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Endocrinology Vol. 149, No. 10 4794-4798
Copyright © 2008 by The Endocrine Society

A New Organotypic Culture of Adipose Tissue Fragments Maintains Viable Mature Adipocytes for a Long Term, Together with Development of Immature Adipocytes and Mesenchymal Stem Cell-Like Cells

Emiko Sonoda, Shigehisa Aoki, Kazuyoshi Uchihashi, Hidenobu Soejima, Sachiko Kanaji, Kenji Izuhara, Seiji Satoh, Noboru Fujitani, Hajime Sugihara and Shuji Toda

Departments of Pathology and Biodefense (E.S., S.A., K.U., S.T.), Biomolecular Sciences (H.S., S.K., K.I.), and Surgery (S.S.), Faculty of Medicine, Saga University, Saga 849-8501, Japan; Chiba Institute of Science (N.F.), Chiba 288-0025, Japan; and International University of Health and Welfare (S.H.), The School of Rehabilitation Sciences, Fukuoka 831-8501, Japan

Address all correspondence and requests for reprints to: Miss Emiko Sonoda, Master of Medicine/Dr. Shuji Toda, Department of Pathology and Biodefense, Faculty of Medicine, Saga University, Nabeshima 5-1-1, Saga 849-8501, Japan. E-mail: todas{at}cc.saga-u.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Adipose tissue that consists of mature and immature adipocytes is suggested to contain mesenchymal stem cells (MSCs), but a culture system for analyzing their cell types within the tissue has not been established. Here we show that three-dimensional collagen gel culture of rat sc adipose tissue fragments maintained viable mature adipocytes for a long term, producing immature adipocytes and MSC-like cells from the fragments, using immunohistochemistry, ELISA, and real time RT-PCR. Bromodeoxyuridine uptake of mature adipocytes was detected. Adiponectin and leptin, and adipocyte-specific genes of adiponectin, leptin, and PPAR-{gamma} were detected in culture assembly, whereas the lipogenesis factor insulin (20 mU/ml) and inflammation-related agent TNF-{alpha} (2 nM) increased and decreased, respectively, all of their displays. Both spindle-shaped cell types with oil red O-positive lipid droplets and those with expression of MSC markers (CD105 and CD44) developed around the fragments. The data indicate that adipose tissue-organotypic culture retains unilocular structure, proliferative ability, and some functions of mature adipocytes, generating both immature adipocytes and CD105+/CD44+ MSC-like cells. This suggests that our method will open up a new way for studying both multiple cell types within adipose tissue and the cell-based mechanisms of obesity and metabolic syndrome.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ADIPOSE TISSUE IS a specific organ that stores excess energy in the form of lipid droplet. Recently, adipose tissue is suggested to be an endocrine organ that affects the biological behavior of various cell types through its production of adipokines (1). In general, adipose tissue consists of mature and immature adipocytes and endothelial cells, but it also has been shown to contain mesenchymal stem cells (MSCs) that produce various mesenchymal cell types, e.g. adipocytes, osteoblasts, myocytes, and chondrocytes (2). Thus, adipose tissue with multiple cell types above seems critical for the maintenance of body homeostasis. However, a culture method for analyzing multiple cell types within adipose tissue has not been established. One of the reasons for this is the difficulty in culturing adipose tissue, which has large lipid droplets and thus does not attach to the surface of culture dish due to its buoyancy in culture medium.

To challenge this interesting issue and to overcome the difficulty in culturing adipose tissue, we developed a new culture system of adipose tissue fragments embedded in a three-dimensional collagen gel, which is able to easily entrap buoyant adipose tissue.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Culture system
All procedures involving animals were performed in accordance with the regulations laid down by the ethical guidelines of Saga University. We mainly used sc adipose tissue from 1-wk-old Wistar rats, but in part the tissue from 6-month-old rats was used as a representative of older rats. After rinsing with MEM (Nissui Co., Ltd., Tokyo, Japan), the adipose tissue was minced at about 0.5 mm diameter. A total of 0.1 ml minced tissue fragments was embedded in 1.0 ml type I collagen gel solution (Nitta Gelatin Co., Ltd., Osaka, Japan), as previously described (3), and cultured in Ham F-12 medium supplemented with 10% fetal calf serum and 50 µg/ml gentamicin. Culture medium was exchanged for fresh medium every 2 d. Figure 1Go illustrates this method. In some cases, 20 mU/ml insulin (Sigma-Aldrich, Inc., St. Louis, MO) was added to the culture medium every medium change, because insulin is a lipogenesis-promoting factor (4). To estimate triglyceride production, we also measured its quantity in the supernatants of 1-wk cultures with or without 20 mU/ml insulin stimulation for 48 h by the standard methods, as described previously (5).


Figure 1
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  		FIG. 1. Three-dimensional collagen gel culture system of adipose tissue fragments named adipose tissue-organotypic culture and its organization procedure. Subcutaneous adipose tissue of Wister rat was minced. The minced tissue fragments (0.5 mm in diameter) were embedded in type I collagen gel and cultured in complete medium. The left lower panel indicates an adipose tissue fragment just after being embedded in collagen gel.

 
Morphology and morphometric analysis
We examined the component cell types of adipose tissue fragments with hematoxylin-eosin (H-E) staining, using deparaffinized sections of the cellular layer gel that was fixed with 4% formalin, routinely processed, and embedded in paraffin (6). To detect both mature and immature adipocytes that have lipid droplets, oil red O staining was carried out, as previously described (7). To examine fine structures of the cells, we also performed transmission electron microscopy by the standard method (6).

The cell types in culture assembly were divided into the following cell types: 1) mature adipocytes, 2) immature adipocytes, and 3) MSC-like cells. The cell type with a single large lipid droplet was judged as mature adipocytes (7). The polygonal to spindle-shaped cell type with fine lipid droplets was determined as immature adipocytes (5). The spindle-shaped cell type, both without lipid droplets and with expression of some MSC markers of CD44 and CD105 (2, 8, 9), was judged as MSC-like cells. According to the criteria, we counted the total number of both immature adipocytes and MSC-like cells per five adipose tissue fragments on the staining sections with both histochemistry and immunohistochemistry. The values represent the number per one tissue fragment. The percentage of immature adipocytes was calculated by the formula (number of immature adipocytes)/(total number of immature adipocytes and MSC-like cells) x 100 (percent), whereas that of MSC-like cells was calculated by the formula (number of MSC-like cells)/(total number of immature adipocytes and MSC-like cells) x 100 (percent). Finally, we estimated the formation of bone, cartilage, and muscle tissues, which may be organized by MSC-like cells, on the basis of their specific morphology (10), using H-E staining of sections of culture assembly.

Cell proliferation
We examined the growth of cell types within adipose tissue fragments at 1 wk in cultures with or without 20 mU/ml insulin, using immunohistochemistry with bromodeoxyuridine (BrdU, Cell Proliferation Kit; Amersham, Arlington Heights, IL), after 48 h incubation with 3 µg/ml BrdU (11). Insulin stimulation was carried out at culture d 2, 4, and 6. The total number of both BrdU-positive mature and immature adipocytes and MSC-like cells per five adipose tissue fragments was counted according to the same method above. The values represent the number per one tissue fragment. To differentiate the BrdU-positive nuclei of mature and immature adipocytes from those of the other cell types, we performed double immunohistochemistry with BrdU and S-100 protein, as described previously (6), because mature and immature adipocytes, but not MSC-like cells and endothelial cells, expressed S-100 protein (12). To detect BrdU-positive MSC-like cells, immunohistochemistry with BrdU, CD44, and CD105 was carried out by the same method as above.

Immunohistochemistry and immunofluorescence
Adipose tissue contains various cell types: mature and immature adipocytes, endothelial cells, and MSCs (13). To detect both mature and immature adipocytes, rabbit polyclonal S-100 protein antibody (DakoCytomation Co., Ltd., Kyoto, Japan) was used. To identify endothelial cells, rabbit polyclonal CD31 (Lab Vision, Fremont, CA) and mouse monoclonal CD54 (ICAM-1; Pierce Biotechnology, Inc., Rockford, IL) were also used (14). To detect MSC-like cells (8, 9), mouse monoclonal CD44 (Cedarlane Laboratories Ltd., Hornby, Ontario, Canada) and CD105 antibodies (NovoCastra Laboratories, Newcastle upon Tyne, UK) were used. Deparaffinized sections were immunostained by the avidin-biotin complex immunoperoxidase (ABC) method, as described previously (6).

To confirm colocalization of CD44 and CD105, we carried out a double-color immunofluorescence analysis with deparaffinized sections, using rhodamine- or fluorescein isothiocyanate (FITC)-conjugated avidin (DakoCytomation), as previously described (15). Briefly, CD44 was immunostained in red by rhodamine according to the ABC method. Then, the section was heated in 0.01 M citrate buffer for 10 min at 90 C. Thereafter, CD105 was immunostained in green with FITC by the ABC method. Rhodamine and FITC fluorescence was detected by a confocal laser scanning microscope (LSM 5 PASCAL; Carl Zeiss Co., Ltd., Oberkochen, Germany).

Total RNA extraction and real-time RT-PCR
We studied gene expression of adiponectin, leptin, and PPAR{gamma} at the mRNA level, using real-time RT-PCR. We extracted total RNA from cultured adipose tissues at 1 wk in culture, using ISOGEN (Nippon Gene, Tokyo, Japan). To increase purity of cDNA, total RNA was reextracted by QIAGEN RNeasy Mini kit (QIAGEN, Valencia, CA). After cDNA (iScript cDNA synthesis kit; Bio-Rad, Carlsbad, CA) was synthesized, real-time PCR was performed in triplicate for each sample with the ABI PRISM 7000 Sequence Detection System (Applied Biosystems, Foster City, CA). Each PCR was conducted in a 20-µl volume of the TaqMan gene expression assays kit (Applied Biosystems, Foster City, CA) (PPAR{gamma}, Rn00440945_m1; leptin, Rn00565158_m1; adiponectin, Rn00595250_m1; β-actin, Rn00667869_m1). Gene expression was normalized as ratio using rat β-actin as internal control.

Adipokine production
We measured adiponectin and leptin in supernatants at 1 wk in culture by ELISA, using rat ELISA kits of adiponectin (assay sensitivity, 50 pg/ml; AdipoGen, Inc., Seoul, South Korea) and leptin (assay sensitivity, 50 pg/ml; BioVendor Laboratory Medicine, Inc., Modrice, Czech Republic). The kit for adiponectin detected its total form.

Effects of several soluble factors on adipokine production and gene expression
We examined effects of the following factors on adipokine production and gene expression above, using the same methods as above: 1) the lipogenesis-promoting factor insulin (Sigma-Aldrich), 2) the lipolysis-related factor β3-agonist BRL-37344 (BRL; Biomol Research Laboratories Inc., Plymouth Meeting, PA), and 3) the inflammation-related agents of rat TNF-{alpha} (R&D Systems, Inc., Minneapolis, MN) and lipopolysaccharide (LPS, Escherichia coli 0127 B8; Sigma-Aldrich). One-week culture assembly stimulated by 20 mU/ml insulin, 0.2 µM BRL (16), 2 nM TNF-{alpha} (17), or 10 µg/ml LPS (16) for 48 h was analyzed by ELISA and real-time RT-PCR above.

Statistical analysis
The data obtained from six to seven independent experiments were analyzed by ANOVA. Values represented the mean ± SD. P < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Adipose tissue-organotypic culture
Just after being embedded in gel, adipose tissue fragments had mature adipocytes and capillaries (Fig. 2AGo). Mature adipocytes were maintained for more than 4 wk (Fig. 2CGo). Capillary network disappeared by 7 d (Fig. 2BGo). Central parts of the fragments had no drastic change, but their peripheral zones underwent prominent changes as follows. After 2 d, two spindle-shaped cell types began to develop at the peripheral zones of the fragments, and thereafter they actively grew (Fig. 3AGo). The one cell type had fine lipid droplets that indicate immature adipocytes (Fig. 3Go, A, B, and D), whereas the other cell type did not show any lipid droplets (Fig. 3Go, A and C). We characterized the phenotype of the cell type without lipid droplets, as described below.


Figure 2
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  		FIG. 2. Histology of adipose tissue fragment in adipose tissue-organotypic cultures of 1-wk-old (A–C) and 6-month-old (D) rat materials. A, Adipose tissue fragment immediately after being embedded in collagen gel has many mature adipocytes that have a large single lipid droplet and a peripherally located nucleus and hence have been called unilocular fat cells. Also, capillary network (arrowheads and inset) is seen in narrow stroma among mature adipocytes. Note that erythrocytes are seen in capillaries. B, At 1 wk in culture, many mature adipocytes are clearly maintained at the center of the tissue fragment, and they have no drastic morphological change. However, capillary network disappears within the tissue fragments. Spindle-shaped cells do not develop at the central part of the fragment. C, Even at 4 wk in culture, mature adipocytes are well retained within the tissue fragment, and spindle-shaped cells are also not observed at the center. D, Mature adipocytes within the tissue fragments derived from 6-month-old rats are also well retained at 1 wk in culture. Notice that the size of 6-month-old rat-originated mature adipocytes is about 1.5–2 times that of 1-wk-old rat-derived mature adipocytes (B). A–D, H-E staining.

 

Figure 3
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  		FIG. 3. Development of two kinds of spindle-shaped cell types at the peripheral parts of adipose tissue fragments derived from 1-wk-old rats. A, At 2 wk in culture, two kinds of spindle-shaped cell types appear actively at the peripheral part of the fragment. One spindle-shaped cell type (arrowheads) has fine lipid droplets in the cytoplasm, whereas another type (arrows) has no lipid droplet. Spindle-shaped cell type with lipid droplets (B) is judged as immature adipocytes (preadipocytes), whereas spindle-shaped cell type without lipid droplets (C) is determined as mesenchymal cells other than immature adipocytes, suggesting its possibility of MSCs. D, Lipid droplets (red) are confirmed by oil red O staining. A–C, H-E staining.

 
Insulin (20 mU/ml) increased immature adipocyte number (supplemental data 1, published as supplemental data on The Endocrine Society’s Journals Online web site at http://endo.endojournals.org). Triglyceride (0.051 ± 0.013 mg/ml) was detected in cultures without 20 mU/ml insulin, whereas insulin increased it (0.16 ± 0.021 mg/ml). Finally, we also cultured 6-month-old rat-derived adipose tissue fragments. Mature adipocytes within these fragments were well maintained (Fig. 2DGo), although the development rate of spindle-shaped cell types decreased to about 30% of that of 1-wk-old rat-derived materials.

Characterization of spindle-shaped cells without lipid droplets
We characterized the phenotype of lipid droplet-noncontaining mesenchymal cells above by immunohistochemistry with CD44 and CD105, which are both expressed in adipose tissue-derived MSCs (8, 9). Spindle-shaped cells without lipid droplets clearly displayed CD44 (red) and CD105 (green) (Fig. 4Go). In electron microscopy (supplemental data 2), CD44+/CD105+ mesenchymal cells had many dilated rough endoplasmic reticula in their cytoplasm without any lipid droplets (18). In contrast, immature adipocytes clearly showed lipid droplets. The ratio of immature adipocytes (85.0 ± 3.4%) to MSC-like cells (15.0 ± 3.4%) was 5.7 to 1 at 1 wk in culture. Finally, we did not detect any of bone, cartilage, and muscle tissues in culture assembly at least on the basis of their specific morphology (10).


Figure 4
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  		FIG. 4. Immunofluorescence analysis of both CD44 and CD105, which are all well known to be expressed in MSCs, in a representative same section (A–C) of adipose tissue-organotypic culture of 1-wk-old rat materials. A, Spindle-shaped cells without lipid droplets express CD44 (red). B, The same cells also express CD105 (green). C, On the same spindle-shaped cells, CD44 and CD105 are merged, suggesting that these CD44+/CD105+ spindle-shaped cells (yellow) without lipid droplets are MSC-like cells. Scale bar, 50 µm.

 
Adipokine production (supplemental data 3)
Production of adiponectin (32.0 ± 1.9 ng/ml) and leptin (149.5 ± 17.0 pg/ml) was detected in cultures without the factors (control), whereas insulin increased about two and three times the production, respectively, of adiponectin (64.0 ± 1.8 ng/ml, P < 0.0001) and leptin (467.7 ± 62.1 pg/ml, P < 0.001). BRL and TNF-{alpha} inhibited the production of both adiponectin (BRL, 23.2 ± 1.6 ng/ml, P < 0.01; and TNF-{alpha}, 22.3 ± 2.3 ng/ml, P < 0.01) and leptin (BRL, 37.2 ± 10.1 pg/ml, P < 0.001; and TNF-{alpha}, 29.6 ± 5.7 pg/ml, P < 0.001), whereas LPS did not affect that of adiponectin (31.3 ± 2.6 ng/ml, P = 0.735) and leptin (98.9 ± 32.2 pg/ml, P = 0.074).

Adipose tissue-specific gene expression (supplemental data 4)
All mRNAs of adiponectin, leptin, and PPAR{gamma} were detected in cultures without the factors (control), whereas insulin enhanced the expression of these genes. TNF-{alpha} and LPS inhibited mRNA expression of adiponectin, leptin, and PPAR{gamma}. BRL prohibited mRNA expression of leptin, whereas BRL did not affect that of adiponectin and PPAR{gamma}.

Cell proliferation (supplemental data 5)
The BrdU uptake of both mature and immature adipocytes was clearly detected. Insulin (20 mU/ml) did not promote BrdU intake of mature adipocytes, whereas insulin significantly increased BrdU uptake of immature adipocytes. Insulin decreased BrdU uptake of CD105+/CD44+ MSC-like cells.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Culturing mature adipocytes in vitro has been technically difficult due to their buoyancy in culture medium. To resolve this problem, we showed the following culture systems of isolated mature adipocytes: 1) ceiling culture (7) and 2) three-dimensional collagen gel culture (3). These methods are useful for studying isolated mature adipocytes, but they do not allow us to investigate multiple cell types within adipose tissues. To challenge this issue, we have established a new adipose tissue-organotypic culture that maintains the proliferative ability and function of mature adipocytes for more than 4 wk. Furthermore, both immature adipocytes and CD44+/CD105+ MSC-like cells develop just around adipose tissue fragments. Our method will open up a new way for studying the multiple cell types within adipose tissue.

BrdU uptake of mature adipocytes was prominently lower than that of immature adipocytes, but the uptake of mature adipocytes was detected. This suggests that mature adipocytes themselves retain their proliferative ability, supporting our previous studies (11). The mature adipocytes produced triglyceride, adiponectin, and leptin. Also, they expressed adipocyte-specific genes of PPAR{gamma}, leptin, and adiponectin. All of these functional properties were enhanced by insulin. The data suggest that mature adipocytes in our system undergo normal structural and functional differentiation with their proliferative ability.

We showed that BRL inhibited the production of adiponectin and leptin, supporting several studies with human, rat, and mouse adipocytes in vitro (19, 20). In our study, TNF-{alpha} suppressed the production of adiponectin and leptin, whereas LPS did not affect their production. One study (21) demonstrated that TNF-{alpha} prohibited the production of adiponectin and leptin in human adipocytes. Another study (22) showed that TNF-{alpha} increased leptin production in mouse adipocytes, whereas LPS did not affect it. Although the precise reason for this discrepancy regarding TNF-{alpha}-affected production of leptin is unclear, differences of species and culture systems may contribute to the discrepancy. As shown in this study, there is a discrepancy between protein and mRNA expression of adiponectin and leptin by BRL or LPS. Although the precise reason for the discrepancy is unclear, the following possibilities may be involved in the cause of the discrepancy: 1) more rapid change of mRNA than that of protein secretion or 2) regulatory issues of protein secretion and mRNA transcription, translation, and posttranslation.

We did not detect formation of bone, cartilage, and muscle tissues in culture assembly (10). Because insulin increased the number of immature adipocytes in this study, it seems possible that some MSC-like cells may differentiate into immature adipocytes in response to its stimulation. In general, the differentiation of MSCs into various mesenchymal cell types requires various factors (23), but most of these factors were not tested in our study. To elucidate whether MSC-like cells in our system are able to differentiate into various mesenchymal cell types and others (8, 9), additional studies are in order.


   Acknowledgments
 
We thank Messrs. H. Ideguchi, S. Nakahara, and F. Mutoh and Mrs. M. Nishida for their helpful technical assistance and Dr. K. Udo for critical suggestions.


   Footnotes
 
This work was supported in part by Grants-in-Aid from the Japanese Ministry of Education, Culture, Sports, Science, and Technology for Scientific Research No. 18591871 (to S.T.).

Disclosure Statement: The authors have nothing to disclose.

First Published Online June 5, 2008

Abbreviations: ABC, Avidin-biotin complex immunoperoxidase; BrdU, bromodeoxyuridine; BRL, BRL-37344; FITC, fluorescein isothiocyanate; H-E, hematoxylin-eosin; LPS, lipopolysaccharide; MSC, mesenchymal stem cell.

Received April 14, 2008.

Accepted for publication May 27, 2008.


   References
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 Introduction
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