2Sydney Medical School, University of Sydney, New South Wales, Australia
3Beatson Institute for Cancer Research, Glasgow, UK
4Department of Pathology, University of Otago, Dunedin, New Zealand
Here we describe a method for growing fibroblasts from human skin explants that increases the number of cells obtained by up to two orders of magnitude, thus increasing the amount of material available for research and diagnostic purposes and potentially for cell-based therapies. Explants can be transferred sequentially up to 80 times, if required, at which point the explants appear to be completely depleted of fibroblasts. Utilizing skin samples obtained from 16 donors, aged 18-66 years old, the first 20 transfers produced cultures with lifespan and growth characteristics that were all very similar to each other, but the cultures derived from later transfers had a decreasing replicative capacity. Final cumulative population doublings did not correlate with donor age, but correlated positively with the telomere length at early passage. We also demonstrated that explants can be transduced directly by lentiviral infection, and that cryopreserved tissue can be explanted successfully using this procedure.
The ease with which normal human diploid fibroblasts can be cultured has meant that for many decades they have been an invaluable resource for studying cellular and molecular biology. They have a limited replicative lifespan (1), which has made them ideal for investigations of the normal controls on proliferative potential and the processes of cellular senescence (2,–3). However, their limited life span also means that they are a finite resource. This limitation can be partly overcome by maximizing the number of fibroblasts obtained initially from tissue explants.
Techniques currently used to obtain fibroblasts from human tissues, such as explant outgrowth and mechanical or enzymatic disaggregation procedures, are relatively inefficient. Only a minority of cells grow out from the explants, and tissue disaggregation usually results in loss of a large proportion of the cells (4). We describe a technique that permits a substantial increase in the number of human skin fibroblasts (HSFs) that can be obtained from human skin tissue explants.Material and methods Donor history
Human skin specimens were obtained, with both informed donor consent and Human Research Ethics Committee approval, from donors undergoing reduction mammoplasty. Tissue samples were obtained from 17 female donors between 18 and 66 years of age (Table 1); one of these samples (Fre148s) was used for lentiviral infection studies.
le> Sequential outgrowth of human fibroblasts from skin biopsy specimens
Skin specimens were placed in a 10 cm Petri dish and washed in F12–10 carrier medium, consisting of F-12 medium supplemented with 10% fetal bovine serum (FBS), 50 µg/mL gentamicin and 50 units/mL fungizone; all components were obtained from Gibco, Life Technologies (Grand Island, NY, USA). The skin specimens were cut to approximately 1 cm2 pieces, then 4 or 5 explants were placed into a 50 mL tube containing 10–15 mL of 0.2% trypsin (Sigma-Aldrich, St. Louis, MO, USA) made up in F-12 medium without FBS, and then left to digest for 2–4 days at 4°C. After digestion, the trypsin was neutralized by transferring the explants to a Petri dish containing carrier medium, and washed at least twice with fresh carrier medium.
For the first transfer (indicated by -1 at the end of the donor number), 3–5 trypsinized explants were placed into an empty 10 cm Petri dish for approximately 10–15 min to promote attachment, then sufficient growth medium DMEM-10, consisting of DMEM (Life Technologies) supplemented with 10% FBS and gentamicin (50 µg/mL) was gently added before the dishes were incubated in a humidified incubator at 37°C and 5% CO2 in air. The medium was exchanged every 2–3 days to remove cell debris and maintain a physiological pH. In order to minimize fungal contamination that might occur in the later transfers, parallel dishes were supplemented with an antimycotic: either fungizone (50 U/mL), or voriconazole (2.5 µg/mL) (Sigma-Aldrich).
The first outgrowth from the explants was usually heterogeneous, comprising skin keratinocytes and HSFs. The second and subsequent transfers were performed every 2–3 weeks, when sufficient cells had migrated from the explants, producing an HSF outgrowth of about 20 mm from the explants. HSFs were collected from such small outgrowths for two reasons: first, to produce homogeneous cell populations and secondly, to obtain HSFs at early population doublings (PDs). The skin explants were then transferred aseptically to a new Petri dish, by inverting the explant before placing it into the dish, leaving it without medium for 10–15 min before adding DMEM-10 to the second transfer. Meanwhile, the remaining HSFs in the original dish from the first outgrowth were removed by trypsinization, counted and either cryopreserved or used to determine the lifespan. Skin explants were then transferred until the desired amount of HSFs was obtained or until no further outgrowth was observed.Cryopreserving tissue specimens
HSFs were grown for lifespan determination from skin specimens that were explanted after cryopreservation in liquid nitrogen for up to two years. In preparation for cryopreservation, the skin specimen was cut into approximately 5 mm × 20 mm pieces, and treated with trypsin as described above. After washing the skin in DMEM-10, it was transferred into a cryovial with freezing medium [10% dimethyl sulfoxide (DMSO) + 90% FBS] and left for 5–10 min at room temperature, to allow the explant to equilibrate before freezing at −80°C. The cryovial was left at −80°C for no more than one week before transfer to liquid nitrogen. Tissues were reconstituted by thawing at 37°C, and then transferred to a Petri dish with DMEM-10 to wash out the DMSO before being used for serial transfer experiments.