METHOD FOR OBTAINING CELL SPHEROIDS

Abstract

The present invention describes a method for obtaining spheroids from differentiated cells, in particular from adipocytes lacking exogenous matrix. These spheroids are particularly suitable for conducting metabolic studies of test compounds by closely mimicking the function of an in vivo organ such as adipose tissue.

Description

Title of the invention: METHOD FOR OBTAINING CELL SPHEROIDS

[0001] The present invention relates to the field of cell culture, in particular, three-dimensional cell culture, methods and uses thereof.

[0002] Toxicological studies based on two-dimensional cell cultures have been used to examine the effects of drugs on cell metabolism, enzyme activity, etc.

[0003] Although the ability to grow cells in flat layers is simple and allows for the study of various aspects of cellular physiology, they do not allow for the complexity and architecture of an entire organ to be accounted for. In particular, in a 2D model, the extracellular matrix, inter-cellular and matrix interactions, allowing the complexity of cellular functions to be reflected, are absent.

[0004] Three-dimensional culture systems can form functional tissue with characteristics close to in vivo structures. Three-dimensional cell culture allows cells to interact with their environment in three dimensions and is and better reflects the integrated functioning of an organ.

[0005] The health, nutrition, cosmetics and aesthetics industries are facing changes in their environment. On the one hand, the tightening of regulations, for example prohibiting animal testing in cosmetics, and on the other hand, the evolution of consumer expectations towards more effective products with scientifically proven results, motivate manufacturers to find new levers of innovation and more relevant tools to evaluate their products, understand their mechanisms of action and optimize their effectiveness.

[0006] Other three-dimensional models such as liver spheroid models have been described. Liver spheroids can be composed of several cell types initially used in two-dimensional cultures to test xenobiotic molecules as well as their hepatic metabolism.

[0007] 3D cell culture can produce specific tissue and cellular structures and mimic the functions and responses of real tissues to external stimuli in a more relevant manner than two-dimensional culture models.

[0008] The reproducible production of 3D cellular spheroids is in high demand. Until now, different techniques (based on protein or polysaccharide supports (scaffold) or via chemical inducers) have been used for the formation of spheroids. The liquid overlay technique (LOT) is one of the methodologies the most explored, due to its low cost and easy handling. This technique has been widely studied to improve its application potential in high-throughput analysis.

[0009] In a particular field, and in particular in the face of the progression of problems of excess weight and the accelerated aging of the population, adipose tissue, known as fatty tissue, which is the site of major alterations during these physiopathological processes, has become one of the major targets of industrial research.

[0010] Manufacturers now consider research on adipose tissue as an area of ​​innovation and increasingly wish to integrate it into the development of their products in order to gain a competitive advantage on the market.

[0011] Adipose tissue is a tissue of very great cellular heterogeneity, containing in particular mature adipocytes responsible for the storage of lipids and their release during energy needs as well as precursors of these cells, preadipocytes, which will be recruited for the formation of new adipocytes and which participate in the alterations of the tissue during obesity and aging.

[0012] Preadipocytes, unlike adipocytes, are cells with adherent properties and easy to cultivate under conventional cell culture conditions in a two-dimensional system. On the other hand, these 2D culture conditions require a large number of cells, significant material resources, a long culture time (two weeks) without allowing optimal maturation of these cells and with, in addition, a significant risk of cell detachment at the end of culture.

[0013] Thus, the preadipocyte has become a target for action in health and beauty products given its role in the expansion of adipose mass and in the fibro-inflammatory processes which characterize the adipose deposits of overweight people, areas of cellulite or even aged skin.

[0014] In the case of preadipocytes, existing 3D system cultures most often integrate a protein matrix of a collagenous nature or involve complex and expensive techniques.

[0015] Ho & al., PLOS one, 2012, vol. 7, describe the formation of spheroids of breast cancer cells by the technique of "liquid overlay cultivation".

[0016] Emon MP et al., Endocrinology; 2015 Dec, describes a simple and reproducible 3D cell culture technique that allows mouse visceral preadipocytes to be differentiated into adipocytes. The preadipocytes are embedded in a collagen gel at a concentration of 300,000 cells per well for 500 μl of gel.

[0017] Daya S et al., Differentiation, 2007, p360-370, describes the use of a three-dimensional culture system for the culture and differentiation of mouse pre-adipoyctes contained in a collagen gel. The cells were added to the gel so as to contain 105 cells / ml of gel and then the mixture was deposited in a plate 24-wells. A 13mm glass slide was placed at the bottom of the wells to make it easier to remove the collagen gel at the end of differentiation.

[0018] Louis F, Biotechnol Bioeng, 2017, volume 114, discloses the use of a bio-functionalized hydrogel containing adipose extracellular matrix components such as collagen I, collagen VI and the cell binding domain of fibronectin, which allows the 3D culture of mature adipocytes derived from pre-adipocyte cell lines, acquiring an in vivo-like organization, forming multicellular adipocyte spheroids.

[0019] J.Borges, Mimimally Invasive Therapy & Allied technologies, 2007, volume 16, describes a method of 3D cell co-culture of preadipocytes and endothelial cells to allow the study of their interactions in a context of proliferation and angiogenesis. The preadipocytes and endothelial cells were cultured in 3D within a fibrin matrix in non-adherent 96-well plates (with a concave round bottom) to allow their grouping into spheroids.

[0020] In Korff & Augustin, Journal of Cell Biology, 1998, volume 143, the technique used to generate endothelial spheroids is a chemical method. Indeed, it is described in the Materials and Methods of this document that the cells are suspended in culture dishes, in culture medium that contains methocel (20%), under temperature conditions of 37 °C, 5% CO2 and 100% humidity. These conditions allow the aggregation of cells in less than 4 hours. It is described that "Variation of the methocel concentration during spheroid formation was therefore used to control the average size of the spheroids", which confirms the fact that methocel is the agent for the formation of the spheroids obtained in this document.

[0021] Turner, Biotechnologies & Bioengineering, 2013, volume 111, describes a method of 3D cell culture of preadipocyte cells that will be induced into differentiation to become adipocytes. This 3D culture of preadipocytes is carried out in 24-well round flat-bottomed plates with a substrate composed of an artificially manufactured polymer: “elastin-like-polypeptide ELP” associated with another synthetic polymer: “polyethyleneimine PEI”.

[0022] Bhang & Cho, Biomaterials, 2011, vol. 32 p.2734-374, discusses the increased efficiency of angiogenesis following the transplantation of spheroids formed from stem cells into ischemic tissue. The spheroids are obtained by placing the cell culture in a flask which is shaken at about 70 rpm for 3 days. This document describes the production of spheroids by a mechanical inducer which is the shaking of the flask for several days.

[0023] WO2016069892 Al, describes a system allowing the 3D culture of cells such as spheroids. This system includes a plate composed of microwells or wells which have a shape that allows the formation of spheroids when cells are in culture, contained in a culture device such as a culture flask. In addition, they are structured and arranged to allow the movement of liquid in and out of the well without trapping air between the substrate and the liquid or lipid droplets that are introduced into the wells, thanks to their capillary structures.

[0024] Generally speaking, the techniques described in the state of the art involve either the use of chemical spheroidization inducers, or exogenous matrix support (scaffold), or complex or lengthy techniques requiring several days of handling in order to obtain the spheroids.

[0025] The present invention thus aims to provide a simple and rapid method for obtaining spheroids of undifferentiated and / or differentiated cells, in particular spheroids of preadipocytes or even spheroids of adipocytes.

[0026] The present invention provides a valuable tool for studying the impact of compounds in a three-dimensional cell culture system that is closer to, and therefore should be more predictive of, the clinical situation and offers a unique, simple and inexpensive three-dimensional solution.

[0027] Thus, according to a first embodiment, the present invention provides a method for obtaining spheroids of undifferentiated cells comprising the following steps:

[0028] Provision of isolated and amplified undifferentiated cells

[0029] Seeding of undifferentiated cells in a container whose bottom has a concave rounded profile, containing a suitable culture medium,

[0030] At least two centrifugations of said container with rotation of 160 to 200°, advantageously 180°, around an axis perpendicular to the plane of said container between each centrifugation,

[0031] Seeding and incubation of newly formed undifferentiated cell spheroids under conditions promoting complete spheroid formation.

[0032] The first step consists of or includes providing isolated and amplified undifferentiated cells.

[0033] By the expression undifferentiated cells is meant undifferentiated cells found within tissues which are composed predominantly of differentiated cells in most adult tissues and organs.

[0034] These undifferentiated cells are generally multipotent cells, in particular they are mesenchymal stem cells. Mesenchymal cells can give rise to adipocytes, osteocytes and chondrocytes.

[0035] They are capable of giving rise to different cell lines of a given tissue. These multipotent stem cells are present in the embryo or in the adult organism, they are at the origin of several types of differentiated cells but retain their ability to self-renew.

[0036] Mesenchymal stem cells can give rise to several types of cells, and they are already committed in a certain direction. Their potential is therefore more restricted than that of embryonic stem cells. Mammalian hematopoietic cells, for example, give rise to red blood cells, platelets, T or B lymphocytes, macrophages, but they cannot give rise to muscle cells. Another example of multipotent stem cells is provided by neural crest cells which emigrate from the neural tube during embryogenesis and which give rise in particular to melanocytes, neurons and glial cells of the peripheral nervous system.

[0037] According to a further particular embodiment of the invention, the undifferentiated cells are preadipocytes.

[0038] Preadipocytes are undifferentiated but determined cells, arising from mesenchymal stem cells, and which can be stimulated to give rise to adipocytes.

[0039] According to a further particular embodiment of the invention, the undifferentiated cells are fibroblasts.

[0040] According to yet another particular embodiment of the invention, the undifferentiated cells are cells of the vascular stroma fraction of adipose tissue.

[0041] According to one embodiment of the invention, the undifferentiated cells originate from one or more human individuals, in particular adults.

[0042] According to a particular and preferred mode, the undifferentiated cells are not embryonic undifferentiated cells.

[0043] Step b) of the method according to the invention provides for the seeding, in a suitable culture medium, of the undifferentiated cells obtained in the previous step, in a container whose bottom has a concave rounded profile.

[0044] The suitable culture medium corresponds to a culture medium allowing the survival of the cells and contains the nutrients specific to the cell type.

[0045] For example, a suitable culture medium may be an EMEM or DEMEM medium supplemented with fetal calf serum (approximately 10% by mass) or ECBM supplemented with bovine serum albumin (approximately 3% by mass). It may also be a rich medium for mammalian cell culture and preferably such that it contains amino acids such as alanine, asparagine, glutamic acid, and proline; vitamins such as B8 and B12, fatty acids such as linoleic and lipoic, and nitrogenous bases such as thymidine; it may preferably be the DMEM / F12 medium marketed by Life Technologies or the ECBM medium marketed by Promocell.

[0046] In the case where the undifferentiated cells are preadipocytes, the suitable culture medium is a medium such as EMEM or DMEM enriched with fetal calf serum. EMEM, or Eagle's minimal essential medium (EMEM), is a cell culture medium developed by Harry Eagle and used to provide nutrients necessary for the maintenance and proliferation of various cell types in vitro. Such a medium contains amino acids, salts (potassium chloride, magnesium sulfate, sodium chloride, and sodium dihydrogen phosphate), glucose, and vitamins (e.g., folic acid, nicotinamide, riboflavin, and vitamin B12). A variation of EMEM exists, DMEM (Dulbecco / Vogt modified Eagle's minimal essential medium). It contains approximately four times more vitamins and amino acids than the original formula and two to four times more glucose. In addition, it may contain iron as well.DMEM medium is suitable for almost all cell types, including human, monkey, hamster, rat, mouse, poultry, and fish cells.

[0047] The container of step b) is a container whose bottom has a concave rounded profile.

[0048] The inventors have in fact found that the formation of spheroids via centrifugation step c) was obtained by the combination of this centrifugation and a concave rounded profile of the seeding container of step b).

[0049] By way of example, such a container whose bottom has a concave rounded profile corresponds to a well such as those found on cell culture plates comprising a plurality of wells, in particular 96-well culture plates. Advantageously, mention may be made of Corning® ULA microplate culture plates comprising 96 or 384 wells, for example.

[0050] Advantageously, the container is treated with “low attachment” or “ultra-low attachment” in order to reduce cell adhesion.

[0051] Step c) of the method according to the invention comprises at least two centrifugations of said container with rotation of 160 to 200°, advantageously 180°, around an axis perpendicular to the plane of said container between each centrifugation.

[0052] Advantageously, the number of centrifugations is three, or even 4, or even 5.

[0053] Between each centrifugation, the container, or preferably the cell culture plate comprising a plurality of wells, undergoes a rotation through an angle of the order of 160 to 200°, more particularly 180°, around an axis perpendicular to the plane of said container.

[0054] This centrifugation can be carried out using a benchtop centrifuge, particularly a cell culture plate centrifuge comprising a plurality of wells.

[0055] As an example, microplate centrifuges may be cited, for example a A centrifuge with a dual-position vertical rotor that supports a wide range of plates. The rotor has plate guides to keep the plates upright and prevent them from tilting toward the rotor. Another example is the MBI PlateFuge® MicroCentrifuge for Plates. This centrifuge has a swing-out rotor for microplates. The easily accessible rotor chamber includes two plate holders that are tilted at a 75° angle. This allows plates to be inserted confidently without the need for sealing bands or capsules. When the centrifuge lid is closed, the rotor accelerates, and centrifugal force forces the plates into an upright position.

[0056] The centrifugation step lasts on average a few minutes, in particular between 1 and 10 minutes, in particular between 1 and 5 minutes, more particularly still between 1 and 3 minutes.

[0057] The centrifugation force is between 100 and 500 g during the centrifugation period, in particular around 200 to 400 g, more particularly around 200 to 300 g.

[0058] According to a particular embodiment of the invention, centrifugation step c) is carried out / repeated 2 to 12 times, in particular 2 to 10 times, or even 2 to 8 times, more particularly 2 to 6 times, or even more particularly 2 to 4 times.

[0059] The combination of several centrifugations with a rotation of the container, i.e. the plate, between each centrifugation, associated with the convex rounded bottom allows the formation of spheroids without the addition of chemical agent, protein or polysaccharide matrix support. In addition, the formation of spheroids does not require several days; it is carried out in a few minutes, or even a few hours.

[0060] The newly formed spheroids are then seeded. Step d) thus aims at seeding and incubating the newly formed undifferentiated cell spheroids under conditions promoting the complete formation of spheroids.

[0061] Advantageously, the conditions of step d) of incubation of the newly formed undifferentiated cell spheroids correspond to a temperature of between 36°C and 38°C and preferably around 37°C and to a percentage of CO2 of between 3 and 6% and preferably around 5%.

[0062] Complete spheroid formation is monitored by assessing spheroid shape, integrity, and growth by transmitted light plate imaging and area measurement (according to the method described in “Scaffold-free generation of uniform adipose spheroids for metabolism research and drug discovery.” Klingelhutz AJ et al. 2018; Nature Scientific Reports - 8:523.).

[0063] According to one embodiment, the method according to the invention aims to obtain spheroids of differentiated cells comprising steps a to d cited above and further includes the following additional steps:

[0064] Differentiation of fully formed undifferentiated cell spheroids into differentiated cell spheroids in a differentiation medium,

[0065] Obtaining spheroids of differentiated cells.

[0066] The differentiation step is carried out according to conventional techniques known to those skilled in the art and according to the cell type.

[0067] According to a particular embodiment, the method according to the invention is characterized in that the undifferentiated cells are pre-adipocytes, that the differentiated cells are adipocytes and in that the medium of step e) leading to the differentiation of the pre-adipocyte spheroids into adipocyte spheroids comprises a buffer, a corticosteroid, a phosphodiesterase inhibitor, an antidiabetic, an iron sensor, and at least one hormone.

[0068] In particular, the medium is a mixture of Hepes, insulin, T3, Dexamethasone, transferrin, rosiglitazone and IBMX.

[0069] Thus, in a particular embodiment where the method according to the invention aims to obtain adipocyte spheroids, the method according to the invention is characterized in that the buffer is chosen from the group comprising bicarbonate and hepes, preferably hepes and at a concentration of between 5 and 30 mM, preferably between 10 and 20 mM.

[0070] According to a particular embodiment where the method according to the invention aims to obtain adipocyte spheroids, the method according to the invention is characterized in that the hormone is chosen from the group comprising insulin and thyroid hormone T3, alone or in a mixture, preferably in a mixture, at concentrations of between 10 and 100 nM, preferably between 30 and 60 nM for insulin and between 0.1 and 2 nM, preferably between 0.5 and 1.5 nM for thyroid hormone T3.

[0071] According to a particular embodiment where the method according to the invention aims to obtain adipocyte spheroids, the method according to the invention is characterized in that the corticosteroid is chosen from the group comprising betamethasone, dexamethasone cortivazol, and their mixtures, preferably Dexamethasone at a final concentration of between 50 and 200 nM, preferably between 80 and 120 nM.

[0072] According to a particular embodiment where the method according to the invention aims to obtain adipocyte spheroids, the method according to the invention is characterized in that the iron sensor / transporter, in particular transferrin, is at a concentration of between 0.01 and 0.2 pg / ml, preferably between 0.05 and 0.15 pg / ml.

[0073] According to a particular embodiment where the method according to the invention aims to obtain adipocyte spheroids, the method according to the invention is characterized in that the antidiabetic is chosen from the group comprising pioglitazone, rosi-glitazone, troglitazone, as well as their mixtures, preferably rosiglitazone at a final concentration of between 50 and 200 nM, preferably between 80 and 120 nM.

[0074] According to a particular embodiment where the method according to the invention aims to obtain adipocyte spheroids, the method according to the invention is characterized in that the non-selective phosphodiesterase inhibitor is chosen from the group comprising caffeine, theophylline, 3-isobutyl-l-methylxantine (IBMX) and mixtures thereof, preferably IBMX at a concentration of between 5 and 35 pM, preferably between 15 and 25 pM.

[0075] Indeed, the spheroids obtained according to the invention constitute an optimal study model because they are produced from primary cell cultures, that is to say cells which are derived directly from fresh human tissues, and placed in conditions which best mimic the physiological conditions found in vivo, in the human body. This is in contrast to models using so-called "immortalized" cells. The metabolism of these cells is artificially modified to facilitate their manipulation and the variability of the results, which, on the other hand, generates different characteristics, further removed from the living and introduces a bias into the studies.

[0076] Furthermore, the absence of exogenous matrix for the formation of these spheroids further reinforces this technical and biological advantage since any study on the matrix remodeling processes of these cells will not be biased by foreign components added for the formation of the spheroids.

[0077] The spheroids obtained according to the invention can be advantageously used for biological and product screening protocols, that is to say either to highlight a biological effect of a given product or to identify a product of interest from a bank of test products.

[0078] The invention thus aims at the use of the spheroids thus obtained according to the invention for the purposes of evaluating test compounds.

[0079] The present invention thus provides a method for evaluating pharmacological properties of test compounds comprising the following steps:

[0080] Providing differentiated cell spheroids obtained by a method according to the present invention;

[0081] Contacting a test compound with the spheroids;

[0082] Evaluate the impact of the test compound on at least one physiological or morphological parameter of the spheroid cells.

[0083] Particularly advantageously, the spheroids according to the invention allow studies of test compounds in different fields, whether in the cosmetics sector to evaluate compounds or active ingredients aimed at weight and silhouette, anti-aging, anti-pollution products or those focused on the quality of the skin and hair.

[0084] The health nutrition sector is also concerned with regard to food supplements with properties similar to those in the cosmetic field as well as products relating to sports nutrition or even immune defenses.

[0085] Finally, the pharmaceutical sector is also particularly targeted with products targeting the phenomena of excess weight, obesity or even associated metabolic disorders such as diabetes for example.

[0086] Also advantageously, the spheroids according to the invention can be used in the field of aesthetic medicine in order to evaluate filling products such as fillers comprising organic polysaccharide or protein polymers for example.

[0087] EXAMPLE of a Method for obtaining preadipocyte spheroids:

[0088] PRINCIPLE:

[0089] Seeding human preadipocytes isolated from adipose tissue in 3D to form spheroids.

[0090] MATERIALS:

[0091] - 96-well round bottom plate (7007 - Costar)

[0092] - Preadipocyte culture medium 37°C (DMEM10% Fetal Calf Serum (SVF) 1% PS)

[0093] - Microscope

[0094] Centrifuge

[0095] ABBREVIATIONS:

[0096] DMEM: Dulbecco's Modified Eagle Medium

[0097] PRELIMINARY:

[0098] Preadipocytes are isolated from human adipose tissue, from biopsies or plasties of human adipose tissue or from liposuction products.

[0099] The tissue is cut and ground or aspirated with a broken-tip pipette to ensure cell separation. A collagenase solution is added to facilitate cell separation. The cells are collected, avoiding blood and / or the oil layer that may have formed following bursting of the adipocytes. The cells are washed with PBS and undergo several centrifugations / washes, at around 300 to 500 g, to remove cellular debris and red blood cells. The cells are washed with PBS. After two centrifugations / washes, the cell pellet is suspended in the preadipocyte culture medium. The cells are then seeded in T175 flasks at a rate of 5 to 10 million cells per flask. The next day, the cells are then washed with PBS and recultured with the Preadipocyte culture medium. At confluence, the cells are treated with trypsin to detach them; then preadipocyte culture medium is added to neutralize the action of the enzyme and the cells are resuspended. They are ready for the spheroid production protocol.

[0100] PROTOCOL FOR OBTAINING SPHEROIDS:

[0101] 1- In a 96-well round-bottom plate, seed the preadipocytes with 1000 cells per well in 150 pL of DMEM 10% FCS medium

[0102] 2- Centrifuge the plate at 200g for 2 minutes at room temperature

[0103] 3- Turn the plate and repeat step 2: 4 times

[0104] 4- After the centrifugations, check the good aggregation of the preadipocytes (start of spheroid formation) and incubate at 37°C 5% CO2 overnight

[0105] 5- The next day, check that the spheroids are forming correctly

[0106] 6- The day after seeding, begin the differentiation treatment (Table 1 below for the preparation of the basal medium and Table 2 below for the differentiation medium). The medium is gently deposited at the edge of the well so as not to disintegrate the spheroid.

[0107] Table 1: Basic medium for differentiation.

[0108] [Tableauxl] Final concentration DMEM / F12 without phenol red QSP Biotin 33 pM Calcium Pantothenate 17 pM Penicillin - Streptomycin 1% (1000 / mL) Stabilized glutamine 2.5 mM

[0109] Table 2: Differentiation medium

[0110] [T ables 2] Volume Final concentration in the well Basal medium 10 mL Hepes Insulin T3 Dexamethasone Transferrin Rosiglitazone IBMX 10 to 30 rnM 30 to 80 nM 0.5 to 2 nM 80 to 150 nM 0.05 to 2 pg / ml 50 to 120 nM 10 to 30 pM

[0111] 7- Change the media every 2-3 days and observe the spheroids every day to check their proper condition.

[0112] 8- Stop differentiation if the spheroids begin to disintegrate.

Claims

Claims

1. Method for obtaining spheroids of undifferentiated cells comprising the following steps: a. Seeding of isolated and amplified undifferentiated cells in a container whose bottom has a concave rounded profile, containing a suitable culture medium, b. At least two centrifugations of said container with rotation of 160 to 200°, advantageously 180°, around an axis perpendicular to the plane of said container between each centrifugation, c. Seeding and incubation of the newly formed spheroids of undifferentiated cells under conditions promoting the complete formation of spheroids comprising an EMEM or DEMEM medium supplemented with fetal calf serum, characterized in that said undifferentiated cells are not human embryonic cells.

2. A method for obtaining differentiated cell spheroids comprising steps a to c according to claim 1 and further comprising the following additional steps: d. Differentiating the fully formed undifferentiated cell spheroids into differentiated cell spheroids in a differentiation medium, e. Obtaining the differentiated cell spheroids.

3. Method according to claim 1 or 2 characterized in that the undifferentiated cells have been taken from one or more human individuals.

4. Method according to claim 1 or 2 characterized in that the container is a well with a concave rounded profile bottom included in a culture device such as a plate comprising a plurality of wells.

5. Method according to one of the preceding claims, characterized in that the concentration of cells seeded in the container corresponds to a quantity of cells per container of between 500 and 1500, advantageously 1000.

6. Method according to one of the preceding claims, characterized in that step b) of centrifugation is carried out 2 to 6 times, in particular 2 to 4 times.

7. Method according to one of the preceding claims, characterized in that the duration of each centrifugation is between 1 and 3 minutes, preferably around 2 minutes with a centrifugation force fugation between 200 and 500g, preferably around 200g.

8. Method according to one of the preceding claims, characterized in that the conditions of step c) of incubation of the newly formed undifferentiated cell spheroids correspond to a temperature between 36°C and 38°C and preferably around 37°C and to a percentage of CO2 between 3 and 6% and preferably around 5%.

9. Method according to one of the preceding claims, characterized in that the undifferentiated cells are chosen from the group consisting of pre-adipocytes, fibroblasts and precursor cells of the cells of the vascular stroma fraction of adipose tissue.

10. Method according to one of claims 2 to 9, characterized in that the undifferentiated cells are pre-adipocytes, that the differentiated cells are adipocytes and in that the medium of step d) leading to the differentiation of the pre-adipocyte spheroids into adipocyte spheroids comprises a buffer, a corticosteroid, a phosphodiesterase inhibitor, an antidiabetic, an iron sensor, and at least one hormone.

11. Method according to claim 10 characterized in that the buffer is chosen from the group comprising bicarbonate and hepes, preferably hepes and at a concentration of between 5 and 30 mM, preferably between 10 and 20 mM.

12. Method according to claim 10 characterized in that the hormone is chosen from the group comprising insulin and thyroid hormone T3, alone or in a mixture, preferably in a mixture, at concentrations of between 10 and 100nM, preferably between 30 and 60 nM for insulin and between 0.1 and 2nM, preferably between 0.5 and 1.5nM for thyroid hormone T3.

13. Method according to claim 10 characterized in that the corticosteroid is chosen from the group comprising betamethasone, dexamethasone cortivazol, and their mixtures, preferably Dexamethasone at a final concentration of between 50 and 200 nM, preferably between 80 and 120 nM.

14. Method according to claim 10 characterized in that the iron sensor / transporter, in particular transferrin, is at a concentration of between 0.01 and 0.2 pg / ml, preferably between 0.05 and 0.15 pg / ml.

15. Method according to claim 10 characterized in that the antidiabetic is chosen from the group comprising pioglitazone, rosiglitazone, tro-glitazone, as well as their mixtures, preferably rosiglitazone at a final concentration of between 50 and 200 nM, preferably between 80 and 120 nM.

16. Method according to claim 10 characterized in that the non-selective phosphodiesterase inhibitor is chosen from the group comprising caffeine, theophylline, 3-isobutyl-l-methylxantine (IBMX) and their mixtures, preferably IBMX at a concentration of between 5 and 35 pM, preferably between 15 and 25 pM.

17. A method for evaluating pharmacological properties of test compounds comprising the following steps: a. implementing a method according to one of claims 2 to 10 in order to obtain spheroids of differentiated cells; b. Bringing a test compound into contact with the spheroids; c. Evaluating the impact of the test compound on at least one physiological or morphological parameter of the cells of the spheroids.