2 was reached at the day 5. Afterwards, TEER values also decreased, but were still higher than the maximal values reached in MG2 (368 Ωcm2) and MG3 (393 Ωcm2) for up to 15 days. Similarly, fluorescein sodium transport was low in a period starting from day 3 up to day 16 (see data file CONCEPTION_WP3_pMECs_TEER_17032021.csv). The transcriptional profile of drug transporters showed a differential level of gene expression ranging from ΔCt values (Ct mean reference genes – Ct interest gene) very negative (lower expression) to ΔCt values less negative (higher expression). Only 3 were not detectable and the level of expression of each gene is similar among the three cell lines (see data file CONCEPTION_WP3_pMECs_RT2ARRAY_17032021.csv). The only gene that shows a variation in ΔCt higher than 5 cycles among cell lines is ABCC2. A single qPCR reaction confirmed the statistically significant higher expression of ABCC2 gene in MG8 pMECs respect to the other cell lines (see data file CONCEPTION_WP3_pMECs_qPCR_17032021.csv) Collectively, the results show that the isolated pMECs: (i) express epithelial cell markers and tight junctions molecule; (ii) are able to create a tight barrier; (iii) express the main drug transporters; demonstrating the validity of the method to obtain primary pMECs lines to study epithelial barrier functions in the pig model.