isolation, culturing and characterization of rat...

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Turk J Biol36 (2012) 658-664 © TÜBİTAKdoi:10.3906/biy-1109-31

Isolation, culturing and characterization of rat adipose tissue-derived mesenchymal stem cells: a simple technique

Mehmet NİYAZ1, Özer Aylin GÜRPINAR2, Serdar GÜNAYDIN1, Mehmet Ali ONUR2

1Department of Cardiovascular Surgery, Faculty of Medicine, Kırıkkale University, Kırıkkale - TURKEY2Department of Biology, Faculty of Science, Hacettepe University, 06800 Beytepe, Ankara - TURKEY

Received: 27.09.2011 ● Accepted: 11.06.2012

Abstract: In this study, our aim was to develop a new simple technique for isolation of mesenchymal stem cells from adipose tissue. For this purpose, mesenchymal stem cells were isolated from rat adipose tissue by using the primary explant culture technique. When the cells became confluent, they were passaged 4 times by using the standard trypsinization method with trypsin/EDTA solution. Cells at second passage were characterized by using immunofluorescence staining against CD13 and CD29 markers. The results showed that these cultured cells were positive for CD13 and CD29 markers. Flow cytometry analysis was also done against CD29, CD90, CD54, MHC Class I, CD45, CD106, and MHC Class II for characterization of mesenchymal stem cells. The results of flow cytometry analysis showed that these cells were mesenchymal stem cells. Half of the cells were cryopreserved at all passages for future applications. It is thought that these mesenchymal stem cells can be used in therapy of cardiovascular diseases as an alternative technique in the near future.

Key words: Adipose tissue, mesenchymal stem cell, CD13, CD29, CD90, CD45, CD54, MHC Class I, MHC Class II, immunofluorescence staining, cryopreservation

IntroductionA mesenchymal stem cell (MSC) is characterized by its self-renewal capacity, long-term viability, and multilineage differentiation (1,2). Therefore, biological and clinical interest in MSCs has risen dramatically over the last 2 decades (3). Ideally, MSCs for clinical applications should meet certain criteria: they should be found in abundant quantities (millions and billions of cells); they should be able to be collected and harvested by a minimally invasive procedure; they should be differentiated along multiple cell lineage pathways in a reproducible manner; and they should be able to be safely and effectively transplanted to either an autologous or allogenic host (3).

The multilineage potential of adult MSCs from bone marrow has been characterized extensively. Adipose tissue is a source of these MSCs that can differentiate into various cell types, like bone, cartilage, adipose, muscle, and endothelium (4-11). These MSCs also meet the criteria previously described. Therefore, adipose tissue-derived MSCs have great potential for novel cellular therapies to repair damaged tissues and in angiogenic therapy.

Heart failure is the terminal stage of many diseases that affect the myocardium (12,13). Coronary artery disease leading to ischemia of the human myocardium is a major cause of heart failure (14,15). However, coronary reperfusion using pharmacological agents and percutaneous coronary intervention reduces

M. NİYAZ, Ö. A. GÜRPINAR, S. GÜNAYDIN, M. A. ONUR

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mortality resulting from myocardial infarction. At present, the only radical treatment for end-stage congestive heart failure is cardiac transplantation; however, a shortage of donors is a severe limitation to this approach, and it requires postoperative immunosuppressive therapy.

Peripheral vascular disease with critical limb ischemia may continue into severe muscle tissue loss or ulcers and be terminated with amputation. Unfortunately, many patients cannot be helped with currently available surgical or endovascular revascularization procedures because of the complex anatomy of the vascular occlusion and/or the presence of other risk factors (16). Therefore, several alternative novel approaches are needed to treat these cardiovascular diseases. Cell transplantation is one possible alternative treatment that may be available in the future. However, this alternative treatment has to be made easier and faster. The present study is important in this because it is the first step toward carrying out such easier and faster alternative treatments.

Materials and methodsCell isolation and cultureIn this study, MSCs were isolated from subcutaneous flank adipose tissue of rats (Wistar albino, male, 250-300 g). Animal procedures related to tissue isolation were approved by the Kırıkkale University Ethical Committee. After the xylazine (Richter Pharma AG, Austria) (10 mg/kg) and ketamine (Richter Pharma AG) (50 mg/kg) anesthesia of rats, flank adipose tissue was collected under sterile conditions. The isolation procedure was repeated 3 times with 3 inbreedings of rats and an average of 0.62 g of adipose tissue was collected per rat (n = 3; n1 = 0.72 g, n2 = 0.57 g, n3 = 0.57 g). Adipose tissue was transferred into transport medium DMEM/F12 (Biochrom AG, Germany) containing 10% (v/v) FBS (Biochrom AG) and 0.4% (v/v) penicillin-streptomycin (Sigma Chemical Co., USA) in a petri dish and then minced into fragments of 4-5 mm in thickness. Tissue fragments were incubated in primary medium (DMEM/F12 containing 20% (v/v) FBS and 0.2% (v/v) penicillin-streptomycin) in a 6-well culture dish under standard culture conditions (in a humidified atmosphere

of 95% air and 5% CO2 at 37 °C). The culture medium was replaced every day to avoid possible differentiative effects of various cytokines originating from MSCs. Cells were passaged using the standard trypsinization method and, as they were passaged, the cell number was counted using the trypan blue staining method (Sigma Chemical Co.). Trypan blue is a vital staining used to selectively blue dead cells. Viable and proliferative cells with intact cell membranes are not colored. In a viable cell, trypan blue is not absorbed. Hence, dead cells are shown with a distinctive blue color under a microscope. The culture medium was removed and the attached cells were harvested using trypsin/EDTA (0.05%/0.02; w/v) solution (Biochrom AG). The cell suspension was then transferred to a centrifuge tube and centrifuged at 800 rpm for 5 min to pellet the cells. The supernatant was removed carefully and the cells were incubated in DMEM/F12 containing 10% (v/v) FBS and 0.2% (v/v) penicillin-streptomycin. The MSCs were passaged 4 times and then cryopreserved for future applications. For cryopreservation, cells were detached from the surface of the culture dish by using a trypsin/EDTA solution. The cell suspension was immediately transferred into a freezing medium containing 50% (v/v) FBS, 40% DMEM/F12, and 10% (v/v) DMSO (Sigma Chemical Co.) in a cryogenic tube. Cryovials were stored at –80 °C for 48 h and then the frozen vials were transferred to a liquid nitrogen container at –196 °C for long-term storage. For standardization of cell concentrations for future applications, all cells harvested from the three 6-well culture dishes were collected in 1 cryogenic tube. Cell morphology and growth were examined under an inverted microscope (IX70, Olympus, Japan).Cell characterizationMesenchymal stem cells were characterized using immunofluorescence staining of CD13 and CD29 molecules (Santa Cruz Biotechnology, Inc., USA) during their second passage. For immunostaining, cells grown in culture dishes were washed in PBS (Biochrom AG) and fixed for 5 min in methanol at –10 °C. After fixation, the methanol was removed and desiccated. For blocking, the cells were incubated for 20 min with blocking serum (normal goat serum). After blocking, the blocking serum was removed, and the cells were washed 3 times in PBS and then


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incubated for 1 h with primary antibody for CD13 and CD29 molecules. After washing for 5 min in PBS, the cells were incubated for 45 min with a secondary antibody (donkey antigoat Ig-TR) and washed 3 times in PBS. After washing, the cells were mounted with mounting medium and visualized under the fluorescence microscope (BH2-RFL-T3 model fluorescence attachment, Olympus). All procedures were done at room temperature. The flow cytometry analysis was done against CD29, CD90, CD54, MHC Class I, CD45, CD106, and MHC Class II for characterization of mesenchymal stem cells. The flow cytometry analysis was performed at the Kocaeli University Center for Stem Cell and Gene Therapies Research and Practice (KOGEM), Kocaeli, Turkey.

Results and discussionCell isolation and cultureIn this study, cell isolation was performed using the primary explant culture technique. No enzymatic digestion method was used. Tissue fragments obtained from the adipose tissue were plated directly into 6-well culture dishes. After 18 h of culture, the emergence of cells with fibroblastic cell morphology was identified among the erythrocytes (Figures 1a and 1b).

After 7 days, the number of the cells with fibroblastic morphology increased and confluency was observed on distinct parts of the culture plate (Figures 2a and 2b). Many of the erythrocytes were

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Figure 1. Morphological appearance of mesenchymal stem cells derived from adipose tissue at 18 h of culture at a) 10× and b) 20×. White arrow indicates mesenchymal stem cell and black arrow indicates erythrocytes.

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Figure 2. Mesenchymal stem cells after a) 7 days of culture, with b) confluency observed on distinct parts of the culture plate (10×).


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removed by changing the culture medium, and just before the first passage, the red blood cells completely disappeared (Figures 3a and 3b).

Although the cells had not become completely confluent, they were passaged on day 7 of primary culture. The cell number was an average of 600,000 cells/mL at the beginning of the first passage for a 6-well culture dish (n = 3; n1 = 400,000 cells/mL, n2 = 640,000 cells/mL, n3 = 750,000 cells/mL). After the first passage, the cell number increased rapidly compared to primary cells The cell number was found to be an average of 900,000 cells/mL (n= 3; n1 = 1,320,000 cells/mL, n2 = 760,000 cells/mL, n3 = 600,000 cells/mL) on day 4 of the first passage.

When the cells became confluent, they were passaged twice a week. While the culture was maintained, half of the cells were cryopreserved after every passage for future applications. The cell-reproducing procedure was maintained for 3 weeks. At the end of this period, a total of 300,000,000 cells had been obtained per rat (900,000,000 cells for 3 rats).

The fibroblastic cell morphology was also maintained through repeated subcultures under nonstimulating conditions after cryopreservation procedure (Figures 4a and 4b).

In the literature, there are a variety of different techniques used to isolate MSCs from adipose tissue,

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Figure 3. Appearance of cultured cells before first passage at a) 10× and b) 20×. The red blood cells completely disappeared.

Figure 4. Morphological appearance of confluent cells after second passage at a) 20× and b) 10×.


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many of which are enzymatic methods. For example, Zuk et al. isolated adipose-derived MSCs from raw human lipoaspirates in this way. In this method, raw aspirates were washed with sterile phosphate-buffered saline (PBS) to remove contaminating debris and red blood cells. Washed aspirates were treated with 0.075% collagenase (Type I) in PBS for 30 min at 37 °C with gentle agitation. The collagenase was inactivated and the cellular pellet was centrifuged for 10 min, resuspended in culture medium, and filtered through a 100-µM mesh filter to remove debris; the cells were then cultured (2). In another study, fatty tissue was harvested from the abdominal subcutaneous tissue of New Zealand white rabbits. The fat was minced and digested with trypsin and collagenase supplemented with albumin for 2.5 h in a 37 °C water bath with a magnetic stir bar. The cells were then centrifuged at 1500 × g for 10 min and the cell pellet was washed and cultured (5). Cao et al. isolated adipose tissue-derived MSCs from human raw lipoaspirates by using an enzymatic digestion method with 0.2% collagenase (6). Katz et al. isolated MSCs from human subcutaneous adipose tissue by treating it with enzyme solution (17). In that study, the dissociated tissue was then filtered to remove debris and the resulting cell suspension was centrifuged. In order to lyse erythrocytes, the cell suspension was treated with an osmotic buffer. In the most recent studies, MSCs were isolated using the collagenase digestion method and red blood cells were removed by treating cells with a lysis buffer (18,19).

In contrast to these studies, in the present study, neither the collagenase nor trypsin digestion methods were used, adipose tissue was not filtered, and a lysis buffer was not used to remove red blood cells. Instead, MSCs were isolated directly using the explant culture method. Therefore, this study has some important advantages in that it is easy, inexpensive, and safe (in accordance with not using any enzymatic method).Cell characterizationAdipose tissue-derived MSCs have a specific surface protein expression, like bone marrow MSCs. Surface protein expression analysis, which allows for a rapid identification of a cell population, has been extensively used (2,20).

In this study, CD13 and CD29 expression was used to identify MSCs. The result of immunofluorescence staining showed that these cultured cells were positive for CD13 and CD29 (Figures 5a and 5b), and these molecules are surface markers for the characterization of MSCs (21,22).

These molecules are not only markers, but are also receptors that play a role in angiogenesis (23). Therefore, we suggest that they could play a role in forming new collaterals and in the improvement of the ischemic myocardium when transplanted into the damaged tissue.

The results of flow cytometry analysis showed that these cells were positive for CD29, CD90, CD54, and MHC Class I, and negative for CD45, CD106, and MHC Class II. The minimum homogeneity of

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Figure 5. Morphological appearance of cells after immunofluorescence staining: a) CD13 staining at 20×, b) CD29 staining at 10×.


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the mesenchymal stem cells was 98.68% (data not shown).

MSCs must express some receptors of cytokines, extracellular matrix components, and growth factors in order to adhere, proliferate, differentiate, and migrate. CD29 (integrin β1) plays a role in MSC adhesion and migration (24). CD90 (Thy-1) is a glycophosphatidylinositol (GPI)-anchored cell surface protein. This protein is a member of the immunoglobulin superfamily and plays a role in cell adhesion, extravasation, and migration. CD90 is commonly used as a positive marker for MSCs (25). CD54 is also known as intracellular cell adhesion molecule-1 (ICAM-1). It plays a role in cell signaling, cell-cell adhesion, and transmigration. CD54 is important for transmigration of MSCs (26). MSCs isolated from adipose tissue express high levels of CD54 (27). MSCs are hypoimmunogenic cells because of the lack in expression of MHC Class II (28). MHC Class II molecules are found only on antigen-presenting cells and lymphocytes. Because of the lack in expression of MHC Class II on MSCs, these cells are hypoimmunogenic cells. Nevertheless, MSCs express the MHC Class I molecules. Although initially it was thought that MSCs could escape natural killer (NK) cells, it was recently shown that NK cells can efficiently lyse MSCs, despite the expression of high levels of HLA-1 by MSCs. However, MHC Class I acts as an inhibitory ligand for NK cells, and MSCs could suppress proliferation of NK cells and decrease the secretion of IFN-g by IL-2-stimulated NK cells. CD45 is a protein

that plays a role in cell growth and differentiation; this molecule is a hematopoietic cell marker and it is not found on MSCs (29). CD106, also known as vascular cell adhesion molecule-1 (VCAM-1), is a protein that mediates the adhesion of lymphocytes, monocytes, eosinophils, and basophils to vascular endothelium. CD106 is negative for MSCs and it is a hematopoietic and endothelial cell marker (30).

Recent studies have shown that MSCs give good results in in vivo applications. However, it is not understood how they do this: by cytokines or by differentiation. This is a question that remains to be answered.

AcknowledgmentThis study was supported by the Kırıkkale University Research Fund (Project Number: 2010/15). We also thank Asım Niyaz for his support with the revision of the article.

Corresponding author:Özer Aylin GÜRPINARDepartment of Biology, Faculty of Science, Hacettepe University, 06800 Beytepe, Ankara - TURKEYE-mail: [email protected]

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