Short tandem repeat (STR) profiling was used to authenticate all cell cultures, comparing STR profiles to the original breast tissue specimen. These analyses were conducted by CellBank Australia (Westmead, Australia). All HSF cultures were authenticated as arising from the correct donor tissue.Cell culture and treatments
For lifespan determination, all HSFs were grown in DMEM-10 until confluent and sub-cultured at a split ratio of 1:8 or 1:16 for young cultures, and then at lower split ratios for later passages when the cultures were approaching senescence. The number of cells was determined at each sub-cultivation using an electronic counter, Coulter Counter Z1 (Beckman Coulter, Luton, UK). The accumulated PDs were determined according to the formula PDs = log n/log 2 where n = total number of cells/cell number seeded. Cultures were terminated when the fibroblasts were unable to reach confluency after four weeks.
All isolated HSFs were routinely monitored for mycoplasma contamination after growth in antibiotic-free medium for 2 weeks. Mycoplasma testing was performed by CellBank Australia using the MycoAlert Mycoplasma Detection Kit (Lonza Group, Basel, Switzerland) and by PCR. No mycoplasma was detected in any of the tested samples.
The stress response in HSFs was assessed using gamma irradiation. HSFs from 16 donors were plated at 500,000 cells per T-25 tissue culture flask (BD Falcon, BD Biosciences, Bedford, MA, USA) and irradiated 24 h later with 5 Gy using a Gammacell 3000 Elan (MDS Nordian, Ottawa, Canada). The protein levels of p53 and its downstream targets were analyzed by Western blot at 0, 4, and 12 h after irradiation.
To assess the efficacy of genetic manipulation, a skin explant was transduced with lentivirus encoding RRLsin.cPPT.CMV.EGFP.WPRE (a gift from Dr. Inder Verma). After the tissue was trypsinized, the tissue was cut into approximately 5 mm × 5 mm pieces, and placed into one well of a 12-well tray with medium containing 8 µg/mL of polybrene and 1× 108 transducing units of the lentivirus. The outgrowing HSFs were visualized seven days after infection using an inverted microscope (Model DMIRB, Leica Microsystems, North Ryde, New South Wales, Australia). Images were captured with a Spot TR-SE6 monochrome camera (Model TRSE, Diagnostic Instruments, SciTech Pty Ltd, Preston, VIC Australia). HSFs were monitored for green fluorescent protein (GFP) expression by flow cytometry as described (5).Western blot analysis
Western blots were performed as previously described (6). The following antibodies were obtained from Santa Cruz Biotechnology, Santa Cruz, CA, USA: p53 (DO1); p53 (ser15); p21WAF1/CIP1 (F-5); and Mdm2 (SMP14). Actin (A2066) antibody was obtained from Sigma-Aldrich. Western blot analyses were quantitated using MultiGauge (Fujifilm, Brookvale, New South Wales, Australia).Terminal restriction fragment (TRF) analysis
Genomic DNA was isolated using the QIAmp blood DNA extraction kit (Qiagen, Doncaster, VIC, Australia) and digested using HinfI and RsaI restriction enzymes (New England Biolabs, Arundel, QLD, Australia). Digested DNA was separated by pulsed field gel electrophoresis (Biorad, Gladesville, New South Wales, Australia) and hybridized in-gel to a γ-32P-ATP-labeled (TTAGGG)4 telomere probe. A PhosphorImager screen was exposed to the gel and scanned on a Typhoon Trio (GE Healthcare, Rydalmere, New South Wales, Australia). Mean telomere length was calculated using ImageQuant (GE Healthcare) as described (7).Results and discussion Serial transfer of explants permits the increased production of HSFs
Standard protocols for harvesting HSFs use skin explants for up to two transfers before discarding the explanted tissue (4). In the current study, the tissues were digested in 0.2% trypsin to enable the fibroblasts to migrate more efficiently from within the extracellular matrix when transferred to a Petri dish. After two to three weeks when sufficient fibroblasts had migrated, the explant was transferred into a new dish while the remaining outgrowth was collected by trypsinization for freezing or lifespan determination. This protocol permits the explant to be transferred until all HSF outgrowth ceases. Explants from two donors were transferred >70 times, and from one donor >80 times, over a period of more than three years.
This protocol was also used to obtain fibroblasts from adult mouse tissues, including skin, tail, bone and liver (data not shown). These experiments were terminated after 10 transfers.