EP3966317A1 - Procede d'amplification in vitro ou ex vivo de cellules souches du tissu adipeux humain - Google Patents
Procede d'amplification in vitro ou ex vivo de cellules souches du tissu adipeux humainInfo
- Publication number
- EP3966317A1 EP3966317A1 EP20700217.1A EP20700217A EP3966317A1 EP 3966317 A1 EP3966317 A1 EP 3966317A1 EP 20700217 A EP20700217 A EP 20700217A EP 3966317 A1 EP3966317 A1 EP 3966317A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- adipose tissue
- extracellular matrix
- human adipose
- cells
- fraction
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
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Classifications
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Definitions
- the present invention relates to a method for in vitro or ex vivo amplification of stem cells from human adipose tissue. It further relates to a method for in vitro or ex vivo amplification of differentiated cells, an extracellular matrix, a composition comprising a mixture of an extracellular matrix and a stroma-vascular fraction, a use of the extracellular matrix or of the composition comprising a mixture of the extracellular matrix and the stroma-vascular fraction, and differentiated cells obtained according to the process of the invention for their use.
- Cell therapy consists of a transplant of cells aimed at restoring the functions of a tissue or an organ when they are impaired by an accident, a pathology or aging. It allows a patient to be treated sustainably by a single injection of so-called “therapeutic” cells. These cells are obtained, in particular, from multipotent stem cells originating from the patient himself.
- Lipofilling is a special cell therapy technique used to transfer fat cells, or adipocytes, from one area of the body to another in order to reshape the body or the face.
- the adipocytes present in certain adipose sites are used for cell therapy and, more particularly, for lipofilling.
- the quantity of adipocytes available in a patient can be limiting. For example, when a patient has too low a body mass index or has undergone chemotherapy, he may not have enough fatty tissue to perform lipofilling.
- the non-enzymatic dissociation of adipose tissue makes it possible to isolate the precursors of adipocytes.
- This type of dissociation appears to be an alternative method that is much less expensive, faster and which has indisputable advantages for the manufacture of a product that complies with the standards of therapeutic grade production (reduced exposure to external products or contaminants).
- the non-enzymatic dissociation presented to date are not satisfactory because the number of adipocyte precursors obtained is low compared to enzymatic dissociation. This then requires their amplification in 2D on a culture dish.
- endothelial cells, the extracellular matrix and the three-dimensional structure of adipose tissue are lost at the end of the process.
- adipocyte precursors therein and thus try to reconstruct the structure of the adipose tissue as well as possible.
- the matrix is also used to orient in vitro the precursors of adipocytes towards a non-adipose cell type (essentially bone or cartilaginous) before implantation.
- Decellularized adipose tissue has also been proposed to increase the differentiation of the precursors and to better mimic the structure of the adipose tissue.
- the manufacture of all these types of matrices requires numerous steps involving enzymatic reactions or long chemical treatments.
- the decellularized tissue loses these endogenous cells but also loses the factors of therapeutic interest which are anchored on the native matrix, which reduces the clinical value of this type of matrix.
- Adipose tissue not decellularized and enriched with adipocyte precursors (previously isolated by enzymatic dissociation) followed by 2D amplification has recently been proposed as a matrix for better bone reconstruction.
- the time to generate this biological matrix is long, requires three weeks of culture in vitro, and does not allow the amplification of the precursors of adipocytes. Only the interest in bone repair was put forward by the authors.
- Three-dimensional (3D) suspension culture represents an alternative method of choice to the standard 2D method because it essentially allows the structure and intrinsic qualities of the tissue to be preserved.
- This advantage is important because, for example, the absence of a relevant human model which best mimics adipose tissue in vitro is a major limitation during trials of preclinical phases for the discovery of new drugs effective in combating obesity and associated metabolic diseases such as type 2 diabetes and cardiovascular disease.
- 3D culture is achievable in a closed system which reduces handling and the risk of contamination.
- a technical problem which the invention proposes to solve is to obtain in vitro or ex vivo a large quantity of stem cells of human adipose tissue or of differentiated cells, of therapeutic grade.
- the first object of the solution of the invention to this technical problem is a method of in vitro or ex vivo amplification of stem cells from human adipose tissue, comprising the following steps: extraction, on the one hand, of a stromal fraction -vascular of human adipose tissue comprising endothelial cells of the vascular network of human adipose tissue and stem cells of human adipose tissue and, on the other hand, of an extracellular matrix of said human adipose tissue, said extracellular matrix comprising cells endothelials of the vascular network of human adipose tissue, stem cells of human adipose tissue and collagen; mixing of said stroma-vascular fraction and said matrix extracellular; and culture of the mixture obtained in the preceding step, in suspension, in a culture medium.
- suspension culture of the strovascular fraction made possible by the presence of the extracellular matrix, allows 3D amplification, giving access to a large number of cells and thus limiting the manipulations which increase the risks of contamination.
- the extraction of the extracellular matrix comprises a non-enzymatic dissociation step and, in particular, the extraction of the extracellular matrix comprises a mechanical dissociation step;
- Extraction of the strovascular fraction and the extracellular matrix comprises the following steps: centrifugation of human adipose tissue to obtain at least two distinct fractions, a fraction A comprising a centrifuged extracellular matrix, and the stroma-vascular fraction; and mechanical dissociation of fraction A to obtain the extracellular matrix;
- the collagen of the extracellular matrix is collagen type I and collagen type III;
- the culture of the mixture of said stromal-vascular fraction and of said extracellular matrix comprises the following steps: transfer of said mixture in a sterile manner into a suspension culture bag comprising culture medium; amplification of said mixture forming cellular aggregates; and mechanical dissociation of said cellular aggregates.
- the invention relates to a method of in vitro or ex vivo amplification of differentiated cells comprising the following steps: extraction, on the one hand, of a stroma-vascular fraction of human adipose tissue comprising, cells endothelials of the vascular network of human adipose tissue and stem cells of human adipose tissue and, on the other hand, of an extracellular matrix of said human adipose tissue, said extracellular matrix comprising endothelial cells of the vascular network of human adipose tissue, cells strains of human adipose tissue and collagen; mixing said stromal fraction and said extracellular matrix; culture of the mixture obtained in the preceding step, in suspension, in a culture medium; and inducing differentiation of adipose tissue stem cells to obtain differentiated cells.
- the differentiated cells are adipocytes or osteoblasts, preferably adipocytes;
- the extraction of the extracellular matrix comprises a step of non-enzymatic dissociation, in particular the extraction of the extracellular matrix comprises a step of mechanical dissociation;
- the extraction of the stroma-vascular fraction and of the extracellular matrix comprises the following steps: centrifugation of human adipose tissue to obtain at least two distinct fractions, a fraction A comprising a centrifuged extracellular matrix, and the stroma-vascular fraction; and mechanical dissociation of fraction A to obtain the extracellular matrix;
- the collagen of the extracellular matrix is collagen type I and collagen type III;
- the culture of the mixture of said stromal-vascular fraction and of said extracellular matrix comprises the following steps: transfer of said mixture in a sterile manner into a suspension culture bag comprising culture medium; amplification of said mixture forming aggregates cell phones; and mechanical dissociation of said cellular aggregates.
- the invention relates to an isolated extracellular matrix capable of being obtained according to the method defined above, comprising endothelial cells of the vascular network of human adipose tissue, stem cells of human adipose tissue, and collagen.
- the collagen is collagen type I and collagen type III; - the extracellular matrix also comprises fibronectin.
- the invention relates to a composition
- a composition comprising the mixture of the extracellular matrix and the stroma-vascular fraction as defined above, the extracellular matrix comprising endothelial cells of the vascular network of human adipose tissue, stem cells. human adipose tissue, and collagen, and the strovascular fraction comprising endothelial cells of the vascular network of adipose tissue and stem cells of adipose tissue.
- the collagen is collagen type I and collagen type III; - the extracellular matrix also comprises fibronectin.
- the invention relates to the in vitro use of the extracellular matrix as defined above or the in vitro use of the composition as defined above for the screening of pharmacological active agents against obesity and associated metabolic diseases.
- the invention relates to differentiated cells obtained according to the process defined above intended for use, or for their use, in cell therapy, in particular in plastic and restorative surgery and, more particularly, for lipofilling.
- FIG. 1A schematically represents the necessary and sufficient steps for the extraction of an extracellular matrix and a stroma-vascular fraction (steps 1 to 3), and their coculture (step 4), according to one invention
- FIG. 1B is a more detailed schematic representation of the method which allows the sequential extraction of extracellular matrices (M1-M4) and of populations of cells (C1-C3) of stromal fraction (steps 1 to 5), and their coculture (step 6), according to the invention;
- FIG. 2 shows the Cl cell population in Endothelial Growth Medium culture medium supplemented with growth factors (EGM +), in suspension, consisting of a majority of endothelial type cells;
- FIG. 3A shows the CD31 + immunofluorescent labeling (endothelial cell marker) of the C1 cell population in adherent culture
- FIG. 3B shows the immunofluorescent labeling PDGFRa + (marker of adipocyte stem cells) of the cell population C1 in adherent culture;
- FIG. 4 illustrates the cell population C2 in EGM + culture medium in suspension showing the formation of a capillary type network consisting of cells CD31 + endothelial cells and the presence of aggregates made up of PDGFRa + adipose tissue stem cells;
- FIG. 5 is a microscopic image of the C3 cell population in EGM + culture medium in suspension showing the presence of aggregates consisting of stem cells from the adipose tissue PDGFRa +;
- FIG. 6A represents a quantitative PCR of the cell populations C2 and C3 making it possible to determine the relative proportion of endothelial cells CD31 + and of the stem cells of the adipose tissue PDGFRa +;
- FIG. 6B is an image of fluorescence microscopy showing the capacity of adipocyte differentiation of C2 cell populations; Cores (dark gray) Lipid droplets (light gray);
- FIG. 6C illustrates, by a fluorescence microscopy image, the adipocyte differentiation capacity of C3 cell populations; Cores (dark gray) Lipid droplets (light gray);
- FIG. 8 shows, by microscopy, that the M2 matrix is heterogeneous in terms of matrix types: fibrous type and type rich in collagen;
- FIG. 9 is a microscopy image illustrating the fibrous type of the matrix M3;
- FIG. 10A shows, by fluorescence microscopy, that the matrix M2 is of the collagen-rich type; collagen labeled with Picro-Sirius Red (light gray) and nucleus labeling (white);
- FIG. 11A is a photograph of the adipose tissue centrifuged from fraction A after mechanical dissociation containing the matrix M4;
- FIG. 11B highlights by fluorescence microscopy, in the M4 matrix, mature adipocytes by Oil red 0 staining (light gray) and a matrix rich in collagen by labeling type I collagen (very light gray);
- FIG. 11C shows, by one CD31 immunostaining, the capillary structures formed by CD31 + endothelial cells (white) in the M4 matrix; core marking (dark gray);
- FIG. 11D illustrates the presence of the network of stem cells of the adipose tissue PDGFRa + (light gray dots) in the matrix M4; core marking (dark gray);
- FIG. 12 shows, by incorporation of Edu, 5-ethylnyl-2 '-deoxyuridine, in the nucleus of proliferating cells, that endogenous cells, in the extracellular matrix of the invention are maintained in proliferation in the EGM + medium in suspension ; nuclei (dark gray), proliferating cells (white) auto fluorescence of the matrix (light gray);
- FIG. 13 shows that stem cells of exogenous adipose tissue, put in coculture with the extracellular matrix of the invention, form structures composed of these stem cells of adipose tissue and endogenous cells present in the matrix; image taken after 3 days of coculture, nuclei (dark gray), collagen (light gray), exogenous stem cells from adipose tissue (light gray / white);
- FIG. 14A shows the formation of a cellular aggregate without proliferation of cells during the cell culture of adipose tissue stem cells and endothelial cells in suspension without an extracellular matrix; image taken after 10 days of co-culture; nuclei (dark gray), proliferating cell nuclei (very light gray);
- FIG. 14B shows a capacity for proliferation of stem cells from adipose tissue and endothelial cells placed in co-culture, in suspension, with the extracellular matrix of the invention; image taken after 10 days of co-culture; nuclei (dark gray), collagen matrix (light gray), nuclei of cells in proliferation by Edu labeling (white);
- FIG. 15A shows the level of expression of the marker for endothelial cells CD31 in the differentiated cell populations obtained by culture, in suspension, with (right) and without (left) the extracellular matrix of the invention
- FIG. 15B shows the level of expression of the adipocyte stem cell marker PDGFRa in the differentiated cell populations obtained by culture, in suspension, with (right) and without (left) the extracellular matrix of the invention
- FIG. 15C shows the level of expression of the marker for mature adipocytes PLN1 in the differentiated cell populations obtained by culture, in suspension, with (right) and without (left) the extracellular matrix of the invention
- FIG. 15D shows the level of expression of the adiponectin mature adipocyte marker in the differentiated cell populations obtained by culture, in suspension, with (right) and without (left) the extracellular matrix of the invention
- - Figure 16 shows an image by fluorescence microscopy of the stroma-vascular fraction after amplification and differentiation in the presence of the extracellular matrix of the invention
- nucleus dark gray
- mature adipocyte light gray
- collagen matrix medium gray
- Figures 17A and 17B are images which demonstrate the activation of the proliferation capacities of the method according to the invention.
- undissociated adipose tissue does not show proliferating cells.
- the composition shows proliferating cells, the nuclei of the proliferating cells being represented in white in this figure;
- FIGS. 18A, 18B, 18C and 18D illustrate the expression of dipeptidyl peptidase-4 (DPP4), which is concentrated in the isolated vascular stroma fraction (SVF), and the expression of ICAM1, which is concentrated in isolated matrix;
- DPP4 dipeptidyl peptidase-4
- FIG. 19A and 19B illustrate the presence of macrophages type Ml and type M2 respectively, in the amplified composition according to the invention.
- FIG. 20 includes a set of photographs which demonstrate the presence of certain proteins in the extracellular matrix according to the invention, and the conservation of a capillary network.
- the adipose tissue is provided to carry out the invention.
- the first object of the invention is a method of in vitro or ex vivo amplification of stem cells from human adipose tissue comprising the following steps: extraction, on the one hand, of a stroma-vascular fraction from human adipose tissue comprising cells endothelials of the vascular network of human adipose tissue and stem cells of human adipose tissue, and on the other hand, of an extracellular matrix of said human adipose tissue, said extracellular matrix comprising endothelial cells of the vascular network of human adipose tissue, cells strains of human adipose tissue and collagen; mixing said stromal fraction and said extracellular matrix; and culture of the mixture obtained in the preceding step, in suspension, in a culture medium.
- This process is also referred to hereinafter as the “ExAdEx process” (for Ex vivo Adipocytes Expansion).
- stroma-vascular fraction is understood to mean the cells present in a sample of human adipose tissue.
- This stromal-vascular fraction comprises endothelial cells of the vascular network of human adipose tissue and stem cells of human adipose tissue.
- extracellular matrix is understood to mean a bioactive matrix, that is to say a matrix which comprises different proteins from adipose tissue and endogenous cells. This extracellular matrix allows cellular amplification in 3D, that is to say the proliferation of cells in three dimensions.
- the extracellular matrix of the invention is also noted below “EndoStem-Matrix” or “EndoStem matrix”.
- the proteins in the extracellular matrix of adipose tissue include collagen. This collagen is type I and type III.
- the proteins in the extracellular matrix of adipose tissue also include fibronectin.
- Extraction of the extracellular matrix comprises a non-enzymatic dissociation step, in particular the extraction of the extracellular matrix comprises a mechanical dissociation step.
- the “mechanical dissociation” of the invention makes it possible to keep the structure of the extracellular matrix intact while an enzymatic digestion generally involves collagenase which destroys it. Mechanical dissociation thus allows the maintenance of the "vasculature" and the micro structure of the extracellular matrix, which consequently presents an organization similar to the organization of adipose tissue in vivo.
- the extraction of the stroma-vascular fraction and of the extracellular matrix comprises the following steps: centrifugation of human adipose tissue to obtain at least two distinct fractions, a fraction A comprising a centrifuged extracellular matrix, and the stroma-vascular fraction; and mechanical dissociation of fraction A to obtain the extracellular matrix.
- the centrifugation step of the human adipose tissue also makes it possible to remove the oil, blood and anesthetic liquid contained in the supplied human adipose tissue. This step also makes it possible to eliminate physiological liquid obtained from prior washes of the human adipose tissue provided.
- the extraction of the stroma-vascular fraction and of the extracellular matrix comprises the following steps: centrifugation of human adipose tissue to obtain at least two distinct fractions, a fraction A comprising a centrifuged extracellular matrix and a fraction B comprising endothelial cells of the vascular network of human adipose tissue and stem cells of the tissue human adipose; mechanical dissociation of fraction A to obtain a fraction A 'comprising a dissociated extracellular matrix; centrifugation of fraction A 'to obtain at least the extracellular matrix and a fraction B' comprising endothelial cells of the vascular network of human adipose tissue and stem cells of human adipose tissue; and mixing fractions B and B 'to obtain the stroma-vascular fraction.
- the step of centrifuging the human adipose tissue also makes it possible to remove the oil, blood and anesthetic liquid contained in the human adipose tissue supplied.
- This step also makes it possible to eliminate physiological liquid obtained from prior washes of the human adipose tissue provided.
- the centrifugation of fraction A ' also makes it possible to remove any residues of oil and physiological fluid. This centrifugation step of fraction A 'is optional.
- Culturing the mixture of said stromal fraction and said extracellular matrix comprises the following steps: transfer of said mixture in a sterile manner into a suspension culture bag comprising culture medium; amplification of said mixture forming cellular aggregates; and mechanical dissociation of said cellular aggregates.
- the transfer in a "sterile manner”, within the meaning of the invention, is a transfer, preferably, carried out in a closed system.
- This sterile transfer makes it possible to limit the number of contaminants during cell culture.
- the mechanical dissociation of the aggregates formed during the amplification does not require opening of the system, thus limiting the exposure of the products cells to possible contamination of the culture by elements of the environment.
- the culture medium in the suspension culture bag, is an EGM + medium.
- This culture medium comprises the basic medium for the proliferation of endothelial cells (EGM) enriched with Epidermal Growth Factor (EGF), Basic Growth Factor (FGF2), Insulin-like Growth Factor, Vascular Endothelial Growth Factor 165, ascorbic acid, heparin and hydrocortisone (EGM +).
- EGM + medium also allows the amplification of adipocyte stem cells without altering their capacity for differentiation into adipocytes.
- the method of the invention allows an amplification of the number of stem cells of adipose tissue with an amplification factor greater than 10, advantageously greater than 20, in particular greater than 30, preferably greater than 35.
- the amplification factor is the ratio between the number of cells obtained after culture of the SVF isolated in the presence of said extracellular matrix and the number of cells before the invention.
- the method of the invention has an amplification factor of 36 in 8 days.
- the invention relates to a method of in vitro or ex vivo amplification of differentiated cells comprising the following steps: in vitro or ex vivo amplification of stem cells of human adipose tissue as defined above; and inducing differentiation of adipose tissue stem cells to obtain differentiated cells.
- the in vitro or ex vivo amplification process of differentiated cells therefore comprises the following steps: extraction, on the one hand, of a fraction stroma-vascular of human adipose tissue comprising, endothelial cells of the vascular network of human adipose tissue and stem cells of human adipose tissue, and secondly, of an extracellular matrix of said human adipose tissue, said extracellular matrix comprising endothelial cells of the vascular network of human adipose tissue, stem cells of human adipose tissue and collagen; mixing said stroma-vascular fraction and said extracellular matrix; culture of the mixture obtained in the preceding step, in suspension, in a culture medium; and inducing differentiation of adipose tissue stem cells to obtain differentiated cells.
- the differentiated cells are adipocytes or osteoblasts.
- the differentiated cells are adipocytes.
- the method of in vitro or ex vivo amplification of differentiated cells comprising the steps related to the in vitro or ex vivo amplification of adipose tissue stem cells, the details given above for the method of in vitro or ex vivo amplification adipose tissue stem cells also apply for the in vitro or ex vivo amplification process of differentiated cells.
- the extraction of the extracellular matrix comprises a non-enzymatic dissociation step, in particular the extraction of the extracellular matrix comprises a mechanical dissociation step.
- the extraction of the stroma-vascular fraction and of the extracellular matrix comprises the following steps: centrifugation of human adipose tissue to obtain at at least two separate fractions, a fraction A comprising a centrifuged extracellular matrix, and the stromal-vascular fraction; and mechanical dissociation of fraction A to obtain the extracellular matrix.
- the extraction of the stroma-vascular fraction and of the extracellular matrix comprises the following steps: centrifugation of human adipose tissue to obtain at least two distinct fractions, a fraction A comprising a centrifuged extracellular matrix and a fraction B comprising endothelial cells of the vascular network of human adipose tissue and stem cells of human adipose tissue; mechanical dissociation of fraction A to obtain a fraction A 'comprising a dissociated extracellular matrix; centrifugation of fraction A 'to obtain at least the extracellular matrix and a fraction B' comprising endothelial cells of the vascular network of human adipose tissue and stem cells of human adipose tissue; and mixing fractions B and B 'to obtain the stroma-vascular fraction.
- the collagen of the extracellular matrix comprises type I collagen and type III collagen revealed by staining with Picro-Sirius Red.
- Culturing the mixture of said stromal fraction and said extracellular matrix comprises the following steps: transfer of said mixture in a sterile manner into a suspension culture bag comprising culture medium; amplification of said mixture forming cellular aggregates; and mechanical dissociation of said cellular aggregates.
- the invention relates to an isolated extracellular matrix capable of being obtained according to the method defined above, comprising cells endothelials of the human adipose tissue vascular network, human adipose tissue stem cells, and collagen.
- Collagen is type I collagen and type III collagen.
- the extracellular matrix further comprises fibronectin.
- the invention relates to a composition
- a composition comprising the mixture of the extracellular matrix and the stroma-vascular fraction as defined above, the extracellular matrix comprising endothelial cells of the vascular network of human adipose tissue, stem cells. human adipose tissue, and collagen, and the strovascular fraction comprising endothelial cells of the vascular network of adipose tissue and stem cells of adipose tissue.
- Collagen is type I collagen and type III collagen.
- the extracellular matrix further comprises fibronectin.
- the invention relates to the in vitro use of the extracellular matrix as defined above or the in vitro use of the composition as defined above for the screening of pharmacological active agents against obesity and associated metabolic diseases such as type 2 diabetes and cardiovascular disease.
- the invention relates to differentiated cells obtained according to the process defined above intended for use, or for their use, in cell therapy, in particular in plastic and repair surgery, and more particularly for lipofilling.
- the extracellular matrix is an autologous matrix, which by definition contains cells specific to the patient from whom the adipose tissue is derived.
- the differentiated cells obtained by the process of the invention are adipocytes.
- Fig. IB The mechanical extraction of the strovascular fraction and the extracellular matrix, from a sample of adipose tissue from a human donor, can be carried out according to the following steps (Fig. IB):
- the syringe is centrifuged at 1600 rcf (relative centrifugal force), 3 min in the collection tube.
- the oil fraction as well as the blood and anesthetic liquid fraction are eliminated.
- the pellet fraction is kept.
- a unit of physiological saline is injected into the syringe, followed by an incubation of 30 min at 37 ° C with shaking.
- the syringe is centrifuged at 1600 rcf, 3 min in the collection tube.
- the physiological liquid fraction and the oil fraction are eliminated.
- the pellet fraction is kept.
- the syringe is connected to another male Luer-Lock syringe connected by a Tulip® connector in order to dissociate the tissue by emulsification.
- Tulip® connector Three types of Tulip® connector, 2.4 mm, 1.4 mm and then 1.2 mm, are successively used, over 30 passages.
- a unit of physiological saline is injected into the syringe, followed by an incubation of 30 min at 37 ° C with shaking.
- the syringe is centrifuged at 1600 rcf 3 min in the collection tube.
- the physiological liquid fraction and the oil fraction are eliminated.
- the pellet fraction is kept.
- the contents of the syringe as well as the contents of the collection tubes previously rid of blood cells are transferred by a sterile connection into a culture bag containing the EGM + culture medium at 37 ° C. for the expansion phase.
- a connector of a brand other than the Tulip® brand can be used.
- the number of connectors used is between 1 and 5.
- the number of passes through these connectors is between 10 and 50.
- the method described above makes it possible to sequentially extract the stroma-vascular fraction into 3 cell populations. These cell populations are characterized in particular by optical microscopy and by fluorescence microscopy.
- the cell population obtained during step 2, here called Cl, is composed of a majority of CD31 + endothelial type cells (Fig. 2 and Fig. 3).
- the cell population obtained during step 3, here called C2 is composed of CD31 + endothelial type cells forming a capillary type network when maintained in 3D and tissue stem cells fat PDGFRa + (Fig. 4 and Fig. 6A).
- the C2 cell population has the capacity to differentiate into mature adipocytes (Fig. 6B).
- the cell population obtained during step 5, here called C3, is composed of a majority of stem cells of the adipose tissue PDGFRa + capable of forming spheres in suspension (Fig. 5 and Fig. 6A).
- the C3 cell population has the capacity to differentiate into mature adipocytes (Fig. 6C).
- Ml is of fibrous type (Fig. 7).
- M2 The matrix obtained during step 3, here called M2 is of fibrous type and rich in collagen (Fig. 8).
- the collagen is revealed by the Picro-Sirius Red (Fig. 10A) which also makes it possible to visualize a structure of the collagen in rods.
- This matrix contains endogenous cells.
- the matrix obtained during step 5 and isolated in the collection tube, named here M3 is of fibrous type (Fig. 9 and Fig. 10B). This matrix also contains endogenous cells.
- the collagen of the isolated matrix is revealed, in FIG. 10B, by Picro-Sirus Red which colors the type I and type III collagen fibers.
- the red coloration (in shades of gray in Fig. 10B) obtained indicates that the collagen remains organized, namely that the collagen present always has a secondary helical structure and a quaternary triple helical structure. It is not degraded. Indeed, disorganized collagen turns green with Picro-Sirus Red.
- the matrix obtained during step 5 and contained in the syringe, named here M4 is composed of a majority mature fat cells and a type I collagen frame (Fig. 11B).
- the M4 matrix is also composed of capillary structures formed by CD31 + endothelial cells (Fig. 11C) and by a network of PDGFRa + adipose tissue stem cells (Fig. 11D).
- FIG. 1A shows a method making it possible to gather in step 2 the populations Cl and C2 as well as the matrices Ml and M2.
- step 3 the population C3 and the matrices M3 and M4 are grouped together.
- a method of ex vivo expansion of adipose tissue stem cells and of differentiation in an environment mimicking adipose tissue comprises the following steps:
- Example 1 The final product obtained in Example 1 containing the C1-C3 populations as well as the so-called EndoStem-Matrix M1-M4 matrices are placed in suspension culture in bags and maintained in the EGM + proliferation medium with stirring for 24 h. at 37 ° C 5% CO2, then maintained under the same conditions, preferably with stirring.
- the EGM + proliferation medium is changed to 50% every two days.
- the EGM + proliferation medium is replaced by the differentiation cocktail I composed of EGM + enriched in 250 mM Dexamethasone; 500 mM IBMX; 1 mM Rosiglitazone; 2 mM T3 and 2.5 pg / ml insulin.
- the differentiation medium I is replaced by the differentiation medium II composed of EGM + enriched in 1 mM Rosiglitazone; 2 mM T3 and 2.5 pg / ml insulin.
- EndoStem-Matrix of the invention The extracellular matrices known as EndoStem-Matrix of the invention were characterized, in particular, by fluorescence microscopy, in the presence of different specific markers. Proliferating cells were thus detected by incorporation, during the DNA replication phase, of fluorescent Edu (5-ethylnyl-2 '-deoxyuridine) in the EndoStem-Matrix matrices of the invention, as illustrated in the Figure 12, proving that these are bioactive. Indeed, Figure 12 shows that cells endogenous to the matrices are maintained in proliferation during the amplification phase.
- FIG. 13 shows the presence of stem cells from the adipose tissue after three days of co-culture with the extracellular matrix of the invention.
- the extracellular matrix therefore makes it possible to provide a support for the proliferation of the strovascular fraction: the added stem cells of the adipose tissue can attach to the EndoStem-Matrix matrix, in suspension.
- the strovascular fraction is amplified by its culture on the EndoStem Matrix of the invention.
- the stroma-vascular fraction when the stroma-vascular fraction is cultured in suspension without the extracellular matrix, cell aggregates without proliferation are observed.
- the extracellular matrix of the invention therefore has the capacity to amplify the added fatty tissue stem cells.
- the cell amplification capacity of the different matrices M1 to M4 obtained during the steps of Example 1 was checked.
- approximately 10 4 stem cells of the adipose tissue were kept in suspension in the presence of the various matrices Ml to M4 in Ultra Low wells.
- the amplification factor is the ratio between the number of cells obtained after culture in the presence of the extracellular matrix and the number of cells obtained in the absence of the extracellular matrix.
- the M2 and M4 matrices have a strong power to amplify stem cells from adipose tissue.
- the volume of matrix M2 obtained is very low compared to the volume of M4 (Fig. 11A).
- the M4 matrix illustrates an extracellular matrix as defined in the invention.
- FIG. 15 shows a comparison of these expression levels with those from a suspension culture of the stroma-vascular fraction without the extracellular matrix of the invention. This study reveals an amplification of the endothelial cells of the vascular network of adipose tissue (FIG. 15A) and of the stem cells of adipose tissue (FIG. 15B).
- FIG. 16 shows the presence of nuclei, mature adipocytes and a collagen matrix after amplification and differentiation of the stromal fraction in the presence of the extracellular matrix of the invention. So the differentiation in the presence of the extracellular matrix of the invention makes it possible to conserve the in vivo structural organization of the adipose tissue.
- an undissociated adipose tissue which can be assimilated to an explant, remains viable for a short time ex vivo.
- the matrix isolated by dissociation contains proliferating cells, unlike non-dissociated tissue.
- Figs. 17A and 17B make it possible to compare cell proliferation in the non-dissociated tissue (Fig. 17A) and in the isolated matrix (Fig. 17B).
- the non-dissociated adipose tissue does not show proliferating cells.
- the composition shows proliferating cells. Indeed, this figure shows, in white, the nuclei of proliferating cells.
- DPP4 is a marker which marks the precursor cells of ICAM1 pre-adipocytes, which have a large proliferation capacity and which are located in the interstitial reticulum of adipose tissue. It is these cells which have the capacity for proliferation in the composition according to the invention. It is important to note that these cells are eliminated following the washes carried out according to the methods of the prior art. As shown in Figs. 18A and 18B, the expression of DPP4 is concentrated in the isolated SVF fraction. The matrix does not express much. On the other hand, and as shown in Figs. 18C and 18D, the expression of ICAM1, is concentrated in the isolated matrix. The cells which carry the amplification in the composition are the cells expressing DPP4 added.
- the adipose tissue in vivo contains macrophages and that the amplified composition according to the invention maintains the presence of type M1 macrophages, as shown in FIG. 19A and type M2 as shown in FIG. 19B.
- the M1 type macrophages are revealed by the marker IL-1b and, in FIG. 19B, macrophages of the type
- M2 are revealed by the marker MRC1.
- the isolated matrix according to the invention comprises proteins of the extracellular matrix, namely in particular, type I collagen, type IV collagen, elastin, fibronectin, laminin.
- the labeling of CD31 endothelial cells shows that a capillary network is preserved.
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FR1900287A FR3091707B1 (fr) | 2019-01-11 | 2019-01-11 | Procédé d’amplification in vitro ou ex vivo de cellules souches du tissu adipeux humain |
PCT/EP2020/050720 WO2020144381A1 (fr) | 2019-01-11 | 2020-01-13 | Procede d'amplification in vitro ou ex vivo de cellules souches du tissu adipeux humain |
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