Ethics, sample collection and storage
All experimental protocols for animal tissues in this study were approved by Institutional Animal Care and Use Committee # SP-R-01-2016. Ears of individual sheep (Katahdin breed; age 2–3 years) were collected from university slaughterhouse. Ears excised from the animal head were brought to the laboratory within an hour, cleaned with 70% alcohol swabs, wrapped in clean sterile paper towels, and stored in plastic bags in a refrigerator set at 4°C.
Explant Preparation And Culture
Ear skin explants were prepared and cultured as described (15). In brief, inner side of the stored ears were cleaned with 70% ethanol swabs, and the skin explants (2–3 mm2) excised aseptically after 0, 10, 20, 27, 30, 35, 38, 41, 45, 50, 55, 60, 65 and 70 days of postmortem storage. Excised explants were adhered onto 60 mm diameter dishes (Falcon, B. D. Biosciences, Oxnard, CA, USA) pre-scratched using a scalpel at 5 uniform spots to facilitate adherence. Adhered explants were cultured in DMEM media supplemented with 10% FBS, 50 units/mL of penicillin, 50 µg/mL of streptomycin, and 2.5 µg/mL of fungizone at 37°C, 5% CO2 in a humidified environment. The media in dishes was changed twice a week and any floating explant and/or contaminated dishes removed from the study. Outgrowth of cells around explants was observed under an inverted microscope. Presence or absence of the outgrowth of cells around explants (clusters of > 50 cells) was recorded and the level of confluence captured on day 10–12 of culture for different postmortem intervals (PMI).
Recovery Of Primary Cells And Cryopreservation
Primary outgrowing cells around the explants were recovered by trypsinization on reaching around 80–90% confluence as described (15). Briefly, the cells in dishes were washed twice with 2.0 mL of the balanced salt solution without calcium and magnesium (Gibco@ Life Technologies, Grand Island, NY, USA), and incubated with 2.0 mL of 0.125% trypsin for 5–10 min at 37°C. The trypsinized cells were neutralized with five volumes of growth media and counted to assess cell-viability using Trypan Blue Dye Exclusion Method (16). The cells were pelleted at 200 X g for 7 min. and suspended in Synth-a-Freeze® (Life Technologies Corp., Carlsbad, CA) media. Cells were aliquoted into cryogenic storage vials (1.0 X 105 cells / vial) and frozen at -80°C deep freezer overnight using Nalgene™ Cryo 1°C Freezing Container (Nalgene, Rochester, NY). Next day the vials were transferred to liquid nitrogen tank and stored until used for further experiments. To study cell characteristics, the frozen vials were quickly thawed at 37°C, mixed slowly with 9.0 mL of the media, pelleted at 200 X g for 7 min, dissolved in complete growth media, and cultured in appropriate culture flasks/dishes. To determine post cryopreservation cell-viability, representative vials in triplicate thawed and viable cells counted manually using a hemocytometer as well as plated for in-vitro culture. Cell-viability was expressed as percentage of live cells from total cell count. The cells passaged at least twice before using for an assay. For mycoplasma testing of the cells used for further characterization, the cells were cultured for a total of 10 d, and the culture supernatants were tested for the contamination at day 4 as well as day 10 of the culture using a MycoFLUOR TM Mycoplasma Detection Kit (Molecular Probes) using the manufacturer's instructions.
Immunofluorescence
An aliquot of actively growing 65-dpm cells (p3) was cultured overnight in chambered polystyrene slides (BD Falcon, Bedford, MA). The cultured cells were labeled with mouse monoclonal anti-vimentin-FITC antibodies (Abcam Inc., Cambridge, MA) to test for their true fibroblastic nature using immunofluorescence technique as per the manufacturer’s instructions.
Gfp Transfection Of Primary Cells
Plasmid pcDNA™3.1/NT-GFP containing GFP gene was prepared using EndoFree Plasmid purification kit (Qiagen Inc.) and DNA was quantified using NanoDrop 2000. We transfected fibroblast cells prepared from 0-dpm and 65-dpm cell-lines at p5 level as per the manufacturer’s instructions, using a Transfectamine 3000 kit from Invitrogen (Life Technologies Inc., CA). GFP gene expressing cells compared in 0-dpm and 65-dpm cell populations by observing under UV light in EVOS fluorescent microscope (EVOS) using GFP filter. GFP positive cells were counted under inverted microscope in at least 10 visible areas for each sample.
Cytogenetic Analysis
Sheep cells recovered from 65-dpm interval at p3 were processed for karyotyping using GTL Banding technique as previously established (18) at Cell-line Genetics (Madison, WI; www.clgenetics.com). Chromosome assignments were made according to the Atlas of Mammalian Cytogenetics (19).
Statistical analysis
Growth curve data were expressed as mean ± standard deviation of cells/mL. Post-freezing cell viability data expressed as % of live cells from total cell count. GFP data were expressed as mean ± SD of GFP expressing cells in at least 10 visible areas. All experiments performed in triplicate and all data analyzed using Excel program (Microsoft). Statistical significance was assessed using unpaired Student’s t-test at p < 0.05.