Immortalization of human nucleus pulposus cells by a recombinant SV40 adenovirus vector: establishment of a novel cell line for the study of human nucleus pulposus cells.
METHODS
Establishment and characterization of a de novo cell line derived from human nucleus pulposus cells using a recombinant simian virus 40 (SV40) adenovirus vector.


OBJECTIVE
To assess the feasibility of human nucleus pulposus cell line procurement and to evaluate the character of the resultant outcome to better understand the nature of human nucleus pulposus cells.


BACKGROUND
Despite recent advances in disc cell biologic research, the fundamental nature of nucleus pulposus cells, especially in the context of human cell lines, is still not well understood. Therefore, a broad-based analysis of these cells is of significant necessity. Because of the limited amount of existing human cells, establishment of an immortal cell line would greatly facilitate resource supply.


METHODS
After release of informed consent, tissue samples of nucleus pulposus were obtained from the lumbar intervertebral disc of a 19-year-old man undergoing anterior fusion for burst fracture. Samples with no apparent damage were selected and digested enzymatically for primary culture and then were infected with recombinant SV40 adenovirus vector (Ad/SV40). The infected cells were maintained in culture for more than 40 population doublings, after which they were considered immortalized. Next, confirmation of expression of T antigen was performed and resultant immortalized cell lines were designated and classified as human nucleus pulposus cell line derived from Ad/SV40 infection-1 (HNPSV-1). HNPSV-1 cells were characterized and compared with their mother cells under two designated culture conditions: monolayer and three-dimensional. Morphologic and immunocytochemical analyses were performed at various intervals. Cell proliferation, DNA synthesis, proteoglycan synthesis, gene expression profiling, and karyotypic analyses were also performed. Moreover, HNPSV-1 cells were injected into rabbit discs to assess the presence of tumorigenesis.


RESULTS
Recombinant SV40 adenovirus vector infected nucleus pulposus cells with relatively high efficiency (90%> at multiplicity of infection 100). HNPSV-1 demonstrated marked prolongation of cell life with continuous cell doublings for over 5 months (60-100 cell population doublings). Despite significant increase in cell proliferation and DNA synthesis when compared with its mother cells, resultant cell lines expressed strikingly similar cell morphology and functional characteristics. Atypical karyotypes were noted; however, no apparent tumorigenesis was seen in rabbit discs 24 weeks after injection of HNPSV-1.


CONCLUSIONS
HNPSV-1 was successfully established using recombinant SV40 adenovirus vector. Results showed that human nucleus pulposus cells are capable of immortalization with maintenance of original cell characteristics. It is anticipated that these cells will be useful for in vitro studies of the biologic nature of human nucleus pulposus cells.
Sakai
Daisuke
D
Department of Orthopaedic Surgery, Tokai University School of Medicine, Bohseidai, Isehara, Kanagawa, Japan.
Mochida
Joji
J
Yamamoto
Yukihiro
Y
Toh
Eiren
E
Iwashina
Tohru
T
Miyazaki
Takeshi
T
Inokuchi
Sadaki
S
Ando
Kiyoshi
K
Hotta
Tomomitsu
T
D000260:Adenoviruses, Human / Q000502:physiology*; D000328:Adult; D000818:Animals; D000952:Antigens, Polyomavirus Transforming / Q000235:genetics; D002455:Cell Division; D002461:Cell Line, Transformed / Q000637:transplantation / Q000648:ultrastructure; D002472:Cell Transformation, Viral; D004261:DNA Replication; D005240:Feasibility Studies; D020869:Gene Expression Profiling; D005822:Genetic Vectors / Q000502:physiology*; D006801:Humans; D007403:Intervertebral Disc / Q000166:cytology*; D007621:Karyotyping; D008159:Lumbar Vertebrae; D008297:Male; D011509:Proteoglycans / Q000096:biosynthesis; D011817:Rabbits; D013539:Simian virus 40 / Q000502:physiology*
D000952:Antigens, Polyomavirus Transforming; D011509:Proteoglycans