The effect of the long-term cultivation on telomere length and morphology of cultured epidermis.
BACKGROUND
Cultured epidermis has been successfully used in clinical treatment such as burns and pigmentary disorders. Although the generation of wide cultured epidermis for clinical use may require repeated passages, especially for allografts, the effects of long-term cultivation on its quality and cell viability are not well known.


OBJECTIVE
To investigate the changes in morphology, telomere length, and telomerase activity during the passages of cultured epidermis and keratinocytes up to the passage limit, and to examine the usefulness of telomere length as a performance criterion for cultured epidermis.


METHODS
The keratinocytes obtained from five patients were used to generate cultured epidermis. At the early passage and after cultivation up to the passage limit, morphology, telomere length and telomerase activity were investigated by using microscopes, southern blot analysis and telomeric repeat amplification protocol assay, respectively.


RESULTS
The cultured cells started to show morphological changes when each passage was close to its limit and the cell sheets assumed an irregular stratification with various sizes of cytoplasm and nuclei. At the passage limit, the telomere length had decreased approximately 80-85%, and the average telomerase activity had declined under serum-free culture conditions.


CONCLUSIONS
The results of this study showed the morphological change and telomere length reduction by long-term cultivation on cultured epidermis. Although the reduction in telomere length and telomerase activity may not be the major cause of the senescence, they could provide a useful information for the quality of the cultured epidermis.
Miyata
Yoshihisa
Y
Department of Dermatology, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan.
Okada
Kunihiko
K
Fujimoto
Atsushi
A
Hata
Ken-Ichiro
K
Kagami
Hideaki
H
Tomita
Yasushi
Y
Ueda
Minoru
M
D002478:Cells, Cultured; D016922:Cellular Senescence; D016895:Culture Media, Serum-Free; D000078404:Epidermal Cells; D006801:Humans; D015603:Keratinocytes / Q000166:cytology* / Q000378:metabolism; D016038:Skin Transplantation; D019098:Telomerase / Q000378:metabolism; D016615:Telomere / Q000502:physiology*; D046509:Tissue Culture Techniques
D016895:Culture Media, Serum-Free; D019098:Telomerase