README file
Data Set Title: “ConcePTION WP3 task 3.2. Isolation of porcine Mammary Epithelial Cells (pMECs). In vitro model for epithelial barrier”
Data Set Authors: Chiara Bernardini (Department of Veterinary Medical Sciences, University of Bologna, 40064, Ozzano dell'Emilia, BO, Italy), orcid.org/0000-0003-3384-0244; Debora La Mantia (Department of Veterinary Medical Sciences, University of Bologna, 40064, Ozzano dell'Emilia, BO, Italy), orcid.org/0000-0002-5649-1852; Roberta Salaroli (Department of Veterinary Medical Sciences, University of Bologna, 40064, Ozzano dell'Emilia, BO, Italy), orcid.org/0000-0002-8819-2857; Augusta Zannoni (Department of Veterinary Medical Sciences, University of Bologna, 40064, Ozzano dell'Emilia, BO, Italy), orcid.org/0000-0002-2972-4921; Nina Nauwelaerts (Dep of Pharmaceutical Sciences, O&N II Herestraat 49 Leuven Belgium), orcid.org/0000-0001-5301-6813; Neel Deferm (KU Leuven Drug Delivery and Disposition Lab, Department of Pharmaceutical and Pharmacological Sciences, O&N II Herestraat, 49 3000, Leuven, Belgium), orcid.org/0000-0002-5014-1691; Domenico Ventrella (Department of Veterinary Medical Sciences, University of Bologna, 40064, Ozzano dell'Emilia, BO, Italy), orcid.org/0000-0002-9330-979X; Maria Laura Bacci (Department of Veterinary Medical Sciences, University of Bologna, 40064, Ozzano dell'Emilia, BO, Italy), orcid.org/0000-0001-6702-5868; Giuseppe Sarli (Department of Veterinary Medical Sciences, University of Bologna, 40064, Ozzano dell'Emilia, BO, Italy), orcid.org/0000-0001-6405-3484; Michele Bouisset-Leonard (Novartis Pharma AG, Novartis Institutes for BioMedical Research, Werk Klybeck Postfach, Basel, CH-4002); Pieter Annaert (Dep of Pharmaceutical Sciences, O&N II Herestraat 49 Leuven Belgium), orcid.org/0000-0003-3525-7351; Monica Forni (Department of Veterinary Medical Sciences, University of Bologna, 40064, Ozzano dell'Emilia, BO, Italy), orcid.org/0000-0003-1310-6202.
Data Set Contact Persons: Augusta Zannoni (Department of Veterinary Medical Sciences, University of Bologna, 40064, Ozzano dell'Emilia, BO, Italy), augusta.zannoni@unibo.it. 
Data Set License: this data set is distributed under a Creative Commons Attribution 4.0 International (CC BY 4.0) license, https://creativecommons.org/licenses/by/4.0/. 
Publication Year: 2021
Project Info: ConcePTION (Building an ecosystem for better monitoring and communicating safety of medicines use in pregnancy and breastfeeding: validated and regulatory endorsed workflows for fast, optimised evidence generation), funded by European Union, Horizon 2020 Programme (call H2020-JTI-IMI2-2017-13-two-stage). Grant Agreement num. 821520; https://www.imi-conception.eu/.  

Data set Contents
The data set consists of a compressed folder, named CONCEPTION_WP3_pMECs_17032021.zip, containing: 
•	6 numerical tabular files in .xlsx or .csv format
CONCEPTION_WP3_pMECs_CellCycle_DNAIndex_17032021.xlsx
CONCEPTION_WP3_pMECs_DoublingTime_17032021.csv
CONCEPTION_WP3_pMECs_FlowCytometer_17032021.csv
CONCEPTION_WP3_pMECs_TEER_17032021.csv
CONCEPTION_WP3_pMECs_RT2Array_17032021.csv
CONCEPTION_WP3_pMECs_qPCR_17032021.csv
•	1 README file saved in .rtf format
CONCEPTION_WP3_pMECs_17032021_README.rtf
Data set Documentation
Abstract
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.
Content of the files 
•	file CONCEPTION_WP3_pMECs_CellCycle_DNAIndex_17032021.xlsx contains data about the cell cycle and the DNA Index of pMECs cell lines MG2, MG3 and MG8 (tree independent experiments). For DNA Index analysis, intestinal porcine enterocytes isolated from the jejunum (IPEC-J2) were used as diploid reference cells;
•	file CONCEPTION_WP3_pMECs_DoublingTime_17032021.csv contains data about the doubling time (from passage 1 to 9) of pMECs cell lines MG2, MG3 and MG8; 
•	file CONCEPTION_WP3_pMECs_FlowCytometer_17032021.csv contains dat from a about E-Cad and CK18 immunophenotyping in pMECs cell lines MG2, MG3 and MG8 (two independent experiments). Human Mammary Epithelial Cells (HMECs) were used as positive control for E-Cad;
•	file CONCEPTION_WP3_pMECs_TEER_17032021.csv contains data about the transepithelial electrical resistance of pMECs cell lines MG2, MG3 and MG8; 
•	file CONCEPTION_WP3_pMECs_RT2Array_17032021.csv contains data about gene expression of drug transporters (a panel of 84 genes) in pMECs cell lines MG2, MG3 and MG8;
•	file CONCEPTION_WP3_pMECs_qPCR_17032021.csv contains data about ABCC2 gene expression (single qPCR) in pMECs cell lines MG2, MG3 and MG8 (tree independent experiments).
File specifics
The field delimiter used for .csv files is « ; » (semicolon).
Notes 
•	hMEC (Human Mammary Epithelial Cell) line was used as positive control in the Flow Cytometer experiments.
•	RT² Profiler™ PCR Array Pig Drug Transporters (Cat. No. PASS-070ZD) were purchased from Qiagen Hilden. 
•	Transport buffer (Tb) for transepithelial electrical resistance (TEER) measurement, consisted of HBSS (Lonza) (with addition of 10 mM HEPES (VWR International) and 25 mM glucose (Thermo Fisher Scientific).
•	Antibody used in Flow Cytometer experiments:
Antibody	P. Number	Species	Supplier	Application	
Brilliant Violet 421TM anti-E-cadherin	147319	Rat monoclonal	BioLegend	FC; 16,6 µl/106	
FITC anti-cytokeratin 18	ab52459	Mouse monoclonal	Abcam	FC: 10 µl/106	
Data sources 
Flow Cytometric data (CONCEPTION_WP3_pMECs_FlowCytometer_17032021.csv and CONCEPTION_WP3_pMECs_CellCycle_DNAIndex_17032021.xlsx) are exported from Flowlogic™ software (Inivai Technologies, Australia).
List of variables 
pMECs= porcine Mammary Epithelial Cells
hMECs= human Mammary Epithelial Cells
MG= Mammary Gland
P= passage
TEER= transepithelial electrical resistance
DT= Doubling Time
Ct= cycle threshold; ΔCt = (Ct mean reference genes – Ct interest gene) 
E-Cad= Epithelial Cadherin
CK18= Cytokeratin 18