A comparative study on two culture methods for adipose-derived stem cells from mice with osteoporosis

doi:10.3969/j.issn.2095-4344.2017.29.011

Abstract

Abstract:

BACKGROUND: To obtain enough adipose-derived stem cells (ADSCs) is the premise of repairing osteoporotic bone defects by autograft transplantation; therefore, how to efficiently, simply and economically harvest primary autologous ADSCs is the key to the treatment of osteoporosis.
OBJECTIVE: To isolate and culture ADSCs from mice with osteoporosis (OP-ADSCs) in vitro by tissue explant culture and collagenase digestion, and to compare the efficacies of these two methods.
METHODS: Adipose tissues were isolated from the inguen of C57BL/6 mice, from which OP-ADSCs were obtained by tissue explant culture and collagenase digestion respectively. Then, the cells were subjected to primary culture and subculture. The surface specific antigens of passage 3 cells were observed using flow cytometry, while the yields and proliferation abilities of passage 3 were compared at 7 days of culture. The adipogenic and osteogenic differentiation of OP-ADSCs at passage 4 was detected.
RESULTS AND CONCLUSION: (1) The expression levels of CD34, CD146, Sca-1 in passage 3 cells were (15.22±1.85)%, (75.55±3.36)% and (83.48±4.22)% for the tissue explant culture and (13.46±2.21)%, (76.62±2.47)% and (84.84±3.56)% for the collagenase digestion method, respectively. (2) The cell yield from each milligram of adipose tissue by tissue explant culture was significantly higher than that by collagenase digestion (P < 0. 05). (3) The proliferation rate of cells in the tissue explant culture group was higher than that in the collagenase digestion group after 24 hours of culture. (4) The OP-ADSCs cultured by the two methods could differentiate into adipocytes and osteoblasts, and the lipid accumulation and the mineralized nodules showed no significant difference between the two groups (both P > 0.05). These experimental findings indicate that the tissue explant culture is more suitable for obtaining OP-ADSCs in vitro as compared with collagenase digestion, which contributes to provide adequate cell sources for studies on ADSCs treatment of osteoporotic fractures and bone defects.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Stem Cells, Osteoporosis, Cell Culture Techniques, Tissue Engineering

CLC Number:

R394.2

Huang Cheng-long, Li Qing, Wang Lei, Huang Kui, Luo Dao-wen, Xiao Jin-gang. A comparative study on two culture methods for adipose-derived stem cells from mice with osteoporosis[J]. Chinese Journal of Tissue Engineering Research, 2017, 21(29): 4654-4659.

Figures/Tables 1

2.1 骨质疏松症小鼠模型构建情况与正常小鼠相比，去卵巢模型小鼠micro-CT可见骨皮质变薄，骨髓腔扩大，骨小梁稀疏或断裂，骨小梁宽度变窄，骨小梁间距变宽，骨小梁占视野面积降低，证明成功构建骨质疏松小鼠(图1)。 2.2 脂肪干细胞生长情况组织块培养法培养第 3 天，少量细胞从组织块边缘爬出，贴壁生长，形态为梭形、纺锤形，也可见多角形；培养第7天，细胞几乎铺满瓶底(图2)，重复培养23次，成功18次，成功率为78%。胶原酶消化法培养第12-24小时，细胞开始贴壁生长，形态为为多角形，第二三天，细胞贴壁伸展，长梭形或多角形；第7天，细胞单层融合率达80% (图2)，重复培养19次，成功6次，成功率为32%。两种方法获取的细胞传至第3代，细胞多为成纤维细胞样，长梭形或纺锤形，形态学无差异，传至第9代，细胞生长状态良好，细胞性状均一，生物学特性稳定。 2.3 流式细胞仪检测结果组织块法培养的第3代脂肪干细胞表达CD34、CD146及Sca-1，表达率分别为(15.22± 1.85)%、(75.55±3.36)%及(83.48±4.22)%。胶原酶消化法培养的第3代脂肪干细胞表达CD34、CD146及Sca-1，表达率分别为(13.46±2.21)%、(76.62±2.47)%及(84.84± 3.56)%。表明两种方法培养的细胞都是脂肪干细胞。 2.4 脂肪干细胞产量情况组织块培养法获得的脂肪干细胞培养至第3天，有细胞从组织块边缘爬出，而胶原酶消化法早期即可获得脂肪干细胞；培养第7天，组织块培养法每毫克脂肪组织获得的细胞产量高于胶原酶消化法(P=0.013)，见图3。 2.5 脂肪干细胞增殖情况将组织块培养法和胶原酶消化法获得的第3代脂肪干细胞分别接种到孔板中，在接种后的24 h换液，随后在孵箱中连续培养6 d，通过xCELLigence system 实时检测细胞增殖情况，并生成生长曲线(图4)，该曲线是3复孔的平均值生成的实时动态曲线，生长曲线均有明显的滞留期、对数生长期和平台期。方面，培养24 h后，组织块培养法培养细胞增殖速率一直高于胶原酶消化法。 2.6 脂肪干细胞多向分化情况两种培养方法所得的第4代脂肪干细胞，于成脂诱导后第 4 天可见细胞胞体回缩，胞浆中有脂滴形成，第7天可见脂滴融合，油红O染色脂滴呈橘红色(图5)；油红O萃取实验表明两组成脂量比较差异无显著性意义(P=0.065)(图6A)。两种培养方法所得的第4代脂肪干细胞，成骨诱导第18天可见类似细胞岛的小结节，第 21天可见白色小点，茜素红染色证明为矿化结节(图7)，两组间成骨量比较差异无显著性意义(P=0.071)(图6B)。未加成脂或成骨诱导剂的脂肪干细胞未见成脂、成骨分化。"

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