cell line: Caco-2 (HTB-37) passage number: between 29-45 growth: grown in a microfluidic device
Treatment protocol
The cells were seeded at a density of ~75,000 cells per cm2 on 12-well Transwell PET inserts (0.4 µm pore size, 1.12 cm2 surface area, Corning Amsterdam, The Netherlands) and cultured in DMEM+ for 21 days. The medium was changed every two to three days. In the microfluidic device, the cells were seeded at a density of 75,000 cell per cm2 and were allowed to attach to the membrane for 24 h, without the fluid flow. The membrane was then inserted in the microfluidic chip and cells were exposed to a continuous flow of 100 µL/h DMEM+ until day 21 of culturing (Fig. 1B). By doing so, the shear stress in the AP compartment was ~0.002 dyne/cm2 at the cell membrane area where the cells were grown. The DMEM+ medium contained sodium bicarbonate (10 mM) to optimize the pH buffering capacity.
Growth protocol
A Caco-2 cell line (HTB-37), derived from a human colorectal adenocarcinoma, was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). The cells were grown (at passage number 29-45) in DMEM supplemented with 10 % FBS, 1 % penicillin-streptomycin, and 1 % MEM non-essential amino acid, further referred to as DMEM+.
Extracted molecule
total RNA
Extraction protocol
Cells were first washed with PBS. RNA was then isolated using Qiagen RNeasy Mini Kit (Qiagen), quantified using NanoDrop, and quality was checked with the Agilent 2100 bioanalyzer (Agilent Technologies). Samples were only included for further analyses in case of intact bands corresponding to 18S and 28S ribosomal subunits and absence of chromosomal peaks or RNA degradation products.
Label
biotin
Label protocol
Total RNA (100ng per sample) was labeled with the Whole-Transcript Sense Target Assay (Affymetrix, Santa Clara, CA, USA; P/N 900652).
Hybridization protocol
Hybridization and washing of the Affymetrix GeneChip Human Gene 2.1 ST peg arrays were performed on a GeneTitan Instrument according to the manufacturer's recommendations (Affymetrix, Santa Clara, CA, USA).
Scan protocol
Arrays were scanned on an Affymetrix GeneTitan instrument (Affymetrix, Santa Clara, CA, USA).
Data processing
Expression estimates were calculated applying the RMA algorithm in the Bioconductor library 'Oligo' (v1.52.1).
Comparative transcriptomics of epithelial cells grown under static and microfluidic gut-on-chip conditions and benchmarked against human in vivo intestinal cells
