Biocompatible composition comprising heterogeneous cellular elements

Abstract

The invention relates to the technical field of biocompatible compositions, in particular injectable biocompatible compositions. In particular, the invention relates to a particular biocompatible composition comprising a solution and heterogeneous cellular elements suspended in said solution, which elements are selected 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.

Description

[0001] BIOCOMPATIBLE COMPOSITION COMPRISING CELLULAR ELEMENTS

[0002] HETEROGENEOUS

[0003] Technical field

[0004] The present invention relates to the field of biocompatible compositions, in particular injectable compositions. The invention relates in particular to a particular biocompatible composition comprising a solution and heterogeneous cellular elements suspended in said solution.

[0005] Prior art

[0006] For over 20 years, regenerative medicine, and more specifically cell therapy, has attracted growing interest among healthcare professionals and the general public. Cell therapy involves transplanting cells to restore the function of a tissue or organ, using an injection of therapeutic cells.

[0007] Today, cell therapy faces many challenges inherent in delivering cells into the body.

[0008] 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.

[0009] In general, drugs can dissolve in liquid and can be administered as a solution, such as in an IV drip. Liquid suspensions containing very small particles also behave like solutions and can also be administered by IV drip.

[0010] On the other hand, as soon as the composition is cellularized, that is to say it includes cells, it is injected in suspension.

[0011] Unlike solutions which form a homogeneous and fluid liquid, cells in suspension naturally sediment over time.

[0012] 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 clumps or microtissue, this relative speed can exceed 100 mm per hour, or even 300 mm per hour in certain cases.

[0013] Particle sedimentation in solid-liquid suspensions is a widely discussed topic in many fields such as chemistry, geology, and biology, particularly through pharmaceutical applications or bioprinting. The sedimentation velocity of solid particles such as cells in a liquid refers to the rate at which particles move downward or settle to the bottom of a container by gravity. This sedimentation phenomenon occurs when particles, due to their mass and density, are heavier than the medium in which they are suspended.

[0014] When using a cell suspension, the cells can be heterogeneous. Indeed, the cells can be found in very varied forms, we then speak of cellular elements. For example, the cellular elements can 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.

[0015] Without control of these parameters, the greatest concentration of cells ends up, by gravity, in the lower part of the device containing the suspension of cellular elements.

[0016] 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 can impair the effectiveness of the cell therapy.

[0017] Furthermore, beyond maintaining the homogeneity of cell distribution in their environment, maintaining biological stability is fundamental to ensure the preservation of living cells in their environment, particularly when injecting 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.

[0018] There is therefore a significant need for a biocompatible composition, comprising heterogeneous cellular elements, capable of maintaining:

[0019] - the homogeneity of distribution of cellular elements within the composition so as to be able to precisely control the quantity of cells contained in a given volume; and

[0020] - the biological stability of cellular elements in suspension.

[0021] Summary of the invention

[0022] 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 their mixtures, at least two of said cellular elements having a different density and / or size and / or number of cells, said composition being characterized in that the difference in density between the density of the solution and the average density of the cellular elements satisfies the following inequality:

[0023] [Math 1]

[0024] V \ < F-2where | | = p s — p0with p srepresenting the density of the composition expressed in kg / m3, and p0 representing the average density of the cellular elements, measured using the density gradient technique expressed in kg / m3, where D represents the largest dimension of the cellular elements expressed in meters, where v c represents the threshold of acceptable sedimentation rate expressed in ms-1; where F = 18. p c / g ~ 9.81 with p c represents the maximum viscosity at rest of the solution expressed in Pa.s, g representing the gravity constant expressed in ms-2; and where v c .= 5.56*10-6 ms-1.

[0025] Advantageously, compliance with the above-mentioned inequality guarantees that the maximum sedimentation rate of the cellular elements relative to the solution is less than 5.56*10-6 ms-1, or 20 mm. h-1.

[0026] The maximum sedimentation rate of less than 20 mm h-1 of the cellular elements compared to the solution provides numerous advantages to the composition according to the invention, namely:

[0027] - an increase in cell viability: A maximum sedimentation rate of 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;

[0028] - increased analytical precision: In specific analyses and measurements, a maximum sedimentation rate of less than 20 mm. h-1 allows for greater precision and sensitivity of the results;

[0029] - better homogeneity: A maximum sedimentation rate of less than 20 mm.h-1 allows the cells to remain evenly distributed in the solution, thus avoiding the formation of concentration gradients;

[0030] - better experimental reproducibility: By maintaining a maximum sedimentation rate below 20 mm. h-1, variations due to differential sedimentation of heterogeneous cellular elements are reduced. The composition according to the invention has a density and / or viscosity particularly suited to heterogeneous cellular elements, promoting a uniform distribution of said cellular elements in suspension in the solution.

[0031] According to a preferred subject, 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 180 and 360 mOsmol.kg-1, preferably between 220 and 360 mOsmol.kg-1. In other words, the composition comprising cellular elements and a solution has an osmolality of between 180 and 360 mOsmol.kg-1, preferably between 220 and 360 mOsmol.kg-1.

[0032] 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.

[0033] Surprisingly, the inventors have developed a biocompatible composition, suitable for many uses, controlling the sedimentation rate of heterogeneous cellular elements suspended in the solution while presenting a biological stability adapted to cells, in particular human or animal cells.

[0034] 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.

[0035] Advantageously, the density of the composition according to the invention is homogeneous, consequently, each cellular element is subject to the same density over the entire volume of the composition.

[0036] According to a preferred embodiment, the composition according to the invention has a viscosity at rest measured at 25°C of between 0.01 and 1.5 Pa.s, preferably between 0.01 and 1 Pa.s, in particular between 400 and 1200 mPa.s, more preferably 400 and 1000 mPa, even more preferably between 500 and 800 mPa.s, in particular between 500 and 900 mPa.s.

[0037] Advantageously, 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.

[0038] The inventors have achieved a biocompatible composition having rheological characteristics adapted to heterogeneous cellular elements based on the density / viscosity couple of the solution comprising said cellular elements. According to one embodiment, the biocompatible composition comprises at least one cellular element contained in a three-dimensional microcompartment comprising an external hydrogel layer.

[0039] Preferably, the biocompatible composition according to the invention 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, cardiac cells, retinal cells, lymphocytes, hepatocytes, endothelial cells, mesenchymal stem cells, keratinocytes, fibroblasts, pre-adipocytes, adipocytes, pancreatic islet cells and mixtures thereof.

[0040] According to one embodiment, the composition according to the invention comprises at least two distinct cell types.

[0041] Advantageously, the composition according to the invention is particularly suitable for all cell types.

[0042] According to another object, the invention relates to the biocompatible composition according to the invention for its use in the treatment of diseases in cell therapy.

[0043] Preferably, the biocompatible composition according to the invention is used as an injectable composition in humans or animals.

[0044] 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:

[0045] - therapeutic efficacy: When injecting cellular elements for therapeutic purposes, precise dosage is essential to achieve the desired result. An insufficient amount of cellular elements may not be effective, while an overdose of cellular elements can lead to adverse effects. By precisely adjusting the dosage, it is possible to maximize therapeutic efficacy while minimizing risks to the patient;

[0046] - experimental precision: Controlling the dosage when injecting cellular elements makes it possible to 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.

[0047] - resource optimization: The resources used for the production of cellular elements, such as cell cultures and culture products, can be expensive. Accurate dosing makes it possible to minimize the waste of these resources by avoiding unnecessary overdoses. The invention also relates to a method for preparing a biocompatible composition according to the invention comprising the implementation of the following steps:

[0048] (a) Preparation or recovery of a composition comprising a solution (A) and a set of cellular elements of which at least two cellular elements have a different density and / or size and / or number of cells;

[0049] (b) Preparation of a biocompatible composition comprising the cellular elements of the composition of step a), a solution (B) and optionally the solution (A) so that the difference in density between the density of the solution and the composition resulting from step

[0050] (b) and the average density of the cellular elements of the composition resulting from step (b) verifies the following inequality:

[0051] [Math 1]

[0052] V \ < F-2where | | = p s — p0with p s representing the density of the composition expressed in kg / m3, and p0 representing the average density of the cellular elements, measured using the density gradient technique expressed in kg / m3, where D represents the largest dimension of the cellular elements expressed in meters, where v c represents the acceptable sedimentation rate threshold expressed in ms-1; where F = 18.p c / g ~ 9.81 with p crepresents the maximum viscosity at rest of the solution expressed in Pa.s, g representing the gravity constant expressed in ms-2; and where v c .= 5.56*10-6 ms-1 ;

[0053] (c) Optionally recovery of the composition resulting from step (b), 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, in which said composition is characterized in that the difference in density between the density of the solution and the average density of the cellular elements verifies the inequality mentioned above in step (b).

[0054] 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 5.56*10-6 ms-1 or 20 mm.h-1.

[0055] Preferably, the composition of step (a) has a maximum sedimentation rate of the cellular elements relative to the solution (A) greater than 20 mm.h-1. Such a method advantageously makes it possible to adapt the rheological characteristics, in particular the density and the viscosity, of a composition so as to make the distribution of the heterogeneous cellular elements within the composition homogeneous. In this way, the composition of step (c) can be used in numerous applications, in particular in cell therapy.

[0056] Finally, according to a last object, the invention relates to a kit comprising:

[0057] - 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 mixtures thereof, including at least two of said cellular elements having a different density and / or size and / or number of cells, said composition being characterized in that the difference in density between the density of the solution and the average density of the cellular elements satisfies the following inequality:

[0058] [Math 1] where | | = p s — p0with p srepresenting the density of the composition expressed in kg / m3, and p0 representing the average density of the cellular elements, measured using the density gradient technique expressed in kg / m3, where D represents the largest dimension of the cellular elements expressed in meters, where v c represents the threshold of acceptable sedimentation rate expressed in ms-1; where F = 18. p c / g ~ 9.81 with p c represents the maximum viscosity at rest of the solution expressed in Pa.s, g representing the gravity constant expressed in ms-2; and where v c .= 5.56*10-6 ms-1; and

[0059] - an injection method, preferably a cannula.

[0060] Advantageously, the rheological characteristics of the composition make it possible to maintain the homogeneity of distribution of the cellular elements in the composition while preventing obstruction of the injection means.

[0061] According to one variant, the invention relates to a kit comprising:

[0062] - a biocompatible preparation (1) comprising at least one biocompatible viscosity agent chosen from PVA, carboxymethyl cellulose (CMC), methylcellulose, hydroxyethylcellulose, ethylhydrocyethylcellulose, hydroxypropylmethylcellulose (HPMC), alginate, pullulan, chitosan, xanthan gum, gellan gum, polyacrilamide, polyvinylpyrrolidone, polyethylene oxide, casein, collagen, sodium hyaluronate and mixtures thereof, or modified forms of these molecules; and at least one biocompatible density agent chosen from dextran, mannitol, NaCl, glycerol, sorbitol and mixtures thereof; and

[0063] - a biocompatible preparation (2) 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 so that the mixture of the preparation (1) and the preparation (2) forms a composition whose difference in density between the density of the preparation (1) and the average density of the cellular elements of the preparation (2) satisfies the following inequality:

[0064] [Math 1]

[0065] V \ < F-2where | | = p s — p0with p srepresenting the density of the composition expressed in kg / m3, and p0 representing the average density of the cellular elements, measured using the density gradient technique expressed in kg / m3, where D represents the largest dimension of the cellular elements expressed in meters, where v c represents the acceptable sedimentation rate threshold expressed in ms-1; where F = 18.p c / g ~ 9.81 with p c represents the maximum viscosity at rest of the solution expressed in Pa.s, g representing the gravity constant expressed in ms-2; and where v c .= 5.56*10-6 ms-1.

[0066] Other characteristics and advantages will emerge from the detailed description of the invention, the examples and the figures which follow.

[0067] Brief description of the Figures:

[0068] [Figure 1] Figure 1 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.

[0069] [Figure 2] Figure 2 represents the comparative study of the impact of prolonged immersion (6h) of cellular elements in a solution, on a panel of production batches of microtissue of interest.

[0070] [Figure 3] Figure 3 is a schematic representation of a comparative study evaluating the impact of an injection procedure on the viability of cellular elements within the composition according to the invention. [Figure 4] Figure 4 is a graphical representation of a study evaluating the different shear deformations of the composition according to the invention.

[0071] Detailed description of the invention

[0072] Definitions:

[0073] For the purposes of the invention, the term "density agent" means 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. More particularly, when a density agent within the meaning of the invention is introduced at a concentration greater than 10% by mass into an aqueous solution, this does not result in a modification of the rheological behavior of the solution. In addition, the addition of such an agent does not increase the osmolality of the solution beyond 360 mOsm. In other words, the addition of a density agent within the meaning of the invention at a concentration greater than 10% by mass in a solution does not result in a phase transition such as gelation.

[0074] 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.

[0075] For the purposes of this 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 organism and / or cellular elements.

[0076] For the purposes of the invention, the term "human cells" means human cells or immunologically humanized non-human mammalian cells. Even when not specified, the cells, stem cells, progenitor cells and tissues according to the invention are constituted by or are obtained from human cells or from immunologically humanized non-human mammalian cells.

[0077] For the purposes of the invention, the term “progenitor cell” means a stem cell already engaged in cell differentiation but not yet differentiated.

[0078] 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.

[0079] 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 original organism, 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").

[0080] 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).

[0081] Rheological behavior, as used herein, refers to the response of a material to stresses and strains applied to it. It describes how the material flows or deforms under the effect of external forces. There are several types of rheological behavior, such as elasticity, plasticity, viscosity, and viscoelasticity.

[0082] For the purposes of the invention, the term "cell layer" or "cell base" means several cells forming a layer or base that can be structured around a lumen; this may be, for example, a cellular tissue or micro-tissue or a three-dimensional grouped culture. The thickness of the cell layer may be variable. This layer is organized in three dimensions in the microcompartment.

[0083] 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 of known density 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, 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 made at the temperature or within the temperature range of intended use because temperature can differentially affect the density of water, solutions and cellular elements.

[0084] By "average density of cellular elements", within the meaning of the invention, is meant the average density of cellular elements such as microcompartments. In the context of the invention, the average density of 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 the tube to form a positive control. The average density is then calculated based on the distribution of the cellular elements within the tube.

[0085] 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.

[0086] 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.

[0087] For the purposes of the invention, the term "cellular element" means a component that comprises at least one human, animal or plant cell and is presented in different forms such as isolated cells, cellular microtissues, cell clusters, encapsulated cells, encapsulated microtissues, cell clusters or not and mixtures thereof. The microtissues and / or clusters, encapsulated or not, may contain one or more lumens. For the purposes of the invention, the term "modified forms of these molecules" means 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 comprises hydroxyl, carboxyl, and N-acetyl functional groups capable of serving as a base for attaching molecular groups.Hydroxyl and carboxyl groups are preferred sites for the attachment of lipids, polymers or other active molecules to the hyaluronic acid monomer. In addition, amine groups can also be added to the N-acetyl functional group. This allows the creation of hyaluronic acid esters, hyaluronic acid dialdehydes, N-deacetylated hyaluronic acids or Cys-hyaluronic acids. Similarly, Dextran, also made of monomer, has hydroxyl functional groups accessible to a wide variety of addition reactions (hydroxyl group chemistry).

[0088] 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.

[0089] 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.

[0090] For the purposes of the invention, the term "microcompartment" or "capsule" also means a partially or totally enclosed three-dimensional structure containing one or more cellular elements. The structure preferably consists of a rigidified hydrogel outer layer and a hollow inner part comprising at least one cellular element and possibly other elements such as an extracellular matrix or an extracellular matrix substitute.

[0091] 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. For the purposes of the invention, a microtissue exhibits a largest dimension of at least 50 pm, more preferably between 50 pm and 1.3 mm, in particular between 100 pm and 800 pm.

[0092] For the purposes of the invention, “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). 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.

[0093] For the purposes of the invention, the term “active ingredient” means one or more substances responsible for a pharmacological or therapeutic activity.

[0094] For the purposes of the invention, “biological stability” means maintaining the integrity of cellular elements.

[0095] 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.

[0096] By "sedimentation velocity threshold" or "maximum sedimentation velocity" within the meaning of the invention, is meant 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 velocity is expressed in mm / h. By convention, the relative velocity is defined as positive in the case where the cellular elements sediment in the solution, and negative if the latter rise in the solution. The term "maximum" qualifies the absolute value of the velocity 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 velocity is, in absolute value, as low as possible.

[0097] For the purposes of the invention, "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, "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 ÿ = 0.1 s. -1 at ÿ = 1000 s -1 .

[0098] By "resting viscosity" we mean the dynamic viscosity measured at rest, i.e. at a very low shear rate ÿ on the fluid, typically ÿ = 0.1 s -1 .

[0099] 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 "CelITiter-GIo® 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 a reference, a viability of 85% for condition B reflects a 15% decrease in viability compared to condition A.

[0100] Biocompatible composition according to the invention

[0101] 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, and the difference in density between the density of the solution and the average density of the cellular elements verifies the following inequality:

[0102] [Math 1] where | | = p s — p0with p srepresenting the density of the composition expressed in kg / m3, and p0 representing the average density of the cellular elements, measured using the density gradient technique expressed in kg / m3, where D represents the largest dimension of the cellular elements expressed in meters, where v c represents the acceptable sedimentation rate threshold expressed in ms-1; where F = 18. g c / g ~ 9.81 with g c represents the maximum viscosity at rest of the solution expressed in Pa.s, g representing the gravity constant expressed in ms-2; and where v c .= 5.56*10-6 ms-1.

[0103] Surprisingly, the inventors have developed a biocompatible composition that allows heterogeneous cellular elements to be kept in suspension while maintaining their biological stability over a long period. Thus, the rheological characteristics of the solution advantageously allow the homogeneity of distribution of the cellular elements within the composition to be maintained.

[0104] According to one embodiment, the sedimentation rate threshold is less than 2.78*10-6 ms-1, or 10 mm.h-1.

[0105] According to another embodiment, the acceptable sedimentation rate threshold is less than 1.39*10-6 ms-1, or 5 mm.h-1.

[0106] According to another embodiment, the acceptable sedimentation rate threshold is less than 2.78*10-7 ms-1, or 1 mm.h-1.

[0107] 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 between 2.78*10-9 and 5.56*10-6 ms-l, i.e. between 0.01 and 20 mm.h-1.

[0108] Thus, the lower the maximum sedimentation rate of the cellular elements, the more the homogeneity of distribution of the cellular elements suspended in the solution is preserved over time.

[0109] Preferably, the osmolality of the cellular elements in the solution is between 180 and 360 mOsml.kg-1, even more preferably between 220 and 360 mOsml.kg-1. 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.

[0110] Preferably, the composition according to the invention comprises a solution comprising at least one ion chosen from sodium, calcium, magnesium, chlorine and potassium ions and mixtures thereof.

[0111] According to a preferred embodiment, the composition according to the invention comprises a solution comprising sodium, calcium, chlorine and potassium ions. 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.

[0112] 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.

[0113] Advantageously, the pH of the solution is particularly suited to the survival of cellular elements.

[0114] 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.

[0115] 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.

[0116] According to a particular embodiment of the invention, the aqueous solution is the culture medium for the cellular elements.

[0117] 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.

[0118] 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.

[0119] Preferably, the composition according to the invention comprises:

[0120] - between 0.01 and 0.9% by mass of NaCl relative to the total mass of the solution; and / or

[0121] - between 0.01 and 0.04% by mass of CaCI2 relative to the total mass of the solution; and / or

[0122] - between 0.01 and 0.05% by mass of KCI relative to the total mass of the solution.

[0123] 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. 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.

[0124] 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.

[0125] 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.

[0126] 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.

[0127] 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.

[0128] 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.

[0129] Advantageously, in all the embodiments according to the invention, the maximum sedimentation rate of the cellular elements relative to the solution is less than 5.56*10-6 ms-1 or 20 mm. h-1.

[0130] 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.

[0131] According to a preferred embodiment, the composition according to the invention has a viscosity at rest measured at 25°C of between 0.01 and 1.5 Pa.s, preferably of between 0.01 and 1 Pa.s, in particular between 400 and 1200 mPa.s, more preferably 400 and 1000 mPa, even more preferably between 500 and 800 mPa.s, in particular between 500 and 900 mPa.s.

[0132] 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.

[0133] According to a particular variant of the invention, the biocompatible composition has:

[0134] - a density between 997 and 1600 kg / m3;

[0135] - a viscosity at rest, measured using a rheometer at 25°C, between 0.01 and 1.5 Pa.s; and

[0136] - a maximum sedimentation rate of cellular elements relative to the solution of less than 5.56*10-6 ms-1 or 20 mm. h-1.

[0137] 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 2.78*10-7 ms-1 or 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.

[0138] 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.

[0139] The composition according to the invention comprises a solution and heterogeneous cellular elements suspended in said solution. Said cellular elements are chosen from isolated cells, cellular microtissues, clusters of cells and mixtures thereof, of which at least two, preferably a majority, even more preferably all of said cellular elements have a different density and / or size and / or number of cells.

[0140] 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 solid ball 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% pD-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 0.5 and 5% by mass, more preferably the concentration is equal to 2% (plus or minus 0.5%) by mass.

[0141] 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. Preferably, the cells constituting all or part of the cellular elements are chosen from neural cells, neuronal cells, glial cells, cardiac cells, retinal cells, lymphocytes, hepatocytes, endothelial cells, mesenchymal stem cells, keratinocytes, fibroblasts, pre-adipocytes, adipocytes, pancreatic islet cells and mixtures thereof.

[0142] Advantageously, the composition according to the invention is suitable for maintaining all cell types in suspension, the cellular elements being encapsulated or not.

[0143] According to one embodiment, the composition according to the invention comprises a solution and cellular elements suspended in said solution, in which the cellular elements consist of at least 2 different cell types.

[0144] 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.

[0145] According to one embodiment, the composition according to the invention comprises between 10 and 55% of the cellular elements in the form of a microtissue.

[0146] According to one embodiment, the composition according to the invention comprises a solution and cellular elements suspended in said solution, in which at least one cellular element is a microtissue preferably having a largest dimension of at least 50 pm, more preferably between 50 pm and 1.3 mm, in particular between 100 pm and 800 pm.

[0147] Thus, in this embodiment, the biocompatible composition according to the invention comprises at least one microtissue whose largest dimension is at least 50 μm. Unexpectedly, the inventors succeeded in maintaining the cellular elements, in this embodiment including at least one microtissue, in suspension in a composition in liquid form. This characteristic makes it possible to make the composition easily injectable.

[0148] In the context of the invention, the composition according to the invention is in liquid form. More particularly, the composition according to the invention is in liquid form and comprises a solution in liquid form and cellular elements suspended in said solution.

[0149] In addition, it is advantageous that the biocompatible composition is not in gel form.

[0150] 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%, less than 4%, less than 3%, less than 2%, less than 1% of the cellular elements are in the form of isolated cells.

[0151] According to one embodiment, the composition according to the invention comprises a solution and cellular elements suspended in said solution in which no cellular element is in the form of an isolated cell.

[0152] In the context of the invention, the percentage of cellular elements in the form of microtissue or isolated cells is expressed as the cumulative volume of cellular elements.

[0153] 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 0.01 and 60%, in particular between 0.01 and 50%, more preferably between 0.01 and 45%, even more preferably between 15 and 30%.

[0154] 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 pm, even more preferably less than 500 pm.

[0155] 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.

[0156] 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 μm, more preferably less than 500 μm. 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.

[0157] According to one embodiment, the composition according to the invention comprises a solution and cellular elements suspended in said solution, in which the composition comprises between 1 and 100 million cells per mL of solution, preferably between 1 and 50 million cells, in particular between 1 and 10 million cells, more preferably between 2 and 6 million cells, even more preferably between 3 and 5 million cells per mL of solution.

[0158] 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:

[0159] - water;

[0160] - at least one biocompatible density agent; and

[0161] - at least one biocompatible viscosity agent, preferably at least one biocompatible viscosity agent having a molecular weight between 0.05 MDa and 5 MDa.

[0162] Advantageously, the density and viscosity agents make it possible to modulate the rheological characteristics of the solution so that the density of the composition verifies the inequality [math 1],

[0163] 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.

[0164] 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.

[0165] 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 at least one biocompatible density agent chosen from dextran, mannitol, sodium chloride, glycerol or sorbitol, and mixtures thereof, or modified forms of these molecules.

[0166] 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 the cellular elements;

[0167] - their high solubility in the solution;

[0168] - their low impact on the viscosity of the solution.

[0169] Preferably, the composition according to the invention comprises a solution and cellular elements suspended in said solution, in which the solution comprises at least one density agent, preferably dextran. 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.

[0170] 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 at least one biocompatible viscosity agent chosen from PVA, carboxymethyl cellulose (CMC), methylcellulose, hydroxyethylcellulose, ethylhydrocyethylcellulose, hydroxypropylmethylcellulose (HPMC), alginate, pullulan, chitosan, xanthan gum, gellan gum, polyacrilamide, polyvinylpyrrolidone, polyethylene oxide, casein, collagen, sodium hyaluronate and mixtures thereof, or modified forms of these molecules.

[0171] The viscosity agents suitable for the composition according to the invention are selected according to several parameters, namely:

[0172] - their biocompatibility, that is to say that they must not impact the survival of cellular elements;

[0173] - their stability at 37°C, injection temperature in humans.

[0174] Preferably, the composition according to the invention comprises a solution and cellular elements suspended in said solution, in which the solution comprises at least one viscosity agent chosen from sodium hyaluronate and / or CMC. When the solution comprises sodium hyaluronate, the latter preferably has a molecular weight of less than 1 MDa, even more preferably between 80 and 1 MDa, in particular between 80 and 600 kDa.

[0175] When the solution includes CMC, it preferably has a molecular weight between 40kDa and 1MDa, even more preferably between 50 and 400kDa.

[0176] 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. According to another embodiment, the composition according to the invention comprises:

[0177] - between 0.01 and 30% by mass of density agent(s) relative to the total mass of the solution

[0178] - between 0.01 and 8% by mass of viscosity agent(s) relative to the total mass of the solution.

[0179] According to a particularly preferred embodiment, the composition according to the invention comprises a solution and cellular elements suspended in said solution, in which the solution comprises:

[0180] - sodium hyaluronate and / or CMC; and

[0181] - dextran.

[0182] Preferably, the composition according to the invention comprises a solution and cellular elements suspended in said solution, in which the solution comprises:

[0183] - between 10 and 25% by mass of dextran with a molecular weight of 20kDa and 80kDa relative to the total mass of the solution;

[0184] - between 0.01 and 7% by weight of CMC having a molecular weight of between 40 kDa and 1 MDa 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 1 MDa relative to the total mass of the solution

[0185] 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 preparation method comprising the following steps:

[0186] (a) Preparation or recovery of a composition comprising a solution (A) and a set of cellular elements of which at least two cellular elements have a different density and / or size and / or number of cells, preferably at least one cellular element is a microtissue having a largest dimension of at least 50 pm;

[0187] (b) Preparation of a biocompatible composition comprising the cellular elements of the composition of step (a), a solution (B) and optionally the solution (A) such that the difference in density between the density of the solution of the composition resulting from step (b) and the average density of the cellular elements of the composition resulting from step (b) verifies the following inequality:

[0188] [Math 1] where | | = p s — p0with p srepresenting the density of the composition expressed in kg / m3, and p0 representing the average density of the cellular elements, measured using the density gradient technique expressed in kg / m3, where D represents the largest dimension of the cellular elements expressed in meters, where v c represents the threshold of acceptable sedimentation rate expressed in ms-1; where F = 18. p c / g ~ 9.81 with p c represents the maximum viscosity at rest of the solution expressed in Pa.s, g representing the gravity constant expressed in ms-2; and where v c .= 5.56*10-6 ms-1 ;

[0189] (c) Optionally recovery of the composition resulting from step (b), 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, preferably at least one cellular element is a microtissue having a larger dimension of at least 50 pm, in which said composition is characterized in that the difference in density between the density of the solution and the average density of the cellular elements verifies the inequality mentioned above in step (b).

[0190] Preferably, when step (a) concerns the preparation of a composition, it comprises the implementation of the following steps:

[0191] 1) Cultivation of at least one cellular element;

[0192] 2) Rinsing of the culture of cellular elements from step 1);

[0193] 3) Centrifugation of the culture of cellular elements from step 2);

[0194] 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, preferably at least one cellular element is a microtissue having a largest dimension of at least 50 pm.

[0195] According to a preferred embodiment, step (b) comprises implementing the following steps:

[0196] (bl) Characterization of at least one rheological parameter chosen from;

[0197] *the density of the cellular element having the lowest density and the density of the cellular element having the greatest density of said cellular elements present in the composition resulting from step (a);

[0198] *the density of the composition resulting from step (a);

[0199] *the viscosity of the composition resulting from step (a); *the largest dimension of the cellular elements present in the composition resulting from step (a)

[0200] (b2) Preparation of a solution (B) comprising at least one density agent and / or at least one viscosity agent and water;

[0201] (b3) Sterilization of the solution (B) resulting from step (b2) so as to obtain a sterilized solution (B);

[0202] (b4) Optionally, adjusting the pH of the sterilized solution (B) from step (b3);

[0203] (b5) Preparation of a biocompatible composition comprising the cellular elements of the composition resulting from step (a) with the sterilized solution (B) resulting from step (b3) or (b4) and optionally with the solution (A) so that the difference in density between the density of the solution resulting from step (b5) and the average density of the cellular elements of the composition resulting from step (b5) verifies the inequality mentioned above in step (b).

[0204] Preferably, step (b2) of preparing the solution (B) is carried out by adding a biocompatible viscosity agent chosen from PVA, carboxymethyl cellulose (CMC), methylcellulose, hydroxyethylcellulose, ethylhydrocyethylcellulose, hydroxypropylmethylcellulose (HPMC), alginate, pullulan, chitosan, xanthan gum, gellan gum, polyacrilamide, polyvinylpyrrolidone, polyethylene oxide, casein, collagen, sodium hyaluronate and mixtures thereof, or modified forms of these molecules.

[0205] Preferably, step (b2) of preparing the solution (B) is carried out by adding at least one biocompatible density agent chosen from dextran, mannitol, NaCl, glycerol, sorbitol and their mixtures, or modified forms of these molecules.

[0206] Preferably, step (b2) of preparing the solution (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 their mixtures.

[0207] According to one embodiment, step (b) comprises implementing the following steps:

[0208] (bl) Characterization of at least one rheological parameter chosen from;

[0209] *the density of the cellular element having the lowest density and the density of the cellular element having the greatest density of said cellular elements present in the composition resulting from step (a);

[0210] *the density of the composition resulting from step (a);

[0211] *the viscosity of the composition resulting from step (a);

[0212] *the largest dimension of the cellular elements present in the composition resulting from step (a) (b2) Preparation of a solution (B) comprising at least one density agent and / or at least one viscosity agent and water;

[0213] (b4) Optionally, adjustment of the pH of the solution (B) resulting from step (b2);

[0214] (b3) Sterilization of the solution (B) resulting from step (b4) so ​​as to obtain a sterilized solution (B);

[0215] (b5) Preparation of a biocompatible composition comprising the cellular elements of the composition resulting from step (a) with the sterilized solution (B) resulting from step (b3) or (b4) and optionally with the solution (A) so that the difference in density between the density of the solution resulting from step (b5) and the average density of the cellular elements of the composition resulting from step (b5) verifies the inequality mentioned above in step (b).

[0216] In this embodiment, step (b4) of adjusting the pH of the solution (B) is carried out upstream of step (b3) of sterilizing the solution (B). Process for preparing a biocompatible composition according to the invention

[0217] 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:

[0218] (a) Preparation or recovery of a composition comprising a solution (A) and a set of cellular elements of which at least two cellular elements have a different density and / or size and / or number of cells, preferably at least one cellular element is a microtissue having a largest dimension of at least 50 pm;

[0219] (b) Preparation of a biocompatible composition comprising the cellular elements of the composition of step a), a solution (B) and optionally the solution (A) so that the difference in density between the density of the solution of the composition resulting from step (b) and the average density of the cellular elements of the composition resulting from step (b) verifies the following inequality:

[0220] [Math 1]

[0221] V \ < F-2where | | = p s — p0with p s representing the density of the composition expressed in kg / m3, and p0 representing the average density of the cellular elements, measured using the density gradient technique expressed in kg / m3, where D represents the largest dimension of the cellular elements expressed in meters, where v c represents the threshold of acceptable sedimentation rate expressed in ms-1; Tl where F = 18. g c / g ~ 9.81 with g crepresents the maximum viscosity at rest of the solution expressed in Pa.s, g representing the gravity constant expressed in ms-2; and where v c .= 5.56*10-6 ms-1 ;

[0222] (c) Optionally recovery of the composition resulting from step (b), 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, preferably at least one cellular element is a microtissue having a larger dimension of at least 50 pm, in which said composition is characterized in that the difference in density between the density of the solution and the average density of the cellular elements verifies the inequality mentioned above in step (b).

[0223] Advantageously, the method according to the invention makes it possible to achieve a balance between density and viscosity as a function of the heterogeneous cellular elements so that the maximum sedimentation rate of the cellular elements relative to the solution is less than 5.56*10-6 ms-1, or 20 mm. h-1.

[0224] According to one embodiment, the composition of step (a) comprises a solution in which said cellular elements are suspended.

[0225] 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 5.56*10-6 ms-1, or 20 mm. h-1.

[0226] Preferably, when step (a) concerns the preparation of a composition, it comprises the implementation of the following steps:

[0227] 1) Cultivation of at least one cellular element;

[0228] 2) Rinsing of the culture of cellular elements from step 1);

[0229] 3) Centrifugation of the culture of cellular elements from step 2);

[0230] 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, preferably at least one cellular element is a microtissue having a larger dimension of at least 50 pm.

[0231] According to one embodiment, the cell culture of step 1) is carried out within a bioreactor.

[0232] 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. Preferably, at least one cellular element of step 1) is contained in a three-dimensional microcompartment comprising an external hydrogel layer.

[0233] 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.

[0234] 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.

[0235] According to one embodiment, the composition of step (a) comprises at least two distinct cell types.

[0236] Preferably, step (b) comprises the implementation of the following steps:

[0237] (bl) Characterization of at least one rheological parameter chosen from;

[0238] *the density of the cellular element having the lowest density and the density of the cellular element having the greatest density of said cellular elements present in the composition resulting from step (a);

[0239] *the density of the composition resulting from step (a);

[0240] *the viscosity of the composition resulting from step (a);

[0241] *the largest dimension of the cellular elements present in the composition resulting from step (a)

[0242] (b2) Preparation of a solution (B) comprising at least one density agent and / or at least one viscosity agent and water;

[0243] (b3) Sterilization of the solution (B) resulting from step (b2) so as to obtain a sterilized solution (B);

[0244] (b4) Optionally, adjusting the pH of the sterilized solution (B) from step (b3);

[0245] (b5) Preparation of a biocompatible composition comprising the cellular elements of the composition resulting from step (a) with the sterilized solution (B) resulting from step (b3) or (b4) and optionally with the solution (A) so that the difference in density between the density of the solution resulting from step (b5) and the average density of the cellular elements of the composition resulting from step (b5) verifies the following inequality:

[0246] [Math 1] V \ < F.2where | | = p s — p0with p s representing the density of the composition expressed in kg / m3, and p0 representing the average density of the cellular elements, measured using the density gradient technique expressed in kg / m3, where D represents the largest dimension of the cellular elements expressed in meters, where v c represents the threshold of acceptable sedimentation rate expressed in ms-1; where F = 18. p c / g ~ 9.81 with p crepresents the maximum viscosity at rest of the solution expressed in Pa.s, g representing the gravity constant expressed in ms-2; and where v c .= 5.56*10-6 ms-1.

[0247] Preferably, step (b2) of preparing the solution (B) is carried out by adding a biocompatible viscosity agent chosen from PVA, carboxymethyl cellulose (CMC), methylcellulose, hydroxyethylcellulose, ethylhydrocyethylcellulose, hydroxypropylmethylcellulose (HPMC), alginate, pullulan, chitosan, xanthan gum, gellan gum, polyacrilamide, polyvinylpyrrolidone, polyethylene oxide, casein, collagen, sodium hyaluronate and mixtures thereof, or modified forms of these molecules.

[0248] Preferably, step (b2) of preparing the solution (B) is carried out by adding at least one biocompatible density agent chosen from dextran, mannitol, NaCl, glycerol, sorbitol and their mixtures, or modified forms of these molecules.

[0249] Preferably, step (b2) of preparing the solution (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 their mixtures.

[0250] Advantageously, the 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 at least one viscosity agent.

[0251] According to a particular embodiment, the solution (B) of step (b2) comprises:

[0252] - between 10 and 20% by mass relative to the total mass of the dextran solution preferably having a molecular weight of 20kDa and 80KDa;

[0253] - between 3 and 6% by mass of CMC preferably having a molecular weight of between 40 and 1 MDa relative to the total mass of the solution and / or between 0.3 and 10% by weight of sodium hyaluronate with a molecular weight of between 80 and 800 kDa.

[0254] According to a preferred embodiment, the solution (B) of step (b2) is produced with stirring, preferably using a paddle mixer or magnetic bar. Preferably, the sterilization of step (b3) is carried out by filtration through meshes of size between 0.01 and 0.22 μm.

[0255] According to one embodiment, step (b4) 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 with the sterilized solution (B) of step (b3) so that the sterilized solution (B) has a pH of between 7 and 7.6.

[0256] According to one embodiment, step (b5) of preparing a composition comprising the cellular elements of the composition resulting from step (a) with the sterilized solution (B) resulting from step (b3) or (b4) comprises a step of extracting the cellular elements from the composition resulting from step (A). In other words, according to this embodiment, step (b5) comprises an extraction step aimed at isolating the cellular elements from the solution (A) present in the composition resulting from step (a).

[0257] Thus, according to one embodiment, the method for preparing a composition according to the invention comprises the implementation of the following steps:

[0258] (a) Preparation or recovery of a composition comprising a solution (A) and a set of cellular elements of which at least two cellular elements have a different density and / or size and / or number of cells, preferably at least one cellular element is a microtissue having a largest dimension of at least 50 pm;

[0259] (bl) Characterization of at least one rheological parameter chosen from;

[0260] *the density of the cellular element having the lowest density and the density of the cellular element having the greatest density of said cellular elements present in the composition resulting from step (a);

[0261] *the density of the composition resulting from step (a);

[0262] *the viscosity of the composition resulting from step (a);

[0263] *the largest dimension of the cellular elements present in the composition resulting from step (a)

[0264] (b2) Preparation of a solution (B) comprising at least one density agent and / or at least one viscosity agent and water;

[0265] (b3) Sterilization of the solution (B) resulting from step (b2) so as to obtain a sterilized solution (B);

[0266] (b4) Optionally, adjusting the pH of the sterilized solution (B) from step (b3); (b5) Preparing a biocompatible composition comprising the cellular elements of the composition resulting from step (a) with the sterilized solution (B) resulting from step (b3) or (b4) and optionally with the solution (A) so that the difference in density between the density of the solution resulting from step (b5) and the average density of the cellular elements of the composition resulting from step (b5) verifies the following inequality:

[0267] [Math 1]

[0268] V \ < F-2where | | = p s — p0with p s representing the density of the composition expressed in kg / m3, and p0 representing the average density of the cellular elements, measured using the density gradient technique expressed in kg / m3, where D represents the largest dimension of the cellular elements expressed in meters, where v crepresents the threshold of acceptable sedimentation rate expressed in ms-1; where F = 18. p c / g ~ 9.81 with p c represents the maximum viscosity at rest of the solution expressed in Pa.s, g representing the gravity constant expressed in ms-2; and where v c .= 5.56*10-6 ms-1

[0269] (c) Optionally recovery of the biocompatible composition resulting from step (b), 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, in which said composition is characterized in that the difference in density between the density of the solution and the average density of the cellular elements verifies the inequality mentioned above in step (b5).

[0270] According to another embodiment, the method for preparing a composition according to the invention comprises the implementation of the following steps:

[0271] (a) Preparation or recovery of a composition comprising a solution (A) and a set of cellular elements of which at least two cellular elements have a different density and / or size and / or number of cells, preferably at least one cellular element is a microtissue having a largest dimension of at least 50 pm, comprising the following steps:

[0272] 1) Cultivation of at least one cellular element;

[0273] 2) Rinsing of the culture of cellular elements from step 1);

[0274] 3) Centrifugation of the culture of cellular elements from step 2);

[0275] 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, preferably at least one cellular element is a microtissue having a larger dimension of at least 50 pm;

[0276] (bl) Characterization of at least one rheological parameter chosen from;

[0277] *the density of the cellular element having the lowest density and the density of the cellular element having the greatest density of said cellular elements present in the composition resulting from step (a);

[0278] *the density of the composition resulting from step (a);

[0279] *the viscosity of the composition resulting from step (a);

[0280] *the largest dimension of the cellular elements present in the composition resulting from step (a)

[0281] (b2) Preparation of a solution (B) comprising at least one density agent and / or at least one viscosity agent and water;

[0282] (b3) Sterilization of the solution (B) resulting from step (b2) so as to obtain a sterilized solution (B);

[0283] (b4) Optionally, adjusting the pH of the sterilized solution (B) from step (b3);

[0284] (b5) Preparation of a biocompatible composition comprising the cellular elements of the composition resulting from step (a) with the sterilized solution (B) resulting from step (b3) or (b4) and optionally with the solution (A) so that the difference in density between the density of the solution resulting from step (b5) and the average density of the cellular elements of the composition resulting from step (b5) verifies the following inequality:

[0285] [Math 1]

[0286] V \ < F-2where | | = p s — p0with p srepresenting the density of the composition expressed in kg / m3, and p0 representing the average density of the cellular elements, measured using the density gradient technique expressed in kg / m3, where D represents the largest dimension of the cellular elements expressed in meters, where v c represents the threshold of acceptable sedimentation rate expressed in ms-1; where F = 18. p c / g ~ 9.81 with p c represents the maximum viscosity at rest of the solution expressed in Pa.s, g representing the gravity constant expressed in ms-2; and where v c .= 5.56*10-6 ms-1

[0287] (c) Optionally recovery of the biocompatible composition resulting from step (b), 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, in which said composition is characterized in that the difference in density between the density of the solution and the average density of the cellular elements verifies the inequality mentioned above in step (b5).

[0288] According to one embodiment, the maximum sedimentation rate of the cellular elements of step (a) in the solution (A) is greater than 5.56*10-6 ms-1, or 20 mm.h-1.

[0289] 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.

[0290] According to one embodiment, step (b) comprises implementing the following steps:

[0291] (bl) Characterization of at least one rheological parameter chosen from;

[0292] *the density of the cellular element having the lowest density and the density of the cellular element having the greatest density of said cellular elements present in the composition resulting from step (a);

[0293] *the density of the composition resulting from step (a);

[0294] *the viscosity of the composition resulting from step (a);

[0295] *the largest dimension of the cellular elements present in the composition resulting from step (a)

[0296] (b2) Preparation of a solution (B) comprising at least one density agent and / or at least one viscosity agent and water;

[0297] (b4) Optionally, adjustment of the pH of the solution (B) resulting from step (b2);

[0298] (b3) Sterilization of the solution (B) resulting from step (b4) so ​​as to obtain a sterilized solution (B);

[0299] (b5) Preparation of a biocompatible composition comprising the cellular elements of the composition resulting from step (a) with the sterilized solution (B) resulting from step (b3) or (b4) and optionally with the solution (A) so that the difference in density between the density of the solution resulting from step (b5) and the average density of the cellular elements of the composition resulting from step (b5) verifies the inequality mentioned above in step (b).

[0300] In this embodiment, step (b4) of adjusting the pH of the solution (B) is carried out upstream of step (b3) of sterilizing the solution (B). om according to the invention its use: According to another aspect, the invention relates to the composition according to the invention for its use as a medicament.

[0301] Advantageously, the biocompatible composition according to the invention has rheological characteristics and a maximum sedimentation rate of the cellular elements particularly suitable for its use in cell therapy.

[0302] Preferably, the composition according to the invention can be used in the treatment of diseases in cell therapy.

[0303] The relative speed of the cellular elements advantageously allows the quantity of cellular elements in a given volume of composition to be controlled.

[0304] According to one embodiment, the composition according to the invention can be used for multiple use. In other words, the composition can be used to carry out several injections while maintaining the viability of the cellular elements, even if the injections are spaced apart by a period of several hours.

[0305] Advantageously, the maximum sedimentation rate of the cellular elements makes it possible to inject, for a constant volume, a quantity of cellular elements presenting a low standard deviation, preferably a standard deviation of less than 20%.

[0306] Preferably, the composition according to the invention can be used as an injectable composition.

[0307] According to one embodiment, the composition according to the invention can be used by injection using an injection means at the injection site.

[0308] Preferably, said injection means is a cannula.

[0309] 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.

[0310] Kit 1:

[0311] According to another aspect, the invention relates to a kit comprising:

[0312] - a biocompatible composition according to any of the embodiments previously described; and

[0313] - an injection method, preferably a cannula.

[0314] 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. According to one embodiment, the kit according to the invention comprises:

[0315] - a cannula

[0316] - 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.

[0317] According to a particular embodiment of the invention, the kit according to the invention comprises a biocompatible composition according to any of the embodiments previously described in solid form.

[0318] Preferably, the kit according to the invention also comprises water.

[0319] 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.

[0320] Kit 2:

[0321] Finally, according to a last aspect, the invention relates to a kit comprising:

[0322] - a biocompatible preparation (1) comprising at least one biocompatible viscosity agent chosen from PVA, carboxymethyl cellulose (CMC), methylcellulose, hydroxyethylcellulose, ethylhydrocyethylcellulose, hydroxypropylmethylcellulose (HPMC), alginate, pullulan, chitosan, xanthan gum, gellan gum, polyacrilamide, polyvinylpyrrolidone, polyethylene oxide, casein, collagen, sodium hyaluronate and mixtures thereof, or modified forms of these molecules, and at least one biocompatible density agent chosen from dextran, mannitol, NaCl, glycerol, sorbitol and mixtures thereof, or modified forms of these molecules.

[0323] - a biocompatible preparation (2) 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, preferably at least one cellular element is a microtissue having a larger dimension of at least 50 μm so that the mixture of the preparation (1) and the preparation (2) forms a composition whose difference in density between the density of the preparation (1) and the average density of the cellular elements of the preparation (2) satisfies the following inequality:

[0324] [Math 1] V \ < F.2where | | = p s — p0with p srepresenting the density of the composition expressed in kg / m3, and p0 representing the average density of the cellular elements, measured using the density gradient technique expressed in kg / m3, where D represents the largest dimension of the cellular elements expressed in meters, where v c represents the threshold of acceptable sedimentation rate expressed in ms-1; where F = 18. p c / g ~ 9.81 with p c represents the maximum viscosity at rest of the solution expressed in Pa.s, g representing the gravity constant expressed in ms-2; and where v c .= 5.56*10-6 ms-1.

[0325] According to a preferred embodiment, the biocompatible preparation (2) comprises a solution.

[0326] Preferably, the kit according to the invention also comprises water.

[0327] According to one embodiment, the kit according to the invention comprises a biocompatible preparation (1) in solid form.

[0328] According to one embodiment, the preparation (1), in solid form, comprises at least one biocompatible viscosity agent chosen from PVA, carboxymethyl cellulose (CMC), methylcellulose, hydroxyethylcellulose, ethylhydrocyethylcellulose, hydroxypropylmethylcellulose (HPMC), alginate, pullulan, chitosan, xanthan gum, gellan gum, polyacrilamide, polyvinylpyrrolidone, polyethylene oxide, casein, collagen, sodium hyaluronate and mixtures thereof, or modified forms of these molecules, and at least one biocompatible density agent chosen from dextran, mannitol, NaCl, glycerol, sorbitol and mixtures thereof, or modified forms of these molecules.

[0329] 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.

[0330] Examples:

[0331] Example 1:

[0332] The objective of this example is to evaluate the sedimentation rate of cellular elements in different compositions, namely a composition comprising cellular elements and a Hank's balanced salt solution (HBSS) (A) (not of 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. After obtaining the cellular elements, these were cryopreserved in vials and stored in nitrogenous liquid. The vials comprising the elements were then thawed with an estimate of 10 million cells.

[0333] In this example, the cellular elements are a mixture of microtissues with a largest dimension of at least 50 pm, isolated cells and aggregates of neuronal cells.

[0334] 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:

[0335] Density of cellular elements: 1056 kg / m3; Largest dimension of cellular elements: 200 pm or 0.0002 m.

[0336] Solution (B) described in Table 1 below was prepared under sterile conditions and characterized in Table 2.

[0337] [Table 1]

[0338] [Table 2] Solution (A) was also prepared under sterile conditions and characterized in Table 3. [Table 3]

[0339] Preparation of the compositions:

[0340] Phase 1: preparation of cellular elements

[0341] The flasks were removed from the liquid nitrogen and placed in a CryoPod at -180°C. The flasks containing the cellular elements were placed in a thermostatically controlled chamber at 37°C and removed when only a small piece of ice remained (=2 min).

[0342] After gentle resuspension of the cells, the contents were transferred to a 25 ml tube containing 9 ml of HBSS (calcium, magnesium, glucose), at room temperature.

[0343] The flasks were rinsed with 1 ml of HBSS (calcium, magnesium, glucose) to collect the maximum amount of cellular elements

[0344] The 25 ml tube was centrifuged at 300 G, 5 minutes, and the maximum of the supernatant was removed with P1000 then P20.

[0345] The cellular elements were resuspended in 1 ml of HBSS (calcium, magnesium, glucose).

[0346] Phase 2: Aliquot the samples

[0347] An undetermined volume and concentration of the cell suspension were aliquoted into two 1.5 ml Eppendorf tubes.

[0348] The 1.5 mL tubes were centrifuged at 300G for 5 min and the maximum of the supernatant was removed with P1000 then P20.

[0349] In one sample, the cellular elements were resuspended in 1 ml of HBSS (solution (A)) and in the other in 1 ml of solution (B).

[0350] Phase 3: Evaluation of the sedimentation rate

[0351] The tubes were installed on the assembled device for taking pictures.

[0352] Pictures were taken every minute for 12 hours, see Figure 1. Using Image J software, manual tracking of the trajectories of the cellular elements, on a time-lapse film recorded with the camera, was performed to assess the sedimentation rate.

[0353] An average of 5 cellular element sedimentation rates in solution (B), over 10 / 12 trajectories, was taken to assess the value of the maximum sedimentation rate. In solution (A), only 3 cellular element rates, over 4 trajectories, were taken to determine the value due to rapid sedimentation and the difficulty in tracking exactly the same cellular element.

[0354] The calculated maximum sedimentation rate (average of 5 maximum rates for solution (B) and average of 3 maximum rates for microtissue solution (A)) is:

[0355] * of approximately 363mm / h in solution (A) outside the invention;

[0356] * 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],

[0357] Table 4 below verifies the inequality [Math 1] for solution (A) and solution (B) with Vc = 5.56*10-6m.sl.

[0358] [Table 4]

[0359] Conclusions:

[0360] 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.

[0361] 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 HBSS (calcium, magnesium, glucose) with a viscosity value at rest of approximately 1 mPa.s and a density of approximately 1.0038 g / cm.3

[0362] Example 2:

[0363] 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.

[0364] To carry out this example, the composition according to the invention comprises the following elements:

[0365] [Table 5]

[0366] The composition was also prepared under sterile conditions and characterized in Table 6.

[0367] [Table 6]

[0368] 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:

[0369] Density of cellular elements: 1065 kg / m3; Largest dimension of cellular elements: 200 pm or 0.0002 m; 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.

[0370] Experimental protocol

[0371] Terms

[0372] 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.

[0373] 1) For each batch, two types of samples are prepared:

[0374] Control: A suspension of cellular elements resuspended in a reference cell culture medium, at a ratio of 20% volume of cellular elements and 80% reference culture medium.

[0375] Composition according to the invention: A suspension of cellular elements resuspended in a solution, at a ratio of 20% volume of cellular elements and 80% solution.

[0376] 2) Once the samples are prepared, they are stored in closed tubes at room temperature for 12 hours.

[0377] 3) The cellular elements are then rinsed in an excess of reference culture medium.

[0378] 4) The cellular elements are then put back into culture for 24 hours in a reference culture medium.

[0379] 5) Viability is measured. The result is expressed by normalizing the viability value of the immersion condition in the solution by the viability obtained in the control condition.

[0380] Results

[0381] The results of this example are described in Figure 2. This experiment shows an average decrease in viability of around 25% after a 12-hour immersion in the solution considered, compared to a 12-hour control in a reference culture medium. In the context of the example, this decrease in viability is acceptable given the acceptance criterion of 70% relative viability.

[0382] Example 3: Evaluation of the impact of the procedure for injecting the composition according to the invention on the viability of cellular elements.

[0383] 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.

[0384] In the context of the invention, it is important to verify that the viscosity of the solution, necessary to maintain the homogeneity of distribution of the cellular elements, does not induce excessive shear forces during injection, which could degrade the viability of the cells.

[0385] 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.

[0386] Since the shearing effects experienced during injection can have a delayed effect on cell viability, the viability of cellular elements having undergone an injection procedure is measured immediately after injection, then after 24 hours of re-culture in a reference culture medium.

[0387] 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.

[0388] The injection protocol comprises the following steps: a) Preparation of the composition according to the invention according to the method according to the invention; b) Filling a syringe with the composition according to the invention; c) Injecting the composition according to the invention using the syringe from step b) into a cannula with an injection flow 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) Rinsing the composition according to the invention and resuspending the cellular elements in a culture medium; f) Putting the culture medium comprising the cellular elements in an incubator for 24 hours.

[0389] 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.

[0390] Results :

[0391] The viability measurements described in Figure 3 do not show any significant impact of the injection procedure on viability under the conditions of the example. The slight 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.

[0392] Therefore, the composition according to the invention can be used as an injectable composition.

[0393]

[0394] Example 3: Characterization of the viscoelastic properties of a composition according to the invention via a rheological measurement in oscillation scanning

[0395] To carry out this example, the composition according to the invention comprises a solution characterized in Table 7 as well as cellular elements. [Table?]

[0396] The cellular elements of the composition used in this example are a mixture of microtissues having a largest dimension of at least 50 pm, isolated cells and aggregates of neuronal cells. Finally, certain physicochemical parameters of the composition according to the invention have been characterized in Table 8 below.

[0397] [Table 8]

[0398] The behavior of the composition according to the invention was verified by conventional rheological measurements in oscillation scanning with an Anton Paar MCR 302 rheometer. The angular frequency is constant and fixed at 10 rad / s. The shear strain varies from 0.001 to 10% and the temperature is constant and regulated at 20°C.

[0399] Two parameters were measured to characterize the viscoelastic properties of the composition: the storage modulus (G') and the loss modulus (G''): -The storage modulus (G') represents the elastic part of the deformation. It is linked to the capacity of the material to store energy during deformation and is proportional to the rigidity of the material. It therefore determines the importance of its elastic response and is therefore associated with an elastic solid behavior, therefore a gel.

[0400] -The Loss Modulus (G'') represents the viscous part of the deformation. It is related to the dissipation of energy in the form of heat and is proportional to the damping behavior of the material. It determines the importance of its viscous response and is therefore associated with fluid behavior.

[0401] In the case of a rigid or structured material, the storage modulus (G') will be greater than the loss modulus (G''). Conversely, in the case of a liquid material, the loss modulus (G'') will be greater than the storage modulus (G').

[0402] The results presented in Figure 4 showed that G 1 ' was constant according to the different shear deformations at constant temperature and always lower than G" reflecting a liquid behavior of the composition.

[0403] Example 4: Study of the viability of cellular elements in a solution comprising CMC.

[0404] The objective of this example is to evaluate the viability of cellular elements after 12 hours of immersion in a solution containing CMC.

[0405] The different solutions used in this example are described in Table 9 below.

[0406] [Table 9]

[0407] To carry out this example, the M4 solution was mixed with cellular elements with a controlled cellular elements / solution volume ratio so as to form a composition according to the invention.

[0408] In this example, the cellular elements are a mixture of microtissues having a largest dimension of at least 50 pm, isolated cells and aggregates of neural cells. The composition according to the invention was stored at room temperature for 12 hours.

[0409] The cellular elements are then rinsed in an excess of reference culture medium. The microtissues are then recovered and viability is measured by a cell viability bioluminometric assay. The result is expressed as luminescence signals renormalized by the CM control signal. To assess cell viability, 7 analytical replicates were performed for each condition.

[0410] The results of this example show a maintenance of the viability of the composition according to the invention comprising the M4 solution and the cellular elements (96% compared to a 12-hour control in a reference culture medium). In conclusion, example 4 demonstrates that the composition according to the invention, comprising the M4 solution and cellular elements, makes it possible to maintain high cell viability after 12 hours of immersion at room temperature.

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, 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 composition being characterized in that the difference in density between the density of the solution and the average density of the cellular elements satisfies the following inequality: \Ap\ < F. v c / D 2 where | | = p s — p0with p s representing the density of the composition expressed in kg / m 3 , and p0 representing the average density of cellular elements, measured using the density gradient technique expressed in kg / m 3, where D represents the largest dimension of the cellular elements expressed in meters, where v c represents the acceptable sedimentation rate threshold expressed in ms -1 ; where F = 18. p c / g ~ 9.81 with p c representing the maximum viscosity at rest of the solution expressed in Pa.s, g representing the gravitational constant expressed in ms -2 ; and where v c .= 5.56*10-6 ms -1 .

2. Composition according to the preceding claim, characterized in that at least one cellular element is a microtissue having a largest dimension of at least 50 pm. [Claim s] Composition according to one of the preceding claims, characterized in that the osmolality of the cellular elements in the solution is between 180 and 360 mOsml .kg -1 .

4. Composition according to one of the preceding claims, characterized by a density of between 800 and 2000 kg / m 3 . [Claim s] Composition according to one of the preceding claims, characterized by a viscosity at rest measured at 25°C, between 0.01 and 1.5 Pa.s.

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

7. Composition according to one of the preceding claims, characterized in that the solution comprises at least one element chosen from sodium chloride (NaCl), calcium chloride (CaCl2), potassium chloride (KCl), magnesium chloride (MgCl2) and mixtures thereof. [Claim s] Composition according to one of the preceding claims, characterized in that it 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 CaCI2 relative to the total mass of the solution; and / or - Between 0.01 and 0.05% by mass of KCI relative to the total mass of the solution.

9. 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.

10. Composition according to one of the preceding claims, characterized in that the cells constituting the cellular elements are mature cells and / or progenitors and / or stem cells.

11. 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, cardiac cells, retinal cells, lymphocytes, hepatocytes, endothelial cells, mesenchymal stem cells, keratinocytes, fibroblasts, pre-adipocytes, adipocytes, pancreatic islet cells and mixtures thereof.

12. Composition according to one of the preceding claims, characterized in that the volume fraction of the cellular elements is between 0.01% and 60% by volume.

13. Composition according to one of the preceding claims, characterized in that the solution comprises: - water, - at least one biocompatible density agent - at least one biocompatible viscosity agent, preferably with a molecular weight between 0.05 MDa - 5 MDa.

14. Composition according to the preceding claim, characterized in that the solution also comprises at least one biocompatible osmolality adjusting agent.

15. Composition according to the preceding claim, characterized in that the at least one density agent is chosen from dextran, mannitol, NaCl, glycerol or sorbitol and their mixtures, or modified forms of these molecules.

16. Composition according to one of claims 13 to 15, characterized in that the biocompatible viscosity agent is chosen from PVA, carboxymethyl cellulose (CMC), methylcellulose, hydroxyethylcellulose, ethylhydrocyethylcellulose, hydroxypropyl methylcellulose (HPMC), alginate, pullulan, chitosan, xanthan gum, gellan gum, polyacrilamide, polyvinylpyrrolidone, polyethylene oxide, casein, collagen, sodium hyaluronate and mixtures thereof, or modified forms of these molecules.

17. Composition according to one of claims 13 to 16, characterized in that it comprises: - between 0.01 and 30% by mass of density agent(s) relative to the total mass of the solution - between 0.01 and 8% by mass of viscosity agent relative to the total mass of the solution.

18. Composition according to one of the preceding claims, characterized in that the composition is obtained by implementing the following steps: (a) Preparation or recovery of a biocompatible composition comprising a solution (A) and 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 biocompatible composition comprising the cellular elements of the composition of step a) and a solution (B) and optionally the solution (A) so that the difference in density between the density of the solution and the average density of the cellular elements of the composition resulting from step (b) verifies the following inequality: \Ap\ < F. v c / D 2 where | | = p s — p0with p s representing the density of the composition expressed in kg / m 3, and p0 representing the average density of cellular elements, measured using the density gradient technique expressed in kg / m 3 , where D represents the largest dimension of the cellular elements expressed in meters, where v c represents the acceptable sedimentation rate threshold expressed in ms -1 ; where F = 18. p c / g ~ 9.81 with p c representing the maximum viscosity at rest of the solution expressed in Pa.s, g representing the gravitational constant expressed in ms -2 ; and where v c .= 5.56*10-6 ms -1 . (c) Optionally recovery of the biocompatible composition resulting from step (b), 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, in which the difference in density between the density of the solution and the average density of the cellular elements verifies the inequality mentioned above in step (b).

19. Composition according to one of the preceding claims for its use in the treatment of diseases in cell therapy.

20. Composition for use according to the preceding claim, by injection using a cannula at the injection site.

21. Process for preparing a 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 solution (A) and 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 biocompatible composition comprising the cellular elements of the composition of step a) and a solution (B) and optionally the solution (A) so that the difference in density between the density of the solution and the average density of the cellular elements of the composition resulting from step (b) verifies the following inequality: \Ap\ < F. v c / D 2 where | | = p s — p0with p s representing the density of the composition expressed in kg / m 3 , and p0 representing the average density of cellular elements, measured using the density gradient technique expressed in kg / m 3, where D represents the largest dimension of the cellular elements expressed in meters, where v c represents the acceptable sedimentation rate threshold expressed in ms -1 ; where F = 18. g c / g ~ 9.81 with g c representing the maximum viscosity at rest of the solution expressed in Pa.s, g representing the gravitational constant expressed in ms -2 ; and where v c .= 5.56*10-6 ms -1 . (c) Optionally, recovery of the biocompatible composition resulting from step (b), 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, in which the difference in density between the density of the solution and the average density of the cellular elements verifies the inequality mentioned above in step (b).

22. Method according to the preceding claim, characterized in that step (a) 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.

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

24. Method according to one of claims 21 to 23, characterized in that the composition of step (a) has a maximum sedimentation rate of the cellular elements relative to the solution (A) greater than 5.56*10-6 m / s, or 20 mm / h.

25. Method according to one of claims 21 to 24, characterized in that step (b) comprises the implementation of the following steps: (bl) Characterization of at least one rheological parameter chosen from; *the density of the cellular element having the lowest density and the density of the cellular element having the greatest density of said cellular elements present in the composition resulting from step (a); *the density of the composition resulting from step (a); *the viscosity of the composition resulting from step (a); * the largest dimension of the cellular elements present in the composition resulting from step (a) (b2) Preparation of a solution (B) comprising at least one density agent and / or at least one viscosity agent and water; (b3) Sterilization of the solution (B) resulting from step (b2) so as to obtain a sterilized solution (B); (b4) Optionally, adjustment of the pH of the sterilized solution (B) from step (b3); (b5) Preparation of a composition comprising the cellular elements of the composition resulting from step (a) with the sterilized solution (B) resulting from step (b3) or (b4) and optionally with the solution (A) so that the difference in density between the density of the solution resulting from step (b5) and the average density of the cellular elements of the composition resulting from step (b5) verifies the following inequality: V \ < p.2where | | = p s — p0with p s representing the density of the composition expressed in kg / m 3, and p0 representing the average density of cellular elements, measured using the density gradient technique expressed in kg / m 3 , where D represents the largest dimension of the cellular elements expressed in meters, where v c represents the acceptable sedimentation rate threshold expressed in ms -1 ; where F = 18.p c / g ~ 9.81 with p c represents the maximum viscosity at rest of the solution expressed in Pa.s, g representing the gravity constant expressed in ms -2 ; and where v c .= 5.56*10' 6 ms -1 -

26. Method according to the preceding claim, characterized in that step (b2) of preparing a solution (B) is carried out by adding at least one biocompatible density agent chosen from dextran, mannitol, NaCl, glycerol, sorbitol and their mixtures, or modified forms of these molecules.

27. Method according to one of claims 25 or 26, characterized in that step (b2) of preparing a solution (B) is carried out by adding a biocompatible viscosity agent chosen from PVA, carboxymethyl cellulose (CMC), methylcellulose, hydroxyethylcellulose, ethylhydrocyethylcellulose, hydroxypropyl methylcellulose (HPMC), alginate, pullulan, chitosan, xanthan gum, gellan gum, polyacrilamide, polyvinylpyrrolidone, polyethylene oxide, casein, collagen, sodium hyaluronate and their mixtures, or modified forms of these molecules.

28. Preparation process according to one of claims 25 to 27, characterized in that step (b2) of preparing a solution (B) also comprises the addition of at least one element chosen from sodium chloride (NaCl), calcium chloride (CaCl 2), potassium chloride (KCl), magnesium chloride (MgCl2) and their mixtures.

29. Preparation process according to one of claims 25 to 28, characterized in that step (b2) is carried out with stirring.

30. Kit comprising: - a biocompatible composition according to one of claims 1 to 20; and - an injection method, preferably a cannula.

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

32. Kit comprising: - A biocompatible preparation (1) comprising at least one biocompatible viscosity agent chosen from PVA, carboxymethyl cellulose (CMC), methylcellulose, hydroxyethyl cellulose, ethylhydrocyethylcellulose, hydroxypropyl methylcellulose (HPMC), alginate, pullulan, chitosan, xanthan gum, gellan gum, polyacrilamide, polyvinylpyrrolidone, polyethylene oxide, casein, collagen, sodium hyaluronate and mixtures thereof, or modified forms of these molecules and at least one biocompatible density agent chosen from dextran, mannitol, NaCl, glycerol, sorbitol and mixtures thereof or modified forms of these molecules; - A biocompatible preparation (2) 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; characterized in that the mixture of the preparation (1) and the preparation (2) forms a composition whose difference in density between the density of the preparation (1) and the average density of the cellular elements of the preparation (2) satisfies the following inequality: where | | = p s — p0with p s representing the density of the composition expressed in kg / m 3 , and p0 representing the average density of the elements cellular, measured using the density gradient technique expressed in kg / m 3, where D represents the largest dimension of the cellular elements expressed in meters, where v c represents the acceptable sedimentation rate threshold expressed in ms -1 ; where F = 18. / z c / g ~ 9.81 with g c represents the maximum viscosity at rest of the solution expressed in Pa.s, g representing the gravity constant expressed in ms -2 ; and where v c .= 5.56*10' 6 ms -1 -

33. Kit according to the preceding claim, characterized in that the preparation (2) comprises a solution.

34. Kit according to one of claims 30 to 33, characterized in that it comprises water.

35. Kit according to one of claims 30 to 34, characterized in that it comprises at least one element chosen from sodium chloride (NaCl), calcium chloride (CaCl2), potassium chloride (KCl), magnesium chloride (MgCl2) and mixtures thereof.

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