高度同源性人脂肪间充质干细胞体外分离培养条件的优化_杨旭芳

Yang XF, He X, Zhang LH, Sun M, Xu H, Gao YH, Li YL.Optimization of isolation and culture conditions of highly homogenous
human adipose-derived mesenchymal stem cells.Zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu. 2009;13(45):
Correspondence to: Li Yu-lin, Doctor, Professor, Key Laboratory of Pathobiology, Ministry of Education, Jilin University, Changchun 130021, Jilin Province, China ylli@
Yang Xu-fang★, Master, Lecturer, Key Laboratory of Pathobiology, Ministry of Education, Jilin University, Changchun 130021, Jilin Province, China; Laboratory of Physiology, Mudanjiang Medical University, Mudanjiang 157001, Heilongjiang Province, China yangxufang12@163. com
doi:10.3969/j.issn.1673-8225.2009.45.011
1Key Laboratory of Pathobiology, Ministry of Education, Jilin University, Changchun 130021, Jilin Province, China; 2Laboratory of Physiology, Mudanjiang Medical University, Mudanjiang 157001, Heilongjiang Province, China
8861-8864.
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摘要 背景：目前关于人脂肪间充质干细胞有效分离培养及扩增的方法尚未统一。 目的：拟在体外建立人脂肪间充质干细胞的有效分离方法。 设计、时间及地点：细胞学体外实验，于 2008-06/12 在吉林大学病理生物学教育部重点实验室完成。 材料：手术切除的人腹部脂肪由吉林大学附属医院提供，提供者对实验均知情同意。 方法：取腹部脂肪，去除结缔组织和血管，用 0.1%Ⅰ型胶原酶 37 ℃振荡消化 60 min，过滤后离心，将位于最下层的片状 沉淀用含体积分数为 10%胎牛血清的 LG-DMEM 条件培养基重悬，调整细胞密度至 1×109 L-1 接种于 6 孔板中，于 37 ℃、 体积分数为 5%的 CO2 孵箱内培养，待细胞接近 80%~90%融合时消化传代。取 P3 代细胞，分别进行成骨及成脂肪诱导。 主要观察指标：应用激光扫描共聚焦显微镜观察人脂肪间充质干细胞形态学特点；采用流式细胞仪和免疫荧光法检测其免 疫学表型、细胞周期、生长曲线；观察其多向分化潜能。 结果：分离培养的人脂肪间充质干细胞为成纤维细胞样，呈漩涡状排列。P3 代人脂肪间充质干细胞呈 CD73，CD105，CD166， CD44 及 CD29 阳性，而 CD31，CD34，CD45 及 HLA-DR 阴性；具有典型的干细胞增殖特点，83.81%细胞处于 G0/G1 期， 仅 16.19%细胞处于 S+G2/M 期；细胞在接种后前 2 d 处于生长潜伏期，第 3~6 天处于对数生长期，第 6 天达峰值，以后细 胞生长速度减慢，进入生长平台期，平均五六天传代 1 次。成骨诱导 2 周后，细胞碱性磷酸酶染色呈阳性；成脂诱导 3 d 后，细胞内有小脂滴出现，油红 O 染色呈阳性。 结论：分离脂肪组织的过程中去除大体可见的血管与结缔组织可减少细胞污染；胶原酶浓度为 0.1%和振荡消化时间为 60 min 时，可使基质细胞和脂肪基质纤维成分有效分离，以及使胶原酶与组织得到充分接触，从而获得高度同源性的人脂肪间充 质干细胞。 关键词：人脂肪间充质干细胞；培养条件；优化
杨旭芳1，2，何 旭1，张丽红1，孙 梅1，徐 红1，高云鹤1，李玉林1
Optimization of isolation and culture conditions of highly homogenous human adipose-derived mesenchymal stem cells
中国组织工程研究与临床康复 第 13 卷 第 45 期 2009–11–05 出版 Journal of Clinical Rehabilitative Tissue ring Research November 5, 2009 Vol.13, No.45
基础医学
高度同源性人脂肪间充质干细胞体外分离培养条件的优化****★◆
Yang Xu-fang1,2, He Xu1, Zhang Li-hong1, Sun Mei1, Xu Hong1, Gao Yun-he1, Li Yu-lin1
Abstract BACKGROUND: Up to date, there is not an acceptable method for isolating, culture and amplifying human adipose-derived mesenchymal stem cells (hADSCs). OBJECTIVE: To explore the most effective way to obtain highly homogenous and undifferentiated hADSCs. DESIGN, TIME AND SETTING: The in vitro cytology experiment was performed at the Key Laboratory of Pathobiology, Ministry of Education, Jilin University from June to December 2008. MATERIALS: Human abdominal adipose tissue resected in the surgery was supplied by the Affiliated Hospital of Jilin University. The informed consent was obtained from patients. METHODS: Human adipose tissue was removed connective tissue and blood vessel, followed by incubation in 0.1% type Ⅰ collagenase for 60 minutes at 37 ℃, filtrated then centrifuged. Consequently, the subnatant precipitation was cultured with LG-DMEM containing 10% fetal bovine serum, incubated at 6-well plate with density of 1×109/L, and placed in incubator of 5% CO2 at 37 ℃. The cultured cells were passaged when the cells reached 80%-90% confluency, and the 3rd passage of cells were induced to osteogenic and adipogenic differentiation. MAIN OUTCOME MEASURES: Morphological characteristics of hADSCs were observed by laser scanning confocal microscope. Immunophenotypes, cell cycle and growth curve of hADSCs were detected by flow cytometry and immunofluorescent techniques. In addition, the multiple differentiation potential of hADSCs was detected. RESULTS: hADSCs presented fibroblast-like morphological feature with a flocked array. The 3rd passage of hADSCs had unique immunophenotypes and they were positive for CD73, CD44, CD166, CD105 and CD29, but negative for CD31, CD34, CD45 and HLA-DR. Cell cycle result showed that they had the typical growth characteristics of stem cells, namely, 83.81% cells stayed at G0/G1 stage, only 16.19% cells were stayed at S+G2/M stage; The latent phase of the primary culture cells was 2 days prior to and after incubation, followed by 3-6 days of logarithmical proliferation, reached a peak at day 6, and entering the growth platform phase with lower growth speed. The alkaline phosphatase was positive expressed at week 2 of osteogenic induction. And the positive expression of oil-O red staining could be seen at day 3 of adipogenic induction. CONCLUSION: Cells contamination can be reduced by removing connective tissue and blood vessel of adipose tissue, and 0.1% type Ⅰ collagenase for 60 minutes can effective separated stroma cell to matrix fiber, furthermore, ensure a sufficient contact between collagenase and tissures, which provide an supportive for harvesting highly homogenous hADSCs.