WO2012004310A1 - Modèle de tissu adipeux et procédé de préparation - Google Patents

Modèle de tissu adipeux et procédé de préparation Download PDF

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WO2012004310A1
WO2012004310A1 PCT/EP2011/061422 EP2011061422W WO2012004310A1 WO 2012004310 A1 WO2012004310 A1 WO 2012004310A1 EP 2011061422 W EP2011061422 W EP 2011061422W WO 2012004310 A1 WO2012004310 A1 WO 2012004310A1
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preadipocytes
population
medium
differentiation
proliferation
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PCT/EP2011/061422
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Valérie ANDRE
Nicolas Bechetoille
Odile Damour
Charlotte Lequeux
Sabine Pain
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Basf Beauty Care Solutions France S.A.S.
Hospices Civils De Lyon
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Publication of WO2012004310A1 publication Critical patent/WO2012004310A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • C12N5/0667—Adipose-derived stem cells [ADSC]; Adipose stromal stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/11—Epidermal growth factor [EGF]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/115—Basic fibroblast growth factor (bFGF, FGF-2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/165—Vascular endothelial growth factor [VEGF]
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/30—Hormones
    • C12N2501/33—Insulin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/30—Hormones
    • C12N2501/38—Hormones with nuclear receptors
    • C12N2501/39—Steroid hormones
    • C—CHEMISTRY; METALLURGY
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/30—Hormones
    • C12N2501/38—Hormones with nuclear receptors
    • C12N2501/395—Thyroid hormones
    • C—CHEMISTRY; METALLURGY
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/998—Proteins not provided for elsewhere

Definitions

  • the invention relates to a method of selecting a cellular population comprising preadipocytes expressing the Pref-1 factor and use thereof for preparing a cellular model containing adipocytes, in particular a model of adipose tissue.
  • Adipose tissue the last tissue to develop in an adult, is among the most abundant in the human body.
  • White adipose tissue is the body's principal site for energy storage in the form of triglycerides and its mass can vary considerably depending on the energy status of the body and in pathological situations such as obesity or cachexia or as part of the ageing process. This variation is due to the increase in size (hyper-hypotrophy) and/or number of adipocytes (hyper- hypoplasia). Lipolysis and lipogenesis are the two mechanisms involved respectively in these processes of release or accumulation of triglycerides by adipocytes and are influenced by numerous hormonal, environmental and metabolic factors.
  • adiponectin which is a protein of 244 amino acids that is only secreted by differentiated adipocytes and which thus constitutes one of the markers of maturation of the adipocytes indicating their functionality.
  • adiponectin seems to take place at the muscular level and to lead to a decrease in the plasmatic levels of free fatty acids and triglycerides (J Clin Endocrinol Metab, 2003. 88(5): 2014-18) .
  • adiponectin causes a decrease in fatty acid transporting proteins as well as a decrease in the hepatic content of triglycerides.
  • Adiponectin is also a hormone that is important because of the negative regulation that it maintains at the level of haematopoiesis and the immune system (Blood, 2000. 96(5): 1723-32). It is thought to inhibit the multiplication of myelomonocyte cell lines by induction of the phenomenon of apoptosis. Thus, it might notably have an anti-inflammatory action by modulating the functions of mature macrophagic cells, for example by moderating their phagocytic action and their production of tumour necrosis factor alpha TNFa.
  • Adiponectin is regulated by a pathway that is dependent on the PPAR Y receptors (Peroxisome proliferator-activated receptors ⁇ . Diabetes, 2001 . 50(9): 2094-09).
  • the preadipocytes are cells of mesenchymal origin that surround the adipocytes. They are contained in the "vascular stromal fraction". Culture of the vascular stromal fraction was first used for studying the differentiation of preadipocytes into mature adipocytes. Several studies indicate that the vascular stromal fraction is a source of multipotent stem cells with abilities for differentiating into mature adipocytes, chondrocytes, myoblasts, osteoblasts and endothelial cells in suitable conditions and a suitable environment (Sang Thrombose Vaisseaux, 2005. 17 (2): 101 -08).
  • adipocytes from mesenchymal precursors are a multistage process, firstly involving adipoblasts.
  • the adipoblast In vitro, after an exponential growth phase, the adipoblast has a fibroblastic morphotype. At confluence, cessation of cell growth seems to be required for involvement of the cells in the differentiation into adipocytes. The adipoblasts then become preadipocytes.
  • the process of differentiation can be divided schematically between early and late events. However, at present, little information is yet available concerning control of the early stage of transition between adipoblast and preadipocyte.
  • PPARs peroxisome proliferator-activated receptors
  • C/EBP CAAT/ Enhancer Binding proteins
  • HLH helix-loop-helix
  • PREF-1 is a transmembrane protein possessing six "EGF-like" motifs in the extracellular part, a transmembrane domain and a cytoplasmic tail.
  • PreMA premembrane protein possessing six "EGF-like" motifs in the extracellular part, a transmembrane domain and a cytoplasmic tail.
  • PreMA Pref-1 B
  • PreM C Pref-1 D
  • Pref-1 D the result of alternative splicing during transcription.
  • These four forms of Pref-1 are then translated and post-translational modifications then occur to give rise to four soluble isoforms of different molecular weights. The most important is that of 41 kDa.
  • Pref-1 is only expressed in the preadipocytes and its expression decreases in the course of differentiation, in contrast to the transcription factors C/EBPS and PPARs, which are only detected when the process of differentiation is underway. Pref-1 is therefore a marker of preadipocytes, completely absent in mature adipocytes. It was demonstrated that Pref-1 inhibits adipocyte differentiation (Molecular mechanisms of adipocyte differentiation and inhibitory action of Pref-1. Smas CM, Sul HS. Crit Rev Eukaryot Gene Expr. 1997; 7(4):281 -98, Pref-1 , a protein containing EGF-like repeats, inhibits adipocyte differentiation. Smas CM, Sul HS. Cell.
  • Pref-1 preadipocyte factor 1 activates the MEK/extracellular signal-regulated kinase pathway to inhibit adipocyte differentiation.
  • Kim KA Kim JH, Wang Y, Sul HS. Mol Cell Biol. 2007 Mar; 27(6):2294-308).
  • Ad ipocyte differentiation is moreover influenced by a large number of hormones, transduction signals, components of the extracellular matrix, and the cellular environment.
  • adipose tissue in particular subcutaneous, are proliferation of preadipocytes, their differentiation into adipocytes and the maturation of adipocytes, reflected in increased storage of lipids.
  • these adipocytes have reached a high level of filling with lipids, they reach a threshold and cannot store more. They then induce proliferation of new preadipocytes to continue storage of lipids.
  • This phenomenon explains why after dieting which enables the mature adipocytes to destock lipids, a new weight gain and storage of lipids may be higher than before, and this occurs after each dieting, as the number of mature adipocytes does not decrease and the capacities for storage after dieting are systematically increased.
  • adipose tissue has been put to good use in the field of skin engineering and various models including adipocyte precursors have been developed in order to study and modulate in particular adipocyte differentiation.
  • the models of adipose tissue engineering described in the prior art are mostly based on the vascular stromal fraction in particular of mesenchymal stem cells derived from adipose tissue obtained by lipectomy or lipoaspiration, after removal of the adipocytes.
  • Modulation between proliferation and adipocyte differentiation is then achieved by a simultaneous or sequential combination of growth factors (FCS, EGF, FGF, PDGF, NGF, SCF etc.) or of adipogenic factors (insulin, transferrin, triiodothyronine (T3), dexamethasone, isobutylmethylxanthine (IBMX) and PPARy agonist during culture (WO2007041869 - US6777231 - FR2843123).
  • patents FR2843123 - US6777231 described the selection of mesenchymal stem cells HLA Class I negative (measured by flow cytometry) according to a complex method based on their capacities for adhesion (before or after 12h), the fraction that adheres before 12 hours then being enriched until a quiescent population is obtained that expresses the transcription factor Oct4 expressed by mouse and human embryonic stem cells and that can auto-renew during 200 population doublings.
  • This population lacking preadipocytes, adipocytes, fibroblasts, endothelial cells, pericytes, mastocytes and smooth muscle cells, can then differentiate into adipocytes, but also into osteoblasts, myocytes, chondrocytes or nerve cells depending on the combination of proliferation/differentiation medium used.
  • Several cell types can be obtained at the same time.
  • the cells can be used in monolayers for evaluating the potential of actives as modulator of adipogenesis, lipolytic or anti-lipolytic substance and can be used for cosmetic or reconstructive surgery, synthesis of recombinant proteins or gene therapy.
  • the screening process for identifying agents that can display lipolytic activity is characterized by 1 - seeding of stem cells in conditions for obtaining adipocytes, 2- contacting with the candidate agent, 3- evaluation of lipolytic activity.
  • anti-lipolytic activity is sought, the same protocol is applied, but it is anti-lipolytic activity that is evaluated for the candidate agent (FR2843123).
  • Culture is carried out in two different culture media, the first containing ascorbic acid between 20 and 200 ⁇ g/ml and the second containing an ad ipogenic stimulus such as insulin, T3 , dexamethasone, I BMX, and a PPARgamma agonist (rosiglitazone or pioglitazone).
  • an ad ipogenic stimulus such as insulin, T3 , dexamethasone, I BMX, and a PPARgamma agonist (rosiglitazone or pioglitazone).
  • Stacking of 3 layers gives a thickness of up to 153.5 ⁇ after some additional days of culture in the presence of vitamin C to obtain strong cohesion between the layers; complete culture therefore took 30 days in this example, but it may take up to 63 days. Moreover, it is mentioned that a layer derived from adipose tissue is obtained and contains a mixture of preadipocytes and adipocytes with a content of adipocytes between 20% and 90% and therefore of preadipocytes from 80% to 10%.
  • preadipocytes no method for detecting preadipocytes is described and only the presence of adipocytes is mentioned by staining with Oil Red O including extraction in nonidet/isopropanol and quantification at 520 nm or synthesis of adiponectin and leptin.
  • Oil Red O Oil Red O
  • a model of reconstructed skin is made in 43 days by seeding keratinocytes on the layer obtained either by stromal cells uniquely differentiated or not by the adipogenic cocktail, or by superposition of 2 dermal layers on 2 stromal layers.
  • the mixture of stromal and dermal cells gives a thicker subepithelial portion. Only induction by the adipogenic cocktail provides visualization of the lipid vacuoles.
  • the models of adipose tissues of the prior art additionally require the preparation of several layers that are to be stacked to reconstitute an adipose tissue, which has many drawbacks, including the need for multiple seedings and culture, manipulations of stacks, increased risk of contamination, lack of reproducibility, and the difficulty of industrialization or automation of the production of these models.
  • the invention therefore has the aim of solving the technical problems mentioned above.
  • the present invention has the aim of shortening the time for making models of adipose tissues, of simplifying their preparation, of improving their quality and reproducibility, and of permitting industrialization.
  • the present invention therefore has the aim of supplying a population of preadipocytes having capacity for differentiation greater than that of the prior art.
  • the present invention therefore has the aim of supplying a method of preparing these tissue models more quickly than those of the prior art and/or for improving the quality of this cellular population.
  • the inventors discovered, particularly surprisingly and unexpectedly, that selection of a population of preadipocytes expressing Pref-1 , despite inhibiting adipocyte differentiation, made it possible to obtain a population of mature adipocytes more quickly, thus reducing the duration of the differentiation phase and at the same time increasing the quality of the tissues thus obtained. This observation is all the more surprising because Pref-1 is known for its opposite action, inhibiting adipocyte differentiation.
  • the present invention thus relates to a method of cell culture for preparing a cellular population comprising preadipocytes, said method comprising:
  • the present invention also relates to the population of preadipocytes thus obtained by this method which has a greater capacity for differentiation than those available on the market, notably the stem cells derived from adipose tissue according to the techniques previously described in the prior art (Adipose-derived Stem Cells called ADSC), as is demonstrated in example 3.
  • ADSC Adipose-derived Stem Cells
  • This method according to the invention makes it possible to supply a new model of adipose tissue, and notably to make a model of reconstructed hypoderm called “hypoderm” according to the invention or a model of reconstructed adipose tissue called “adipose tissue” according to the invention more quickly and therefore also of higher quality.
  • This method in fact makes it possible to prepare models that do not have the drawbacks of the prior art, notably not necessarily requiring the stacking of several layers, which reduces the risk of contamination, increases reproducibility, and allows for the possibility of industrialization or automation for making said models.
  • the present invention thus relates to these cellular and/or tissue models.
  • models are particularly useful as a model tissue for testing in the cosmetic, dermatological and/or pharmaceutical industry, notably for screening active principles and in particular for detecting their properties on adipose tissues.
  • These models can also constitute a support for tissue engineering, surgical repair, reconstructive and cosmetic surgery, cosmetic dermatology, notably for the filling of soft tissues, for example for the filling of wrinkles, loss of substance and scars.
  • these models constitute alternatives models to testing on animals within the scope of tests of pharmacotoxicology and/or activity of active ingredients, notably in cosmetics and pharmaceutics.
  • models constitute models that are particularly advantageous for investigating the mechanisms involved in the phenomena of lipogenesis and which may be deregulated in certain pathologies such as obesity.
  • the invention also relates to a cellular population essentially of preadipocytes, enriched with preadipocytes expressing the marker Pref-1 .
  • the present invention relates to a method of cell culture for preparing a cellular population comprising preadipocytes, said method comprising: - isolation of a cellular population containing preadipocytes, and preferably obtained from adipose tissue of a human being;
  • Cellular population containing preadipocytes means a population of various types of cells, at least a proportion of which is constituted of preadipocytes.
  • Population of preadipocytes enriched with preadipocytes expressing Pref-1 means a cellular population containing preadipocytes enriched with preadipocytes expressing Pref-1.
  • Enriched with preadipocytes expressing Pref-1 means that the cellular population is specially selected for increasing the number of preadipocytes expressing the preadipocyte factor-1 cellular marker (Pref-1 ). The method thus comprises selection of at least one population of preadipocytes expressing Pref-1 .
  • Preadipocytes according to the preferred embodiment of the invention means cells extracted from adipose tissue and expressing the preadipocyte factor-1 cellular marker (Pref-1 ).
  • the stage of isolation of a cellular population containing preadipocytes comprises cellular extraction from adipose tissue, and preferably by lipectomy of human adipose tissues or lipoaspiration of human adipose tissues preferably the vascular stromal fraction, then tissue separation in order to obtain stromal cells, notably by fractional enzymatic digestion, preferably with a collagenase.
  • the adipocytes and erythrocytes are preferably withdrawn.
  • a population of preadipocytes enriched with preadipocytes expressing Pref-1 is selected.
  • Various conventional techniques of cellular separation known by a person skilled in the art can be used for this selection stage.
  • a population of preadipocytes expressing Pref-1 is a population of preadipocytes adhering spontaneously to the cell culture support, preferably plastic plate, dishes and/or flashes. As demonstrated in the examples, expression of Pref-1 is then maintained in the population of preadipocytes thus selected.
  • the stage of selection of the preadipocytes comprises contacting the preadipocytes with a cell culture support, preferably plastic plate, dishes and/or flashes, said selection stage comprising selection of preadipocytes adhering to the cell culture support, preferably after 30 minutes, and preferably after 45 minutes, and more preferably after or at about 1 hour of culture in contact with the cell culture support, preferably plastic plate, dishes and/or flashes.
  • this contacting is not more than 6 hours, preferably not more than 2 hours.
  • the population of preadipocytes enriched with preadipocytes expressing Pref-1 contains at least 50% of preadipocytes expressing Pref-1 , more preferably at least 70% and more preferably at least 90% of preadipocytes expressing Pref-1.
  • the population of preadipocytes is constituted essentially of preadipocytes expressing Pref-1.
  • the population of preadipocytes is constituted exclusively of preadipocytes expressing Pref-1 .
  • the invention thus relates, according to another aspect, to a cellular population essentially comprising preadipocytes, characterized in that it is enriched with preadipocytes expressing the marker Pref-1 .
  • the stage of proliferation of the preadipocytes comprises very advantageously the seeding and culture of the preadipocytes in the presence of a proliferation medium comprising Fibroblast Growth Factor (FGF) or Epidermal Growth Factor (EGF), preferably at a concentration of about 10 ng/ml, and preferably with FGF, and more preferably with beta-FGF.
  • FGF Fibroblast Growth Factor
  • EGF Epidermal Growth Factor
  • the proliferation medium is also called amplification medium.
  • the population of preadipocytes enriched with preadipocytes expressing Pref-1 is amplified in the proliferation medium from 1 to 5 times usually designated as passage 0 to passage 5.
  • Culture in the proliferation medium is advantageously carried out for 12 to 1 8 days, preferably for 14 and 16 days and optimally for 14 days, advantageously in the proliferation medium according to the invention described below. It was in fact discovered that at 14 days of culture after seeding in this medium, the preadipocytes have optimum capacity for differentiation. Surprisingly, a longer culture time does not improve this capacity.
  • the cellular population obtained at the end of the proliferation stage contains predominantly preadipocytes expressing Pref-1 ; preferably at least 90 % of the cell ula r population expresses Pref-1.
  • the method of cell culture according to the invention is useful for preparing a cellular population containing preadipocytes expressing Pref-1 havi ng opti m um capacities for differentiation. This population is called undifferentiated.
  • This population can be amplified when renewing culture in the proliferation medium with larger cell culture support, for instance double surface to obtain higher number of preadipocytes expressing Pref-1 in the cellular population containing preadipocytes. As demonstrated in examples, even after 5 and 6 passages, Pref-1 expression is stable and thus amplified cells keep optimum ability to differentiate into adipocytes.
  • the method comprises a stage of differentiation of at least a proportion of the population of preadipocytes into adipocytes.
  • This stage advantageously comprises culture of the preadipocytes in the presence of a differentiation medium comprising at least one of the following substances: a calf serum, preferably fetal (FCS), hydrocortisone, dexamethasone, and an activator of cAMP for example 3- isobutyl-1 -methylxanthine (IBMX), and preferably a mixture comprising a calf serum, preferably fetal (FCS), hydrocortisone, and dexamethasone and optionally I BMX.
  • a differentiation medium comprising at least one of the following substances: a calf serum, preferably fetal (FCS), hydrocortisone, dexamethasone, and an activator of cAMP for example 3- isobutyl-1 -methylxanthine (IBMX), and preferably a mixture comprising a calf serum,
  • the cells are preferably seeded and cultivated in a differentiation medium according to the invention described below.
  • Culture in the differentiation medium is advantageously carried out for 3 to 20 days, preferably for 4 to 10 days and more preferably for 7 days.
  • the method comprises culture of the population of preadipocytes in the proliferation medium carried out for 12 to 18 days, then in the differentiation medium for 3 to 20 days, preferably for 4 to 10 days and more preferably for 7 days.
  • the method according to the invention gives a cellular population containing preadipocytes and adipocytes at a controlled content.
  • the mixed population of preadipocytes and adipocytes contains predominantly mature adipocytes, preferably at least 70% of mature adipocytes.
  • the method can be implemented without matrix substrate such as a gel, a sponge notably based on collagen or collagen, chitosan, glycosaminoglycans (GAG) or any three-dimensional network for supporting the cell culture, and does not require intermediate combining of different sub-parts or layers of the model in order to obtain the desired state of differentiation and thickness for preparing a tissue model according to the invention.
  • matrix substrate such as a gel, a sponge notably based on collagen or collagen, chitosan, glycosaminoglycans (GAG) or any three-dimensional network for supporting the cell culture.
  • GAG glycosaminoglycans
  • the models according to the invention include in particular a monolayer of cells, a tissue model, in particular a model of hypoderm or a model of adipose tissue.
  • hypooderm means a tissue obtained by proliferation and differentiation of at least a proportion of the population of preadipocytes according to the method of the invention, optionally in the presence of other cell types, preferably fibroblasts and/or keratinocytes and/or melanocytes and/or endothelial cells and/or interstitial dendritic cells (IDC) and/or Langerhans' cells (LC) optionally on an inert substrate.
  • fibroblasts and/or keratinocytes and/or melanocytes and/or endothelial cells and/or interstitial dendritic cells (IDC) and/or Langerhans' cells (LC) optionally on an inert substrate.
  • IDC interstitial dendritic cells
  • LC Langerhans' cells
  • model of adipose tissue means a reconstructed 3D tissue, being in layers or in a substrate of the sponge type, notably based on collagen, or based on collagen, chitosan, glycosaminoglycans (GAG) at least one of which comprises adipocytes obtained after proliferation and differentiation of preadipocytes according to the method of the invention.
  • the model of adipose tissue contains or is a hypoderm according to the invention.
  • the model of adipose tissue is commonly called “model of reconstructed adipose skin” when it contains fibroblasts and keratinocytes.
  • a model of reconstructed adipose skin containing melanocytes is commonly called “model of pigmented reconstructed adipose skin”.
  • the invention thus relates to a hypoderm comprising a population of preadipocytes and adipocytes obtained according to the method of the invention.
  • the adipocytes are mature and differentiated.
  • the hypoderm contains predominantly mature adipocytes, preferably at least 70% of mature adipocytes.
  • the method according to the invention makes it possible to obtain, surprisingly, hypoderm having a greater thickness than the models of the prior art.
  • the hypoderm obtained according to the invention has a thickness of at least 70 ⁇ (micrometers), preferably more than 100 ⁇ (micrometers), and more preferably more than 130 ⁇ (micrometers), notably obtained in less than 28 days from a population of preadipocytes according to the invention.
  • This very significant advantage relative to the model of the prior art makes it possible to avoid superposition of layers for preparing adipose tissue, which is a drawback of the models of the prior art.
  • This method of culture which is easy and can be automated, makes it possible to obtain an adipose tissue or reconstructed hypoderm after a phase of proliferation of the population of preadipocytes in a first medium called proliferation medium permitting the formation of an adipose tissue or three-dimensional undifferentiated hypoderm then in a so-called differentiation medium permitting transformation of the preadipocytes into adipocytes.
  • proliferation medium permitting the formation of an adipose tissue or three-dimensional undifferentiated hypoderm then in a so-called differentiation medium permitting transformation of the preadipocytes into adipocytes.
  • the model of hypoderm can contain fibroblasts.
  • the method according to the invention in fact makes possible the co-seeding of fibroblasts and its coculture with the population of preadipocytes according to the invention.
  • the seeding of fibroblasts can be carried out on the hypoderm after it is obtained. Generally, seeding of the fibroblasts can therefore be done before, at the same time or after that of the preadipocytes.
  • the model of hypoderm can also contain endothelial cells, interstitial dendritic cells (IDC) and/or Langerhans' cells (LC), preferably a population of mixed IDC/LC cells such as described in patent application WO03/050271 .
  • IDC interstitial dendritic cells
  • LC Langerhans' cells
  • This embodiment has the advantage of supplying a cellular support that is particularly favourable for culture of the population of preadipocytes owing to the capacity of the fibroblasts to secrete and organize the components of the extracellular matrix.
  • the model of hypoderm or the model of fibroblast hypoderm can be epidermized by keratinocytes cultivated in immersed phase for a sufficient time to permit covering of the hypoderm or of the fibroblast hypoderm then in emerged phase for a sufficient time to permit epidermal differentiation.
  • the adipodermal proliferation medium is used first, then that for adipodermal differentiation.
  • a medium of the Green's medium type is used, which is then depleted during the phase of raising to the air-liquid interface.
  • the invention is implemented in the cell culture device described in patent (FR2881434) comprising at least one cell culture well intended to receive cells with their culture medium containing at least one layer of porous cell substrate material, said layer being obtained by dehydration of an aqueous gel poured directly into the bottom of said well, or into the bottom of a carriage or insert of suitable size so that it can be inserted in the well.
  • This method of preparation of the cell culture device makes possible the reproducible, safe and reliable high- throughput screening of active principles by a platform that can be automated.
  • the culture substrate corresponds to a gel comprising a mixture of collagen, at least one polysaccharide and chitosan, optionally modified, for example having a degree of acylation, preferably of acetylation, adjusted depending on the application envisaged, different degrees of acetylation being well known by a person skilled in the art and in particular described in the European document (EP 0296 078).
  • the models of hypoderm and of fibroblast hypoderm can be made in a sponge notably based on collagen or based on collagen, chitosan, glycosaminoglycans (GAG) in particular comprising by weight of the aqueous composition:
  • a second alternative described in patent US6777231 consists of using biocompatible matrices composed of monomers of glycolic and lactic acids, propyl fumarate, caprolactone, glycosaminoglycans, proteins, polysaccharides, polydihydroxy acids, polyorthoesters, polyanhydrides, polyphaophazenes, and other biocompatible synthetic polymers.
  • These agents can be used mixed either to form a crosslinked hydrogel or a sponge notably based on collagen or on collagen, chitosan, glycosaminoglycans (GAG) or a network with porosity between 100 and 300 ⁇ .
  • These models can be used in particular for making filling kits for surgery, in particular human surgery, such as the filling of wrinkles and of scars as well as for the grafting of adipose tissue in cases of deficiency pathologies.
  • hypoderm The functionality of the hypoderm was demonstrated in the examples notably by the expression of markers of maturation of the adipocytes, for example adiponectin, and in the case of a fibroblast hypoderm, from the synthesis of the constituents of the extracellular matrix. Moreover, the functionality of the hypoderm was demonstrated by its capacity for response to conventional slimming agents, notably caffeine, in particular from measurements of changes in thickness and secretion of adiponectin in response to the agents.
  • conventional slimming agents notably caffeine
  • the invention thus makes it possible to produce a reliable, economical, and reproducible model very quickly, and production can be automated.
  • - no matrix substrate such as a gel, a sponge notably based on collagen or on collagen, chitosan, glycosaminoglycans (GAG) or any three-dimensional network is used for supporting the cell culture, which can be performed in multi-well culture plates with from 6 to 96 wells in immersed phase
  • the culture phase can be performed using only 3 different media sequentially, renewal of which can be controlled by an automated station or robot
  • the invention also relates to a model of adipose tissue comprising a hypoderm defined previously.
  • the hypoderm, optionally fibroblastic and/or epidermalized has a sufficient thickness to be used alone for making a model of adipose tissue without the need for self- assembly of 3 layers, which was the major drawback of the models of the prior art.
  • self- assembly of different layers greatly complicates the method of production of models of adipose tissue or even makes them impossible and does not allow certain pharmaceutical applications to be envisaged, in particular injection.
  • the model of adipose tissue according to the invention makes its use possible by injection, for filling in surgery.
  • the invention also relates to a method for screening active principles, characterized in that it employs a model according to the invention, preferably a hypoderm or a model of adipose tissue.
  • the method according to the invention allows a model to be made that is particularly suitable for the screening of active principles, and notably make it possible to take into account the response of the two populations of preadipocytes and adipocytes. It thus makes it possible to supply active principles acting on the preadipocytes and/or adipocytes part of skin in order to modulate the degree of proliferation and differentiation of preadipocytes, as well as the state of maturity of the adipocytes and hence select slimming active principles and/or those for the prevention and/or treatment of obesity.
  • the present invention permits identification of substances having the capacity to reduce the proliferation and differentiation of preadipocytes and/or only the level of differentiation/maturity of adipocytes in subjects notably having a skin whose hypoderm is excessive, in particular persons who are overweight or obese or suffer from lipodystrophies, whether or not they have hormonal disorders or fluctuations, and whether or not they have problems of drainage, in particular lymphatic.
  • the models according to the invention can be used for screening active principles and identifying those that act on the preadipocytes and/or adipocytes part in order to modulate the degree of proliferation and/or differentiation of the preadipocytes and/or on the state of maturity of the adipocytes with the aim of increasing or reducing an individual's adipose skin mass.
  • the growth of subcutaneous adipose tissue is a function both of the proliferation of the preadipocytes and of their potential for maturation. It is therefore advantageous to evaluate both the effect of a substance on cellular viability, cellular proliferation and differentiation of the preadipocytes in order to evaluate the overall effect of a substance.
  • the stages of measurement can be carried out during the method of culture according to the invention or on the models obtained following application of the method according to the invention.
  • the method of cell culture comprises one or more stages of measurement of the gene and/or protein expression of Pref-1 and/or can be coupled to other criteria for investigation, notably:
  • the method of evaluation of gene expression is a method of RT-PCR, preferably quantitative.
  • the method of evaluation of protein expression is a method of Western blot, flow cytometry or immuno-localization on cells or on histological sections.
  • the quality of the reconstructed hypoderm is evaluated after staining with haematoxylin-phloxine-saffron or Masson trichrome or haematoxyl i n-eosin and permits measurement of thickness.
  • evaluation of the lipid vacuoles is carried out after staining the lipids for example with Oil Red O.
  • lipolysis is evaluated from secretion of glycerol after stimulation of the reconstructed hypoderm with an adrenergic agonist.
  • lipogenesis is evaluated in particular by measurement of adiponectin secretion by ELISA.
  • Investigations of cellular viability and of cellular proliferation can be performed on the preadipocytes after their characterization from the expression of the PREF-1 markers using methods well known by a person skilled in the art for example manual or automatic cell counting, t e s t i n g w i t h M T T ( 3-(4-5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium) or PNPP (paranitrophenyl phosphate).
  • the investigations of proliferation are performed on preadipocytes in a medium containing optimum concentrations or depleted of proliferation factors but not containing adipogenic factors.
  • the preadipocytes are in the proliferative phase and cannot differentiate owing to lack of adipogenic factors; for example the substance screened is thus considered to be an active principle:
  • the substance screened can also be toxic to the preadipocytes. Toxicity and decrease in potential for proliferation can be differentiated by the observation of phenomena of necrosis or apoptosis.
  • Increase in gene expression means, according to the invention, an increase greater than or equal to 20% (or 1 .2 times), preferably 50% , preferably 1 00% (or 2 times) the level of expression of the cells in culture without the screened molecule, at the same incubation time.
  • Decrease in gene expression means, according to the invention, a decrease greater than or equal to 20% (or 0.8 times), preferably 50% (or 0.5 times) the level of expression of cells in culture without the screened molecule, at the same incubation time.
  • Increase in protein expression means, according to the invention, an increase greater than or equal to 10% (or 1 .1 times), preferably 30% (or 1 .3 times) the level of expression of the cells in culture without the screened molecule, at the same incubation time.
  • Decrease in gene expression means, according to the invention, a decrease greater than or equal to 10% (or 0.9 times), preferably 30% (or 0.7 times) the level of expression of the cells in culture without the screened molecule, at the same incubation time.
  • the investigations of differentiation can be performed on preadipocytes in a medium containing optimum concentrations or depleted of proliferation factors with or without adipogenic factors.
  • the preadipocytes are in the proliferative phase and can differentiate depending on the presence of adipogenic factors.
  • the substance screened is thus considered as being an active principle:
  • adipocyte dedifferentiation has never been clearly demonstrated since it is necessary to start from a population no longer containing preadipocytes or mesenchymal precursors. It would optionally be possible to study this phenomenon on mature adipocytes no longer expressing Pref-1 but expressing at least one marker such as adiponectin, in differentiation media preferably without proliferative agent and adipogenic factors.
  • the adipocytes may possibly dedifferentiate, and for example the screened substance is considered as being an active principle:
  • Pref- 1 reversing the maturation of the adipocytes if the gene and/or protein expression of Pref- 1 is increased by more than 10%, preferably 30%, relative to the gene expression of cells not treated with the screened substance during the same incubation time.
  • the substances thus selected for their capacities for stimulating the proliferation of the preadipocytes, their differentiation into adipocytes and the maturation of the adipocytes can be used as active principle for preparing a composition for modulating the reactivity of an individual's cutaneous adipose tissue, in particular for increasing the proliferation and differentiation of the preadipocytes and/or only the level of differentiation/maturity of the adipocytes in subjects notably having skin whose hypoderm is not sufficiently thick or firm, such as elderly human beings or those suffering from cachexia or lipodystrophies, whether or not they have hormonal disorders or fluctuations.
  • the invention further relates to the use of a model according to the invention, in particular a hypoderm or a model of adipose tissue according to the invention, as a support for tissue engineering, surgical repair, reconstructive surgery, cosmetic surgery, cosmetic dermatology, notably for the filling of soft tissues, for example for the filling of wrinkles, loss of substance and scars.
  • the present invention also relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a population of preadipocytes according to the invention.
  • Said composition advantageously comprises a pharmaceutically acceptable excipient, and optionally other pharmaceutically active substances.
  • the present invention also relates to an injectable pharmaceutical product containing a population of pread ipocytes accord i ng to the invention in suspension in a suitable pharmaceutical support such as an injectable gel or liquid or other injectable pharmaceutical vehicle.
  • a suitable pharmaceutical support such as an injectable gel or liquid or other injectable pharmaceutical vehicle.
  • the present invention also relates to the use of said product or composition for filling wrinkles and loss of substance, prevention and/or repair of scars, and/or of lipodystrophies by subcutaneous or intramuscular injection.
  • the invention also relates to the use of a model according to the invention, in particular a hypoderm or a model of adipose tissue according to the invention, as a model for performing tests for pharmacotoxicology and/or activity of active ingredients, notably in cosmetics and pharmaceutics.
  • the invention also relates to a medium for cellular proliferation of the population of preadipocytes enriched with preadipocytes expressing Pref-1 according to the invention, characterized in that it comprises preadipocytes and FGF or EGF, preferably at a concentration between 5 and 20 ng/ml, more preferably between 8 and 12 ng/ml.
  • This medium is particularly advantageous for maintaining expression of Pref-1 and constitutes the preferred proliferation medium (experiment 1 B/D).
  • the population of cells isolated and/or selected according to the method of the invention can be frozen in a suitable medium.
  • a medium that is particularly suitable for freezing preadipocytes and preserving their undifferentiated character has been identified, and comprises fetal calf serum to more than 50%, preferably to more than 75%, and preferably to more than 80% , and preferably to about 90% , by volume relative to the total volume of the medium.
  • the inventors have in fact demonstrated that said freezing medium permits expression of Pref-1 to be maintained (experiment 2).
  • the invention thus relates to a population of preadipocytes, optionally enriched with preadipocytes expressing Pref-1 in a freezing medium according to the invention.
  • the invention also relates to a med ium for cellular differentiation of preadipocytes, characterized in that it comprises a population of preadipocytes, preferably expressing Pref-1 , and fetal calf serum, preferably at a concentration between 5 and 20% by volume relative to the total volume of the medium, hydrocortisone, dexamethasone, and an activator of cAMP for example 3-isobutyl-1 -methylxanthine (I BMX), and preferably a mixture comprising calf serum, preferably fetal (FCS), hydrocortisone, and dexamethasone, and optionally IBMX.
  • I BMX 3-isobutyl-1 -methylxanthine
  • FCS fetal
  • hydrocortisone fetal
  • dexamethasone dexamethasone
  • IBMX optionally IBMX
  • Example 1 Protocol for optimization of extraction and proliferation of preadipocytes
  • the subcutaneous adipose tissue is collected from the operating theatre during cosmetic or reconstructive surgery, lipectomy, preferably lipoaspiration.
  • the preadipocytes are extracted for example according to Bjorntorp's method or by a gentler method by syringe:
  • the adipose tissue can also be removed by a technique similar to the lipostructure of Coleman et al.
  • the basic principle is the atraumatic character of manipulation of the adipose tissue. Extraction must be done using a special suction cannula with a diameter of 3 mm, a length of 15 cm, having a tip made of foam and a double orifice wide enough for the adipocytes to pass through.
  • the vacuum in the syringe is created manually and progressively, so that the adipocytes are not subjected to excessive vacuum. Making multiple tunnels during extraction can reduce the risk of haemorrhage and trauma.
  • Tissue separation to obtain the stromal cells is carried out in a stage of fractional digestion. Briefly, the adipose tissue is digested in a solution of collagenase (EC 3.4.24.3 Roche 0.18-0.22 U/ml) at varying concentration from 0.2 to 1 mg/ml with constant agitation for 15 to 60 minutes in order to select the optimum conditions for obtaining the best rate of growth and extraction yield of cells expressing Pref-1 .
  • collagenase EC 3.4.24.3 Roche 0.18-0.22 U/ml
  • the action of the enzyme is then stopped by adding a solution of DMEM supplemented with 10% by volume of FCS.
  • the floating adipocytes are withdrawn.
  • the cell pellet is taken up in PBS to remove all traces of serum and then it is filtered at 70 ⁇ .
  • the remaining erythrocytes are lysed by adding a lysis buffer (NH4CI at 8.7 g/L) for 10 minutes at 37°C. Repeat centrifugation removes the residues of erythrocytes from the cells of the vascular stromal fraction.
  • EGF EGF
  • FGF FGF
  • VEGF vascular endothelial growth factor
  • a base medium containing: a DMEM/Ham's F12 mixture, supplemented with 10% of fetal calf serum, 0.4 ⁇ g/ml of hydrocortisone, 0.12 I U/ml of insulin, 24.3 ⁇ g/ml (or 18.5 nM) of adenine, 2 nM of triiodothyronine, isoprenaline (Isuprel at 0.4 ⁇ g/ml) or cholera toxin 10 nM and of a mixture of antibiotics (100 lU/ml of penicillin, 20 ⁇ g/ml of gentamicin and 1 ⁇ g/ml of Fungizone).
  • antibiotics 100 lU/ml of penicillin, 20 ⁇ g/ml of gentamicin and 1 ⁇ g/ml of Fungizone.
  • the preadipocytes are seeded in plates at a density of about 7000 cells per cm 2 .
  • the medium is changed every three days until confluence of the cells.
  • the cells are detached with trypsin 0.0125%/EDTA 0.005%, centrifuged at 1000 rpm, and taken up in 10 ml of medium.
  • the cells are counted on Malassez cells.
  • the population doubling time (PD) and growth rate (GR) are calculated.
  • the population doubling time (PD) corresponds to the number of multiplications that occurred during the culture time and the growth rate (GR) corresponds to the number of population doublings per day.
  • the cells are seeded in the appropriate culture medium and cultivated at 37°C and 5% CO2. Culture is performed in triplicate for 6 passages (from P0 to P5) on precursors obtained from three different donors. C- Characterization of the preadipocytes by Western blot of the marker Pref-1
  • Selection of the proliferation medium was therefore carried out taking into account expression of Pref-1 in the preadipocytes in this medium.
  • the selection criteria were to maintain protein expression of Pref-1 obtained on the day of extraction throughout the passages in the different proliferation media tested.
  • the cells of non-adherent fraction, adherent for 1 hour and of the total fraction were washed once with phosphate buffer (PBS), and the proteins were extracted for 30 min at 4°C in the lysis buffer (TBS - Tris 50 mM, NaCI 250 mM), pH 7.5, 0.1 % sodium dodecyl sulphate (SDS), 2 mM dithiothreitol (DTT), in the presence of protease inhibitors.
  • the lysates were centrifuged for 15 min at 13 000 g at 4°C.
  • the supernatants are diluted with Laemmli buffer in the presence of beta mercaptoethanol before electrophoresis.
  • sample equivalent content in total proteins
  • the proteins are separated by electrophoresis on SDS-polyacrylamide 4-12% gel.
  • the proteins were transferred onto a nitrocellulose membrane.
  • the membranes are then saturated for one hour at room temperature in TBS buffer in the presence of 3% of bovine serum albumin (BSA).
  • BSA bovine serum albumin
  • the proteins are finally immunodetected after incubation of the goat or mouse anti-human PREF-1 primary antibody, diluted in a 1 % solution of TBS/BSA at 4°C overnight.
  • the primary antibody was then detected with an anti-goat or monkey or-mouse secondary antibody coupled to peroxidase using a chemiluminescent substrate or detected with a secondary antibody coupled to Alexa 488 revealed in fluorescence.
  • the intensity of the bands can be evaluated by image analysis and referred to the content of total proteins in the sample.
  • An anti-actin Western blot is used for normalizing the results.
  • the optimum collagenase concentration is selected at 0.5 mg/ml since the cumulative results for times of 15 and 30 min are the most advantageous at this concentration.
  • This protocol which includes a stage of adhesion, makes it possible to select preadipocyte cells expressing Pref-1 from the stromal cells extracted. Adhesion for one hour is sufficient to enrich the population in preadipocytes expressing Pref-1 , but a longer time does not give a significant difference.
  • beta-FGF and EGF at 10 ng/ml exert a strong positive influence, whereas vEGF does not lead to a response, contrary to what was expected according to the prior art.
  • the medium containing EGF or FGF makes it possible to maintain expression of Pref-1 .
  • the preadipocyte phenotype is maintained in media containing EGF or FGF at 10 ng/ml at least after 5 passages.
  • the Pref-1 antibody does not recognize any of the proteins extracted from the fibroblasts (negative control), thus demonstrating its specificity.
  • Example 2 Selection of a freezing medium for preadipocytes for preserving their undifferentiated character
  • the preadipocytes are trypsinized and the cellular suspension is taken up in 1 ml of freezing medium. 3 freezing media were tested in order to select the one giving the best cellular uptake after thawing.
  • Table 3 Composition of the 3 freezing media as percentage by volume
  • the preadipocytes obtained from two different donors are resuspended in the proliferation medium, and the live and dead cells are counted to evaluate the percentage cellular viability as well as the yield from 2 passages.
  • the cells are seeded in 3 plates of 25 cm 2 at a rate of about 10 000 cells/cm 2 in the proliferation medium. At confluence, the cells were trypsinized, and the population doubling time (PD) and growth rate (GR) were calculated according to the formula given in example 1 .
  • the preadipocytes were also seeded in an additional plate to verify the preadipocyte phenotype of the cells thawed at the 3rd passage in comparison with preadipocytes cultivated up to P3 but without freezing according to the protocol described in example 1 .
  • the characteristic band of Pref-1 is present as in the cells in the 3rd passage that did not undergo freezing (Fig. 3). All the results show that the medium containing 90% by volume of serum is optimum for preserving the preadipocytes and maintaining their undifferentiated character after thawing.
  • Example 3 Selection of a medium for differentiation of preadipocytes into mature adipocytes
  • the preadipocytes in passage 3 were seeded at a density of about 40 000 cells/cm 2 in plates of 25 cm 2 in various media.
  • the results were interpreted according to 3 criteria: the number and size of the mature adipocytes, and the secretion of adiponectin.
  • Mathematical analysis made it possible to study the factors separately and in interaction two by two.
  • the calf serum used is not supplemented with ingredients known to have a PPARy agonist effect (Hyclone supplier certificate).
  • the base medium is a medium derived from the composition of Green's medium used for the culture of reconstructed skin, the formula of which was adapted on the basis of three successive experimental matrices firstly for inducing adipogenic differentiation.
  • the first experimental matrix comprises 1 1 media, which were compared with the proliferation medium, the following four ingredients of which were evaluated at variable concentrations relative to Green's medium.
  • FCS Fetal calf serum
  • I BMX at 0.5 mM or isoprenaline (Isuprel) at 0.4 ⁇ g/ml.
  • I BMX is added for three days only because of its recognized cytotoxicity, whereas isoprenaline can be left throughout the culture in the differentiation medium.
  • Table 6 Composition of ingredients evaluated in the differentiation media of the first experimental matrix Observation in optical microscopy (Fig. 4)
  • the proliferation medium control medium
  • the cells organize spontaneously into thick layers, which are easily removed. No mature adipocyte within this layer is observed on the surface.
  • the preadipocytes cultivated in a medium without serum have difficulty adhering.
  • the adipocytes are smaller, with small lipid lobules.
  • the media with serum the cells adhere and differentiate.
  • the number of mature adipocytes is counted and they are distributed for each medium into medium or large adipocytes.
  • the assay is performed using a commercial kit (Linco research) for ELISA with fixed concentration of antigen labelled with iodine 125.
  • the control is a Green's medium.
  • the medium must contain serum (indispensable for all the evaluation criteria), IBMX (indispensable to obtain medium and large adipocytes), hydrocortisone and dexamethasone at 25 nM (indispensable for maximum secretion of adiponectin). This medium corresponds to medium 6.
  • the first experimental matrix was able to show the strong influence of fetal calf serum at 10% by volume and of IBMX at 0.5 mM on all the criteria investigated.
  • the experiment shows that there are differences according to the type of serum, for example between newborn calf serum or fetal calf serum .
  • the optimum dose has yet to be refined for dexamethasone and hydrocortisone.
  • the second experimental matrix comprises 7 media, which were compared with the proliferation medium, the following three ingredients of which were evaluated at variable concentrations or grades relative to Green's medium containing IBMX at 0.5 mM.
  • NCS Newborn calf serum
  • FCS fetal calf serum
  • Dexamethasone 0 or 1 ⁇
  • Table 8 Composition of ingredients evaluated in the differentiation media of the second experimental matrix Observation in optical microscopy
  • the cells cultivated in medi um with NCS have a fi broblastic appearance and are organized in a thick layer.
  • FCS 3, 4, 7
  • fetal calf serum was selected at 10% by volume, hydrocortisone at 10 nM, IBMX at 0.5 mM and T3 at 2 nM.
  • Dexamethasone was re-tested between 25 and 1000 nM and insulin between 0.150 and 1.5 Ill/ml.
  • the fol lowi ng med i um was the medium adopted for optimum differentiation of preadipocytes into adipocytes, i.e. a medium containing FCS, hydrocortisone, dexamethasone, and IBMX.
  • Example 4 Comparison of adiponectin synthesis during differentiation between a cell population derived from ADSC stem cells and a cell population derived from preadipocytes selected according to the method of the invention
  • the population of preadipocytes (PAA07085) is isolated and selected according to example 1.
  • ADSC stem cells The population of ADSC stem cells is a classical population that is commercially available (hADSC, Invitrogen®).
  • the cells are cultured in the same conditions.
  • the proliferation medium is that described in example 1 (optimized proliferation medium) for 15 days before induction of differentiation (DO) and the differentiation medium is the optimized medium described in example 3.
  • Adiponectin synthesis is followed by ELISA colorimetric assay (R&D System) and the presence of adipocytes is determined with Oil Red O.
  • the population obtained from preadipocytes expressing Pref-1 according to the invention (PAA07085) is capable of synthesizing adiponectin more quickly than one and the same population obtained from stem cells (ADSC). Moreover, the cells obtained according to the method of the invention have, at D14, larger vacuoles than the cells obtained from ADSC.
  • the rate of proliferation is greater for the population obtained according to the method of the invention than the population derived from ADSC that has a lower growth rate. It was in fact noted that at the same seeding rate, the population derived from ADSC reaches confluence on D4 vs D3 for the population derived from preadipocytes according to the invention.
  • preadipocytes could organize into layers when they were cultivated in certain media.
  • the conditions of preparation of the hypoderm were optimized so as to obtain a cellular population in which preadipocytes and mature adipocytes coexist in a controlled manner.
  • the protocol followed uses preadipocytes seeded for example in passage 3 at a density of about 40 000 cells/cm 2 in plates of 75 cm 2 .
  • the cells are cultivated for 2, 3 or 4 weeks in the proliferation medium supplemented with vitamin C (50 g/ml) as well as for 19 days in a differentiation medium composed for example of 10% by volume of fetal calf serum (FCS), 2 nM of triiodothyronine, 0.15 lU/ml of insulin, 1 ⁇ of dexamethasone, 10 nM of hydrocortisone and 0.5 mM of I BMX and without vitamin C.
  • FCS fetal calf serum
  • the cells are observed in optical microscopy at 5 days in differentiation medium.
  • the layers were fixed in 10% formol solution and embedded in paraffin. 5 ⁇ sections were deparaffined and the antigenic sites were unmasked with an enzyme specific to each type of antibody. The sections were then incubated in a solution of PBS/NGS 3% for one hour.
  • the primary antibody (rabbit polyclonal anti-human collagen I and V antibodies, mouse monoclonal anti-human collagen III and VI) was deposited on slides overnight at room temperature. Rabbit and mouse anti-lgGs coupled to peroxidase were used for detecting the immunocomplexes, using diaminobenzidine as substrate. Counter-staining was carried out with Harris haematoxylin. For the negative controls, the primary antibody was omitted.
  • the results show that regardless of the culture time in the proliferation medium alone, the cells have a fibroblastic appearance. Starting from 1 week of culture, the cells self-assemble into a layer but at this culture time the layer is too thin to be removed easily.
  • culture for 14 days in the proliferation medium is optimum, in that a longer culture time in the proliferation medium does not give better results for thickness and differentiation of preadipocytes into mature adipocytes.
  • culture for 19 days in the differentiation medium i.e. 33 days total culture time, it is the condition that makes it possible to obtain the largest amount of mature adipocytes.
  • the mature adipocytes appear very early, since at 3 days after the change to the differentiation medium, the cells have small lipid vacuoles, which increase in size throughout the culture.
  • the total thickness of the hypoderm is about 70 ⁇ .
  • the assays of adiponectin show that the cells' capacity to differentiate is optimum after a culture time in the proliferation medium of 14 days, since a longer culture time in the proliferation medium did not give greater secretion of adiponectin during differentiation.
  • Cells derived from 3 strains from different donors are seeded for example at 7000 cells per cm 2 in a 6-well plate.
  • the active substance tested is a hydrolysate of kappa-carrageenan corresponding to the product marketed under the name Slim-excessTM by BASF BEAUTY CARE SOLUTIONS FRANCE and described in patent application WO2009000935.
  • the active substance is added starting from the first change of culture medium, which corresponds to the second day of culture.
  • the preadipocytes are cultivated up to confluence, i.e. 7 days of culture, and are detached with trypsin/EDTA. After centhfugation, the cells are counted on Malassez cells and population doubling (PD) and the growth rate (GR) are calculated (formula described in example 1 ).
  • Table 13 Number of millions of cells after 7 days of culture of preadipocytes in the proliferation medium
  • Table 14 Evaluation of the growth rate (GR) of the preadipocytes after 7 days of proliferation of the preadipocytes and evaluation of the growth rate The results show that the active substance evaluated (at 0.2% and at 1 %) decreases the proliferation of the preadipocytes significantly relative to the negative control (Student test p ⁇ 0.05).
  • the active substance therefore limits the proliferation of the preadipocytes without cytotoxicity.
  • Example 7 Investigation of effects of active substances on a hypoderm
  • hypoderm is prepared according to the protocol described in example 5 (optimized protocol).
  • the cells were cultivated for 14 days in the previous proliferation medium described in example 5 then for 14 days in the differentiation medium previously described in example 5, the 2 media being supplemented with vitamin C at 50 ⁇ g/ml.
  • the ingredients tested namely the active substance and caffeine, were added at various times (2, 14 and 21 days) and until the end of the experiment.
  • the active substance was tested at two concentrations (0.2% and 1 %) versus the positive control (caffeine at 1 mM) and the negative control (neither active substance nor caffeine) on a population according to the invention.
  • the model developed allows the ingredients to be added at three different times:
  • hypoderms were investigated histologically, by staining with HPS and Oil Red O and with immunolabelling of collagen I, III, V according to the protocols described in example 5.
  • FIG. 7 Histological examination (Fig. 7) permitted measurement of the thickness of each of the hypoderms and the immunolabelling for characterization of the presence of the collagen extracellular matrix for the conditions "untreated", “caffeine” and “active substance” at concentration of 0.2% on several cellular strains.
  • first line results are control
  • second line results are obtained with caffeine
  • third line with active at 0.2%.
  • 1 st column results obtained when ingredient was added after 2 days and 2 nd column when ingredient was added after 14 days.
  • Staining H PS was performed after 28 days total culture and scale is 50 micrometers.
  • the model has a thickness of more than 150 ⁇ after 28 days of culture
  • the active substances when the ingredients are added after the proliferation phase, the active substances induce a decrease in differentiation since the final thickness of the hypoderms is less than that of the untreated hypoderms.
  • Example 8 Selection of modulators of adipocyte differentiation by evaluation of Pref-1 in quantitative RT-PCR
  • Pref-1 and of actin were analysed by real-time RT-PCR (quantitative reverse transcriptase polymerase chain reaction).
  • the preadipocytes are cultivated for 24 hours in the proliferation medium and their differentiation is induced or not for 24h at least in the differentiation medium.
  • the media are removed, the cells lawns are rinsed and the total RNAs are purified with the "SV 96Total RNA Isolation System" kit (Promega, Charbonnieres, France).
  • the purified RNAs are eluted in 100 ⁇ _ of water (Promega, Charbonnieres, France), assayed and distributed in plates (96-well, 10 ml. of total RNAs at 5 ng/mL).
  • the primers selected for performing this work are as follows:
  • Pref-1 is weakly expressed i n the preadipocytes in a monolayer but disappears after differentiation.
  • the active principles are selected when the degree of expression is increased or decreased by 20%, i.e. a factor of 1.2 or 0.8 times the degree of expression in the case of the untreated control after normalization with actin.
  • the active principles stimulating adipogenesis are selected when the degree of expression of Pref-1 is decreased by 20%, i.e. a factor of 0.8 times the degree of expression in the case of the untreated control after normalization with actin.
  • the active principles that cause adipogenesis to decrease are selected when the degree of expression of Pref-1 is stable or is increased by 20%, i.e. a factor of 0.8 times the degree of expression in the case of the untreated control after normalization with actin.

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  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Analytical Chemistry (AREA)
  • Rheumatology (AREA)
  • Physics & Mathematics (AREA)
  • Food Science & Technology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • General Engineering & Computer Science (AREA)
  • Toxicology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne un procédé de sélection d'une population cellulaire comprenant des préadipocytes exprimant le facteur Pref-1 et son utilisation pour la préparation d'un modèle cellulaire contenant des adipocytes, en particulier un modèle de tissu adipeux.
PCT/EP2011/061422 2010-07-06 2011-07-06 Modèle de tissu adipeux et procédé de préparation WO2012004310A1 (fr)

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FR1055489 2010-07-06
FR1055489A FR2962443B1 (fr) 2010-07-06 2010-07-06 Modele de tissu adipeux et procede de preparation
US201061425787P 2010-12-22 2010-12-22
US61/425,787 2010-12-22

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CN103060267A (zh) * 2012-12-26 2013-04-24 上海市内分泌代谢病研究所 3t3-l1脂肪前体细胞诱导分化方法
CN107345205A (zh) * 2017-09-06 2017-11-14 阜阳师范学院 一种成熟脂肪细胞去分化培养的装置及方法

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Publication number Priority date Publication date Assignee Title
DE102018120466B4 (de) 2018-08-22 2022-09-22 GEDIA Gebrüder Dingerkus GmbH Gehäuse mit einer quaderartigen Form

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103060267A (zh) * 2012-12-26 2013-04-24 上海市内分泌代谢病研究所 3t3-l1脂肪前体细胞诱导分化方法
CN107345205A (zh) * 2017-09-06 2017-11-14 阜阳师范学院 一种成熟脂肪细胞去分化培养的装置及方法

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FR2962443B1 (fr) 2017-11-17

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