Biocompatible composition suitable for the administration of cell therapies and its use in particular Parkinson's disease

Abstract

The invention relates to the technical field of the treatment of Parkinson's disease. In particular, the invention relates to a particular biocompatible composition comprising a solution and heterogeneous cellular elements suspended in said solution.

Description

Title of the invention: Biocompatible composition suitable for the administration of cellular therapies and its use in particular Parkinson's disease Technical field

[0001] The present invention relates to the treatment of Parkinson's disease. In particular, the subject of the invention is a particular biocompatible composition comprising a solution and heterogeneous cellular elements suspended in said solution. Prior art

[0002] Parkinson's disease is a neurodegenerative disorder characterized by a progressive decrease in dopamine production in the brain. This decrease in dopamine results in motor symptoms such as slowness in initiating movements (akinesia), muscle stiffness, and tremor at rest.

[0003] Currently, one of the most promising treatments for Parkinson's disease is the injection of dopaminergic neurons into the putamen, a brain region involved in motor control. This approach, known as cell therapy, aims to restore impaired dopaminergic function by providing functional cells capable of producing and releasing dopamine. Cell therapy via the injection of dopaminergic neurons offers many benefits, including prolonged symptom reduction, reduced medication use, and improved quality of life for patients with Parkinson's disease.

[0004] Today, cell therapy faces many challenges inherent in the administration of cells into the body.

[0005] In addition to the difficulties of large-scale cultivation of therapeutic cells adapted to the targeted organ, the composition of the injection medium comprising the cells to be inoculated is a fundamental challenge for any injectable therapy, particularly in the brain.

[0006] In general, drugs can dissolve in a liquid and can be distributed as a solution, especially in an infusion. Liquid suspensions comprising very small particles also behave as solutions and can also be administered by infusion.

[0007] On the other hand, as soon as the composition is cellularized, that is to say it comprises cells, it is injected in suspension.

[0008] Unlike solutions which form a homogeneous and fluid liquid, cells in suspension naturally sediment over time.

[0009] For example, "normal" erythrocytes sediment in the blood at a relative speed of up to 20 mm per hour, and when certain cells are aggregated into clusters or microtissue, this relative speed can exceed 100 mm per hour, or even 300 mm per hour in certain cases.

[0010] The sedimentation of particles in solid-liquid suspensions is a subject widely addressed in many fields such as chemistry, geology and biology, particularly via pharmaceutical applications or bioprinting.

[0011] The settling rate of solid particles such as cells in a liquid refers to the rate at which the particles move downward or settle to the bottom of a container by gravity. This sedimentation phenomenon occurs when the particles, due to their mass and density, are heavier than the medium in which they are suspended.

[0012] When using a cell suspension, the cells may be heterogeneous. Indeed, the cells may be in very varied forms, in which case they are referred to as cellular elements. For example, the cellular elements may be in the form of isolated cells, cellular microtissues or clusters of cells having a different density and / or size and / or number of cells. Consequently, when using suspensions comprising heterogeneous cellular elements, the cells are distributed heterogeneously in their medium, depending on several parameters, such as the size, shape and density of the cells, as well as the physicochemical properties of the medium.

[0013] Without control of these parameters, the greatest concentration of cells is found, by gravity, towards the lower part of the device comprising the suspension of cellular elements.

[0014] For example, in cell therapy, when injecting a suspension comprising heterogeneous cellular elements, the first injection volume may contain many more cells than those distributed subsequently. This density gradient creates heterogeneity in the different injection volumes which may impair the effectiveness of the cell therapy.

[0015] In the context of the treatment of Parkinson's disease, cell therapy consists of directly injecting cellular elements, preferably functional dopaminergic neurons, into the putamen. The success of such a technique depends heavily on the ability to precisely control the quantity of cellular elements injected. Indeed, it is necessary to inject an equivalent quantity of cells into several precisely defined regions of the putamen during the surgical procedure.

[0016] Furthermore, beyond maintaining the homogeneity of the distribution of cells in their medium, maintaining biological stability is fundamental to guarantee the preservation of living cells in its medium, in particular during the injection of viable cells for use in cell therapy. Indeed, the osmolality, concentration and diversity of ionic species is essential to maintain the homeostasis of cells in suspension.

[0017] There is therefore a significant need for a biocompatible composition, comprising heterogeneous cellular elements, capable of maintaining:

[0018] - the homogeneity of distribution of cellular elements within the composition of so as to be able to precisely control the quantity of cells contained in a given volume; and

[0019] - the biological stability of cellular elements in suspension. Summary of the invention

[0020] To meet this need, the invention proposes a novel biocompatible composition, comprising a solution and cellular elements suspended in said solution, said cellular elements being chosen from isolated cells, cellular microtissues, clusters of cells and mixtures thereof, at least two of said cellular elements having a different density and / or size and / or number of cells, said composition comprising at least: - hyaluronic acid and / or carboxymethyl cellulose (CMC), and - at least one biocompatible density agent, - and / or at least one modified form of one or more of these molecules

[0021] Hyaluronic acid and CMC are biocompatible viscosity agents particularly suited to the context of the invention.

[0022] Indeed, hyaluronic acid is a substance naturally present in the human body, mainly in the skin, joints and connective tissues. It is made up of a chain of sugars and plays an essential role in maintaining the hydration, elasticity and firmness of the skin.

[0023] Sodium hyaluronate, a soluble form of hyaluronic acid, is widely used in aesthetic and regenerative medicine, particularly to increase the volume and shape of certain parts of the body, such as the lips, cheeks or deep wrinkles. Sodium hyaluronate is a biocompatible molecule, which considerably reduces the risks of adverse effects and intolerance.

[0024] Carboxymethylcellulose (CMC) is a biocompatible and biodegradable polymer derived from cellulose.

[0025] Preferably, the composition according to the invention has a viscosity at rest of between 0.01 and 1.5 Pa.s, preferably of between 0.01 and 1 Pa.s, especially between 400 and 1000 mPa.s, even more preferably between 500 and 900 mPa.s.

[0026] According to a preferred embodiment, the composition according to the invention comprises dextran, as a density agent.

[0027] Dextran is a biocompatible, water-soluble polymer of glucose derived from starch and can be produced from plant or microbial sources.

[0028] Preferably, the composition according to the invention has a density of between 800 and 2000 kg / m3, preferably between 997 and 1600 kg / m3, preferably between 1025 and 1100 kg / m3, in particular between 1047 and 1090 kg.m3.

[0029] The rheological characteristics of the composition according to the invention make it possible to use the composition according to the invention in injection devices without the fluid friction or shear forces encountered during injection deteriorating the precision of the injection and impacting the viability and integrity of the cells. Indeed, excessively high viscosity and / or density can lead to increased resistance during the flow of the composition through the injection means, associated with viscous friction forces likely to damage the cellular elements.

[0030] The inventors have achieved a biocompatible composition having rheological characteristics adapted to heterogeneous cellular elements based on the density / viscosity pair of the solution comprising said cellular elements.

[0031] Furthermore, hyaluronic acid, CMC and dextran are biocompatible and are particularly suitable for the context of the invention. Indeed, these molecules modify the rheological characteristics of the composition according to the invention so as to reduce the maximum sedimentation rate of the heterogeneous cellular elements present in the solution and thus guarantee a uniform distribution of the cellular elements within the solution.

[0032] According to a preferred subject of the invention, the composition according to the invention comprises a solution and cellular elements suspended in said solution, in which the cellular elements have a maximum sedimentation rate of less than 20 mm.h 1 relative to the solution.

[0033] The maximum sedimentation rate of less than 20mm.h 1 of the cellular elements compared to the solution makes it possible to provide numerous advantages to the composition according to the invention, namely:

[0034] - an increase in cell viability: A maximum sedimentation rate less than 20 mm.h 1 reduces the mechanical stress exerted on cellular elements, thus minimizing the risks of alteration of their structure or metabolism, which can improve their viability;

[0035] - increased analytical precision: In the context of analyses and measurements specific, a maximum sedimentation rate lower than 20 mm.h 1 allows better precision and greater sensitivity of the results;

[0036] - better homogeneity: A maximum sedimentation rate lower than 20 mm.h 1 allows cells to remain homogeneously distributed in the solution, thus avoiding the formation of concentration gradients;

[0037] - better experimental reproducibility: By maintaining a speed of maximum sedimentation less than 20 mm.h', variations due to differential sedimentation of heterogeneous cellular elements are reduced.

[0038] According to a preferred object, the composition according to the invention comprises a solution and cellular elements, in which the osmolality of the cellular elements in the solution is between 220 and 360 mOsmol / kg.

[0039] Advantageously, the osmolality of the cellular elements makes it possible to form an environment favorable to the survival of the cellular elements such as an isotonic environment.

[0040] Surprisingly, the inventors have developed a biocompatible composition, suitable for numerous uses, controlling the sedimentation rate of heterogeneous cellular elements suspended in the solution while presenting a biological stability adapted to the cells, in particular to human or animal cells.

[0041] According to a preferred object of the invention, the composition comprises a solution and cellular elements suspended in said solution, in which the cells constituting the cellular elements are chosen from neural cells, neuronal cells, glial cells and mixtures thereof.

[0042] Preferably, the composition according to the invention comprises a solution and cellular elements suspended in said solution, in which at least one cell, preferably a majority of cells, constituting the cellular elements is / are one or more dopaminergic neuron(s).

[0043] According to another object, the invention relates to the biocompatible composition according to the invention for its use in the treatment of Parkinson's disease.

[0044] Preferably, the biocompatible composition according to the invention is used as an injectable composition in humans or animals.

[0045] Advantageously, by guaranteeing a uniform distribution of the cellular elements within the solution, the composition according to the invention makes it possible to obtain a precise dosage of the injected cellular elements, thus making it possible to improve: - therapeutic efficacy: When injecting cellular elements for therapeutic purposes, precise dosage is essential to achieve the desired result. A Insufficient amounts of cellular elements may not be effective, while an overdose of cellular elements may lead to adverse effects. By precisely adjusting the dosage, it is possible to maximize therapeutic efficacy while minimizing risks to the patient; - experimental precision: Controlling the dosage when injecting cellular elements helps minimize variations between samples and obtain reliable and reproducible data. This is particularly important during comparative experiments or when testing the efficacy of new cell therapies. - resource optimization: The resources used for the production of cellular elements, such as cell cultures and culture products, can be expensive. Accurate dosing helps minimize the waste of these resources by avoiding unnecessary overdoses.

[0046] The invention also relates to a method for preparing a biocompatible composition according to the invention comprising the implementation of the following steps: (a) Preparation or recovery of a composition comprising a set of cellular elements of which at least two cellular elements have a different density and / or size and / or number of cells; (b) Preparation of a solution comprising: - hyaluronic acid and / or carboxymethyl cellulose (CMC), and - at least one biocompatible density agent, -and / or at least one modified form of one or more of these molecules; (c) Sterilization of the solution from step (b) so as to obtain a sterilized filtrate; (d) Mixing of the cellular elements from step (a) with the sterilized filtrate from step (c); (g) Optionally, recovery of a biocompatible composition, comprising a solution and mesoscopic objects suspended in said solution, at least two of which mesoscopic objects have a different density and / or size, and said composition comprising the following molecules: - Hyaluronic acid and / or Carboxymethyl cellulose (CMC); - At least one biocompatible density agent; -and / or at least one modified form of one or more of these molecules.

[0047] Advantageously, the method according to the invention makes it possible to prepare a biocompatible composition having the combination of an optimal density and viscosity to maintain the heterogeneous cellular elements in suspension at a maximum sedimentation rate relative to the solution of less than 20 mm.h'.

[0048] Finally, according to a last object, the invention relates to a kit comprising:

[0049] * a biocompatible composition, comprising a solution and elements cellular elements suspended in said solution, said cellular elements being chosen among isolated cells, cellular microtissues, clusters of cells and mixtures thereof, at least two of said cellular elements having a different density and / or size and / or number of cells, said composition comprising at least:

[0050] - hyaluronic acid and / or carboxymethyl cellulose (CMC), and

[0051] - at least one biocompatible density agent,

[0052] - and / or at least one modified form of one or more of these molecules

[0053] ; and

[0054] * an injection means, preferably a cannula.

[0055] Other characteristics and advantages will emerge from the detailed description of the invention, the examples and the figures which follow. Brief description of the drawings

[0056] [Fig-1] [Fig.l] represents a sequence of images showing the stability in suspension of cellular elements over time in a composition according to the invention, and a composition outside the invention.

[0057] [Fig.2] [Fig.2] represents the comparative study of the impact of immersion prolonged (6h) of cellular elements in an administration medium, on a panel of production batches of cellular aggregates of interest.

[0058] [Fig.3] [Fig.3] is a schematic representation of a comparative study evaluating the impact of an injection procedure on the viability of the cellular elements within the composition according to the invention. Description of the invention

[0059] Definitions:

[0060] By "density agent" within the meaning of the invention, we mean a biocompatible chemical species, soluble in aqueous solution and used to increase the density thereof while not significantly altering the biocompatibility of the solution with the cellular elements in suspension.

[0061] For the purposes of the invention, the term “cell cluster” means a three-dimensional grouping of cells held together by adhesive interactions or by the presence of interstitial tissue.

[0062] For the purposes of the invention, "biocompatible" means the ability of a substance or composition to interact favorably with biological systems, such as tissues, organs or living organisms, without causing harmful reactions, excessive irritation or unwanted damage. When a substance or composition is described as biocompatible, this means that it is designed and formulated in such a way as to minimize the risks of toxicity, inflammation, rejection or other unwanted responses from the body and / or cellular elements.

[0063] For the purposes of the invention, the term "human cells" means human cells or immunologically humanized non-human mammalian cells. Even when this is not specified, the cells, stem cells, progenitor cells and tissues according to the invention are constituted or are obtained from human cells or from immunologically humanized non-human mammalian cells.

[0064] For the purposes of the invention, the term “progenitor cell” means a stem cell already engaged in cell differentiation but not yet differentiated.

[0065] For the purposes of the invention, the term "embryonic stem cell" means a pluripotent stem cell derived from the inner cell mass of the blastocyst. The pluripotency of embryonic stem cells can be assessed by the presence of markers such as the transcription factors OCT4, NANOG and SOX2 and surface markers such as SSEA4 / 5, Tra-1-60 and Tra-1-81. The embryonic stem cells used in the context of the invention are obtained without destroying the embryo from which they originate, for example using the technique described in Chang et al. (Cell Stem Cell, 2008, 2(2)): 113-117). Optionally, embryonic stem cells of human beings can be excluded.

[0066] For the purposes of the invention, the term "pluripotent stem cell" or "pluripotent cell" means a cell that has the capacity to form all the tissues present in the entire organism of origin, without being able to form an entire organism as such. Human pluripotent stem cells may be referred to as hPSCs in the context of the present invention. In particular, they may be induced pluripotent stem cells (iPSCs or hiPSCs for human induced pluripotent stem cells), embryonic stem cells or MUSE cells (for "Multilineage-differentiating Stress Enduring").

[0067] For the purposes of the invention, the term "induced pluripotent stem cell" means a pluripotent stem cell induced to pluripotency by genetic reprogramming of differentiated somatic cells. These cells are in particular positive for pluripotency markers, such as alkaline phosphatase staining and expression of the proteins NANOG, SOX2, OCT4 and SSEA4 / 5. Examples of methods for obtaining induced pluripotent stem cells are described in the articles Yu et al. (Science 2007, 318 (5858): 1917-1920), Takahashi et al. (Cell, 207, 131(5): 861-872) and Nakagawa et al. (Nat Biotechnol, 2008, 26(1): 101-106).

[0068] By "layer of cells" or "base of cells" within the meaning of the invention, we mean several cells forming a layer or a base which can be structured around a lumen, it can be for example a tissue or a cellular micro-tissue or a three-dimensional grouped culture. The thickness of the cell layer can be variable. This layer is organized in three dimensions in the microcompartment.

[0069] For the purposes of the invention, the term "density" means the density of the entities concerned (cellular elements or solutions) expressed in mg / mL. In the context of the invention, the density of the solutions is measured at a temperature of 20°C using a densimeter. In the case of cellular elements, the density is measured by the density gradient method. This consists of preparing solutions calibrated in density and isotonic. These solutions with known densities are then introduced in successive layers into a tube so as to form a density gradient from bottom to top, each layer being denser than the layer directly above. Typically, the density gradients are prepared in density increments of 5 kg / m3. The density of the cellular elements is therefore given at a resolution of + / -5 kg / m3. Once the density gradient has been formed, the cellular elements are injected onto the upper part of the tube.These sediment and stabilize in or between the layer(s) whose density is closest to them. The reading is taken once all the cellular elements have stabilized, typically after 1 h. Advantageously, colored synthetic beads of calibrated densities can be introduced simultaneously, their equilibrium position providing a positive control of the measurement of the position of the cellular elements. All these measurements are carried out at the temperature or within the temperature range of intended use because the temperature can differentially affect the density of water, solutions and cellular elements.

[0070] By "average density of the cellular elements", within the meaning of the invention, is meant the average density of the cellular elements such as microcompartments. In the context of the invention, the average density of the cellular elements is measured by the density gradient method. This method, well known to those skilled in the art, consists of preparing calibrated solutions with known densities. These solutions are then introduced into a tube so as to form a density gradient from bottom to top, each layer being denser than the layer directly above. Once the density gradient is formed in the tube, the cellular elements are introduced into said tube and sediment by stabilizing in or between the layers whose density is closest to them. The reading is taken once all the cellular elements are stabilized, typically after 1 hour.In parallel with the injection of the cellular elements, colored synthetic beads of known density are also introduced into said tube so as to form a positive control. The average density is then calculated based on the distribution of cellular elements within the said tube.

[0071] By "Feret diameter" of a cellular element within the meaning of the invention, we mean the distance between two tangents, these two tangents being parallel, such that the entire cellular element is between these two parallel tangents.

[0072] For the purposes of the invention, “differentiated” cells are understood to mean cells which have a particular phenotype, as opposed to pluripotent stem cells which are not differentiated or progenitor cells which are in the process of differentiating.

[0073] For the purposes of the invention, the term "cellular element" means a component which comprises at least one human, animal or plant cell and which is in different forms such as isolated cells, cellular microtissues, clusters of cells, encapsulated cells, encapsulated microtissues, clusters of cells or not and mixtures thereof. The microtissues and / or clusters, encapsulated or not, may contain one or more lumens.

[0074] By "modified forms of these molecules" within the meaning of the invention, it is meant that the molecules have been modified by adding one or more molecular groups to one or more functional groups present on the molecules. For example, hyaluronic acid is made up of a monomer assembly, each of the monomers comprising hydroxyl, carboxyl, and N-acetyl functional groups capable of serving as a base for attaching molecular groups. The hydroxyl and carboxyl groups are preferred sites for attaching lipids, polymers or other active molecules to the hyaluronic acid monomer. In a complementary manner, amine groups can also be added to the N-acetyl functional group. It is thus possible to create hyaluronic acid esters, hyaluronic acid dialdehydes, hyaluronic acid N-deacetylated or Cys-hyaluronic acids.Similarly, Dextran, also consisting of monomer, has hydroxyl functional groups accessible to a wide variety of addition reactions (hydroxyl group chemistry).

[0075] For the purposes of the invention, the term “largest dimension” means the value of the largest Feret diameter. The dimension of the cellular elements can be obtained by various techniques. In particular, it can be characterized using a Coulter effect measuring device. Alternatively, it can be determined optically by microscopy.

[0076] By “keeping the cellular elements in suspension” within the meaning of the invention, we mean slowing down the sedimentation rate of the cellular elements without impacting their survival.

[0077] For the purposes of the invention, the term “microcompartment” or “capsule” also means a partially or totally closed three-dimensional structure containing one or more cellular elements. The structure preferably consists of a rigidified external hydrogel layer and a hollow internal part comprising at least one cellular element and possibly other elements such as an extracellular matrix or an extracellular matrix substitute.

[0078] For the purposes of the invention, the term "microtissue" means a community of cells exhibiting cellular interactions, optionally mechanical cohesion and at least partially recapitulating a cellular composition present in a human tissue in a physiological or pathological situation. Typically, a microtissue exhibits a specific topology of distribution of the cells that compose it.

[0079] For the purposes of the invention, the term “osmolality of cellular elements” means a measurement of the total concentration of dissolved particles within the cellular elements, expressed in osmoles per kilogram of water (osmol / kg H2O).

[0080] It includes active solutes, such as electrolytes and non-ionic molecules. In the context of the invention, osmolality can be measured using a freezing point osmometer of the “Osmotech XT” type. It should be noted that the use of different models of freezing point osmometers may make the measurement impossible. Indeed, the composition of the solutions and their viscosity make the measurement of osmolality complex and require high-power devices. Alternatively, the osmolality measurement can be calculated by extrapolation based on a measurement of a dilution of the solution, which allows the use of standard devices.

[0081] By “active principle” within the meaning of the invention, we mean one or more substance(s) responsible for a pharmacological or therapeutic activity.

[0082] By “biological stability” within the meaning of the invention, we mean the maintenance of the integrity of cellular elements.

[0083] By "tissue" or "biological tissue" within the meaning of the invention, we mean the common meaning of tissue in biology, that is to say the intermediate level of organization between the cell and the organ. A tissue is a set of similar cells of the same origin (most often from a common cell lineage, although they can find their origin by association of distinct cell lineages), grouped in clusters, networks or bundles (fibers). A tissue forms a functional whole, that is to say that its cells contribute to the same function. Biological tissues regenerate regularly and are assembled together to form organs.

[0084] By "sedimentation velocity threshold" or "maximum sedimentation velocity" within the meaning of the invention, we mean the maximum measured relative velocity of vertical displacement within the solution relative to the reference frame of the system, container or fluidic element in which the suspension is contained. The maximum relative speed is expressed in mm / h. By convention, the relative speed is defined as positive in the case where the cellular elements sediment in the solution, and negative if they rise in the solution. The term "maximum" qualifies the absolute value of the speed regardless of its sign, in the sense that it is important in the context of the invention that the cellular elements remain in suspension, which implies that their relative speed is, in absolute value, as low as possible.

[0085] For the purposes of the invention, the term “viscosity” means the dynamic viscosity that characterizes the resistance of a fluid to laminar flow. It is expressed in Pa.s. In the case of non-Newtonian fluids, this viscosity may depend on the shear stresses applied to the fluid. In particular, the term “shrew-thinning fluid” means a fluid whose viscosity decreases with the shear rate. Similarly, a “threshold fluid” is a fluid whose viscosity is very significant at low shear rates, then drops sharply beyond a threshold shear rate value. These two categories of fluids are advantageous in the context of the invention since the high viscosity at rest prevents sedimentation, while fluidization at high shear rates facilitates the flow of the solution and limits the viscous friction forces on the biological elements, for example during injection.In the context of the invention, the viscosity can be measured at 25°C using a rheometer by the shear viscosity curve method measured by confining the fluid in a Cone-Plane geometry of 25mm diameter, an angle of 1°, sanded surface and over a range of shear rates from r = 0.1 s-' to t = 1000 s1. .

[0086] By "viscosity at rest" is meant the dynamic viscosity measured at rest, i.e. at a very low shear rate r on the fluid, typically r = 0.1 s*1.

[0087] For the purposes of the invention, “viability” means the proportion of healthy cells in a sample of cellular elements. In the context of the invention, the proportion of healthy cells is evaluated with regard to the metabolic activity of a sample of cells. Metabolic activity is quantified by the concentration of ATP measured in a suspension of lysed cellular elements. The technique used is marketed under the name “CellTiter-Glo® 3D Cell Viability Assay”. It is specifically designed to determine the viability of cells in 3D microtissue spheroids. The test reagent penetrates large spheroids and has an increased lytic capacity, which allows for a more accurate determination of viability compared to other test methods. Preferably, viability is expressed as a percentage relative to a control condition. In this case, viability is said to be “relative”.For example, if condition A is taken as reference, . a viability of 85% for condition B reflects a 15% decrease in viability compared to condition A.

[0088] Biocompatible composition according to the invention

[0089] The subject of the present invention is a biocompatible composition, comprising a solution and cellular elements suspended in said solution, said cellular elements being chosen from isolated cells, cellular microtissues, clusters of cells and their mixtures, at least two of said cellular elements having a different density and / or size and / or number of cells, said solution comprising the following molecules: - hyaluronic acid and / or carboxymethyl cellulose (CMC); - dextran; and / or - a salt of one or more of these molecules.

[0090] Surprisingly, the inventors have developed a biocompatible composition making it possible to maintain heterogeneous cellular elements in suspension while maintaining their biological stability over a long period. Thus, the choice of compounds as well as the rheological characteristics of the solution advantageously make it possible to maintain the homogeneity of distribution of the cellular elements within the composition without altering the viability of the cellular elements.

[0091] According to a particular embodiment of the invention, the composition comprises at least one encapsulated cellular element, preferably contained in a three-dimensional microcompartment comprising an external hydrogel layer. The microcompartment may be a ball full of hydrogel. Preferably, the composition comprises at least one encapsulated cellular element, preferably contained in a hollow three-dimensional microcompartment comprising an external hydrogel layer and a hollow internal part containing the encapsulated cellular element(s). The hollow internal part may also contain extracellular matrix and / or one or more extracellular matrix substitutes and / or a solution and / or other elements. According to a particular embodiment, the external hydrogel layer of the microcompartment may comprise or consist exclusively of alginate.The alginate may in particular be a sodium alginate, composed of 80% α-L-guluronate and 20% β-D-mannuronate, having a Young's modulus greater than 10 kPa, preferably greater than 60 kPa, more preferably greater than 100 kPa. According to one embodiment, the external hydrogel layer comprises alginate, said alginate having an average molecular weight of 100 to 400 kDa, more preferably a molecular weight of between 150 and 250 kDa. When the hydrogel of the external layer of the microcompartment is alginate, the concentration of the alginate solution intended to form said external layer of the microcompartment is preferably between . between 0.5 and 5% in mass, more preferably the concentration is equal to 2% (plus or minus 0.5%) in mass.

[0092] Preferably, the composition according to the invention comprises a solution and cellular elements, encapsulated or not, suspended in said solution, in which the cells constituting the cellular elements are mature cells and / or progenitors and / or stem cells.

[0093] Preferably, the composition comprises a solution and cellular elements suspended in said solution, in which the cells constituting the cellular elements are chosen from neural cells, neuronal cells, glial cells and mixtures thereof.

[0094] When the composition according to the invention comprises cellular elements, in which the cells constituting the cellular elements are glial cells, these may be chosen from astrocytic cells and / or oligodendrocyte cells.

[0095] Preferably, the composition according to the invention comprises a solution and cellular elements suspended in said solution, in which at least one cell constituting the cellular elements is a dopaminergic neuron.

[0096] According to one embodiment, the composition according to the invention comprises a solution and cellular elements suspended in said solution, in which a majority of cells constituting the cellular elements are dopaminergic neurons.

[0097] According to one embodiment, the composition according to the invention comprises cellular elements comprising astrocytic cells, oligodendrocyte cells and dopaminergic neurons.

[0098] According to one embodiment, the composition according to the invention comprises a solution and cellular elements suspended in said solution, in which the cells constituting the cellular elements belong to at least 2 distinct cell types.

[0099] According to one embodiment, the composition according to the invention comprises a solution and cellular elements suspended in said solution, in which said cellular elements are in the form of microtissues comprising dopaminergic neurons and optionally astrocytic cells.

[0100] Advantageously, the cellular elements present in the composition according to the invention are heterogeneous. They may in particular be in the form of microtissues, encapsulated or not, comprising several different cell types recreating the tissue architecture and the cellular interactions necessary to promote the regeneration and functionality of the tissues.

[0101] According to one embodiment, the composition according to the invention comprises a solution and cellular elements suspended in said solution, in which at least 20%, preferably at least 30%, in particular at least 40% of the cellular elements are in the form of a microtissue.

[0102] According to one embodiment, the composition according to the invention comprises between 10 and 100% of the cellular elements in the form of a microtissue.

[0103] According to another embodiment, the composition according to the invention comprises a solution and cellular elements suspended in said solution, in which less than 15%, preferably less than 10%, in particular less than 5% of the cellular elements are in the form of isolated cells.

[0104] In the context of the invention, the percentage of cellular elements in the form of microtissues or isolated cells is expressed as the cumulative volume of cellular elements.

[0105] According to one embodiment, the composition according to the invention comprises a solution and cellular elements suspended in said solution, in which the volume fraction of the cellular elements is between 0.01 and 80% by volume, preferably between 10 and 50%, in particular between 40 and 80%.

[0106] According to another embodiment, the composition according to the invention comprises a solution and cellular elements suspended in said solution, in which the cellular elements have a largest dimension less than 1.3 mm, preferably less than 1 mm, in particular less than 700 μm, even more preferably less than 500 μm.

[0107] Preferably, the composition according to the invention comprises a solution and cellular elements suspended in said solution, in which the cellular elements have a largest dimension of between 50 pm and 1.3 mm, preferably between 100 pm and 800 pm.

[0108] In other words, the composition according to the invention comprises a solution and cellular elements suspended in said solution, in which the cellular element having the largest dimension has a largest dimension less than 1.3 mm, preferably less than 700 pm, more preferably less than 500 pm.

[0109] According to a particular embodiment, the composition according to the invention comprises a solution and cellular elements suspended in said solution, in which the composition comprises between 1 million and 500 million cells per mL of solution, preferably at least 5 million cells, in particular 50 million cells.

[0110] Preferably, the composition according to the invention comprises a solution and cellular elements suspended in said solution, in which the composition comprises between 500 and 10,000 dopaminergic neurons per pL of solution, in particular at least 2,000 dopaminergic neurons per pL of solution.

[0111] According to another embodiment, the osmolality of the cellular elements in the solution is between 220 and 360 mOsml.kg In this way, the cellular elements and the solution are close to osmotic equilibrium, which means that the ion concentration gradients of the composition are balanced and do not cause osmotic shock.

[0112] Preferably, the composition according to the invention also comprises at least one biocompatible osmolality adjusting agent. Said at least one osmolality adjusting agent makes it possible to adapt the osmolality of the solution in order to make the solution compatible with the survival of the cellular elements.

[0113] Preferably, at least one agent for adjusting the osmolality of the solution is chosen from mannitol, sorbitol, sodium chloride and their mixtures, or modified forms of these molecules.

[0114] According to one embodiment, the composition according to the invention comprises a solution comprising at least one type of ion chosen from sodium ions, calcium, chlorine and potassium and their mixtures.

[0115] Preferably, the composition according to the invention comprises a solution comprising sodium, calcium, chlorine and potassium ions.

[0116] Preferably, the composition according to the invention comprises a solution and cellular elements suspended in said solution, in which the solution is an aqueous solution.

[0117] According to one embodiment, the composition comprises a solution and cellular elements suspended in said solution, in which the solution has a pH of between 7.0 and 7.6.

[0118] Advantageously, the pH of the solution is particularly suited to the survival of cellular elements.

[0119] According to one embodiment, the composition according to the invention comprises a solution and cellular elements suspended in said solution, in which the solution comprises a biocompatible pH buffer, preferably chosen from phosphate buffered saline (PBS), citric acid or Hepes, and preferably the PBS buffer referenced at number 4004200 of the European Pharmacopoeia.

[0120] According to one variant, the composition according to the invention comprises a solution and cellular elements suspended in said solution, in which the solution also comprises at least one active ingredient and / or a pharmaceutically acceptable excipient.

[0121] According to a particular embodiment of the invention, the aqueous solution is the culture medium of the cellular elements.

[0122] According to another embodiment, the composition according to the invention comprises a solution comprising at least one element chosen from sodium chloride (NaCl), calcium chloride (CaCl2), potassium chloride (KCl), magnesium chloride (MgCl2) and mixtures thereof.

[0123] Advantageously, the diversity of ions contained in the solution makes it possible to ensure a balanced environment conducive to the proper functioning of the cellular processes of the cellular elements within the composition according to the invention.

[0124] Preferably, the composition according to the invention comprises: - between 0.01 and 0.9% by mass of NaCl relative to the total mass of the solution; and / or - between 0.01 and 0.04% by mass of CaCl2 relative to the total mass of the solution; and / or - between 0.01 and 0.05% by mass of KC1 relative to the total mass of the solution.

[0125] Advantageously, the solution and the cellular elements suspended in said solution are close to osmotic equilibrium, guaranteeing a stable environment for the cellular elements, without risk of osmotic shock.

[0126] Preferably, the composition according to the invention comprises a solution and cellular elements suspended in said solution, in which the solution is isotonic with respect to the cellular elements.

[0127] According to one embodiment, the composition according to the invention has a density of between 800 and 2000 kg / m3, preferably between 997 and 1600 kg / m3, even more preferably between 1025 and 1100 kg / m3, in particular between 1047 and 1090 kg / m3

[0128] Advantageously, the density of the composition according to the invention contributes to reducing the maximum sedimentation rate of the cellular elements compared to the solution.

[0129] According to a variant, the composition according to the invention comprising a solution and cellular elements in suspension, has a density of the solution between the density of the cellular element having the lowest density and the density of the cellular element having the greatest density.

[0130] According to another variant, the composition according to the invention comprising a solution and cellular elements in suspension, has a density of the solution lower than the density of the cellular element having the lowest density.

[0131] Finally, according to another variant, the composition according to the invention comprising a solution and cellular elements suspended in said solution, has a density of the solution greater than the density of the cellular element having the greatest density.

[0132] Advantageously, in all the embodiments according to the invention, the maximum sedimentation rate of the cellular elements relative to the solution is less than 20 mm.h

[0133] According to another variant, the composition according to the invention has a density equal to the average of the densities of all the cellular elements present in the composition, with a tolerated variation of 20 kg / m3, preferably 10 kg / m3. In this way, the density of the composition is centered on the density of the average of the densities of all the cellular elements.

[0134] According to one embodiment, the composition according to the invention comprises at least one biocompatible density agent having a molar mass greater than 400g / mol.

[0135] According to a preferred embodiment, the composition according to the invention comprises at least one biocompatible density agent chosen from dextran, hypromelose, Iodixanol, Polyethylene glycol (PEG), chitosan, pullulan, or molecules from the glycosaminoglycan family, or modified forms of these molecules, and mixtures thereof.

[0136] The density agents suitable for the composition according to the invention are selected according to several parameters, namely: - their biocompatibility, that is to say that they must not impact the survival of cellular elements - their high solubility in the solution; - their low impact on the viscosity of the solution.

[0137] Preferably, the composition according to the invention comprises a solution and cellular elements suspended in said solution, in which the solution comprises dextran, as a biocompatible density agent. When the solution comprises dextran, the latter preferably has a molecular weight of between 20 and 80 KDa, in particular 30, 35 or 40 kDa.

[0138] Carboxymethyl cellulose, hyaluronic acid or modified forms of these molecules are present in the composition according to the invention and play a role as viscosity agent, that is to say molecules aimed at adjusting the viscosity without impacting the biocompatibility of the composition according to the invention.

[0139] These molecules were selected based on several parameters, namely: - their biocompatibility, i.e. they must not impact the survival of cellular elements; - their stability at 37°C, injection temperature in humans.

[0140] Furthermore, carboxymethyl cellulose, hyaluronic acid or modified forms of these molecules are particularly suitable when the cells constituting the cellular elements are neural cells, neuronal cells, glial cells and their mixtures.

[0141] When the solution comprises sodium hyaluronate, the latter preferably has a molecular weight lower than IMDa, even more preferably between 80 and IMDa, in particular between 80 and 600 kDa.

[0142] When the solution comprises carboxymethyl cellulose, it preferably has a molecular weight of between 40kDa and IMDa, even more preferably between 50 and 400kDa.

[0143] Advantageously, the components of the solution are all biocompatible, thus ensuring that the composition according to the invention will be well tolerated by the body, particularly in the case of injection.

[0144] According to another embodiment, the composition according to the invention comprises: - between 0.01 and 30% by mass of at least one biocompatible density agent, preferably dextran, relative to the total mass of the solution - between 0.01 and 7% of carboxymethyl cellulose and / or hyaluronic acid and / or modified forms of these molecules by mass relative to the total mass of the solution.

[0145] According to one embodiment, the composition according to the invention comprises a solution and cellular elements suspended in said solution, in which the solution comprises: - sodium hyaluronate with a molecular weight between 80kDa and IMDa and / or carboxymethyl cellulose with a molecular weight between 40 and IMDa; and / or - dextran with a molecular weight between 40 and 80kDa.

[0146] Preferably, the composition according to the invention comprises a solution and cellular elements suspended in said solution, in which the solution comprises: - between 10 and 25% by mass of dextran with a molecular weight of 20 and 80kDa relative to the total mass of the solution; - between 0.1 and 7% by weight of carboxymethyl cellulose having a molecular weight of between 40kDa and IMDa relative to the total mass of the solution and / or between 0.1 and 7% by weight of sodium hyaluronate with a molecular weight of between 80kDa and IMDa relative to the total mass of the solution

[0147] According to one embodiment, the composition according to the invention has a viscosity at rest of between 0.01 and 1.5 Pa.s, preferably of between 0.01 and 1 Pa.s, in particular between 400 and 1000 mPa.s, even more preferably between 500 and 900 mPa.s.

[0148] The viscosity of the composition makes it possible to prevent the sedimentation of the cellular elements suspended in the solution while allowing the use of the composition according to the invention, in particular the injection of said composition. Consequently, the composition according to the invention has a viscosity high enough to keep the cellular elements in suspension while having a viscosity low enough to minimize frictional stresses when the composition is injected, in particular using an injection means such as a syringe or a cannula.

[0149] According to a particular variant of the invention, the biocompatible composition has: - a density between 997 and 1600 kg / m3; - a viscosity at rest, measured using a rheometer at 25°C, between 0.01 and 1.5 Pa.s.

[0150] Advantageously, the components of the solution are all biocompatible, thus ensuring that the composition according to the invention will be well tolerated by the body, particularly in the case of injection.

[0151] According to another embodiment, the composition according to the invention comprises a solution and cellular elements suspended in said solution, in which the solution comprises: - between 10 and 25% by mass of dextran with a molecular weight of 20kDa and 80kDa relative to the total mass of the solution; - between 0.01 and 7% by weight of CMC having a molecular weight of between 40 kDa and IMDa relative to the total mass of the solution and / or between 0.0 and 7% by weight of sodium hyaluronate with a molecular weight of between 80 kDa and IMDa relative to the total mass of the solution; - between 0.01 and 0.9% by mass of NaCl relative to the total mass of the solution; and / or - between 0.01 and 0.04% by mass of CaCl2 relative to the total mass of the solution; and / or - between 0.01 and 0.05% by mass of KC1 relative to the total mass of the solution.

[0152] Preferably, the composition according to the invention comprises a solution and cellular elements suspended in said solution, in which the maximum sedimentation rate of the cellular elements relative to the solution is less than 10 mm.h ', preferably less than 5 mm.h ', in particular less than 1 mm.h 1

[0153] According to a variant, the composition according to the invention comprises a solution and cellular elements suspended in said solution, in which the relative speed maximum density of cellular elements relative to the solution is between 0.01 and 20 mm.h '.

[0154] Thus, the lower the maximum relative speed of the cellular elements, the more the homogeneity of distribution of the cellular elements suspended in the solution is maintained over time.

[0155] According to a particular embodiment, the composition according to the invention having a maximum sedimentation rate of the cellular elements relative to the solution of less than 1 mm.h 1 can be stored for up to 6 hours before use. Thus, in this embodiment, the composition according to the invention maintains the uniform distribution of the heterogeneous cellular elements for 6 hours.

[0156] Advantageously, the composition according to the invention can be stored for a long period without affecting the uniform distribution of the heterogeneous cellular elements, thus ensuring precise dosage, in particular during its injection.

[0157] The composition according to the invention can be obtained by any suitable method known to those skilled in the art. However, according to a particularly preferred embodiment, the composition according to the invention is a biocompatible composition obtained according to a method comprising the implementation of the following steps: a) Preparation or recovery of a composition comprising a set of cellular elements of which at least two cellular elements have a different density and / or size and / or number of cells; (b) Preparation of a solution comprising: - hyaluronic acid and / or carboxymethyl cellulose (CMC), and - at least one biocompatible density agent, -and / or at least one modified form of one or more of these molecules; (c) Sterilization of the solution resulting from step (b) so as to obtain a sterilized filtrate; (d) Mixing the cellular elements from step (a) with the sterilized filtrate from step (c); (e) Optionally recovery of the biocompatible composition resulting from step (d), comprising a solution and cellular elements suspended in said solution, of which at least two cellular elements have a different density and / or size and / or number of cells, said composition comprising the following molecules: - Hyaluronic acid and / or Carboxymethyl cellulose (CMC); - At least one density agent; -and / or at least one modified form of one or more of these molecules.

[0158] According to one embodiment, the set of cellular elements of step (a) comprises neural cells, neuronal cells and / or glial cells, preferably dopaminergic neurons.

[0159] Preferably, when step (a) concerns the preparation of a composition, it comprises the implementation of the following steps: 1) Cultivation of at least one cellular element; 2) Rinsing of the culture of cellular elements from step 1); 3) Centrifugation of the culture of cellular elements from step 2); 4) Recovery of a composition comprising cellular elements resulting from the centrifugation of step 3), of which at least two cellular elements have a different density and / or size and / or number of cells.

[0160] According to one embodiment, the composition of step (a) comprises a solution in which said cellular elements are in suspension.

[0161] Preferably, the solution of step (b) comprises: - between 4 and 7% by mass of sodium hyaluronate with a molecular weight of between 80kDa and IMDa relative to the total mass of the solution and / or between 0.1 and 7% by mass of carboxymethyl cellulose with a molecular weight of between 40 and IMDa relative to the total mass of the solution; and - between 10 and 25% by mass of at least one biocompatible density agent relative to the total mass of the solution.

[0162] Preferably, step (b) also comprises the addition of at least one element chosen from sodium chloride (NaCl), calcium chloride (CaCl2), potassium chloride (KCl), magnesium chloride (MgCl2) and mixtures thereof.

[0163] Process for preparing a biocompatible composition according to the invention

[0164] According to another aspect, the invention also relates to a process for preparing a composition according to the invention comprising the implementation of the following steps: (a) Preparation or recovery of a composition comprising a set of cellular elements of which at least two cellular elements have a different density and / or size and / or number of cells; (b) Preparation of a solution comprising: - hyaluronic acid and / or carboxymethyl cellulose (CMC), and - at least one biocompatible density agent, -and / or at least one modified form of one or more of these molecules; (c) Sterilization of the solution resulting from step (b) so as to obtain a sterilized filtrate; (d) Mixing the cellular elements from step (a) with the sterilized filtrate from step (c); (e) Optionally, recovery of the biocompatible composition resulting from step (d), comprising a solution and cellular elements suspended in said solution, of which at least two cellular elements have a different density and / or size and / or number of cells, said composition comprising the following molecules: - Hyaluronic acid and / or Carboxymethyl cellulose (CMC); - At least one density agent; -and / or at least one modified form of one or more of these molecules.

[0165] Advantageously, the method according to the invention makes it possible to obtain an injectable biocompatible composition having a homogeneity of distribution of the cellular elements and biological stability, and this, for a period of up to 12 hours.

[0166] According to one embodiment, the composition of step (a) comprises a solution in which said cellular elements are in suspension.

[0167] According to a preferred embodiment, the composition of step (a) has a maximum sedimentation rate of the cellular elements relative to the solution greater than 20 mm.li1.

[0168] Preferably, the composition of step (a) comprises cellular elements comprising: - neuronal cells, preferentially dopaminergic neurons; and / or - glial cells, preferentially astrocytic cells and / or oligodendrocyte cells; and / or - neural cells such as progenitor cells.

[0169] According to one embodiment, the composition of step (a) comprises at least two distinct cell types.

[0170] Preferably, when step (a) concerns the preparation of a composition, it comprises the implementation of the following steps: 1) Cultivation of at least one cellular element; 2) Rinsing of the culture of cellular elements from step 1); 3) Centrifugation of the culture of cellular elements from step 2); 4) Recovery of a composition comprising cellular elements resulting from the centrifugation of step 3), of which at least two cellular elements have a different density and / or size and / or number of cells.

[0171] According to one embodiment, the cell culture of step 1) is carried out within a bioreactor.

[0172] According to a particular embodiment of the invention, at least one cellular element of step 1) comprises at least one encapsulated cell, preferably in alginate.

[0173] Preferably, at least one cellular element from step 1) is contained in a three-dimensional microcompartment comprising an external hydrogel layer.

[0174] When step 1) comprises the culturing of at least one encapsulated cellular element, step (a) may also comprise a step 2') aimed at removing the capsule so as to recover the cellular element before step 3) of centrifugation.

[0175] Step 2') consists of removing the capsule, preferably removing the external hydrogel layer. Preferably, step 2') can be carried out in particular by hydrolysis, dissolution, piercing and / or rupture by any biocompatible means, i.e. non-toxic to the cells. For example, the removal can be carried out using a saline phosphate buffer, a divalent ion chelator, an enzyme such as alginate lyase if the hydrogel comprises alginate and / or laser microdissection.

[0176] According to one embodiment, step (b) comprises the preparation of a solution in which at least one biocompatible density agent has a molar mass greater than 400g / mol.

[0177] Preferably, at least one biocompatible density agent of step (b) is chosen from dextran, hypromelose, Iodixanol, Polyethylene glycol (PEG), chitosan, pullulan, or molecules from the glycosaminoglycan family, or modified forms of these molecules, and mixtures thereof.

[0178] According to a particularly preferred embodiment, the solution resulting from step (b) comprises dextran, as a density agent.

[0179] According to a preferred embodiment, the solution resulting from step (b) comprises: - between 4 and 7% by mass of sodium hyaluronate with a molecular weight of between 80kDa and IMDa relative to the total mass of the solution and / or between 0.1 and 7% by mass of carboxymethyl cellulose having a molecular weight of between 40 and IMDa relative to the total mass of the solution; and - between 10 and 25% by mass of at least one biocompatible density agent relative to the total mass of the solution.

[0180] According to another particular embodiment, the mixture of step (b) comprises: - between 10 and 20% by mass relative to the volume of the solution of dextran preferably having a molecular weight of 20kDa and 80KDa;

[0181] - between 3 and 7% by mass of CMC preferably having a molecular weight between 40 and IMDa relative to the volume of the solution and / or between 0.3 and 7% by weight of sodium hyaluronate with molecular weights between 80 and 800kDa.

[0182] Preferably, step (b) also comprises the addition of at least one element chosen from sodium chloride (NaCl), calcium chloride (CaCl2), potassium chloride (KCl), magnesium chloride (MgCl2) and mixtures thereof.

[0183] According to one embodiment, the solution resulting from step (b) comprises: - between 0.01 and 0.9% NaCl mass relative to the total mass of the solution; and / or - between 0.01 and 0.04% by mass of KC1 relative to the total mass of the solution; and / or - between 0.01 and 0.05% by mass of CaCl2 relative to the total mass of the solution.

[0184] Preferably, the sterilization of step (c) is carried out by filtration through meshes of size between 0.01 and 0.22 pm.

[0185] According to one embodiment, the method according to the invention comprises a step (c'), carried out after step (c), of adjusting the pH of the sterilized filtrate resulting from step (c), so that the filtrate resulting from step (c') has a pH of between 6.8 and 8, preferably between 7 and 7.6.

[0186] According to a preferred embodiment, step (c') consists of adding a biocompatible pH buffer, preferably chosen from phosphate buffer, citric acid or Hepes, and preferably the saline phosphate buffer referenced at number 4004200 of the European Pharmacopoeia in the sterilized filtrate of step (b3) so that the sterilized filtrate has a pH of between 6.8 and 8, preferably between 7 and 7.6.

[0187] According to one embodiment, step (d) is carried out with stirring, preferably using a paddle mixer or a magnetic bar.

[0188] According to one embodiment, step (d), of mixing the cellular elements of the composition of step (a) with the sterilized filtrate of step (c) or optionally (c') comprises a step of extracting the cellular elements from the composition of step (a).

[0189] According to one embodiment, the mixture resulting from step (d) has: - a density of between 800 and 2000 kg / m3, preferably between 1047 and 1090 kg / m3; and - a viscosity, measured using a rheometer at 25°C, between 0.01 and 1.5 Pa.s, preferably between 500 and 900mPa.s.

[0190] Advantageously, a person skilled in the art will be able to adapt the density and viscosity by varying the concentrations of at least one density agent and / or dextran.

[0191] Thus, according to one embodiment, the method for preparing a composition according to the invention comprises the implementation of the following steps:

[0192] (a) Preparation of a composition comprising a set of cellular elements in a solution in which at least two cellular elements have a different density and / or size and / or number of cells comprising the following steps:

[0193] 1) Cultivation of at least one cellular element; 2) Rinsing of the culture of cellular elements from step 1); 3) Centrifugation of the culture of cellular elements from step 2); 4) Recovery of a composition comprising cellular elements resulting from the centrifugation of step 3), of which at least two cellular elements have a different density and / or size and / or number of cells; (b) Preparation of a solution comprising:

[0194] - hyaluronic acid and / or carboxymethyl cellulose (CMC), and

[0195] - at least one biocompatible density agent,

[0196] -and / or at least one modified form of one or more of these molecules;

[0197] (c) Sterilization of the solution from step (b) so as to obtain a filtrate sterilized;

[0198] (c') Adjustment of the pH of the sterilized filtrate from step (c), so that the filtrate from step (c') has a pH between 6.8 and 8;

[0199] (d) Mixing, with stirring, the cellular elements from step (a) with the filtrate sterilized from step (c);

[0200] (e) Optionally, recovery of the biocompatible composition resulting from step (d), comprising a solution and cellular elements suspended in said solution, at least two of which cellular elements have a different density and / or size and / or number of cells, said composition comprising the following molecules:

[0201] - Hyaluronic acid and / or Carboxymethyl cellulose (CMC);

[0202] - At least one density agent;

[0203] -and / or at least one modified form of one or more of these molecules.

[0204] Preferably, step (b) is carried out so that the solution resulting from step (b) presents: - a density between 0.997 and 1.6 g / ml; and - a viscosity, measured using a rheometer at 25°C, between 0.01 and IPa.s.

[0205] According to one embodiment, the method according to the invention comprises a step of cryopreservation of the composition resulting from step (a). When the method according to the invention comprises a step of cryopreservation of the composition resulting from step (a), said method also comprises a step of thawing the cryopreserved composition.

[0206] Composition according to the invention for its use:

[0207] According to another aspect, the invention relates to the composition according to the invention according to any one of the embodiments previously described for its use as a medicament.

[0208] Advantageously, the biocompatible composition according to the invention has rheological characteristics and a maximum sedimentation rate of the cellular elements which are particularly suitable for its use in cell therapy.

[0209] Preferably, the composition can be used in the treatment of diseases in cell therapy.

[0210] According to a preferred embodiment, the composition can be used in the treatment of Parkinson's disease.

[0211] According to one embodiment, the composition according to the invention comprises cellular elements, preferably in the form of microtissue(s), comprising at least neural cells, neuronal cells, glial cells, or mixtures thereof for its use in the treatment of Parkinson's disease.

[0212] According to a preferred embodiment, the composition according to the invention comprises cellular elements comprising at least one dopaminergic neuron for its use in the treatment of Parkinson's disease.

[0213] Advantageously, the biocompatible composition according to the invention has rheological characteristics and a maximum sedimentation rate of cellular elements, preferably dopaminergic neurons, particularly suitable for its use in cell therapy.

[0214] According to another embodiment, the composition according to the invention comprising a solution and cellular elements suspended in said solution, in which at least 70% of the cellular elements are in the form of microtissue comprising dopaminergic neurons can be used in the treatment of Parkinson's disease.

[0215] According to a variant of the invention, the composition according to the invention comprises a solution and cellular elements suspended in said solution, in which: - at least 50% of the cellular elements are in the form of microtissue; - less than 20% of the cellular elements are in the form of isolated cells; and - at least 30% of the cellular elements are dopaminergic neurons can be used in the treatment of Parkinson's disease.

[0216] Advantageously, when the composition according to the invention comprises microtissues, said microtissues recreate the tissue architecture and cellular interactions necessary to promote tissue regeneration. The cell clusters make it possible to provide a microenvironment conducive to cell survival and differentiation. Furthermore, the injection of a composition comprising heterogeneous cellular elements makes it possible to provide cellular diversity and potentially better plasticity.

[0217] In the context of the invention, the composition according to the invention is particularly suitable for its use as an injectable biocompatible composition for treating Parkinson's disease.

[0218] According to one embodiment, the composition according to the invention can be used for multiple uses. In other words, the composition can be used to make multiple injections control the quantity of cells injected while maintaining the viability of the cellular elements, even if the injections are spaced several hours apart.

[0219] Advantageously, the maximum sedimentation rate of the cellular elements makes it possible to inject, for a constant volume, a quantity of cellular elements having a low standard deviation, preferably a standard deviation of less than 20%.

[0220] According to one embodiment, the composition according to the invention can be used by injection using an injection means at the injection site.

[0221] Preferably, said injection means is a cannula.

[0222] Advantageously, the control of the rheological characteristics of the composition according to the invention makes it possible to optimize the administration of the cellular elements and, consequently, of the therapeutic cells in a precise and efficient manner. The control of these parameters makes it possible to adjust the injection rate, the injection volume and thus to ensure a homogeneous distribution of the cellular elements in the area of interest.

[0223] According to one embodiment, the composition according to the invention can be used by injection using a cannula at the injection site, preferably at the putamen in humans or animals.

[0224] Kit J.

[0225] According to another aspect, the invention relates to a kit comprising: - a biocompatible composition according to any of the embodiments previously described; and - an injection method, preferably a cannula.

[0226] Advantageously, the kit according to the invention allows the practitioner to have all the elements necessary to optimize the effectiveness of the injection of the cellular elements.

[0227] According to one embodiment, the kit according to the invention comprises: - a cannula - a biocompatible composition according to any of the embodiments previously described in which the cellular elements have a largest dimension less than 0.8 times the smallest internal diameter of the cannula, preferably less than 0.75, in particular between 0.2 and 0.7.

[0228] According to a particular embodiment of the invention, the kit according to the invention comprises a biocompatible composition according to any one of the embodiments previously described in solid form.

[0229] Preferably, the kit according to the invention also comprises water.

[0230] According to one embodiment, the kit according to the invention also comprises an element chosen from sodium chloride (NaCl), calcium chloride (CaCl2), potassium chloride (KCl), magnesium chloride (MgCl2) and mixtures thereof.

[0231] Kit 2:

[0232] Finally, according to a last aspect, the invention relates to a kit comprising: *a preparation (1), in solid form, comprising - hyaluronic acid and / or carboxymethyl cellulose (CMC), and - at least one biocompatible density agent, - and / or at least one modified form of one or more of these molecules. * a preparation (2) comprising cellular elements, said cellular elements being chosen from isolated cells, cellular microtissues, clusters of cells and their mixtures, at least two of said cellular elements having a different density and / or size and / or number of cells.

[0233] Preferably, the kit according to the invention also comprises water.

[0234] According to one embodiment, the preparation (1), in solid form, comprises at least one biocompatible density agent chosen from dextran, hypromelose, Iodixanol, Polyethylene glycol (PEG), chitosan, pullulan, or molecules from the glycosaminoglycan family, or modified forms of these molecules, and mixtures thereof.

[0235] Preferably, preparation (1) comprises dextran, as a biocompatible density agent.

[0236] According to one embodiment, the kit according to the invention also comprises an element chosen from sodium chloride (NaCl), calcium chloride (CaCl2), potassium chloride (KCl), magnesium chloride (MgCl2) and mixtures thereof. Examples#:

[0237] Example 1:

[0238] The objective of this example is to evaluate the sedimentation rate of the cellular elements in different compositions, namely a composition comprising cellular elements and a Dulbecco's phosphate buffered saline (DPBS) solution (A) (not included in the invention), and the composition according to the invention comprising a solution (B) developed to maintain the homogeneity of distribution of the cellular elements and prevent obstruction of the injection system.

[0239] After obtaining the cellular elements, these were cryopreserved in vials and stored in nitrogenous liquid. The vials containing the elements were then thawed with an estimate of 10 million cells.

[0240] The results of the characterization of the cellular elements made it possible to measure the density and the largest dimension of the cellular elements, namely:

[0241] Density of cellular elements: 1056 kg / m3;

[0242] Largest dimension of the cellular elements: 200pm or 0.0002 m.

[0243] The solution (B) described in Table 1 below was prepared under sterile conditions and characterized in Table 2. [Tables 1] Reagent Reference Test Concentration HA100 PHI4468P02E (VI: 0.22%) 48 mg / mL Dextran40 CAS 9004-54-0 130 mg / mL KC1 CAS 7447-40-7 0.30 mg / mL CaCl2,2H O2 CAS 10035-04-8 0.15 mg / mL CH O687 CAS 77-92-9 0.25 mg / mL Na2 HPO4 CAS 7558-79-4 2.50 mg / mL

[0244] [ Table 2 ] Physicochemical parameter of the solution (B) Value Viscosity at rest 0.454 Pa.s Density 1049 kg / m3 PH 7.37 Osmolarity 216 mOsmol

[0245] Solution (A) was also prepared under sterile conditions and characterized in Table 3.

[0246] [Tables3] Physicochemical parameter of the solution (B) Value Viscosity at rest 0.0012 Pa.s Density 1002 kg / m3

[0247] Preparation of the compositions:

[0248] Phase 1: preparation of cellular elements

[0249] The vials were removed from the liquid nitrogen and placed in a CryoPod at -180°C. The vials containing the cellular elements were placed in a water bath at 37°C and removed when only a small piece of ice remained (“2 min”).

[0250] After slight resuspension of the cells, the contents were transferred to a 15 ml tube containing 9 ml of DPBS (calcium, magnesium, glucose, pyruvate) with Tween 80, at room temperature.

[0251] The flasks were rinsed with 1 ml of DPBS (calcium, magnesium, glucose, pyruvate) with Tween 80 to collect the maximum number of cellular elements

[0252] The 15 ml tube was centrifuged at 300 G, 5 minutes, and the maximum of the supernatant was removed with P1000 then P20.

[0253] The cellular elements were resuspended in 1 ml of DPBS (calcium, magnesium, glucose, pyruvate) with a Tween 80 surfactant.

[0254] Phase 2: Aliquot the samples

[0255] An undetermined volume and concentration of the cell suspension were aliquoted into two 1.5 ml Eppendorf tubes.

[0256] The 1.5 mL tubes were centrifuged at 300G for 5 min and the maximum of the supernatant was removed with P1000 then P20.

[0257] In one sample, the cellular elements were resuspended in 1 ml of DPBS (solution (A)) and in the other in 1 ml of solution (B).

[0258] Phase 3: Evaluation of the sedimentation rate

[0259] The tubes were installed on the assembled device for taking pictures.

[0260] Pictures were taken every minute for 12 hours, see [Fig. 1].

[0261] With the Image J software, manual tracking of the trajectories of microtissues, on a A time-lapse film recorded with the camera was taken to assess the sedimentation rate.

[0262] An average of 5 sedimentation rates of cellular elements in solution (B), during 10 / 12 trajectories, was taken to evaluate the value of the maximum sedimentation rate. In solution (A), only 3 microtissue rates, during 4 trajectories, were taken to determine the value due to the rapid sedimentation and the difficulty in following exactly the same cellular element.

[0263] The calculated maximum sedimentation rate (average of 5 maximum rates for solution (B) and average of 3 maximum rates for microtissue solution (A)) is: * of approximately 363mm / h in solution (A) outside the invention; * of approximately 1.66 mm / h in the solution (B) according to the invention, in which the difference in density between the solution and the average density of the cellular elements of the composition resulting from step (b5) verifies the inequality [Math 1].

[0264] Table 4 below verifies the inequality [Math 1] for solution (A) and solution (B) with Vc = 5.56*10 6m.s '.

[0265] [Tables4] 1i Pmt P s F.vjD2 | Ap| <FvjD2 Solution (A) Hors i nvention 54 0,03 NON Solution (B) Invent ion 7 11,6 OUI

[0266] Conclusions:

[0267] The composition according to the invention comprising the cellular elements and the solution (B) makes it possible to maintain said cellular elements in suspension in said solution (B) for several hours. Within the composition according to the invention, certain cellular elements are still in suspension after 12 hours of sedimentation.

[0268] On the other hand, the composition outside the invention comprises the cellular elements and the solution (A) does not allow the cellular elements to be maintained in suspension in said solution (A) for a long time. The cellular elements sediment very quickly in low viscosity solutions with no density match, such as DPBS (calcium, magnesium, pyruvate, glucose) with a viscosity value at rest of approximately 1 mPa.s and a density of approximately 1.0038 g / cm.3

[0269] Example 2:

[0270] The objective of this example is to evaluate the viability of the cellular elements after 12 hours of sedimentation within the composition according to the invention.

[0271] For the realization of this example, the composition according to the invention comprises the following elements:

[0272] [Tables5] Reagent Reference Test concentration HA100 PHI4468P02E (VI: 0.22%) 5.4% Dextran40 CAS 9004-54-0 12% KC1 CAS 7447-40-7 0.03% CaCl2,2H O2 CAS 10035-04-8 0.015% c6h8 07 CAS 77-92-9 0.025% Na2 HPO4 CAS 7558-79-4 0.25%

[0273] The composition was also prepared under sterile conditions and characterized in Table 6.

[0274] [Tableauxô] Physicochemical parameter Value Viscosity at rest 0.62 Pa.s Density 1073.5 kg / m3 PH 7.34 Osmolarity 211 mOsmol

[0275] The results of the characterization of the cellular elements made it possible to measure the density and the largest dimension of the cellular elements, namely:

[0276] Density of cellular elements: 1065 kg / m3;

[0277] Largest dimension of the cellular elements: 200pm or 0.0002 m;

[0278] In the context of this example, the sedimentation time is 12 hours, which corresponds to a maximum stability limit in the context of the use of the composition according to the invention in cell therapy.

[0279] Table 7 below verifies the inequality [Math 1] for solution (A) and solution (B) with Vc = 5.56*10 6m.s '.

[0280] [Tables7] F.vjD2 |Ap| <F.vjD2 Solution (A) Hors i nvention 8,5 158 OUI

[0281] Experimental protocol

[0282] Conditions

[0283] Production batches are referenced by the prefix “PAR”, followed by an incremental code ranging from 19 to 22. They correspond to production batches of successive cellular elements.

[0284] 1) For each batch, two types of samples are prepared: Control: A suspension of cellular elements resuspended in a reference cell culture medium, at a ratio of 50% volume of cellular elements and 50% reference culture medium. Composition according to the invention: A suspension of cellular elements resuspended in a solution, at a ratio of 50% volume of cellular elements and 50% administration medium. 2) Once the samples are prepared, they are stored in closed tubes at room temperature for 12 hours. 3) The cellular elements are then rinsed in an excess of reference culture medium. 4) The cellular elements are then put back into culture for 24 hours in a reference culture medium. 5) Viability is measured. The result is expressed by normalizing the viability value of the immersion condition in the administration medium by the viability obtained in the control condition.

[0285] Results

[0286] The results of this example are described in [Fig.2]. This experiment shows an average decrease in viability of around 25% after 12 hours of immersion in the medium administration considered, compared to a 12-hour control in a reference culture medium. In the context of the example, this drop in viability is acceptable given the acceptance criterion of 70% relative viability.

[0287] Example 3: Evaluation of the impact of the procedure for injecting the composition according to the invention on the viability of the cellular elements.

[0288] The objective of this example is to evaluate the impact of the procedure for injecting the composition according to the invention on the viability of the cellular elements.

[0289] In the context of the invention, it is important to verify that the viscosity of the administration medium, necessary for maintaining the homogeneity of distribution of the cellular elements, does not induce excessively high shear forces during the injection, which could degrade the viability of the cells.

[0290] The experiment consists of comparing the viability of a known volume of composition according to the invention having undergone an injection procedure under the conditions of the invention, with the viability of a known volume of composition according to the invention having not undergone an injection procedure.

[0291] Since the shearing effects experienced during the injection can have a delayed effect on the viability of the cells, the viability of the cellular elements having undergone an injection procedure is measured immediately after the injection, then after 24 hours of re-culture in a reference culture medium.

[0292] The composition according to the invention used during this example has the same composition (Table 5) and the same rheological characteristics (Table 6) as the composition according to the invention described in Example 2.

[0293] The injection protocol includes the following steps: a) Preparation of the composition according to the invention according to the process according to the invention; b) Fill a syringe with the composition according to the invention; c) Inject the composition according to the invention using the syringe from step b) into a cannula with an injection rate of 50 pL.min; d) Injecting the composition according to the invention using the cannula onto a matrix at a flow rate of between 18 pL and 2 pL per minute; e) Rinse the composition according to the invention and resuspend the cellular elements in a culture medium; f) Place the culture medium including the cellular elements in an incubator for 24 hours.

[0294] The viability of the cellular elements is measured at the end of step e) and at the end of step f). The experiment was carried out 3 times.

[0295] Result s:

[0296] The viability measurements described in [Fig.3] do not show any significant impact of the injection procedure on viability under the conditions of the example. The slight The decrease observed directly after injection could reflect a transient decrease in the metabolic activity of the cells, which is however not found after 24 hours of culture.

[0297] Therefore, the composition according to the invention can be used as an injectable composition.

Claims

Claims

1. Biocompatible composition, comprising a solution and cellular elements suspended in said solution, said cellular elements being chosen from isolated cells, cellular microtissues, cell clusters and mixtures thereof, at least two of said cellular elements having a different density and / or size and / or number of cells, said composition comprising at least: - hyaluronic acid and / or carboxymethyl cellulose (CMC), and - at least one biocompatible density agent, - and / or at least one modified form of one or more of these molecules.

2. Composition according to the preceding claim, characterized in that at least one biocompatible densifying agent has a molar mass greater than 400g / mol.

3. Composition according to one of the preceding claims, characterized in that at least one biocompatible density agent is chosen from dextran, hypromelose, Iodixanol, Polyethylene glycol (PEG), chitosan, pullulan, or molecules from the glycosaminoglycan family, or modified forms of these molecules, and mixtures thereof.

4. Biocompatible composition according to one of the preceding claims, characterized in that the cells constituting the cellular elements are chosen from neural cells, neuronal cells, glial cells and their mixtures.

5. Biocompatible composition according to one of the preceding claims, characterized in that at least one cell constituting the cellular elements is a dopaminergic neuron.

6. Biocompatible composition according to one of the preceding claims, characterized in that the cells constituting the cellular elements belong to at least 2 distinct cell types.

7. Biocompatible composition according to one of the preceding claims, characterized in that the cells constituting the cellular elements comprise astrocytic cells, oligodendrocyte cells and dopaminergic neurons.

8. Biocompatible composition according to one of the preceding claims, characterized in that it comprises at least one cellular element contained in a three-dimensional microcompartment comprising an external hydrogel layer.

9. Biocompatible composition according to one of the preceding claims, characterized in that the volume fraction of the cellular elements is between 0.01 and 80% by volume.

10. Biocompatible composition according to one of the preceding claims, characterized in that it comprises: - sodium hyaluronate having a molecular weight of between 80kDa and IMDa and / or carboxymethyl cellulose having a molecular weight of between 40 and IMDa; and / or - dextran having a molecular weight of between 40 and 80kDa.

11. Composition according to one of the preceding claims, characterized in that it comprises a solution comprising at least one ion chosen from sodium ion, calcium ion, chlorine ion, potassium ion and mixtures thereof.

12. Composition according to one of the preceding claims, characterized in that it comprises a solution comprising at least one element chosen from sodium chloride (NaCl), calcium chloride (CaCl2), potassium chloride (KC1), magnesium chloride (MgCl2) and their mixtures.

13. Biocompatible composition according to one of the preceding claims, characterized in that the composition comprises: - between 4 and 7% by mass of sodium hyaluronate with a molecular weight of between 80kDa and IMDa relative to the total mass of the solution and / or between 0.1 and 7% by mass of carboxymethyl cellulose having a molecular weight of between 40 and IMDa relative to the total mass of the solution; and - between 10 and 25% by mass of at least one density agent relative to the total mass of the solution; and - between 0.01 and 0.9% by mass of NaCl relative to the total mass of the solution; and / or - between 0.01 and 0.04% by mass of KC1 relative to the total mass of the solution; and / or

14.

15.

16.

17.

18.

19.

20.

21. - between 0.01 and 0.05% in mass of CaC12 relative to the total mass of the solution. Biocompatible composition according to one of the preceding claims, characterized in that the composition has a density of between 800 and 2000 kg / m3. Biocompatible composition according to one of the preceding claims, characterized in that the composition has a viscosity at rest measured using a rheometer at 25°C of between 0.01 and 1.5 Pa.s. Biocompatible composition according to one of the preceding claims, characterized in that the osmolality of the solution is between 220 and 360 mOsml.kg Biocompatible composition according to one of the preceding claims, characterized in that the maximum sedimentation rate of the cellular elements is between 0.1 and 20 mm.h '. Composition according to one of the preceding claims for its use in the treatment of diseases in cell therapy. Composition for its use according to the preceding claim, in the treatment of Parkinson's disease. Composition for its use according to one of claims 18 or 19, by injection using a cannula at the level of the putamen in humans or animals. Composition according to one of the preceding claims, characterized in that it is obtained by implementing the following steps: (a) Preparation or recovery of a composition comprising a set of cellular elements of which at least two cellular elements have a different density and / or size and / or number of cells; (b) Preparation of a solution comprising: - hyaluronic acid and / or carboxymethyl cellulose (CMC), and - at least one biocompatible density agent, -and / or at least one modified form of one or more of these molecules; (c) Sterilization of the solution resulting from step (b) so as to obtain a sterilized filtrate; (d) Mixing the cellular elements from step (a) with the sterilized filtrate from step (c); (e) Optionally recovering the biocompatible composition from step (d), comprising a solution and cellular elements suspended in said solution, of which at least two cellular elements have a different density and / or size and / or number of cells, said composition comprising the following molecules: - Hyaluronic acid and / or Carboxymethyl cellulose (CMC); - At least one biocompatible density agent; - and / or at least one modified form of one or more of these molecules.

22. Process for preparing a biocompatible composition according to one of the preceding claims, characterized in that it comprises the implementation of the following steps: (a) Preparation or recovery of a composition comprising a set of cellular elements of which at least two cellular elements have a different density and / or size and / or number of cells; (b) Preparation of a solution comprising: - hyaluronic acid and / or carboxymethyl cellulose (CMC), and - at least one biocompatible density agent, -and / or at least one modified form of one or more of these molecules; (c) Sterilization of the solution resulting from step (b) so as to obtain a sterilized filtrate; (d) Mixing the cellular elements resulting from step (a) with the sterilized filtrate resulting from step (c);(e) Optionally, recovery of the biocompatible composition from step (d), comprising a solution and cellular elements suspended in said solution, of which at least two cellular elements have a different density and / or size and / or number of cells, said composition comprising the following molecules: - Hyaluronic acid and / or Carboxymethyl cellulose (CMC); - At least one biocompatible density agent; -and / or at least one modified form of one or more of these molecules.

23. Method according to the preceding claim, characterized in that step (a) comprises the implementation of the following steps: 1) Culturing at least one cellular element; 2) Rinsing the culture of cellular elements from step 1); 3) Centrifugation of the culture of cellular elements from step 2); 4) Recovery of a composition comprising cellular elements from the centrifugation of step 3), of which at least two cellular elements have a different density and / or size and / or number of cells.

24. Method according to the preceding claim, characterized in that at least one cellular element comprises at least one encapsulated cell, preferably in alginate.

25. Method according to one of claims 22 to 24, characterized in that the composition of step (a) comprises a solution in which said cellular elements are in suspension.

26. Method according to one of claims 22 to 25, characterized in that the composition of step (a) has a maximum sedimentation rate of the cellular elements relative to the solution greater than 20 mm.h '.

27. Method according to one of claims 22 to 26, characterized in that the solution of step (b) comprises: - between 0.1 and 7% by mass of sodium hyaluronate with a molecular weight of between 80kDa and IMDa relative to the total mass of the solution and / or between 0.1 and 7% by mass of carboxymethyl cellulose having a molecular weight of between 40 and IMDa relative to the total mass of the solution; and - between 10 and 25% by mass of at least one biocompatible density agent relative to the total mass of the solution.

28. Method according to one of claims 22 to 27, characterized in that step (b) also comprises the addition of at least one element chosen from sodium chloride (NaCl), calcium chloride (CaCl2), potassium chloride (KCl), magnesium chloride (MgCl2) and mixtures thereof.

29. Method according to the preceding claim, characterized in that the solution resulting from step (b) also comprises: - between 0.01 and 0.9% by mass of NaCl relative to the total mass of the solution; and / or - between 0.01 and 0.04% by mass of KC1 relative to the total mass of the solution; and / or - between 0.01 and 0.05% by mass of CaC12 relative to the total mass of the solution.

30. Method according to one of claims 22 to 29, characterized in that it comprises a step (c'), carried out after step (c), of adjusting the pH of the sterilized filtrate resulting from step c), so that the filtrate resulting from step c') has a pH of between 6.8 and 8.

31. Method according to one of claims 22 to 30, characterized in that step (d) is carried out with stirring.

32. Kit comprising: - a biocompatible composition according to one of claims 1 to 21; and - an injection means, preferably a cannula.

33. Kit according to the preceding claim, characterized in that the biocompatible composition is in solid form.

34. Kit comprising: - A preparation, in solid form, comprising the following molecules: - Hyaluronic acid and / or Carboxymethyl cellulose (CMC); and at least one density agent and / or a modified form of these molecules; - A preparation comprising cellular elements, said cellular elements being chosen from isolated cells, cellular microtissues, clusters of cells and mixtures thereof, at least two of said cellular elements having a different density and / or size and / or number of cells.

35. Kit according to one of claims 32 to 34, characterized in that it comprises water.

36. Kit according to one of claims 32 to 35, characterized in that it comprises at least one element chosen from sodium chloride (NaCl), calcium chloride (CaCl2), potassium chloride (KCl), magnesium chloride (MgCl2) and their mixtures.

37. Kit according to one of claims 32 to 36, characterized in that one or more cells constituting the cellular elements are chosen from neuronal cells, glial cells and their mixture.

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