Zhu Ming, Gao Jian-Hua, Lu Feng
Department of Plastic Surgery, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China.
Zhonghua Zheng Xing Wai Ke Za Zhi. 2008 Mar;24(2):138-44.
To explore an approach to isolate and culture the Adipose derived stem cells (ASCs) from the fatty and the fluid portions of liposuction aspirates, and to investigate the growth kinetics, morphology, differentiation capability, cell senescence, surface marker profiles of the ASCs.
The liposuction aspirates were divided into fatty portion and liquid portion. ASCs were isolated from each portion by collagenase digestion and directly centrifugate and cultured to observe the morphology and biology characters in vitro. Cell activity was studied by MTT chromatometry and analyzed statistically. Cell cycle was detected by flow cytometry. Cells were randomly selected from the 3rd, 4th, 6th, 8th generation cells to detect senescence of ASCs by acridine orange staining. The cell surface markers were detected by flow cytometry and immunohistochemistry. Adipogenic and osteogenic lineage differentiation of ASCs was assessed by Oil Red O and alizarin bordeaux staining respectively.
A large amount of ASCs could be isolated and cultured both from the fatty portion and the liquid portion, including PLA cells and LAF cells which had fibroblastic characters with strong viability and proliferative activity. The statistical result indicated that the cell activity of PLA cells and LAF cells was very similar. ASCs from passage 3, 4, 6, 8 didn't show insenecence. CD29, CD44, CD34, which were the markers of mesenchymal stem cells, vWF, CD31, CD105, SMA were all expressed in ASCs. Adipogenic differentiation of ASCs was assessed by Oil Red O staining after 2 weeks. The cells contained many lipid-filled droplets. After 2 weeks' osteogenic induction, cells were positively stained by alizarin Bordeaux.
The method can isolate ASCs by directly centrifugate from the fatty and the fluid portions of human liposuction aspirates. The way of culture is convenient and economical. ASCs isolated from the liquid and fatty portions of liposuction aspirates show identical in cells numbers and quality. LAF cells and PLA cells have similar characters in growth dynamics, morphology, cell senescence, surface marker profiles and differentiation ability, etc. Expression of the cell surface marker of stem cells is also observed in ASCs. ASCs can differentiate into adipose and osteogenesis directionally. The results suggest that the ASCs, which are isolated with minimum intervention, may be the ideal seed cells for adipose tissue engineering in future.
探索从抽脂吸出物的脂肪部分和液体部分分离培养脂肪来源干细胞（ASCs）的方法，并研究ASCs的生长动力学、形态学、分化能力、细胞衰老及表面标志物谱。
将抽脂吸出物分为脂肪部分和液体部分。通过胶原酶消化、直接离心从各部分分离ASCs并进行培养，观察其体外形态和生物学特性。采用MTT比色法研究细胞活性并进行统计学分析。通过流式细胞术检测细胞周期。从第3、4、6、8代细胞中随机选取细胞，采用吖啶橙染色检测ASCs的衰老情况。通过流式细胞术和免疫组织化学检测细胞表面标志物。分别采用油红O染色和茜素红染色评估ASCs的成脂和成骨谱系分化。
从脂肪部分和液体部分均可分离培养出大量ASCs，包括具有成纤维细胞特征、活力和增殖活性强的PLA细胞和LAF细胞。统计学结果表明PLA细胞和LAF细胞的细胞活性非常相似。第3、4、6、8代的ASCs未显示衰老。间充质干细胞标志物CD29、CD44、CD34，vWF、CD31、CD105、SMA在ASCs中均有表达。2周后通过油红O染色评估ASCs的成脂分化，细胞内含有许多充满脂质的小滴。成骨诱导2周后，细胞茜素红染色呈阳性。
该方法可通过直接离心从人抽脂吸出物的脂肪部分和液体部分分离ASCs。培养方式简便、经济。从抽脂吸出物的液体部分和脂肪部分分离的ASCs在细胞数量和质量上相同。LAF细胞和PLA细胞在生长动力学、形态学、细胞衰老、表面标志物谱及分化能力等方面具有相似特征。在ASCs中也观察到干细胞表面标志物的表达。ASCs可定向分化为脂肪和成骨细胞。结果表明，以最小干预分离得到的ASCs可能是未来脂肪组织工程的理想种子细胞。
