The telomere length of early passage (5 to 10 PDs) cultures from early transfers was also analyzed for the 16 donors. No correlation was observed between the mean telomere length and donor age but there was a significant association (r2 = 0.51) between the mean telomere length and lifespan (Figure 2C and D). We also tested whether there was a correlation between mean telomere length and lifespan with increasing transfer number, because the lifespan decreases with increasing transfer for all donors examined (Figure 1A and Supplementary Figure 1). Telomere length was analyzed by TRF analysis for Fre96s at early passage over 70 transfers. The telomere length of Fre96s shortened over 70 transfers reflecting a strong association between telomere length and the replicative capacity of HSF cultures (Figure 1C). However, the relationship between telomere length and lifespan was less clear for cultures arising from serial transfers of Fre96s.
Next, we investigated whether the stress response in HSFs was affected by donor age by irradiating the 16 donor cultures at early passage with 5 Gy. The optimal response to radiation stress was determined to be 4 and 12 h after irradiation (data not shown). The levels of p53-serine15 and two downstream targets of p53, Mdm2 and p21WAF1/CIP1, were compared at 0, 4, and 12 h after irradiation (Figure 3; data not shown). All donors showed an increase in the levels of p53, p53-serine15, and p21WAF1/CIP1 at 4 and 12 h after irradiation, although the response was variable. The increase in protein levels was 1.4–5.7-fold for p53, 1.4–11-fold for p53-serine15, and 1.5–11-fold for p21WAF1/CIP1. The Mdm2 levels were also elevated after irradiation but maximal at 4 h for all donors. These data indicate that the overall response to radiation stress was normal and there was no observable effect of donor age. This is in contrast to a report for mouse fibroblasts (12). We conclude that HSFs obtained using the protocol described here retain normal characteristics with respect to growth kinetics, telomere shortening and response to DNA damage.
Cryopreservation of tissue samples prior to explant culture
To avoid the necessity for immediate culture of tissue upon its arrival in the laboratory, we developed a procedure for storing tissues in liquid nitrogen and found that explant cultures could be established successfully in 90% of cases after two years of cryopreservation, and that the explants could be transferred many times. The lifespans of frozen and fresh explants were compared using the 2nd, 10th and 20th transfers (Figure 4A). The lifespans of cultures from the 2nd transfer of frozen and fresh explants were similar, but at later transfers the frozen explants gave rise to cultures with considerably shorter lifespans. These data indicate that there may have been significant cell death during the freezing procedure (Figure 4B). Nevertheless, the ability to freeze skin samples would enable laboratories to store tissues for later culture and generation of large numbers of cells from patients with rare conditions. In addition, frozen biopsy specimens could potentially be used to produce induced pluripotent stem cells for therapeutic purposes.
Lentiviral infection of explant tissue
One of the limitations of working with normal cells such as fibroblasts is that the number of available PDs is insufficient to permit multiple rounds of gene transfer. We demonstrated here that genes can be transferred into cells that are still inside explant tissues via lentiviral infection. Explant tissue from donor Fre148s-2 was infected with lentivirus encoding GFP. Seven days later, images show the presence of GFP in both the explants and migrating cells (Figure 4C). Approximately 30% of outgrowing cells were positive for GFP as measured by flow cytometry (data not shown). Fibroblasts growing on the plastic surface retain a similar spindle-shaped morphology to those that are located in situ. The ability to transfer genes directly into explant tissue may increase the number of genetic manipulations that may be conducted within the proliferative lifespan of normal cells.
In summary, we have described a technique that allows large numbers of early passage HSFs to be obtained from explants of fresh or frozen tissue. This should extend the usefulness of normal human fibroblasts for studies of cell biology and functional genomics involving multiple rounds of genetic manipulations.
We thank Dr. Amanda Capes-Davis for her comments on the manuscript and Dr. Inder Verma for the lentiviral construct. HGC and AWB were supported by Cancer Institute NSW (CINSW) RLP 05/01, AYMA by CINSW 07/ECF/1-01, and RRR by a Cancer Council NSW Program Grant.
The authors declare no competing interests.
1.) Hayflick, L. 1965. The limited in vitro lifetime of human diploid cell strains. Exp. Cell Res. 37:614-636. 2.) Goldstein, S. 1990. Replicative senescence: the human fibroblast comes of age. Science 249:1129-1133. 3.) Campisi, J. 2005. Senescent cells, tumor suppression, and organismal aging: good citizens, bad neighbors. Cell 120:513-522. 4.) Freshney, R.I. 2011.Primary culture. In R.I. Freshney (Ed.) Culture of animals cells: a manual of basic technique and specialized applications. Wiley-Liss Inc, New York:163-186. 5.) Huschtscha, L.I., J.D. Moore, J.R. Noble, H.G. Campbell, J.A. Royds, A.W. Braithwaite, and R.R. Reddel. 2009. Normal human mammary epithelial cells proliferate rapidly in the presence of elevated levels of the tumor suppressors p53 and p21WAF1/CIP1. J. Cell Sci. 122:2989-2995. 6.) Toouli, C.D., L.I. Huschtscha, A.A. Neumann, J.R. Noble, L.M. Colgin, B. Hukku, and R.R. Reddel. 2002. Comparison of human mammary epithelial cells immortalized by simian virus 40 T-Antigen or by the telomerase catalytic subunit. Oncogene 21:128-139. 7.) Harley, C.B., A.B. Futcher, and C.W. Greider. 1990. Telomeres shorten during ageing of human fibroblasts. Nature 345:458-460. 8.) Balin, A.K., A.J. Fisher, M. Anzelone, I. Leong, and R.G. Allen. 2002. Effects of establishing cell cultures and cell culture conditions on the proliferative life span of human fibroblasts isolated from different tissues and donors of different ages. Exp. Cell Res. 274:275-287. 9.) Martin, G.M., C.A. Sprague, and C.J. Epstein. 1970. Replicative life-span of cultivated human cells. Effects of donor's age, tissue, and genotype. Lab. Invest. 23:86-92. 10.) Yun, J., C. Rago, I. Cheong, R. Pagliarini, P. Angenendt, H. Rajagopalan, K. Schmidt, J.K. Willson. 2009. Glucose deprivation contributes to the development of KRAS pathway mutations in tumor cells. Science 325:1555-1559. 11.) Maier, A.B., and R.G. Westendorp. 2009. Relation between replicative senescence of human fibroblasts and life history characteristics. Ageing Res. Rev. 8:237-243. 12.) Feng, Z., W. Hu, A.K. Teresky, E. Hernando, C. Cordon-Cardo, and A.J. Levine. 2007. Declining p53 function in the aging process: a possible mechanism for the increased tumor incidence in older populations. Proc. Natl. Acad. Sci. USA 104:16633-16638.