Collagen was obtained from Cohesion Corporation, Palo Alto, CA. Cells were removed from tissue culture plates using trypsin and mixed with DMEM, 10% FBS and collagen (final collagen concentration 0.5 mg/ml). This mixture was polymerized in a water bath at 37° C, aliquoted into 3.5 cm tissue culture dishes, and placed into an incubator at 37º until harvest. Final cell density was approximately 200,000 cells/ml. Tissue culture plates for non-contractile gels had been pre-coated with collagen 100 μg/ml in phosphate-buffered saline (PBS) in order to prevent matrix contraction. Cells in these non-contractile collagen matrices were incubated at 37ºC for 6 hours. Contractile matrices were prepared as above; they were allowed to polymerize in uncoated tissue culture dishes for two hours, and then the gel was released by tapping the side of the dish. They were placed in an incubator for four more hours before harvesting (for a total 6-hour incubation time, equal to non-contractile matrices). Degree of collagen matrix contraction was not significantly different between IPF and control myofibroblasts.
Growth protocol
Lung tissue explants were cultivated in 35 mm tissue culture dishes in explant medium (DMEM + 20% FBS + antibiotics and antimycotics) at 37°C in 95% air, 5% CO2. Outgrowth was evident in 5 to 7 days, and cells filled the dish in 2 to 3 weeks. Cells from each 35 mm dish were released with trypsin-EDTA and placed in 100 mm tissue culture dishes after trypsin was neutralized with fresh explant medium. These cells, designated passage 1, were cultivated in growth medium (DMEM + 10% FBS + antibiotics) at 37°C in 95% air, 5%CO2. Medium was replaced twice weekly, and cells were subcultivated weekly at a 1:4 split ratio. Cells designated myofibroblasts in both IPF and control samples had typical spindle morphology, were vimentin- and alpha smooth muscle actin-positive; and factor VIII- and surfactant C-negative. Cells used in this study were between passage 4 and 9.
Extracted molecule
total RNA
Extraction protocol
Cells were harvested in log phase using trypsin and incorporated into collagen gels as described above. Myofibroblasts were collected from the gels at the predetermined time point using collagenase 5 mg/ml (Sigma, St. Louis, MO) containing cycloheximide (100 μg/ml) and collected by centrifugation. A small portion of non-homogenized cells was retained for Trireagent (Sigma) processing to isolate total cellular RNA (designated “total RNA”) for microarray analysis. The remaining cells were used for polyribosome preparations as described (Larsson, O., Perlman, D. et al. Nucleic Acids Research 2006). Ten 0.5 ml fractions were collected from each sample into tubes containing 50 μl of 10% SDS. RNA from each fraction was processed using Trireagent according to the manufacturer’s directions and precipitated with isopropanol. Fractions 7-10, consisting of mRNA with four or more bound ribosomes, designated ”heavy” were pooled for microarray analysis.
Label
Biotin
Label protocol
Biotinylated cRNA were prepared according to the single round amplification protocol (Affymetrix) from 10 ug RNA
Hybridization protocol
Following fragmentation, 20 ug of cRNA were hybridized for 16 hr at 45C on HG-U133Plus2 gene chips. GeneChips were washed and stained in the Affymetrix Fluidics Station 450.
Scan protocol
GeneChips were scanned using the Gene Chip Scanner 3000.
Description
Comparison of lung myofibroblasts from patients with Idiopatic Pulmonary Fibrosis (IPF, 6 donors) to control (6 donors) in contractile and non-contractile matrices using both total and polyribosomal RNA.
Data processing
The data was normalized using GCRMA and updated probe sets definitions “RefSeq v7” as defined in GPL6732.
