WO1998033889A1

WO1998033889A1 - Adipocyte culture

Info

Publication number: WO1998033889A1
Application number: PCT/US1998/001989
Authority: WO
Inventors: Christopher J. Lynch
Original assignee: The Penn State Research Foundation
Priority date: 1997-01-31
Filing date: 1998-01-30
Publication date: 1998-08-06
Also published as: AU6054698A

Abstract

A method for culturing primary adipocytes suspended in a gelled medium yields multi-cellular adipose clusters having a structure that resembles adipose tissues in vivo. The clusters produced in the method can be surgically implanted subcutaneously and, after implantation, will mature into fat pads that can shore up depressions caused by loss of soft tissue.

Description

ADIPOCYTE CULTURE

CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of Provisional Patent Application Number 60/036,030, filed 01/31/97, entitled "Method of Producing Fat Cell Organoids for Autologous Transplant in Plastic and Reconstructive Surgery" .

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT Not applicable.

BACKGROUND OF THE INVENTION In vivo in animals, adipocytes are not randomly distributed throughout the body, but rather are organized into vascularized and innervated fat pads (or depots) . Animals, particularly mammals, can use the fat pads to efficiently mobilize and deliver fuels to remote locations in the body as needed, to facilitate the storage of excess circulating fuels, to bring the storage and mobilization of these fuels under the control of the central nervous system and, in turn, to provide the CNS with input concerning the adequacy of these stores (9) . Organogenesis, the concerted assembly of adipose, vascular and nervous tissues into fat pads, is a complex process. For instance, to form adipose tissue, the major cellular component of fat pads, mesodermal stem cells commit to a preadipocyte lineage and then preadipocytes differentiate into adipocytes. During differentiation, the genes for fatty acid and triglyceride metabolism are induced. These and other changes lead to rounding and the formation of the intracellular lipid droplet that leads to an obvious cellular morphogenesis. In addition to these more apparent cellular changes, the amount of secreted basement membrane components (such as laminin, proteoglycans and type IV collagen, a networking type of collagen) increases, and the amount of secreted fibrillar type I collagen decreases during differentiation.

Either at the same time or following this cellular morphogenesis, tissue morphogenesis occurs. In tissue morphogenesis newly differentiated or differentiating adipocytes give rise to cellular extensions, migrate from stroma, form cell-cell junctions and organize themselves into spherical clusters (34) called "primitive organs." These primitive organs are observed wherever adipose tissue is eventually found. Sufficient basement membrane components accumulate in the extracellular space during tissue morphogenesis to organize a surrounding basal lamina. An early stage in the formation of the primitive organs must also involve the secretion of factors needed to initiate vascularization and innervation of the forming tissue (i.e., organogenesis) .

Much has been learned about the commitment of mesodermal stem cells and the differentiation (cellular morphogenesis) of preadipocytes by examining in vi tro model systems, e.g., C3H- 10T-1/2 and 3T3-L1 cell lines, respectively (for review see 7) . However, no known in vi tro systems support adipose tissue morphogenesis, and relatively little is known about the formation and maintenance of adipose tissue structure. For instance, it is unclear, for instance, whether the structure of adipose tissue arises simply as a result of adipoblasts localizing into "primitive organs (34)" during early development, or whether the structure is actively maintained and stabilized by adipocyte adhesion molecules involved in cell-matrix and cell-cell interactions.

The culture conditions of many model systems used to study tissue morphogenesis of non-adipose cells include basement membrane components. When cultured on (or suspended in) basement membrane components, a number of cell types migrate and organize into multicellular structures that are similar to tissue or primitive tissue in vivo (13, 20, 25, 26) . In some case cell polarity lost when isolating the cells from tissue can be restored as part of these in vi tro reorganizations. A medium described in US Patent No. 5,405,772 for long-term proliferation and development of cells, which are said to include adipocytes, includes extracellular matrix materials that can be purified components or less pure sources of extracellular matrix such as MATRIGEL^® (Collaborative Biomedical Products, USA, commercially available from

Boehringer Ingelheim Bioproducts Partnership) . The medium of 5,405,772 requires an adherent layer of bone marrow stromal cells, which is said to include adipocytes, as a supporting network for hematopoietic cells. There is no indication in 5,405,772 that the culture conditions can support a suspended culture of adipocytes in the absence of an adherent bone marrow stromal cell layer. Rather, the non-adherent cells are said to be primarily neutrophilic and monocyte/macrophage .

An important use for adipose tissue is as an augmenting filler in surgical procedures for depressions of a body surface resulting from loss of soft tissue. See United States Patent No. 5,681,561. Typically, fat containing primary adipocytes is aspirated from a subcutaneous layer and is injected without culture into subcutaneous tissues that overlie the depression in the body surface. US Patent No. 5,681,561 mentions a number of shortcomings in the use of autologous fat implants. For example, it is estimated empirically that only some (about 15- 20%) of the injected fat survives re-injection and consequently the amount of fat injected is in excess of that needed for filling the depression. Three, four, or more fat injection procedures may be required before the desired goal of completely overcoming the depression is reached. US Patent No. 5,681,561 discloses certain compositions for improving autologous fat grafting procedures, where the compositions include autologous primary adipocytes and at least one of a number of specified anabolic hormones in combination with a defined nutrient medium in an aqueous solution.

Other prior efforts to implant donor-fat into a patient are summarized in Kitagawa, Y. et al . , "De novo formation in mice of adipose tissue and muscle fibers at the site of injecting MATRIGEL^® and growth factors," in Regulation of Gene Expression and Morphogenesis by Extracellular Matrix, Third Internet World Congress on Biomedic and Sciences, available at http : //www.3iwc . rike . go . jp/CONGRESS/SYMPO/SYMPOSP/SAI0109. HTM . Green and Kehind (1979, cited in Kitagawa et al . ) established a preadipocyte cell line that was susceptible to adipose conversion. The established cell line was injected subcutaneously into nude mice and a soft pad of mature adipose cells was formed after 6 weeks, demonstrating that an established cultured preadipocyte cell line can differentiate in vivo. Kitagawa et al . produced vascularized fat pads after injecting 107 3T3-F442A cells into nude mice. Smaller numbers of cells were inadequate to produce fat pads . Although the 3T3-F442A cell line can differentiate in vi tro into adipocyte- like cells with intracellular lipid separated into small droplets, the cells formed after injection were typical adipocytes with a single lipid droplet filling the entire cell. Kitagawa et al . also co-injected the 3T3-F442A cell line with MATRIGEL^® reconstituted basement membrane and with bFGF and observed heavier fat pads, although adipose tissue formation from endogenous precursors of adipocytes was also noted even when the 3T30F442A cell line was not present in the injected suspension.

Hazen, S.A. et al . , "Monolayer cell culture of freshly isolated adipocytes using extracellular basement membrane components," J. Lipid Research 36 (4) : 868-875 (1995) used MATRIGEL^® to attach freshly isolated rodent adipocytes floating on a culture medium surface to a plastic surface for subsequent cell biological techniques such as monolayer cell culture, microspectrofluorometry and confocal microscopy. Hazen et al . noted that adipocytes maintained in culture for six days did not display any change in size or differentiated microscopic appearance .

The related arts of adipocyte culture and free-fat implantation are limited by an inability to effectively culture primary adipocytes ex vivo to a stage in which the adipocytes organize into three-dimensional, multicellular clusters or into adipose tissues which can be implanted. Non-adherent primary adipose cells have not heretofore been cultured to form three- dimensional, multicellular structures when suspended in a matrix. Although adipose cells can be cultured in floating cell cultures on a surface of a medium or suspended in fibrillar type I collagen gels (30), they tend to lyse when attached to charged, non-biological surfaces.

BRIEF SUMMARY OF THE INVENTION The present invention is summarized in that primary adipocytes from an animal are cultured to form three- dimensional, multicellular structures in basement membrane component gels. The adipocytes thus cultured can be implanted subcutaneously and can form fat pads in vivo after implantation.

The method of the present is summarized in that primary adipocytes in a mixture with liquid ice-cold (between about 0 and 4°C) basement membrane components are evenly distributed onto a surface of a container having a flat surface, where the container surface is first coated with a uniform layer of solidified undiluted basement membrane components . The temperature of the container is then raised to a temperature at which the basement membrane components solidify or gel to form a gelled matrix comprising the adipocytes suspended therein. The gelling process takes place over a period of time that is insufficient for the adipocytes to float to the top of the culture. After the basement membrane components gel, a suitable adipocyte-sustaining culture medium is added to each dish. The cultures are maintained at a physiological temperature in a humidified atmosphere effective to maintain the cells in culture until the cells form three-dimensional multicellular clusters, or organoids, suitable for harvesting and surgical subcutaneous implantation in plastic and reconstructive surgery.

It is an object of the present invention to culture three- dimensional adipocyte clusters which are suitable for surgical subcutaneous implantation. It is another object of the present invention to produce adipocyte cultures wherein the adipocytes do not float on the surface of the culture media but rather are suspended in a gelled medium.

It is a feature of the present invention that the cells are placed into culture in a mixture with ice-cold basement components that are subsequently permitted to gel before the adipocytes have an opportunity to float to the culture surface.

It is an advantage of the present invention that the adipocyte culture are more protected from damage and degradation than floating cell cultures of adipocytes. It is another advantage of the present invention that the adipocyte cultures can be safely transported without risking damage to the cultured cells .

Other objects, advantages, and features of the present invention will become apparent upon consideration of the following detailed description.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS Not applicable.

DETAILED DESCRIPTION OF THE INVENTION Primary adipocytes are obtained from fat pads of an animal. A preferred animal is a mammal. The mammal can be, for example, human, monkey, bovine, ovine, equine, and urine . Cells from other mammalian species can also be used. The preferred source of the adipocytes is a human. The adipocytes are readily harvested from fat pads such as those found in the abdomen. The culture method of the present invention can also be practiced on adipocytes which are not primary adipocytes.

The basement membrane components themselves are known to those skilled in the art. In particular, the basement membrane components typically include laminin, proteoglycans, and type IV collagen. The basement membrane components contain only small amounts of fibrillar type I collagen. A commercially available source of basement membrane components is MATRIGEL^® EHS Solution (Collaborative Biomedical Products, USA, commercially available from Boehringer Ingelheim Bioproducts Partnership.) The adipocyte-sustaining culture medium is preferably Dulbecco's modified Eagle's medium (DMEM) supplemented with 25 mM NaHC0₃, 1 mM N- 2 -hydroxy ethyl-piperazine-W -2-ethane sulfonic acid, 1.5 mM glutamine, 1 U/ml penicillin, 1 μg/ml streptomycin, and 10% calf serum. The medium can optionally include fatty acid-free bovine serum albumin (BSA) , although this can be omitted without noticeable effect . Any medium known to sustain a floating culture of adipocytes, such as DMEM:Ham' s-12 medium in 1:1 ratio, is suitable. In the present invention, the medium should sustain the culture of primary adipocytes until the multicellular clusters described herein form, which is typically at least about 6 days in culture.

When the cell-basement membrane component mixture is added to the coated surface, as described, the mixture can form a meniscus along the edges of the cell culture dish, and tends to aggregate there if care is not taken to avoid the edges of the plates while dispersing the mixture or if the dishes are swirled to distribute the mixture. Instead, a pipette should be used to distribute the cells. It is also advantageous to begin to distribute the cells on the coated surface quickly after forming the mixture, since the cells are vulnerable to breakage at the low temperatures required to keep the basement membrane components in liquid form.

After the cell-basement membrane component mixture is added the mixture is allowed to gel. If MATRIGEL^® is used, the gelling is accomplished by raising the temperature of the mixture to a temperature of at least about 12°C, but preferably room temperature or a physiological temperature such as 37°C. The gelling process proceeds faster as the temperature increases.

The cells can readily be cultured for at least 21 days, although it may be possible to culture the cells for longer periods. By about six days in culture, nearly all of the cells migrate through the basement membrane and organize into three dimensional clusters of adipocytes that appear very similar to developing adipose tissue in vivo . Specifically, the cells initially form intensive ramifications and then later organize into spherical clusters that are the approximate size and shape of primitive organs. Primitive organs are early spherical clusters of fat cells found during development wherever adipose tissue eventually develops. Also, the adipocytes so cultured remain in a post-mitotic state. This can be important for transplant purposes since a dividing fat cell population might cause the inappropriate formation of lipomas or fat cell tumors or masses.

When the organized clusters are about 15 mm long and between about 0.5 and 3 mm wide, they are appropriately sized for harvesting and subcutaneous implantation. In these structures, the cells in culture are sufficiently dense as to prevent light transmission. Numerous empty spaces exist between the clusters in which few if any cells can be observed. The inter-cluster spaces are devoid of the original basement membrane. Some of the clusters float above the plastic and are connected to the plastic and to one another by fibrillar collagen-like strands. A number of adipocytes are attached to, and appear to be migrating on, these strands. Other adipocyte clusters seem to be encapsulated within a hazy material which may also provide some attachment to the plates .

Primary cells cultured in the method of the present invention are distinguished from those cultured using previous culture methods in that mitotic structures are not observed in the cultured primary cells nor do the adipocytes convert into fibroblast-like cells. The clustered multicellular masses remain positive for CAIII and ALBP, two differentiation state- specific adipocyte markers (2,21).

The clusters are further characterized in that they secrete a matrix protease activity that is associated with bands on a zymogram that migrate near a molecular weight marker for BSA, and which have approximate sizes of 72 and 62 kilodaltons, on the basis of comparisons with molecular weight markers. The elution profile of this matrix protease activity from gelatin-sepharose 4B columns is similar to that of matrix metaloproteinase 2 (MMP-2) , a 72-kilodalton matrixin with a 62 kilodalton mature form. The dimethyl sulfoxide eluent from these columns contains MMP-2 immunoreactivity . The activity of this protease appears to be important for multicellular clustering of the adipocytes.

Other characteristic properties of the multicellular adipocyte clusters are reported in Brown, L.M. et al, "Role of the matrixin MMP-2 in multicellular organization of adipocytes cultured in basement membrane components," Am. J. Physiol . 272 (Cell Physiol. 41):C937-949 (1997), incorporated herein in its entirety by reference. The incorporated paper also includes photomicrographs of the clusters and of the cell-cell interaction between adipocytes during and after cluster formation.

Clusters produced according to the method of the invention can be removed from the containers in which they are cultured and implanted subcutaneously, in the manner described in US Patent No. 5,681,561, incorporated herein in its entirety by reference. The clusters formed according to the method of the present invention are particularly well suited for implantation in that they bear a remarkable resemblance to adipose tissue in vivo and in that they are already shown to be competent to form multicellular structures. The structures are also sufficiently small so as not to become necrotic when implanted. The cultures are poised, upon implantation, to vascularize and innervate when placed into a subcutaneous environment . Depending upon the implantation site, it may or may not be important to simultaneously implant one or more growth factors as well as additional membrane basement components.

Examples Preparation of Adipocytes . Male Sprague-Dawley rats (7-15 weeks old) from Charles River were housed in the animal facility at the Pennsylvania State University College of Medicine and fed ad libitum. Rats were anesthetized with sodium pentobarbital, and the epididymal fat pads were removed. Adipocytes were isolated from the tissue by collagenase digestion as previously described (16) . Collagenase lots were purchased based on information kindly provided by Dr. Samuel Cushman's laboratory (National Institutes of Health, Bethesda, MD) . Following tissue digestion, the cells were filtered through 250 micron mesh into a 50 ml conical tube. The cells were resuspended in 40 ml of KRH buffer (16) and centrifuged for 30 s at 200 x g. Cells were concentrated by inserting a tube below the layer of floating cells and removing the buffer. The cells were then washed twice more by centrifugation.

Cell Cul ture . The bottom of 35 mm petri dishes (or 6 x 35 mm well Falcon dishes) were first coated with 0.25 ml of undiluted ice-cold basement membrane components. The Matrigel was applied in a uniform layer with a wide mouth pipette tip. Dishes were incubated for 30 minutes in a cell culture incubator at 37°C in an atmosphere of 95% humidified air and 5% C0₂ to allow the basement membrane components to solidify. Following solidification of the first layer, a mixture of adipocytes and cold basement membrane components were made for the second layer. The cells were first concentrated by removing the buffer underneath the cells until the total volume was between 1.5 and 3.5 ml . A mixture was then made of the concentrated cells (20-150 ml as indicated) and ice cold liquid basement membrane components to a final volume of 600 ml. This was mixed on ice in a microfuge tube by repeatedly drawing up and gently expelling the mixture from a wide mouth Eppendorf pipette tip. Wide mouth pipette tips were used throughout the procedure to help prevent damage to the cells. The mixture of cells and basement membrane components was slowly pipetted into the center of the dish while moving the tip outward with a slow and gentle swirling motion to help facilitate even distribution of the cells. The suspension was then allowed to disperse across the bottom. The dishes were then carefully transferred to the cell culture incubator without media and incubated at 37°C for 30 minutes. Once gelled, 2-3 ml of Dulbecco ' s Modified Eagle Medium supplemented with 25 mM NaHC0₃, 1 mM HEPES, 1.5 mM glutamine, 1 unit/ml penicillin, 1 mg/ml streptomycin, and 10% calf serum, were added to each dish. Cultures were maintained at 37°C in an atmosphere of 95% humidified air/ 5% C0₂, and media were changed daily or every other day. In early experiments 4% fatty acid-free BSA was added to the cell culture media as is done in floating fat cell cultures which are in direct contact with floating lipid droplets. However this does not seem to be necessary for the viability of this type of culture or for multicellular cluster formation. Since no effect was seen by omitting BSA, it was subsequently omitted.

Western blot analysis . Cell lysates or immunoprecipitates were analyzed for adipocyte lipid binding protein (ALBP) and carbonic anhydrase III (CA III) as previously described (16) using ¹⁵I-F (ab' ) ₂ goat anti-rabbit fragments from Dupont New England Nuclear as the second antibody. MMP-2 levels in adipocyte conditioned media were analyzed following affinity purification of the enzyme as described below. MMP-2 was detected with a rabbit polyclonal anti-MMP-2 sera that was a generous gift from Dr. William Stetler-Stevenson (National Cancer Institute). MMP-2 antisera was used at a 1:500 dilution and the second antibody was either ¹⁵I-F (ab' ) ₂ goat anti-rabbit fragments (0.2 mCi/ml) followed by autoradiography or a 1:4000 dilution of donkey anti-rabbit horseradish peroxidase conjugate (Amersham) using the ECL detection kit from Amersham according to the manufacturer's directions.

Zymographic analysis of secreted matrix proteases . Cultures were serum starved by exchanging the media three times during a one to two hour period with 3 ml of serum-free, BSA- free media and then, 24-48 hours after the last exchange, the conditioned media was collected and frozen at -84°C. A 20-40 ml aliquot of media was then solubilized in an equal volume of a non-reducing SDS-PAGE sample buffer (5% SDS, 2% sucrose, 0.2% bromophenol blue and 20% glycerol) without boiling.

Zymographic analysis was performed as previously described (4) to determine the molecular size and relative activity of protease (s) secreted by adipocytes in primary culture. Changes in the depth and width of the bands of clarification were quantitated by densitometry using the NIH Image program. Affini ty purification of MMP-2. MMP-2 was affinity purified by one of two procedures as indicated in the figure legends . These procedures were derived from previous methods (e.g., 4, 5, 24). In procedure A. adipocyte conditioned media (75 ml) was applied to a 0.5 x 6 cm bed of gelatin sepharose 4B at 4°C. The column bed was washed with PBS until the light absorption (A₂₈₀) declined to background levels. Some proteins were then eluted with six column volumes of a high salt buffer [0.5 M NaCl, 50 mM Tris HCL (pH 8.0), 5 mM CaCl₂, 0.02% NaN₃ and 0.05% Brij 35], and MMP-2 was subsequently eluted by. applying six bed volumes of high salt buffer containing 7.5% DMSO (5, 37) . The high salt and DMSO-high salt eluants were subsequently concentrated to 1 ml by vacuum dialysis against 10 mM Tris HCL (pH 7.5) .

Alternatively, in procedure B 1 ml aliquots of conditioned media from different plates of cells were added to different 0.5 x 2 cm columns previously equilibrated with 10 ml of high salt buffer (see above) . The columns were then washed with 5 x 1 ml aliquots of high salt buffer and the eluant discarded. Bound MMP-2 was then eluted with 2 x 1.25 ml of 7.5% DMSO- containing high salt buffer. The MMP-2 containing eluant was desalted on Pharmacia PD-10 columns equilibrated with water. The desalted fraction, containing highly purified MMP-2, was lyophilized and used as indicated.

Characterization of adipocytes suspended in basement membrane components . Cells were plated at a density of approximately 1.5 x 10⁶ cells per dish and were photographed at various magnifications. An initial layer of basement membrane components was found to be important for attachment of the cell-matrix mixture. As a liquid, the basement membrane-cell mixture can form a meniscus along the edges of the cell culture dish when plating. The adipocytes/ matrix mixture tended to aggregate there if care was not taken to avoid the edges of the plates during the dispersal process or if the dishes were swirled to distribute the basement membrane components-cell mixture instead of using the pipette to distribute the cells. After six days in primary culture nearly all of the cells from 7-8 week-old animals had migrated through the basement membrane and organized into three-dimensional clusters of adipocytes that appeared very similar to adipose tissue in vivo . These structures were up to 15 mm in length and 0.5-3 mm in width. The number of cells in these structures prevented light transmission in the microscopes. Numerous empty spaces existed between the clusters in which few if any cells could be observed and scraping these spaces with a spatula indicated that they were devoid of the original basement membrane. Some but not all of these adipocyte clusters were floating just above the plastic, connected to it and to one another by fibrillar collagen-like strands. A number of adipocytes were attached and appeared to be migrating on these strands . Other structures seemed to be encapsulated within a hazy material just visible in the micrographs which may also provide some attachment to the plates. During primary culture in basement membrane, neither mitotic structures nor the conversion of adipocytes into fibroblast like-cells was observed, as previously seen by others using various other culture methods (e.g., 29, 30) . Despite the fact that the cells were organized into large multicellular clusters, they remained positive for CA III and ALBP, two differentiation-state specific adipocyte markers (2, 21) .

To characterize the early stages of colonization, cells were suspended and plated at a lower density. By day three to four, neighboring cells in these cultures had given rise to cellular extensions that formed points of contact between the cells. These cell-cell interactions appeared as either narrow intercellular tubular plasma membrane extensions or as a thickening of the pericellular cytoplasm in the vicinity of neighboring cells that seemed to mold to those cells. The cell nuclei were visible as small circles within the parabolic bulge of the pericellular cytoplasmic ring that surrounds the lipid droplet. In other microscopic fields, structures which we termed "seed clusters" were visible. These may represent the early stages of cluster formation. "Seed clusters" usually contained 3-10 adipocytes that appeared to be extending filopodia and migrating towards an optically-dense material extending from neighboring cells. Interestingly, the adipocytes closest to this optically dense material were typically on three but not four sides of it.

At later times, many of the adipocytes had organized into discrete, relatively symmetrical and spherical clusters of adipocytes. These could be seen without magnification. For convenience these were designated "intermediate clusters" to distinguish them from the smaller seed clusters that were asymmetrical and not visible without a microscope. Around the light -dense intermediate clusters there was an area of clearing, in which there were few if any cells. From the bottom of the plates, these intermediate clusters (1 mm or greater in size) appeared like bacterial colonies on agar plates, and thus, when counted on a per dish basis, provided a means to quantitate and statistically analyze the rate of this process (Table I) . Many cells around the intermediate clusters extended filopodia- like structures that were vectored toward the clusters. Other lines in the matrix were thought to represent remodeling of the basement membrane. The apparent filopodia appeared to contain some small lipid droplets.

Typically, cells located between two adipocyte clusters had two apparent filopodia with bipolar distribution vectored towards each cluster. During the formation of the intermediate clusters, adipocytes migrated towards the core of the structures either individually, as lines of cells, or as clusters that contained smaller numbers of cells. Finally, larger structures were formed from the continued migration and joining of intermediate clusters, smaller clusters and/or individual cells. Thus it is demonstrated that when primary adipocytes are cultured in the method of the present invention, the adipocytes migrate, associate with one another, and form multicellular clusters akin to those seen in adipose tissue morphogenesis in vivo . The clusters that form are appropriately dimensioned for use in subsequent subcutaneous surgical implantation in a manner comparable to that practiced in the art using primary adipocytes . REFERENCES

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Claims

CLAIM OR CLAIMS I CLAIM:

1. A method for in vi tro culture of adipose cells in a container having a surface, the method comprising the steps of: coating the surface of the container with basement membrane components; distributing on the coated surface a liquid comprising basement membrane components and a plurality of adipocytes; gelling the liquid mixture before the adipocytes float to the top of the liquid to form a culture of adipocytes suspended in a gelled matrix; and adding an adipose-sustaining culture medium to the container.

2. A method as claimed in Claim 1 wherein the basement membrane components comprise at least one ingredient selected from the group consisting of laminin, proteoglycans, and type IV collagen.

3. A method as claimed in Claim 1 wherein the liquid is evenly distributed on the coated surface.

4. A method as claimed in Claim 1 wherein the basement membrane components are a liquid at a temperature between 0 and 4┬░C.

5. A method as claimed in Claim 4 wherein the gelled matrix is formed by warming the liquid mixture to a temperature of at least about 12┬░C.

6. A method as claimed in Claim 1 further comprising the step of culturing the adipocytes in the gelled matrix under conditions of temperature and humidity effective to maintain the adipocytes, until the adipocytes organize into multicellular clusters.

7. A method for implanting adipocytes into an animal, the method comprising the steps of: coating a surface of a container with basement membrane components; distributing on the coated surface a liquid comprising basement membrane components and a plurality of adipocytes; gelling the liquid mixture before the adipocytes float to the top of the liquid to form a culture of adipocytes suspended in a gelled matrix; culturing the adipocytes in the gelled matrix under conditions of temperature and humidity effective to maintain the adipocytes, until the adipocytes organize into a multicellular cluster; and implanting the multicellular cluster into the animal.

8. A method as claimed in Claim 7, wherein the multicellular cluster is implanted subcutaneously.

9. A method as claimed in Claim 7 wherein the basement membrane components comprise at least one ingredient selected from the group consisting of laminin, proteoglycans, and type IV collagen.

10. A method as claimed in Claim 7 wherein the liquid is evenly distributed on the coated surface.

11. A method as claimed in Claim 7 wherein the basement membrane components are a liquid at a temperature between 0 and 4┬░C.

12. A method as claimed in Claim 11 wherein the gelled matrix is formed by warming the liquid mixture to a temperature of at least about 12 ┬░C.