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Cell Biology International (2012) 36, 519–527 (Printed in Great Britain)

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Establishment of ultra long-lived cell lines by transfection of TERT into normal human fibroblast TIG-1 and their characterization

Mizuna Kamada, Tsutomu Kumazaki, Taira Matsuo, Youji Mitsui and Tomoko Takahashi¹

Laboratory of Physiological Chemistry, Faculty of Pharmaceutical Sciences at Kagawa, Tokushima Bunri University, 13141 Shido, Sanuki, Kagawa, 7692193, Japan

To establish useful human normal cell lines, TERT (telomerase reverse transcriptase) cDNA was transfected into normal female lung fibroblast, TIG-1. After long-term-sub-cultivation of 74 individual clones selected for resistance to G418, we obtained 55 cultures with normal range of life span [75 PDL (population doubling level)], 16 cultures with extended life span (75–140 PDL). In addition, 3 immortal cell strains and unexpectedly, one ultra long-lived cell line (ULT-1) with life span of 166 PDL were established. IMT-1, one of the immortal cell strains was confirmed to maintain long telomere length, high telomerase activity and an extremely low level of p16^INK4A. They also showed moderate p53 and p21^CIP1 expression, keeping vigorous growth rate even at 450 PDL. High level of fibronectin and collagen 1α expression confirmed IMT-1 as normal fibroblasts, although one X chromosome had been lost. ULT-1, however, kept a near normal karyotypes and had shortening of telomere length, high expression of p16^INK4A, moderate levels of senescence associated-β-galactosidase positive cells and decreased growth rate only after 150 PDs (population doublings), and finally reached senescence at 166 PDL with morphology of normal senescent fibroblasts. As resources of standard normal human cell, abundant vials of early and middle passages of ULT-1 have been stocked. The use of the cell line is discussed, focusing on isograft of artificial skin and screening of anti-aging or safe chemical agents.

Key words: cell transplantation, immortal cell strains, non-tumorigenic, replicative senescence, telomere

Abbreviations: FBS, fetal bovine serum, GAPDH, glyceraldehyde-3-phosphate dehydrogenase, HRP, horseradish peroxidase, PD, population doubling, PDL, population doubling level, RT–PCR, reverse transcription PCR, SA-β-Gal, senescence associated-β-galactosidase, SCID, severe combined immune-deficient, TERT, telomerase reverse transcriptase, TRF, telomere restriction fragment

¹To whom correspondence should be addressed (email t-takahashi@kph.bunri-u.ac.jp).

Received 26 August 2011/9 December 2011; accepted 25 January 2012

Published as Cell Biology International Immediate Publication 25 January 2012, doi:10.1042/CBI20110460

© The Author(s) Journal compilation © 2012 International Federation for Cell Biology