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

doi:10.19586/j.2095-2341.2024.0079

Current Biotechnology ›› 2024, Vol. 14 ›› Issue (5): 857-867.DOI: 10.19586/j.2095-2341.2024.0079

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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

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

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)

Abstract

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

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

CLC Number:

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.

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.

Figures/Tables 9

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

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

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

Fig. 4 Identification by flow cytometry

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

Fig. 5 Morphological observation of chondroblasts in culture

Fig. 6 Identification by Alcian blue staining of chondrogenic induction

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

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

References 25

1	LIU Y S, WANG G, WANG Y, et al.. Highly efficient and rapid generation of human pluripotent stem cells by chemical reprogramming[J]. Cell Stem Cell, 2023, 30(4):450-459[2024-05-10]. doi: 10.1016/j.stem.2023.02.008 .
2	WANG J, ZHANG X, CHEN H, et al.. Engineered stem cells by emerging biomedical stratagems[J]. Sci. Bull., 2024, 69(2): 248-279.
3	OTTAVIANI D, HUURNE MTER, ELLIOTT DA, et al.. Maturing differentiated human pluripotent stem cells in vitro: methods and challenges[J/OL]. Development, 2023, 150(11):dev201103[2024-05-10]. .
4	ABDELRAZIK H. Mesenchymal stem cells: a hope or a hype?[J/OL]. Int. J. Mol. Sci., 2023, 24(17): 13218[2024-05-10]. .
5	QIN Y, GE G, YANG P, et al.. An update on adipose-derived stem cells for regenerative medicine: where challenge meets opportunity[J/OL].Adv. Sci. (Weinh). 2023, 10(20): e2207334[2024-05-10]. .
6	SONG Y P, WANG N, SHI H X, et al.. Biomaterials combined with ADSCs for bone tissue engineering: current advances and applications[J/OL].Regen Biomater., 2023, 10:rbad083[2024-05-10]. .
7	YI K, LI H, XU C, et al.. Feature recognition of different differentiation stages of induced ADSCs based on deep learning[J/OL]. Comput. Biol. Med., 2023, 159:106906[2024-05-10]. .
8	FARHANA S, KAI Y C, KADIR R, et al.. The fate of adipose tissue and adipose-derived stem cells in allograft[J]. Cell Tissue Res., 2023, 394(2): 269-292.
9	DONG L, LI X, LENG W, et al.. Adipose stem cells in tissue regeneration and repair: from bench to bedside[J]. Regen. Ther., 2023, 24: 547-560.
10	CZOP J K, JAŁOWSKA M. Stem cells in plastic surgery and aesthetic medicine[J]. Postepy Dermatol. Alergol., 2023, 40(4): 504-509.
11	WANG B Z, WANG M M, LI Y, et al.. Functional enhancement of acute infracted heart by coinjection of autologous adipose-derived stem cells with matrigel[J/OL]. Turk. J. Biol., 2023, 47(3):170-185[2024-05-10]. .
12	BINIAZAN F, STOIAN A, HAYKAL S. Adipose-derived stem cells: angiogenetic potential and utility in tissue engineering[J].Int. J. Mol. Sci., 2024, 25(4):2356[2024-05-10]. .
13	GUO W, XU Y, LIU X, et al.. Therapeutic effect of adipose-derived stem cells injected into pericardial cavity in rat heart failure[J]. ESC Heart Fail., 2024, 11(1):492-502.
14	Pavlov-Dolijanovic S, Bogojevic M, Nozica-Radulovic T, et al.. Elderly-onset rheumatoid arthritis: characteristics and treatment options[J/OL].Medicina (Kaunas). 2023, 59(10):1878[2024-05-10]. .
15	BROWN P, PRATT A G, HYRICH K L. Therapeutic advances in rheumatoid arthritis[J/OL]. Br. Med. J., 2024, 384: e070856[2024-05-10]. .
16	LI F F, LU J H, SHI X L, et al.. Effect of adipose tissue-derived stem cells therapy on clinical response in patients with primary Sjogren's syndrome[J]. Random. Control. Trial Sci. Rep., 2023, 13(1):13521[2024-05-10]. .
17	SHIMIZU Y, NTEGE E H, AZUMA C, et al.. Management of rheumatoid arthritis: possibilities and challenges of mesenchymal stromal/stem cell-based therapies[J/OL]. Cells, 2023, 12(14):1905[2024-05-10]. .
18	CLIM Y, JUNG J I, HONG I K. A novel method for human adipose-derived stem cell isolation and cryopreservation[J]. Cell. Reprogram., 2023, 25(4): 171-179.
19	YOSHIDA M, NAKASHIMA A, ISHIUCHI N, et al.. Comparison of the therapeutic effects of adipose- and bone marrow-derived mesenchymal stem cells on renal fibrosis[J/OL]. Int. J. Mol. Sci., 2023, 24(23):16920[2024-05-10]. .
20	潘媛,顾航,肖涵,等.泛素特异性蛋白酶42调节人脂肪干细胞成骨向分化[J].北京大学学报(医学版),2024,56(1):9-16.
	PAN Y, GU H, XIAO H, et al.. Ubiquitin-specific protease 42 regulates osteogenic differentiation of human adipose-derived stem cells[J]. J. Peking Univ. Health Sci., 2024, 56(1): 9-16.
21	郭佳佳. 炎症微环境下线粒体动力学在人脂肪干细胞成软骨分化中的作用及机制研究[D].贵阳:贵州大学,2023.
	Guo J J. The role and mechanism of mitochondrial dynamics in chondrogenic differentiation of human adipose stem cells under inflammatory microenvironment[D]. Guiyang: Guizhou University, 2023.
22	张爽,郑冬,马月辉.脂肪干细胞在再生医学中的应用[J].生物技术进展,2015,5(4):291-296.
	ZHANG S, ZHENG D, MA Y H. The application of adipose stem cells in regenerative medicine[J]. Curr. Biotechnol., 2015, 5(4): 291-296.
23	汪晶晶,马月辉,关伟军.脂肪干细胞促进肌腱损伤修复研究进展[J].生物技术进展,2018,8(1):7-13.
	WANG J J, MA Y H, GUAN W J. Advances of adipose-derived stem cells enhancing the healing of tendon injury[J]. Curr. Biotechnol., 2018, 8(1): 7-13.
24	钱燕,刘启颂.调整细胞培养条件提高间充质干细胞外泌体治疗骨关节炎的潜能[J].中国组织工程研究,2025,29(1):164-174.
	QIAN Y, LIU Q S. Enhancing potential of mesenchymal stem cell exosomes for osteoarthritis by adjusting cell culture condition[J]. Chin. J. Tissue Eng. Res., 2025, 29(1): 164-174.
25	单鸿哲,齐鹏坤,商岚清等. 中药干预骨髓间充质干细胞成软骨分化治疗膝骨性关节炎研究进展[J]. 实用中医内科杂志, 2024(3):71-73.
	SHAN H Z, QI P K, SHANG L Q, et al.. Research progress on traditional Chinese medicine intervention for chondrogenic differentiation of bone marrow mesenchymal stem cells in the treatment of knee osteoarthritis[J]. J. Pract. Tradit. Chin. Intern. Med., 2024(3): 71-73.

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

脂肪干细胞的分离培养、生物学特性及其应用研究

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