脂肪源性干细胞的体外分离培养与鉴定

doi:10.3969/j.issn.2095-4344.2013.40.006

中国组织工程研究 ›› 2013, Vol. 17 ›› Issue (40): 7054-7060.doi: 10.3969/j.issn.2095-4344.2013.40.006

• 脂肪干细胞 adipose-derived stem cells • 上一篇下一篇

脂肪源性干细胞的体外分离培养与鉴定

杜郭佳，陈小红，朱国华，范雁东，王昀，党木仁

新疆医科大学第一附属医院神经外科，新疆维吾尔自治区乌鲁木齐市 830054

出版日期:2013-10-01 发布日期:2013-10-31
通讯作者: 党木仁，博士，主任医师，博士生导师，新疆医科大学第一附属医院神经外科，新疆维吾尔自治区乌鲁木齐市 830054
作者简介:杜郭佳★，男，1979年生，新疆维吾尔自治区昭苏县人，蒙古族，2008年新疆医科大学毕业，硕士，主治医师，主要从事颅底外科、神经内镜及干细胞方面的研究。
基金资助:
新疆维吾尔自治区青年科学基金资助项目(2011211B31)*

In vitro isolation, culture and identification of adipose-derived stem cells

Du Guo-jia, Chen Xiao-hong, Zhu Guo-hua, Fan Yan-dong, Wang Yun, Dang Mu-ren

Department of Neurosurgery, the First Affiliated Hospital of Xinjiang Medical University, Urumqi 830054, Xinjiang Uygur Autonomous Region, China

Online:2013-10-01 Published:2013-10-31
Contact: Dang Mu-ren, M.D., Chief physician, Doctoral supervisor, Department of Neurosurgery, the First Affiliated Hospital of Xinjiang Medical University, Urumqi 830054, Xinjiang Uygur Autonomous Region, China damrjab@163.com
About author:Du Guo-jia★, Master, Attending physician, Department of Neurosurgery, the First Affiliated Hospital of Xinjiang Medical University, Urumqi 830054, Xinjiang Uygur Autonomous Region, China dgrjav@163.com
Supported by:
the Youth Science Foundation of Xinjiang Uygur Autonomous Region, No. 2011211B31*

摘要/Abstract

摘要：

背景：脂肪间充质干细胞来源丰富、取材方便、体外有较强增殖能力并具有多向分化的潜能，有望成为组织工程的种子细胞。
目的：体外分离培养SD大鼠脂肪干细胞并进行鉴定。
方法：取SD大鼠腹股沟区皮下脂肪组织，0.075%I型胶原酶消化分离、培养脂肪源性干细胞，倒置相差显微镜下观察脂肪源性干细胞的细胞形态和增殖特征。取第3代细胞用MTT比色法描绘生长曲线，免疫荧光法鉴定干细胞标志物CD44，含有体积分数10%胎牛血清、地塞米松和胰岛素的DMEM/F12定向诱导成脂分化，油红“O”染色成脂分化鉴定。免疫组化法鉴定向神经细胞诱导分化的结果。
结果与结论：分离出的大鼠脂肪源性干细胞生长曲线呈“S”形，强烈表达干细胞标志物CD44。成脂诱导分化经油红“O”染色呈橘红色。向神经细胞诱导分化后表达神经元标志物神经元特异性烯醇化酶。说明SD大鼠脂肪源性干细胞在体外具有易于分离培养和扩增，表达间充质干细胞相关表型，特定条件下可诱导分化的特点。

关键词: 干细胞, 脂肪干细胞, 细胞培养, 成脂诱导, 免疫荧光法, 干细胞鉴定, 省级基金, 干细胞图片文章

Abstract:

BACKGROUND: Adipose-derived stem cells are easily collected and abundantly cultured, which can proliferate rapidly when being cultured in vitro. With multi-directional differentiation potential, adipose-derived stem cells are expected as seed cells for tissue engineering.
OBJECTIVE: To isolate, culture and identify of adipose-derived stem cells from Sprague-Dawley rats in vitro.
METHODS: The subcutaneous adipose tissue was obtained from the iliac region of rats under the aseptic condition, and then was digested with 0.075% type Ⅰ collagenase and cultured in vitro. The morphology and proliferation characteristics of the cells were observed under an inverted phase contrast microscope. The third passage was put into gauge for growth curve by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, and the cells were also identified by CD44, a stem cell marker, with immunofluorescence staining. Adipose-derived stem cells were induced and differentiated into adipocytes in Dulbecco’s modified Eagle’s medium/Ham’s nutrient mixture F-12 containing 10% fetal bovine serum, dexamethasone and insulin, and then the cells were identified with oil red “O” staining. Adipose-derived stem cells were induced and differentiated into neural cells, and then the cells were identified with immunohistochemical staining.
RESULTS AND CONCLUSION: The growth curve of adipose-derived stem cells was opposite-like “S” shape, and it strongly expressed CD44 that can designate a stem cell. The passage cells were exposed to a defined medium for adipocyte differentiation, and then could be stained with oil red. After being induced and differentiated into nerve cells, the cells expressed neuron-specific enolase. The adipose-derived stem cells of Sprague-Dawley rats are characterized by easy isolation, culture and proliferation in vitro, expressing related phenotypes of mesenchymal stem cells, as well as induction and differentiation under certain conditions.

Key words: stem cells, adult stem cells, cell differentiation, mesenchymal stem cells, adipogenesis, collagenases

中图分类号:

R318

杜郭佳，陈小红，朱国华，范雁东，王昀，党木仁. 脂肪源性干细胞的体外分离培养与鉴定[J]. 中国组织工程研究, 2013, 17(40): 7054-7060.

Du Guo-jia, Chen Xiao-hong, Zhu Guo-hua, Fan Yan-dong, Wang Yun, Dang Mu-ren. In vitro isolation, culture and identification of adipose-derived stem cells[J]. Chinese Journal of Tissue Engineering Research, 2013, 17(40): 7054-7060.

图/表（结果） 4

Morphology of cultured cells

Under the inverted phase contrast microscope, primarily cultured adipose-derived stem cells were round or oval, adherent cells were spindle-shaped and cells were proliferated to form colonies in fish-like or spiral arrangement. After primary culture for 8-10 days, more than 90% of cells were confluent. After passage, fusiform cells appeared and grew fast with the same shape and size, and no significant difference in the morphology was found between different cell generations (Figure 1).

Growth curve of cultured adipose-derived stem cells

Growth curves obtained by MTT assay showed active adipose-derived stem cells with strong proliferation ability were in logarithmic growth. At 1 or 2 days, the cells were in the incubation period; from the 3rd day, the cells began to enter the logarithmic phase, and reached a peak at day 8 followed by entering the plateau phase. The cell growth curve was in “S” shape, shown in Figure 2.

Cultured adipose-derived mesenchymal stem cells possess stem cell properties

Immunofluorescence staining showed that adipose-derived stem cells strongly expressed CD44 that existed in the cytoplasm. Cell outline was clear shown by green fluorescence, nuclei were stained with 4,6-diamino-2-phenyl indole (blue), in line with mesenchymal stem cells properties (Figure 3).

Adipogenic differentiation of cultured adipose-derived stem cells

After adipogenic differentiation, the polygonal cells were seen. Small intracellular lipid droplets were visible at day 3, and became significantly larger at day 7, which are positive for oil red “O” staining. In the control group, no lipid droplets appeared (Figure 4).

Neuronal differentiation of cultured adipose-derived stem cells

After neuronal differentiation, the cells were extended, and the longer protrusions formed and were mutually connected, all of which showed neuron-like cell morphology. Immunohistochemistry examination showed that the induced cells expressed neurons specific enolase (Figure 5).

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引言

材料方法

Design

In vitro cytological observation.

Time and setting

The experiment was completed at the Medical Research Center, the First Affiliated Hospital of Xinjiang Medical University from June 2011 to January 2012.

Materials

Animals: Two healthy male Sprague Dawley rats, specific pathogen-free grade, weighing 160-180 g, were provided by the Experimental Animal Center, the First Affiliated Hospital of Xinjiang Medical University. All animal procedures were in accordance with animal ethics standards^[10].

Main reagents and instruments for isolation, culture and identification of adipose-derived stem cells are as follows.

Methods

In vitro isolation, culture and passage of adipose-derived stem cells

Sprague Dawley rats were anesthetized by intraperitoneal injection of 10% chloral hydrate

(3 mL/kg) under sterile conditions, to extract the inguinal adipose tissue. After removing soft tissue and small blood vessels, the tissues were washed with PBS three times. Then, according to the method of Zuk

et al ^[2], the tissues were cut into pieces by a microsurgery scissor, digested with 0.075% type Ⅰ collagenase at 37 ℃ for 40 minutes and centrifuged at 1 000 r/min (centrifugal radius, 150 mm) for 10 minutes. After the removal of supernatant and PBS resuspension, the samples were filtered using 400-mesh filter and then centrifuged at 1 000 r/min (centrifugal radius, 150mm) for 5 minutes to harvest sediment. The sediment was even mixed with low-glucose DMEM medium containing 10% fetal bovine serum, and cultured in a 37 ℃, 5% CO₂ incubator. After 24 hours, the medium was changed for the first time, and then it was changed every 3 days. When the cells reached over 90% confluence, the cells were digested with 0.25% trypsin/ethylene diamine tetraacetie acid and then passaged at a ratio of 1:2.

MTT determination of growth curve of adipose-derived stem cells

The third generation of adipose-derived stem cells were digested with trypsin to prepare single cell suspension, and then seeded in 96-well plates at a concentration of 5 × 10³ per well. After cultured 1-8 days, 20 μL of 5 g/L MTT solution was added for continuous culture in the dark for 4 hours. After liquid removal, 150 μL dimethyl sulfoxide solution was added per well, and the samples were placed on the micro-oscillator at room temperature for 10 minutes. Absorbance value at 490 nm was detected using the microplate reader, and cell growth curve was drawn.

Identification of stem cell surface markers

The third generation of cells were seeded on 6-well plates with sterilized coverslip. The sample was placed in 60 mm petri dish, rinsed with PBS three times, fixed in cold acetone for 8 minutes, washed with PBS

5 minutes×3, fixed with 3% H₂O₂ for 10 minutes, washed with PBS 5 minutes×3. The diluted rabbit anti-rat CD44 antibody was added to the sample overnight at 4 ℃ wet box. After washed with PBS

5 minutes×3, the diluted FITC-labeled rabbit anti-rat IgG was added to the sample at room temperature for 1 hour. Then, the sample was washed with PBS

(pH 7.4) 5 minutes×3, and cemented with 4,6-diamino-2-phenyl indole. Fluorescence microscope (Olympus) was used to take photos in the darkroom.

Adipogenic differentiation of adipose-derived stem cells and identification using oil red "O" staining

The third generation of adipose-derived stem cells were divided into experimental and control groups, three wells per group. After digestion, the cells were collected and counted. Cells were seeded into 6-well plates with sterilized coverslip at a density of 5×10⁴ cells per well, and then cultured in adipogenic induction medium: DMEM/F12 medium containing 10% fetal bovine serum, 1 μmol/L dexamethasone, and

10 μmol/L insulin (experimental group), or DMEM/F12 medium containing 10% fetal bovine serum (control group). The culture medium was exchanged every 3 days. After 14 days of induction, the culture slide was rinsed with distilled water, and incubated and sealed with oil red O dilutions in the dark for 10-15 minutes. Before usage, 6 mL stock solution was taken, 4 mL distilled water was added for standing 5-10 minutes, and then, the sample was filtered twice and used within 2 hours. The sample was differentiated after addition of 60% ethanol under microscopy till the gap was clear, and then washed with water. Mayer’s hematoxylin nuclear staining was done for 1 minute followed by washing. The sample was mounted with glycerogelatin glue, and observed under the phase contrast microscope. Digital camera was used to take photos. In the control group, the same procedures were done.

Neuronal differentiation of adipose-derived stem cells and identification

After centrifugation, the third generation of cells were resuspended using neural stem cell culture medium and seeded into 6-well plates for neuronal differentiation. After 6 days of directional induction, the cell culture slide was placed in 60 mm petri dish, rinsed with PBS (pH 7.4) three times, fixed in cold acetone for 8 minutes, washed with PBS (pH 7.4) 5 minutes×3, fixed with 3% H₂O₂ for 10 minutes, washed with PBS (pH 7.4) 5 minutes×3. The diluted first antibody was added to the sample overnight at 4 ℃ wet box. After washed with PBS (pH 7.4) 5 minutes×3, 50-100 μL horseradish peroxidase anti-mouse/anti-rabbit polymer was added to the sample at room temperature for 10 minutes. Then, the culture slide was washed with PBS (pH 7.4) 5 minutes×3. After removal of PBS, 100 μL fresh 3,3'-diaminobenzidine was added to the culture slide for coloration under microscope. After rinsing with tap water, the culture slide was couterstained with hematoxylin, and treated with 0.1% HCl for differentiation. After rinsing with tap water, the culture slide was dehydrated, transparent, mounted with neutral balsam, and observed under phase contrast microscope. Digital camera was used to take photos.

Main outcome measures

(1) In vitro morphology of adipose-derived stem cells and growth curve reflecting the proliferative capacity; (2) CD44 expression in adipose-derived stem cells; (3) identification results of adipogenic and neuronal differentiation of adipose-derived stem cells.

讨论

Stem cells are a class of self-renewal and pluripotent cells in human and animal body. According to the developmental stage, the stem cells are divided into embryonic stem cells and adult stem cells^[11-22] . Embryonic stem cells are highly undifferentiated cell derived from the inner cell mass of a blastocyst, with developmental pluripotency, but embryonic stem cells are limited in clinical application because of its limitations in regulation of cell differentiation and ethical problem^[23] . Adult stem cells, in certain conditions, have mesodermal differentiation capacity, which brings new hope for clinical treatment of various diseases[^24]. Therefore, many scholars focus on “adult stem cells”. Adipose-derived stem cells as a new kind of adult stem cells are found in recent years, and have been paid more attentions because of its wide variety of sources, easily obtained, less pain to patients, rapid proliferation and no ethical issues^[25-26] .

There are mainly four methods for isolation and purification of adipose-derived stem cells: immunomagnetic bead method, flow cytometry method, density gradient centrifugation, enzyme digestion and adherent separation^[27-28] . In this study, we successfully isolated and cultured adipose-derived stem cells from lnguinal adipose tissue of Sprague-Dawley rats using enzymatic digestion and adherent separation, and then the cultured cells were passaged and purified. It has been confirmed that these cells demonstrated strong proliferation and differentiation capacity. MTT assay showed the growth curve of adipose-derived stem cells was similar to “S” shape, in line with the growth rule of normal stem cells^[29] . Adipose-derived cells are most active for proliferation at passage 3 and remain a stronger proliferative capacity at passage 10. CD44 is a marker which is expressed most widely in adult stem cells^[30] , and distributed in the cell membrane and cytoplasm. Presence of CD44 can further identify stem cells. In our study, adipose-derived stem cells strongly expressed CD44.

We found that adipose-derived stem cells could differentiate into adipocytes when only cultured in DMEM/F12 medium containing 10% fetal bovine serum, dexamethasone and insulin, without adding of other ingredients, which are inconsistent with national reports^[31-32]. During the whole process of induced differentiation, adipose-derived stem cells gradually change from long spindle to round or polygonal at 3 days, and small lipid droplets were seen; at the end of 1 week, small lipid droplets could been found in the majority of induced cells. After 2 weeks, lipid droplets which were positive for oil red “O” staining increased in number and merged mutually, indicating that the induced cells had differentiated into adipocytes. Oil red “O” could be dissolved in lipid droplets showing orange. In this study, staining methods were also improved. Without formalin fixation, the direct dyeing effect was good only after washing with distilled water. Studies have shown that the specific mechanisms underlying adipogenic differentiation of adipose-derived stem cells may be related to the adipogenic medium that stimulate the differentiation of pluripotent stem cells to fat mother cells, and activate specific gene expression regulating adipogenic differentiation^[33-34] . Another study has shown that adipose-derived stem cells can differentiate into adipocytes possibly through intracellular signaling pathways^[35-36] . Neuron-specific enolase is a marker of neurons, and the expression of this marker can confirm that the adipose-derived stem cells can be induced in vitro into neuron-like cells under specific inducers. Ashjian et al^[37] not only found neuronal morphology and neuron-specific expression of the signal, but also discovered electrophysiological phenomena after neuronal differentiation of adipose-derived stem cells. After induction, potassium channels were observed and delayed rectifier potassium currents were recorded, which prompts that the early development of ion channels generates after neural induction. It is similar to the function of mature nerve cells.

In conclusion, with the continuous in-depth study of functional properties and molecular mechanisms, adipose-derived stem cells, based on their own merits, can have broad application prospects in tissue engineering construction.

脂肪源性干细胞的体外分离培养与鉴定

In vitro isolation, culture and identification of adipose-derived stem cells

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