세포 치료를 위한 인간 추간판 세포의 적절한 체외 계대 배양 횟수 -세포의 성장 및 표현형- = Adequate Serial Monolayer Passage Number of Human Intervertebral Disc Cells for Cell Therapy -Growth and Phenotype of Cells-

다국어 초록 (Multilingual Abstract)

Study Design: This is an in-vitro experimental study Objectives: We wanted to analyze the changes in the growth and phenotype of human degenerative intervertebral disc cells depending on the frequency of subculture in an in vitro monolayer culture system.
Summary of the Literature Review: A subculture of disc cells is needed to obtain an adequate amount of disc cells for cell therapy,tissue engineering and analysis of the biological characteristics of degenerative disc cells Materials and Methods: The obtained intervertebral discs were divided into the nucleus pulposus (NP) and the annulus fibrosus (AF).
The AF and NP cells were cultured in a monolayer manner, respectively. At each subculture time, we analyzed the morphological changes,the adhesion rate, the proliferation rate and the viability. The expressions of types I and II collagen and proteoglycan were analyzed at the mRNA gene level.
Results: Both the AF and NP cells gradually showed a fibroblast-like spindle shape while undergoing subculture. The adhesion rate was higher at the second and third times of subculture. The cell proliferation was the highest at the second subculture time. The viability was markedly lower prior to the subculture. On RT-PCR, the type II collagen expression was gradually decreased in the NP cells. In the AF cells, Type II collagen was not expressed from the second time of subculture. The expression of proteoglycan was gradually decreased in both.
Conclusions: Following the 3rd subculture, the degenerative disc cells had completely changed their original growth and phenotypic characteristics. Therefore, we believe that it is not desirable for us to do passage cultures more than three times for cell therapy.

국문 초록 (Abstract)

연구 계획: 시험관 실험적 연구목적: 사람의 퇴행성 추간판 세포를 체외에서 배양하면서 계대 배양의 횟수에 따른 성장 및 표현형의 변화를 분석하고자 한다.
선행문헌의 요약: 세포 치료, 조직공학 및 추간판 세포의 생물학적 특성의 분석을 위해서는 퇴행성 추간판 세포의 계대 배양을 거쳐 많은 양의 세포를 획득하여야 한다.
대상 및 방법:. 적출된 추간판 조직을 수핵과 섬유륜 부분으로 나누어서 단층 배양하였다. 계대 배양을 거치면서 세포의 형태 변화, 부착율, 증식율 및 생존율 분석을 진행하였다. 또한 RT-PCR 검사를 시행하여 제 1, 2형 교원질 (type I, II collagen) 및 단백다당 (proteoglycan) mRNA 발현을 유전자 수준에서 분석하였다.
결과: 세포의 모양은 수핵 및 섬유륜 세포 모두 점차적으로 방추형인 섬유모세포 모양을 나타냈으며, 부착율은 2차 및 3차 계대 배양에서 상대적으로 증가하였고, 증식율은 2차 계대 배양에서 가장 우수했으며, 세포의 생존율은 배양을 거치기 전에는 현저히 낮았다. RT-PCR 분석 상 수핵 세포의 제2형 교원질 발현은 점차적으로 감소하는 양상을 보였으며, 섬유륜 세포의 제 2형 교원질은 2차 계대 배양 시부터 발현이 되지 않았다. 단백 다당의 발현은 수핵 및 섬유륜 세포 모두에서 점차 감소하는 양상을 보였다.
결론: 퇴행성 변화를 일으킨 추간판의 섬유륜 및 수핵 세포는 체외에서 계대 배양의 횟수가 3차 계대 배양에서는 고유의 성장과 표현형을 잃어버렸다. 이에 세포치료에 있어서 체외에서 단층 배양 시 3번 이상의 계대 배양을 거치지 않는 것이 바람직할 것으로 사료된다.

참고문헌 (Reference)

• 1 Bayliss MT, "Volvo award in basic science. Proteoglycan synthesis in the human intervertebral disc. Variation with age, region and pathology" 13 : 972-981, 1988
• 2 Brittberg M, "Treatment of deep cartilage defects in the knee with autologous chondrocyte transplantation" 331 : 889-895, 1994
• 3 Freeman BJ, "Total disc replacement in the lumbar spine: a systematic review of the literature" 15 : 439-447, 2006
• 4 Johnstone B, "The large proteoglycans of the human intervertebral disc. Changes in their biosynthesis and structure with age, topography, and pathology" 20 : 674-684, 1995
• 5 Eie N, "The knee-elbow position in lumbar disc surgery: a review of complications" 8 : 897-900, 1983
• 6 Chou AI, "The effect of serial monolayer passaging on the collagen expression profile of outer and inner anulus fibrosus cells" 31 : 1875-1881, 2006
• 7 Eyre DR, "Quantitative analysis of types I and II collagens in human intervertebral discs at various ages" 492 : 29-42, 1977
• 8 Yeung AT, "Posterolateral endoscopic excision for lumbar disc herniation: Surgical technique, outcome, and complications in 307 consecutive cases" 27 : 722-731, 2002
• 9 Hoppenfeld S, "Percutaneous removal of herniated lumbar discs. 50 cases with ten-year follow-up periods" 238 : 92-97, 1989
• 10 Lee JY, "New use of a three-dimensional pellet culture system for human intervertebral disc cells: initial characterization and potential use for tissue engineering" 26 : 2316-2322, 2001
• 11 Yoon ST, "Molecular therapy of the intervertebral disc" 5 : 280-286, 2005
• 12 White AH, "Lumbar laminectomy for herniated disc: a prospective controlled comparison with internal fixation fusion" 12 : 305-307, 1987
• 13 Gan JC, "Intervertebral disc tissue engineering I: characterization of the nucleus pulposus" 411 : 305-314, 2003
• 14 Wang JY, "Intervertebral disc cells exhibit differences in gene expression in alginate and monolayer culture" 26 : 1747-1751, 2001
• 15 Loeser RF, "Integrin expression by primary and immortalized human chondrocytes: evidence of a differential role for alpha1beta1 and alpha2beta1 integrins in mediating chondrocyte adhesion to types II and VI collagen" 8 : 96-105, 2000
• 16 Tullberg-Reinert H, "In situ measurement of collagen synthesis by human bone cells with a sirius red-based colorimetric microassay: effects of transforming growth factor beta2 and ascorbic acid 2-phosphate" 112 : 271-276, 1999
• 17 Farndale RW, "Improved quantitation and discrimination of sulphated glycosaminoglycans by use of dimethylmethylene blue" 883 : 173-177, 1986
• 18 Kluba T, "Human anulus fibrosus and nucleus pulposus cells of the intervertebral disc: effect of degeneration and culture system on cell phenotype" 30 : 2743-2748, 2005
• 19 Yashiki S, "Evaluation of attachment and growth of anchorage-dependent cells on culture surfaces with type I collagen coating" 92 : 385-388, 2001
• 20 Urban JP, "Degeneration of the intervertebral disc" 5 : 120-130, 2003
• 21 Benya PD, "Dedifferentiated chondrocytes reexpress the differentiated collagen phenotype when cultured in agarose gels" 30 : 215-224, 1982
• 22 Lemare F, "Dedifferentiated chondrocytes cultured in alginate beads: restoration of the differentiated phenotype and of the metabolic responses to interleukin-1beta" 176 : 303-313, 1998
• 23 Freemont AJ, "Current understanding of cellular and molecular events in intervertebral disc degeneration: implications for therapy" 196 : 374-379, 2002
• 24 Eie N, "Comparison of the results in patients operated upon for ruptured lumbar discs with and without spinal fusion" 41 : 107-113, 1978
• 25 Hong Y, "Collagen-coated polylactide microspheres as chondrocyte microcarriers" 26 : 6305-6313, 2005
• 26 Wang H, "Chondrocytes attach to hyaline or calcified cartilage and bone" 12 : 56-64, 2004
• 27 An HS, "Biological repair of intervertebral disc" 28 : 86-92, 2003
• 28 Baums MH, "Autologous chondrocyte transplantation for treating cartilage defects of the talus" 88 : 303-308, 2006
• 29 Sato M, "An atelocollagen honeycomb-shaped scaffold with a membrane seal (ACHMS-scaffold) for the culture of annulus fibrosus cells from an intervertebral disc" 64 : 248-256, 2003
• 30 Ganey TM, "A potential role for cell-based therapeutics in the treatment of intervertebral disc herniation" 11 : 206-214, 2002