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1.
Primary cultures of unilocular fat cells: Characteristics of growth in vitro and changes in differentiation properties 总被引:6,自引:0,他引:6
Hajime Sugihara Nobuhisa Yonemitsu Shinichi Miyabara Kankatsu Yun 《Differentiation; research in biological diversity》1986,31(1):42-49
Mature white fat tissue consists primarily of unilocular fat cells. Clearly, the study of the biology of these cells would be most helpful for the elucidation of the mechanism of obesity. We describe a new method termed 'ceiling culture' for culturing in vitro unilocular fat cells obtained from humans and rats. These cells can be maintained in culture for long periods of time and, under such conditions, continue to exhibit specific functions such as lipogenesis and lipolysis. Under the culture conditions described, unilocular fat cells change into multilocular fat cells or cells with a fibroblast-like appearance. These cells then proliferate, form a cell monolayer attached to the substratum, and after becoming confluent, exhibit accumulations of intracytoplasmic lipid droplets. These attached dedifferentiated cells continue to exhibit lipogenesis and lipolysis. 相似文献
2.
Proliferation of unilocular fat cells in the primary culture 总被引:8,自引:0,他引:8
Mature white fat cells (unilocular fat cells) have generally been considered to be in terminal differentiation and, hence, to have no proliferative ability. A new method, referred to as "ceiling culture," has been devised in our laboratory to culture unilocular fat cells in vitro. Under such culture conditions, the fat cells continue to exhibit specific functions of lipid metabolism and proliferate extensively. Intracytoplasmic lipid droplets did not inhibit division of the cells. There were two modes of proliferation of unilocular fat cells: "loculus-dividing" cell division, in which the single loculus of fat in the dividing cell was broken down into multiple droplets and distributed evenly between the daughter cells, and "loculus-preserving" cell division, in which the loculus in the dividing cell was minimally broken down and inherited with its shape preserved by one of the daughter cells with the other getting only a small number of fine lipid droplets. Such findings suggest that unilocular fat cells in mature fat tissue in vivo are probably capable of proliferation in such modes under some conditions. 相似文献
3.
Generation of mature fat pads in vitro and in vivo utilizing 3-D long-term culture of 3T3-L1 preadipocytes 总被引:1,自引:0,他引:1
Fischbach C Spruss T Weiser B Neubauer M Becker C Hacker M Göpferich A Blunk T 《Experimental cell research》2004,300(1):54-64
Tissue-inherent factors such as cell-cell and cell-extracellular matrix interactions are regarded to exert a potentially large impact on adipogenesis as well as on secretory functions of adipose tissue. However, an appropriate 3-D adipogenesis model useful for addressing such interactions is still lacking. In this study, using tissue-engineering techniques, we demonstrate for the first time the development of coherent fat pads consisting of unilocular signet-ring cells in vitro. The constructs were generated by differentiating 3T3-L1 preadipocytes on 3-D polymeric scaffolds for either 9, 21, or 35 days in vitro. Only long-term culture yielded uniform tissues histologically comparable to native fat. Light and scanning electron microscopy provided direct evidence of 3-D tissue coherence and cell-cell contact in a tissue context, which was in strong contrast to conventional 2-D monolayer culture. Further differences between the two culture systems included enhanced secretion of leptin in 3-D tissue culture and differences in laminin expression (mRNA and protein level). Increase of triglyceride content over culture time and mRNA expression of other adipocyte genes, such as PPARgamma and Glut-4, were found to be similar. Implantation of long-term differentiated tissue constructs in nude mice resulted in further development and maintenance of fat pads. The presented model system is suggested to contribute to a better understanding of adipose tissue development and function facilitating studies on tissue-inherent interactions in vitro and in vivo. 相似文献
4.
Jeffrey R. Laduca Dilip K. Sinha 《In vitro cellular & developmental biology. Animal》1996,32(4):204-210
Summary Chemical carcinogenesis is a lengthy process that involves the rather loosely defined stages of initiation, promotion, and
progression. Several model systems of mammary carcinogenesis have been designed to elucidate the mechanisms of chemical carcinogenesis.
Most of these systems have included animal models. While organ specific chemical carcinogenesis can be initiated in these
systems, the subsequent stages of promotion and progression are difficult to study in detail. Investigations onin vitro carcinogenesis have shown transformation of mammalian cells in culture; the transformational event, however, is difficult
to discern within the monolayer culture. We have recently reported the development of anin vitro carcinogenesis system that allows both the initiation as well as the progression of mammary cells in a collagen gel matrix
culture system. The cells transformed by a chemical carcinogen develop into discernible microtumors with the three dimensions
of a collagen gel culture. Isolation of these microtumors from the collagen gel an subsequent culture in monolayer has produced
cells capable of colony formation in soft agar. The present study further characterizes these microtumors originatedin vitro by analysis of cell growth kinetics versus parallel control cells. In addition, flow cytometric and cytogenetic studies have
been performed to investigate the chromosomal stability of these cells. It was also observed that the microtumors, producedin vitro from mammary epithelial cells of an inbred strain of rats, show the ability to form tumors upon transplantation into the
fat pad of syngeneic hosts. 相似文献
5.
M Néchad 《Experimental cell research》1983,149(1):119-127
The stroma of mature brown fat has been shown to contain cells which can proliferate and accumulate fat in monolayer cultures, and which have inherent characteristics distinct from those of white fat precursor cells. The purpose of the present investigation was to characterize by electron microscopic analysis these brown fat cells and their subsequent development when they were grown in vitro. By comparison with the existing ultrastructural data on brown fat in situ, it could thus be determined whether or not the precursor cells have the capacity to differentiate in culture. The stromal-vascular fraction isolated from the brown fat of weaned rats was identified as containing adipocyte stem cells, preadipocytes, endothelial cells and a few mature adipocytes. During the first week in culture (i.e., growth phase to confluence), when multilocular fat accumulation occurred, the mitochondria of the preadipocytes developed cristae and matrix granules, as they do in differentiating brown fat in situ. Such granules have been shown to be a sign of intense inner membrane synthetic activity. After confluence, the mitochondria regressed in internal structure and became morphologically more similar to white fat mitochondria. It was concluded that mature brown fat contains precursor cells which can differentiate in vitro. However, this differentiation was incomplete, and the necessity of specific factors for a full mitochondrial development in brown fat is discussed. 相似文献
6.
《Organogenesis》2013,9(2):50-56
Adipose tissue consists of mature adipocytes, preadipocytes and mesenchymal stem cells (MSCs), but a culture system for analyzing their cell types within the tissue has not been established. We have recently developed “adipose tissue-organotypic culture system” that maintains unilocular structure, proliferative ability and functions of mature adipocytes for a long term, using three-dimensional collagen gel culture of the tissue fragments. In this system, both preadipocytes and MSCs regenerate actively at the peripheral zone of the fragments. 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. Thus, it seems to be a promising model for investigating adipose tissue biology and regeneration. In this article, we introduce adipose tissue-organotypic culture, and propose two theories regarding the mechanism of tissue regeneration that occurs specifically at peripheral zone of tissue fragments in vitro. 相似文献
7.
An important application of primary hepatocyte cultures is for hepatotoxicity research. In this paper, gel entrapment culture of rat hepatocytes in miniaturized BAL system were evaluated as a potential in vitro model for hepatotoxicity studies in comparison to monolayer cultures. After exposure for 24 and 48 h to acetaminophen (2.5 mM), gel entrapped hepatocytes were more severely damaged than hepatocyte monolayer detected by methyl thiazolyl tetrazolium (MTT) reduction, intracellular glutathione (GSH) content, reactive oxygen species (ROS) levels, urea genesis and albumin synthesis. CYP 2E1 activities detected by 4-nitrocatechol (4-NC) formation were higher in gel entrapped hepatocytes than in hepatocyte monolayers while the addition of CYP 2E1 inhibitor, diethyl-dithiocarbamate (DDC), more significantly reduced acetaminophen-induced toxicity in gel entrapped hepatocytes. In addition, protective effects of GSH, liquorice extract and glycyrrhizic acid against acetaminophen hepatotoxicity were clearly observed in gel entrapped hepatocytes but not in hepatocyte monolayer at an incubation time of 48 h. Overall, gel entrapped hepatocytes showed higher sensitivities to acetaminophen-induced hepatotoxicity than hepatocyte monolayer by a mechanism that higher CYP 2E1 activities of gel entrapped hepatocytes could induce more severe acetaminophen toxicity. This indicates that gel entrapped hepatocytes in hollow fiber system could be a promising model for toxicological study in vitro. 相似文献
8.
J. L. Vierck J. P. McNamara M. V. Dodson 《In vitro cellular & developmental biology. Animal》1996,32(9):564-572
Summary The responsiveness of progeny of sheep-derived unilocular fat cells (adipofibroblasts) to dexamethasone, insulin, insulinlike
growth factor I (IGF-I), growth hormone (GH), and basic fibroblast growth factor (FGF) was determined in a clonal culture
system. Primary cultures of mature adipocytes were obtained from intermuscular adipose tissue (semimembranosus/semitendinosus
seam depot) of sheep by ceiling culture techniques. Following degeneration of unilocular fat droplets and re-establishment
of fibroblasticlike adipofibroblasts, all adipofibroblasts adhering to upper flask surfaces were collected and isolated away
from fibroblasts (which had no multilocular vesicles) by Percoll? gradient centrifugation. Progeny derived from a single adipofibroblast were isolated and tested for the ability to proliferate,
differentiate, and accumulate lipids. Stock cultures of adipofibroblasts reached confluence in 5 d and were induced to differentiate
from 7 to 9 d with dexamethasone-methyl isobutylxanthine-insulin (DMI). Incubation with insulin, IGF-I, GH, or FGF prior to
confluence followed by induction with DMI produced no direct (priming) effect on subsequent differentiation. When substituted
individually in place of DMI during the 2 d differentiation/induction period, all factors induced differentiation of cultured
adipofibroblasts as determined by lipogenesis (P<.05) and lipoprotein lipase activity (P<.05). Thus, isolated adipofibroblasts from sheep muscle may be induced by hormones and growth factors to display mature adipocyte
morphology in cell culture. Further definition of the adipofibroblast culture system may aid in the identification of mechanisms
regulating adipocyte development in sheep skeletal muscle, as well as in the study of intercommunication between fat and muscle
cells.
With technical assistance from B. Mathison. 相似文献
9.
Shuji Toda Kazuyoshi Uchihashi Shigehisa Aoki Emiko Sonoda Fumio Yamasaki Meihua Piao Akifumi Ootani Nobuhisa Yonemitsu Hajime Sugihara 《Organogenesis》2009,5(2):50-56
Adipose tissue consists of mature adipocytes, preadipocytes and mesenchymal stem cells (MSCs), but a culture system for analyzing their cell types within the tissue has not been established. We have recently developed “adipose tissue-organotypic culture system” that maintains unilocular structure, proliferative ability and functions of mature adipocytes for a long term, using three-dimensional collagen gel culture of the tissue fragments. In this system, both preadipocytes and MSCs regenerate actively at the peripheral zone of the fragments. 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. Thus, it seems to be a promising model for investigating adipose tissue biology and regeneration. In this article, we introduce adipose tissue-organotypic culture, and propose two theories regarding the mechanism of tissue regeneration that occurs specifically at peripheral zone of tissue fragments in vitro.Key words: adipose tissue-organotypic culture, three-dimensional, tissue fragments, peripheral zone, central zone, mature adipocytes, preadipocytes (immature adipocytes), mesenchymal stem cells, adipokines, tissue regeneration 相似文献
10.
H. Z. Kupchik R. S. Langer C. Haberern S. El Deriny M. O''Brien 《Experimental cell research》1983,147(2):454-460
A method has been developed which allows, for the first time, the in vitro growth of human tumor cells in calcium alginate gel microcapsules. The three-dimensional matrix is permeable to macromolecules of at least 200 000 molecular weight (MW) (e.g., carcinoembryonic antigen) and provides an environment for organized growth and differentiation more comparable to in vivo growth than monolayer culture. This inexpensive, nondestructive procedure allows studies of morphological, biochemical and immunochemical parameters of human tumor cells within a single model. 相似文献
11.
12.
It has been reported that 3-D cultures of hepatocytes or HepG2 cells were less susceptible to methotrexate (MTX) than their 2-D counterparts. Such a mechanism was addressed in this study by investigation of MTX hepatotoxicity in gel entrapped (3-D) rat hepatocytes vs. traditional monolayer culture (2-D). Similarly, gel entrapped hepatocytes showed higher drug resistance to MTX than hepatocyte monolayers in whatever culture medium with or without modification by hormone supplements (dexamethasone, glucagon and insulin). It was also found that medium modification by hormones greatly increased drug resistance of hepatocyte monolayers but has only a slight effect on 3-D cultured hepatocytes. These differential MTX toxicities regarding culture medium and culture models were assumed to correlate with multidrug resistance associated protein 2 (Mrp2). The involvement of Mrp2 was confirmed directly by the fact that MTX intracellularly accumulated less in gel entrapped hepatocytes than in hepatocyte monolayer but could be enhanced by Mrp2 inhibitors accompanied by reduced drug resistance. Furthermore, the expression of Mrp2 on gene level and transportation activity together with bile-duct-like structure were more significantly evidenced in 3-D gel entrapment culture than in 2-D monolayer culture. In conclusion, the highly preserved Mrp2 in 3-D gel entrapped hepatocytes determines its high drug resistance to MTX. Gel entrapped hepatocytes could be useful for investigation of hepatic transportation and hepatotoxicity. 相似文献
13.
The aim of this study was to examine the effects of Vero cells and other somatic cells on in vitro maturation of bovine oocytes. Both denuded oocytes and oocytes with intact cumuli (COCs) were cultured on monolayer of Vero cells, cumulus cells and granulosa cells. The effect of gonadotropins was investigated after the addition of gonadotropins to the culture medium. The evaluation using analysis of variance revealed that removal of cumulus cells generally reduced the percentage of oocytes completing their maturation in vitro and that this effect could not be overcome by the addition of gonadotropins to the culture medium. However, in individual experiments, when oocytes were co-cultured with different monolayers of somatic cells, Vero cells were able significantly support the maturation of denuded oocytes, and their beneficial effect was further enhanced by the addition of gonadotropins (76 vs 80.9%). We did not observe a similar effect after the co-culture of oocytes with a monolayer of cumulus cells (65.3 and 53%, respectively). Granulosa cell monolayer delayed maturation in the both COCs and denuded oocytes (10.5 and 16.5%, respectively). In vitro fertilization was successful in most of the experimental groups. However, when denuded oocytes were cultured without any somatic cell support, they did not decondense the penetrated sperm head after in vitro fertilization. This study demonstrates that 1) Vero cells beneficially affect the in vitro maturation of bovine oocytes; 2) cumulus cells in the form of monolayer lose their beneficial influence on in vitro maturation of bovine oocytes; and 3) granulosa cells and FSH and LH alone (without somatic cells) do not show positive effects on in vitro maturation of bovine oocytes. 相似文献
14.
The aim of the present work was to study the morphological characteristics of neonatal adipose tissue using rats as an animal model. The results revealed that the subcutaneous adipose tissue of newborns consists of packets of unilocular adipose cells (one large lipid drop occupying the whole cell and pushing the cytoplasm and the nucleus to the cell periphery) and some multilocular fat cells (several lipid droplets of different size and an almost centrally located nucleus). All the adipocytes demonstrated positive immunohistochemical expression for leptin, whereas the multilocular adipose cells were positive for cyclin D1. These findings suggest that the multilocular adipose cells are preadipocytes that have not yet finished proliferation and differentiation and could under some external and/or internal stimuli conclude their development and become mature unilocular adipocytes, thus increasing fat mass. 相似文献
15.
Rapid, Direct Tissue Culture Test for Toxigenicity of Corynebacterium Diphtheriae 总被引:4,自引:0,他引:4
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A method for testing toxigenicity of Corynebacterium diphtheriae in tissue culture is described. The technique, called the colony overlay test (COT), involves inoculating material from an isolated colony of C. diphtheriae to a small area on the surface of an agar medium which overlays a monolayer of toxin-susceptible HeLa cells. If toxin is produced during incubation at 37 C, it diffuses to the tissue monolayer and destroys the cells below the inoculation site. Twenty-four hours after inoculation, organisms are killed and tissue cells are fixed with formaldehyde. The agar overlay is then removed, and the monolayer is stained with crystal violet. Toxin-affected areas fail to stain or stain poorly. A second plate with antitoxin incorporated in the overlay serves as a control for specificity. Forty-eight strains of C. diphtheriae were tested by the COT, guinea pig, and in vitro, gel diffusion tests. The COT is as specific as the other two tests, is easy to read, and can be used to test large numbers of isolates for toxin action more conveniently than by animal inoculation. 相似文献
16.
O Kuperman G W Fortner Z J Lucas 《Journal of immunology (Baltimore, Md. : 1950)》1975,115(5):1277-1281
A method is described for the consistent in vitro generation cytotoxic cells by incubating Fischer 344 rat spleen cells on monolayers of a syngeneic mammary adenocarcinoma. Significant cytotoxicity by in vitro culture is generated as early as 3 days after initiation and effector cells are cytolytic only toward target cells of the sensitizing monolayer. Reciprocal sensitization with allogeneic fibroblasts as the immunizing monolayer yielded effector cells cytolytic for the fibroblasts but without effect on the mammary tumor. The consistency in the generation of cytotoxic cells by in vitro culture should permit its standardized use in following other related immune phenomena such as blocking by serologic factors and suppression, recritment of memory for cytotoxic function. 相似文献
17.
A. M. Tsoy D. S. Zaytseva-Zotova E. F. Edelweiss A. Bartkowiak J. -L. Goergen E. L. Vodovozova E. A. Markvicheva 《Biochemistry (Moscow) Supplemental Series B: Biomedical Chemistry》2010,4(3):243-250
To generate multicellular tumor spheroids (MTS) based on human breast adenocarcinoma MCF-7 cells and to study them as a novel
in vitro model for anticancer drug screening, a technique for cell microencapsulation in biocompatible alginate-chitosan microcapsules
has been used in this study. Using the MTS based on the MCF-7 cells methotrexate (MTX) cytotoxicity has been investigated.
A set of MTS with an average size of 150, 200 and 300 μm was prepared as a function of cultivation time. Cell viability was
evaluated after MTS incubation in cultivation medium containing various MTX concentrations (1, 2, 10, 50 and 100 nM) for 48
h. MTS were shown to be markedly more resistant to MTX than the monolayer culture. The increase of the spheroid size was in
correlation with the enhanced MTS resistance to MTX. Thus, at 100 nM MTX a number of viable cells in MTS with the size of
300 μm was 2.5-fold higher than that in the monolayer culture. It is suggested that the cells microencapsulated into MTS can
better mimic cell behavior in small solid tumors compared to the monolayer culture. In the future MTS could be proposed as
a novel in vitro model for anticancer drug screening. 相似文献
18.
Yuichi Adachi Tadashi Mio Keiichi Takigawa Ilja Striz Debra J. Romberger John R. Spurzem Stephen I. Rennard 《In vitro cellular & developmental biology. Animal》1998,34(3):203-210
Summary
In vivo, fibroblasts are distributed in a three-dimensional (3-D) connective tissue matrix. Fibronectin is a major product of fibroblasts
in routine cell culture and is thought to regulate many aspects of fibroblast biology. In this context, we sought to determine
if the interaction of fibroblasts with a 3-D matrix might affect fibronectin production. To examine this hypothesis, fibronectin
production by fibroblasts cultured in a 3-D collagen gel or on plastic dishes was measured by ELISA. Fibroblasts in 3-D gel
culture produced more fibronectin than those in monolayer culture. Fibroblasts in 3-D culture produced increasing amounts
of fibronectin when the collagen concentration of the gel was increased. The 3-D nature of the matrix appeared to be crucial
because plating the fibroblasts on the surface of a plastic dish underneath a collagen gel was not different from plating
them on a plastic dish in the absence of collagen. In addition to increased fibronectin production, the distribution of the
fibronectin produced in 3-D culture was different from that of monolayer culture. In monolayer culture, more than half of
the fibronectin was released into the culture medium. In 3-D culture, however, approximately two-thirds remained in the collagen
gel. In summary, the presence of a 3-D collagen matrix increases fibroblast fibronectin production and results in greater
retention of fibronectin in the vicinity of the producing cells. 相似文献
19.
Ke Zheng Tian-Qing Liu Ming-Shu Dai Dan Ge Xiang-Qin Li Xue-Hu Ma Zhan-Feng Cui 《Cytotechnology》2006,52(3):209-218
Neural stem cells (NSCs) can be cultured in two modes of suspension and monolayer in vitro. The cultured cells are different
in both the ability to proliferate and heterogeneity. In order to find the appropriate methods for large-scale expansion of
NSCs, we systematically compared the NSCs cultured in suspension with those cultured in monolayer. The forebrain tissue was
removed from embryonic day 14 (E14) mice, then the tissue was dissociated into single-cell suspension by Accutase and mechanical
trituration. The cells were cultured in both suspension and monolayer. The NSCs cultured in suspension and in monolayer were
compared on viability, ability to proliferate and heterogeneity by fluorescent dyes, immunofluorescence and flow cytometry
on DIV21 (21 days in vitro), DIV56 and DIV112, respectively. The results indicated that the NSCs cultured in both suspension
and monolayer represented good viability in long-term cultures. But they displayed a distinct ability to proliferate in long-term
cultures. The NSCs cultured in monolayer preceded those cultured in suspension on the ability to proliferate on DIV21 and
DIV56, but no obvious difference on DIV112. The NSCs population cultured in suspension displayed more nestin-positive cells
than those in monolayer during the whole process of culture. The NSCs population cultured in monolayer, however, displayed
more βIII tubulin-positive cells than those in suspension in the same period. The suspension culture mode excels the monolayer
culture mode for large-scale expansion of NSCs. 相似文献
20.
Jean Mauchamp Aline Mirrione Christian Alquier Frdric Andr 《Biology of the cell / under the auspices of the European Cell Biology Organization》1998,90(5):369-380
The thyroid follicle, the morphofunctional unit of thyroid gland, is a spheroidal structure formed by a monolayer of polarized cells surrounding a closed cavity in which thyroglobulin accumulates. Newly isolated porcine thyroid cells reorganize into two types of structures which differ by the orientation of cell polarity: in follicle-like structures, obtained in the presence of TSH, the apical pole delineates a closed cavity and cells express most parameters characteristic of thyroid function; in inside-out follicles the apical pole is oriented towards the culture medium and cells do not express properly the thyroid function. The organization of newly formed follicles can be modified by stimulation of cell migration or by interaction of their apical poles with a new cell environment. Seeded on a hard surface (glass, plastic), cells of follicle-like structures or inside-out follicles formed in suspension migrate giving a monolayer. On the contrary, cells organized into a monolayer treated with hexamethylene bisacetamide, reorganize into follicle-like structures. Inside-out structures reoganize upon interaction of their apical poles with collagen I gel, a coherent matrix, or with a reconstituted basement membrane (RBM), a soft matrix. Overlaid with collagen I, monolayers reorganize into follicles. Embedded in collagen I or in RBM, inside-out follicles reorient their polarity giving functional follicles. On the RBM surface, cells pull on the gel and embed themselves in the soft matrix gel, finally reorienting their polarity to inside-in polarity. When comparing thyroid cells with other epithelial cell types (mammary cells, Sertoli cells), it appears that the obtention in culture of follicle-like structures, ie closed inside-in polarized cell organization, is the best way to express in culture both morphology and function of any specific epithelial tissue, the polarized monolayer in porous bottom culture chamber coming just behind. 相似文献