脂肪干细胞的分离培养、生物学特性及其应用研究

doi:10.19586/j.2095-2341.2024.0079

生物技术进展 ›› 2024, Vol. 14 ›› Issue (5): 857-867.DOI: 10.19586/j.2095-2341.2024.0079

• 研究论文 • 上一篇

脂肪干细胞的分离培养、生物学特性及其应用研究

林锦梅¹(), 陈少锐¹, 陈麒烨², 李永伟³()

^1.广州烨善生物科技有限公司，广州 510670
^2.广州市黄埔区开元学校，广州 511356
^3.中山大学附属第三医院，广州 510630

收稿日期:2024-04-15 接受日期:2024-07-02 出版日期:2024-09-25 发布日期:2024-10-22
通讯作者: 李永伟
作者简介:林锦梅 E-mail: 460785658@qq.com；
基金资助:
全国名老中医药专家传承工作室建设项目(140000020132)

Study on Isolation, Culture, Biological Characteristics and Applications of ADSCs

Jinmei LIN¹(), Shaorui CHEN¹, Qiye CHEN², Yongwei LI³()

^1.Guangzhou Yeshan Biotechnology Co. ，Ltd. ，Guangzhou 510670，China
^2.Guangzhou Huangpu District Kaiyuan School，Guangzhou 511356，China
^3.Third Affiliated Hospital of Sun Yat-sen University，Guangzhou 510630，China

Received:2024-04-15 Accepted:2024-07-02 Online:2024-09-25 Published:2024-10-22
Contact: Yongwei LI

摘要/Abstract

摘要：

对人体脂肪干细胞（adipose-derived stem cells, ADSCs）进行原代分离、培养和鉴定，期望建立一套以自体移植治疗为目标的脂肪干细胞原代分离与培养扩增的标准方法，为研究类风湿性关节炎的移植治疗提供基础。使用K-SFM、M-199和DMEM-低糖3种培养基，分别培养分离的脂肪干细胞，比较细胞形态与传代、复苏活力；通过流式细胞仪检测细胞表面标志物CD34、CD45、CD73、CD105和HL-ABC，判断分离培养细胞的特异性与纯度，比较不同培养状态下细胞特征是否有差异；对几种培养条件下的细胞分别进行成软骨和成脂肪诱导，判断和比较不同处理之间干细胞特性是否有改变。经此方法得到的细胞通过流式细胞仪检测细胞表面标志物，符合脂肪干细胞的表面抗原特征，且干细胞纯度较高；而不同的培养体系下，细胞都有较好的适应性，传代或者冻存复苏后仍有较高的细胞活力；在成软骨和成脂肪的诱导实验中，细胞显示出较好的干细胞分化潜能。根据此优化方法，得到的干细胞通过质量检测，无异源物污染、无交叉污染、无遗传变异危险，并保持高活力与明显干细胞特性，可用于进行自体干细胞移植治疗类风湿性关节炎的基础研究。

关键词: 脂肪干细胞, 分离, 培养, 生物学特性

Abstract:

This study aims to perform primary isolation， culture， and identification of adipose-derived stem cells （ADSCs）， with the objective of establishing a standardized method for the primary isolation， culture， and expansion of adipose stem cells for autologous transplantation therapy， providing a foundation for the transplantation treatment of rheumatoid arthritis. Three types of culture media， namely K-SFM， M-199， and DMEM-low glucose， were used to culture the isolated adipose stem cells， and comparisons were made regarding cell morphology， passage， and recovery vitality. Flow cytometry was employed to detect cell surface factors including CD34， CD45， CD73， CD105， and HL-ABC， in order to determine the specificity and purity of the isolated and cultured cells， as well as to compare whether there are differences in cell characteristics under different culture conditions. Chondrogenic and adipogenic induction experiments were conducted on cells under several culture conditions to assess and compare whether there are changes in stem cell characteristics between different treatments. The cells obtained through this method， upon flow cytometry detection of cell surface factors， conformed to the surface antigen characteristics of adipose stem cells and exhibited a high degree of stem cell purity. Under different culture systems， the cells showed good adaptability， maintaining high cell vitality even after passage or cryopreservation and recovery. In the chondrogenic and adipogenic induction experiments， the cells demonstrated significant stem cell differentiation potential. According to this optimized method， the obtained stem cells， having passed quality inspection and confirmed to be free from foreign matter contamination， cross-contamination， and genetic variation risks， while maintaining high vitality and distinct stem cell characteristics， can be utilized for basic research on autologous stem cell transplantation for the treatment of rheumatoid arthritis.

Key words: adipose stem cells, isolation, culture, biological characteristics

中图分类号:

林锦梅, 陈少锐, 陈麒烨, 李永伟. 脂肪干细胞的分离培养、生物学特性及其应用研究[J]. 生物技术进展, 2024, 14(5): 857-867.

Jinmei LIN, Shaorui CHEN, Qiye CHEN, Yongwei LI. Study on Isolation, Culture, Biological Characteristics and Applications of ADSCs[J]. Current Biotechnology, 2024, 14(5): 857-867.

图/表 9

图1 不同培养条件脂肪干细胞原代分离培养比较（100×）

Fig. 1 Comparing of different cultivation conditions for fat primitive stem cells (100 ×)

图2 DMEM低糖培养基持续培养形态观察（100×）

Fig. 2 DMEM diet culture medium for cultivating morphological observation (100 ×)

图3 不同培养基细胞传代形态观察（100×）

Fig. 3 The morphology observation of the different culture medium cells extend (100×)

图4 流式检测细胞鉴定注：每图右上角的数字代表检测细胞指标细胞阳性比例。

Fig. 4 Identification by flow cytometry

表1 细胞鉴定流式检测分析表

Table 1 Flow cytometric analysis of cell identification

	CD34	CD45	CD73	CD105	HLA-ABC
P2-K-SFM	0.06±0.00**	0.03±0.00**	95.60±3.35	97.60±3.01	96.80±2.56
P2-DMEM-L	0.72±0.02	0.86±0.04	92.70±3.12	99.80±2.38	98.10±4.45
P2-M199	0.94±0.03	0.61±0.02	98.00±1.84**	99.40±1.68	99.50±1.70**
P6-DMEM-L	0.21±0.00	0.24±0.00	95.00±3.37	99.40±3.64	98.40±1.58

图5 成软骨细胞培养形态观察

Fig. 5 Morphological observation of chondroblasts in culture

图6 成软骨诱导阿尔新蓝染色鉴定

Fig. 6 Identification by Alcian blue staining of chondrogenic induction

图7 不同培养条件下成脂细胞诱导形态A：成脂诱导形态比较（100×）；B：成脂诱导脂滴（200×）

Fig. 7 Celluler morphology of lipoblast induction under different culture condition

图8 成脂诱导鉴定油红O染色

Fig. 8 Identification of oil red O staining by adipogenic induction

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脂肪干细胞的分离培养、生物学特性及其应用研究

Study on Isolation, Culture, Biological Characteristics and Applications of ADSCs

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