SPECIFIC LIVER MICRO-TISSUES COMPRISING AT LEAST THREE DIFFERENT PHENOTYPES OF HEPATOCYTES AND USES THEREOF - Patent application

Abstract

The present invention relates to specific liver microtissues comprising hepatocytes of at least three different phenotypes derived from induced pluripotent stem cells encapsulated in a single three-dimensional closed microcompartment. The present invention also relates to methods for preparing such liver microtissues and their use in the treatment or prevention of liver failure.

Description

[Technical field]

[0001] The present invention relates to the treatment of liver failure by using liver microtissues obtained from specific cellular microcompartments.The present invention particularly relates to specific liver microtissues comprising at least three different phenotypes of liver cells obtained from induced pluripotent stem cells encapsulated in a single three-dimensional closed microcompartment. [Background technology]

[0002] The liver is one of the most complex organs in the human body. As an integral part of the digestive system, it is constantly supplied with nutritious or toxic substances produced by digestion. The processing of these substances by the liver is essential for the organism and serves the following purposes: -Storage and distribution of nutrients produced by digestion -Decomposition of toxic substances -Synthesis of most blood proteins, as well as - Bile production has.

[0003] To perform these functions, the liver is composed of a wide variety of cells, including hepatocytes, bile duct cells (cholangiocytes), stellate cells (Ito cells), Kupffer cells, mesenchymal stem cells, and endothelial cells.

[0004] Hepatocytes are the most representative liver cells and are responsible for most of the liver functions, from fatty acid synthesis to urea production and plasma protein synthesis.

[0005] Biliary epithelial cells are polarized epithelial cells that form the walls of the bile ducts. These cells are responsible for regulating the secretion of bile and for collecting bile from the hepatocytes for transport to the intestine.

[0006] Other cells provide good vascularization as well as signaling functions and interactions with other liver cells and cells of the immune system.

[0007] When the liver is no longer able to perform its function, it is called liver failure. The main causes of liver failure are viral infection, drug overdose, immune disorder, genetic disease, or blood circulation disorder. When the damage to liver function is irreversible, the recommended treatment is liver transplantation.

[0008] Liver transplantation is therefore the standard treatment for patients with end-stage liver disease.

[0009] Today, it is very difficult to find organ donors who can provide livers of sufficient quality for transplantation.

[0010] Like any organ transplant, liver transplants can only be performed if the liver is of good quality, to avoid the risks associated with transplants, such as infection, cancer, long-term immunosuppression, and major surgery.

[0011] Today, only two-thirds of patients benefit from a transplant, and then only if their quality of life has deteriorated significantly. In addition, the expected cost of a liver transplant is approximately 1 million euros (or $1 million) in the United States.

[0012] Faced with these problems, several innovative solutions have emerged.

[0013] Transplantation of isolated hepatocytes has emerged as an attractive approach, but clinical trials have been few and inconclusive. This is limited by the poor survival, integration, and proliferation of isolated hepatocytes after transplantation in vivo, resulting in multiple factors limiting short-term and long-term therapeutic efficacy. In fact, hepatocytes isolated from donors have very limited proliferation capacity both in vivo and in vitro. Moreover, isolated hepatocytes placed in culture tend to enter a process of dedifferentiation, thus reducing the possibility of obtaining a sufficient number of mature hepatocytes.

[0014] Although this technique makes it possible to treat a large number of patients, the dose administered is very often insufficient and there is a risk of cells leaking into the systemic circulation during infusion.

[0015] This is mainly due to the difficulty of integrating single cells into the liver, as well as the poor quality caused by the preparation of primary cells and their culture in vitro, and the rejection of a portion of liver cells despite immunosuppressive conditions.

[0016] As a result, the low number of hepatocyte donors and the limited stability and functionality of these hepatocytes represent barriers to their use.

[0017] However, the development of differentiation induction protocols for pluripotent stem cells, namely embryonic stem cells and induced pluripotent stem cells, has made it possible to obtain a nearly inexhaustible source of hepatocytes.

[0018] Although promising, pluripotent cell-derived hepatocytes are extremely difficult to culture at scale and incur prohibitive production costs: for example, the projected cost of generating an autologous liver graft from induced pluripotent stem cells is approximately $9.7 million.

[0019] To date, differentiation protocols have failed to generate a range of functional cell phenotypes, particularly fully mature hepatocytes, which retain characteristics of fetal liver hepatocytes, in particular the persistent expression of α-fetoprotein and low production of albumin.

[0020] Nevertheless, certain protocols can obtain liver cells with better functional characteristics. These protocols are still complex and require steps of dissociation, reaggregation, or co-culture, especially with mesenchymal stem cells and endothelial cells. These additional steps add additional risks, increase the total cost, and make it difficult to control the final product.

[0021] For cell therapy applications, existing methods require that they are compatible with the following objectives: i) achieve production in a limited number of steps to be effective on a large scale, and ii) improve the integrity of the transplanted cells. No technology developed today is capable of producing liver microtissues derived from induced pluripotent stem cells that are suitable for large-scale culture and yield functional hepatocytes.

[0022] Current methods for generating liver microtissues remain too complicated, involve multiple steps, and are therefore very costly and difficult to scale up.

[0023] Therefore, to meet the essential demand for liver grafts, there is a strong need for a solution that allows for the large-scale production of liver microtissues containing several phenotypes of liver cells that can be produced on a large scale and directly transplanted.

[0024] Therefore, it is an object of the present invention to fulfill all of these needs and overcome the disadvantages and limitations of the prior art. Summary of the Invention

[0025] To this end, the present invention proposes specific liver microtissues suitable for use in cell therapy, in particular in the fight against liver failure.

[0026] To this end, the present invention relates to a three-dimensional liver microtissue comprising liver cells of at least three different phenotypes, all of said cells of the microtissue being derived from induced pluripotent stem cells encapsulated in a single three-dimensional closed microcompartment.

[0027] Advantageously, the liver microtissues have sufficient cellular diversity to restore and / or improve liver function in a sustained manner.

[0028] Preferably, the liver microtissue comprises at least immature hepatocytes, mature hepatocytes and bile duct epithelial cells.

[0029] According to a preferred object of the present invention, the liver microtissue comprises at least: - liver cells, at least 40% of which express cytokeratin 19 (CK19); - cells expressing CD73 and CD90; Includes.

[0030] Preferably, the cells expressing CD73 and CD90 are mesenchymal stem cells, and the cells expressing CK19 are bile duct epithelial cells.

[0031] Advantageously, the phenotypic composition of the liver microtissue approaches that of healthy human liver.

[0032] Preferentially, the liver microtissue according to the invention comprises: - at least one lumen, - at least one cell of the microtissue in contact with both the lumen and the medium outside the microtissue, and at least one cell that is surrounded only by cells, Includes.

[0033] Advantageously, the organization of the liver microtissues is close to that of liver tissue at the developmental stage, making it possible in particular to promote the integration of the liver microtissues into the liver and the proper execution of metabolic functions in the treated liver.

[0034] According to particularly preferred embodiments, the liver microtissue is ovoid, cylindrical, spheroidal or spherical, or substantially ovoid, cylindrical, spheroidal or spherical or elliptical in shape. Preferably, the liver microtissue is elliptical in shape.

[0035] Advantageously, the elliptical shape of the microtissues makes it possible to promote the survival of the liver microtissues, so that a larger portion of the liver microtissues is integrated by the treated liver.

[0036] Preferably, a liver microtissue according to the present invention comprises at least one bile duct and / or at least one glycogen granule.

[0037] According to a preferred object of the present invention, the liver microtissue is - 50-99% liver cells, of which 20-60% express cytokeratin 19; - 1-20% of cells expressing CD73 and CD90; Includes.

[0038] According to one variation, the liver microtissue is a three-dimensional liver microtissue having a greatest dimension of 500-700 μm and / or expressing CYP3A4 monooxygenase with an activity of at least 75,000 RLU per million cells and / or producing at least 18 μg of urea per million cells per 24 hours.

[0039] Advantageously, the activity of CYP3A4 associated with urea production can ensure a liver microtissue that contains at least functional liver cells.

[0040] Preferably, the liver cells secrete at least 75 μg of albumin per million cells per 24 hours.

[0041] Advantageously, liver microtissues exhibit metabolic activity similar to that of healthy liver.

[0042] The subject of the present invention is also a set of several three-dimensional liver microtissues in a culture medium, at least one of which is a liver microtissue according to the present invention.Preferably, at least 50% (based on number) of the liver microtissues of the set of liver microtissues is a liver microtissue according to the present invention.

[0043] According to another aspect, the present invention relates to a three-dimensional closed cellular microcompartment comprising an outer hydrogel layer defining an interior portion, said interior portion comprising at least one liver microtissue according to the present invention.

[0044] The microcompartments according to the invention make it possible to guarantee a suitable microenvironment for the culture of pluripotent stem cells and their differentiation into the cells that constitute the microtissues according to the invention. Indeed, such microcompartments make it possible to reproduce the in vivo conditions of the cellular microenvironment during liver organogenesis.

[0045] The present invention also relates to a set of cellular microcompartments according to the invention.

[0046] According to another aspect, the present invention relates to a method for preparing a microcompartment or a set of microcompartments, comprising performing at least the following steps: generating a microcompartment comprising induced pluripotent stem cells, inducing cell differentiation within the microcompartments to obtain liver cells of at least three different phenotypes.

[0047] Finally, the present invention relates to liver microtissues according to the invention or microcompartments containing them, or to a set of liver microtissues according to the invention or a set of microcompartments containing them, for use as a medicament, preferably for use in the prevention or treatment of liver failure caused by diseases such as liver fibrosis and cirrhosis, fatty liver, non-alcoholic fatty liver, hepatitis, metabolic diseases of the liver, diseases associated with the secretion of factor VIII, alpha-1 antitrypsin, factor IX and / or VWF, Wilson's disease, and hereditary hemochromatosis.

[0048] Other features and advantages will become apparent from the detailed description of the invention and the examples that follow. [Brief description of the drawings]

[0049] [Figure 1a]Comparative expression of genes of interest (EOMES, CXCR4, HHEX, PROX1, AFP, ASGR1) during differentiation via the differentiation protocol. [Figure 1b] Comparative expression of genes of interest (SOX17, FOXA2, TBX, HNF4A, ALB, KT18) during differentiation via the differentiation protocol. [Figure 1c] Comparative expression of genes of interest (GATA4, HNF1B, SOX9, KT19, TAT) during differentiation via the differentiation protocol. [Diagram 2] The expression of genes of interest (EOMES, CXCR4, FOXA2, SOX17) is compared according to culture conditions. [Diagram 3] The expression of the proteins of interest (SOX17, FOXA2) is compared according to culture conditions 5 days after the initiation of differentiation. [Figure 4a] The expression of the genes of interest (PROX1, TBX, AFP, KT18, KT19, HNF4A) is compared according to the culture conditions. [Figure 4b] The expression of the genes of interest (HNF1B, SOX9, ASGR1, ALB, TAT) is compared according to the culture conditions. [Diagram 5] The growth coefficients over a 30-day period are compared according to the culture conditions. [Figure 6] FIG. 1 is a graph depicting albumin secretion during differentiation as a function of culture conditions. [Figure 7] FIG. 13 is a graph showing urea production during differentiation as a function of culture conditions. [Figure 8] 1 is a graph showing CYP3A4 activity depending on culture conditions. [Figure 9] 1 is a series of images obtained by confocal microscopy of microcompartments bearing hepatocytes. [Figure 10a] A series of confocal microscopy images of microcompartments from days 0 to 9 after the start of differentiation. [Figure 10b] A series of confocal microscopy images of microcompartments from days 12 to 30 after the start of differentiation. [Figure 10c]FIG. 1 is a series of images obtained by confocal microscopy of microcompartments 15 days after the start of differentiation. [Figure 10d] FIG. 1 is a series of images obtained by confocal microscopy of microcompartments 20 days after the start of differentiation. [Figure 10e] FIG. 1 is a series of images obtained by confocal microscopy of microcompartments 30 days after the start of differentiation. [Figure 11] 1 is a series of images obtained by confocal microscopy of microsections identifying specific liver cell phenotypes. [Figure 12a] A series of images obtained by confocal microscopy of single cell grafts (recipients 1 and 2, top row) and microtissue grafts (recipients 4 and 5, bottom row) two days after transplantation into Balb / c mice. [Figure 12b] A series of confocal microscopy images of a single cell graft (recipient 3, top row) 6 days after transplantation into a -Balb / c mouse, and a microtissue graft (recipient 6, bottom row) 6 days after transplantation into a -Balb / c mouse. [Figure 12c] 1 is a series of images obtained by confocal microscopy of tissues in Balb / c mice at the site of injection of vehicle alone (basal medium containing 50% Matrigel) without grafts. [Figure 12d] 1 is a series of images obtained by confocal microscopy of microtissues according to the present invention prior to implantation. [Figure 13a] Graph showing A1AT (alpha 1 antitrypsin) secretion before implantation of 2D differentiated single cells (2D) and microtissues according to the invention (present invention). [Figure 13b] FIG. 1 is a graph showing detection of A1T (alpha 1 antitrypsin) in serum of Balb / c mice before (day 0), 2 and 6 days after transplantation of 2D differentiated single cells (SC), microtissues according to the invention (MT) and vehicle only (control). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0050] definition For purposes of the present invention, "alginate" refers to a linear polysaccharide formed from β-D-mannuronate and α-L-guluronate, their salts, and derivatives.

[0051] For the purposes of the present invention, "hydrogel capsule" or "hydrogel microcompartment" refers to a three-dimensional structure formed from a matrix of polymer chains swollen with a liquid, preferentially water.

[0052] For the purposes of the present invention, "human cells" means human cells or immunologically humanized non-human mammalian cells. Even if this is not specified, the cells, stem cells, progenitor cells, and tissues according to the present invention consist of or are derived from human cells or immunologically humanized non-human mammalian cells.

[0053] For the purposes of the present invention, "embryonic stem cells" refers to pluripotent stem cells derived from the inner cell mass of a blastocyst. The pluripotency of embryonic stem cells can be assessed by the presence of markers such as transcription factors OCT4, NANOG, and SOX2, and surface markers such as SSEA3 / 4, Tra-1-60, and Tra-1-81. Embryonic stem cells used in the context of the present invention can be obtained without destroying the embryo from which they are derived, for example, using the techniques described in Chang et al. (Cell Stem Cell, 2008, 2(2)): 113-117). Alternatively, human embryonic stem cells can be excluded.

[0054] Within the meaning of the present invention, "cells surrounded only by cells" in a microtissue means cells that are not in contact with either the lumen or the outside of the microtissue.

[0055] For purposes of the present invention, the term "mutated cell" refers to a cell that has at least one mutation.

[0056] For the purposes of the present invention, "pluripotent stem cells" or "pluripotent cells" refers to cells that have the potential to form all tissues present in the whole organism of origin, but are unable to form the whole organism itself. Human pluripotent stem cells may be referred to as hPSCs in this application. These may in particular be induced pluripotent stem cells (iPSCs (induced pluripotent stem cells) or, in the case of human induced pluripotent stem cells, hiPSCs (human induced pluripotent stem cells)), embryonic stem cells, or MUSE cells ("multilineage differentiation stress resistant").

[0057] For the purposes of the present invention, "induced pluripotent stem cells" refers to pluripotent stem cells induced to become pluripotent by genetic reprogramming of differentiated somatic cells. These cells are positive for pluripotency markers, such as, inter alia, staining with alkaline phosphatase and expression of the proteins NANOG, SOX2, OCT4, and SSEA3 / 4. Examples of methods for obtaining induced pluripotent stem cells are described in the articles by 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).

[0058] Within the meaning of the present invention, "progenitor cells" refers to stem cells that are already committed to differentiating into liver cells but have not yet differentiated.

[0059] According to the invention, the "Ferret diameter" of a microcompartment (or a part of a microcompartment) or a microtissue according to the invention means the distance "d" between two tangents to said microcompartment (or a part of said microcompartment) or microtissue, said two tangents being parallel, so that the entire projection of said microcompartment (or a part of said microtissue) lies between said two parallel tangents. The Feret diameter of an internal part of a microcompartment is measured between the two interfaces of the internal part of the microcompartment with the external layer, i.e. the distance "d" between two tangents to said internal part, said two tangents being parallel, so that the entire projection of said internal part lies between said two parallel tangents.

[0060] Within the meaning of the present invention, "variable thickness" of a layer refers to the fact that the same microcompartment or layer of the same microtissue does not have the same thickness everywhere.

[0061] For the purposes of the present invention, "microcompartment" or "capsule" means a partially or completely enclosed three-dimensional structure that contains several cells.

[0062] Within the meaning of the present invention, "microtissue" or "liver microtissue" refers to a three-dimensional human tissue that contains at least liver cells and has a greatest dimension of less than 1 mm.

[0063] Within the meaning of the present invention, "medium" means an aqueous solution containing cells or microtissues suitable for the survival, development and / or metabolism of the cells. It may be a culture medium.

[0064] For purposes of the present invention, "convective culture medium" means a culture medium that is agitated by internal movement.

[0065] For the purposes of the present invention, the term "mutation" refers to a genetic or epigenetic mutation, preferentially a functional mutation, which may in particular involve a point modification of the gene sequence, a structural variant, an epigenetic modification, or a modification of the mitochondrial DNA.

[0066] The term "functional mutation" within the meaning of the present invention refers to a transmissible genetic or epigenetic modification that results in a potential gain or loss of function or a potential loss of function for the relevant mutant cells. This preferably involves a mutation that causes a modification of the phenotype of the relevant mutant cells. Very preferentially, this is a change in the genomic and / or epigenomic sequence that alters the therapeutic potential of the cell population by increasing the risk associated with the generated therapy or decreasing the benefit that the generated therapy brings.

[0067] According to the present invention, the "smallest dimension" of a microcompartment or cell layer means the value of the smallest Feret's diameter of said microcompartment.

[0068] For purposes of the present invention, "lumen" refers to the volume of aqueous solution topologically surrounded by a cell, the contents of which are preferentially not in diffusion equilibrium with the volume of convective liquid present outside the microcompartment.

[0069] According to the invention, the "minimum radius" of an inner portion of a microcompartment means half the value of the minimum Feret's diameter of the inner portion of the microcompartment.

[0070] According to the invention, the "average radius" of the interior portion of a microcompartment means the average of the radii of the smallest compartments, each radius corresponding to half the value of the Feret's diameter of the interior portion of the microcompartment.

[0071] According to the present invention, "proliferation rate at day X" refers to the measurement of cell proliferation at time t = X. The proliferation rate is measured by taking the ratio of the number of cells counted at day X of culture divided by the number of cells at the start of culture (day of encapsulation or day of placement in culture medium).

[0072] According to the present invention, "large-scale culture" means a cell culture method suitable for production batches of liver microtissues that allow the treatment of at least one patient, preferably 10 patients, more preferably 100 patients, and even more preferably more than 1,000 patients.

[0073] According to the present invention, the "functional phenotype" of the microtissue refers to the presence of mature hepatocytes characterized by the expression of albumin and the absence of alpha-fetoprotein and cytokeratin 19 expression.

[0074] According to the present invention, "liver bud" refers to a cellular organization characterized by cellular outgrowths of the endoderm of the embryonic foregut that give rise to the liver and bile duct parenchyma, a specific conformation that gives rise to hepatocytes and bile duct epithelial cells.

[0075] Liver microtissue Thus, the present invention relates to a three-dimensional liver microtissue comprising liver cells of at least three different phenotypes, wherein all cells of the microtissue are entirely derived from induced pluripotent stem cells encapsulated in a single three-dimensional closed microcompartment.

[0076] Preferably, the liver microtissue comprises liver cells of at least four different phenotypes, and even more preferably, liver cells of at least five different phenotypes.

[0077] Advantageously, the liver microtissue according to the invention has a great cellular diversity that allows it to reproduce the liver microenvironment, the diversity and the close proximity to the cells present in the liver microtissue allowing a large number of cellular interactions, which in turn cooperate in the execution of many metabolic and transport functions.

[0078] The cellular diversity found in the liver microtissues according to the present invention is derived from induced pluripotent stem cells in a single three-dimensional closed microcompartment, in that different cell types can be organized within a microcompartment that recapitulates the liver microenvironment.

[0079] According to a preferred embodiment, all cells of the microtissue are all derived by differentiation of induced pluripotent stem cells encapsulated in a single three-dimensional closed microcompartment, said induced pluripotent stem cells preferably all of the same lineage.

[0080] Highly preferably, all cells of the microtissue are derived from induced pluripotent stem cells encapsulated in a single three-dimensional closed microcompartment, all by a single differentiation method carried out within said microcompartment.

[0081] Preferentially, the induced pluripotent stem cells before differentiation in the microtissue according to the invention form cysts within the microcompartment.Thus, preferably, all cells of the microtissue are obtained from at least one cyst of induced pluripotent stem cells enclosed in a single three-dimensional closed microcompartment, preferably by differentiation, in particular by a single differentiation method carried out within said microcompartment.

[0082] According to one variant, the liver microtissue according to the invention may be obtained from stem cells, progenitor cells and / or cells capable of differentiating into liver cells.

[0083] Advantageously, the microenvironment of the microcompartments in which the microtissues according to the invention are obtained reproduces the conditions of liver organogenesis: indeed, the microcompartments according to the invention make it possible to limit various physical and / or stress constraints and to promote interactions between the various cell types within the microcompartments.

[0084] According to a particularly preferred embodiment, the liver microtissue according to the invention comprises at least immature hepatocytes, mature hepatocytes and bile duct epithelial cells. Preferentially, it comprises at least: - immature hepatocytes characterized by the expression of alpha-fetoprotein (AFP) and albumin (ALB) and the absence of expression of cytokeratin 19 (CK19), - mature hepatocytes characterized by the expression of albumin and the absence of expression of alpha-fetoprotein and cytokeratin 19, biliary epithelial cells characterized by the expression of cytokeratin 19 and the absence of expression of albumin and alpha-fetoprotein, Includes.

[0085] According to one variant, the liver microtissue according to the invention comprises hepatoblasts, which can be characterized by the expression of alpha-fetoprotein, albumin and cytokeratin 19.

[0086] The presence of these liver cell phenotypes ensures pleiotropic effects on liver function, which can restore or optimize specific liver functions.

[0087] In certain embodiments, the liver microtissue comprises at least 50% mature and / or immature hepatocytes.

[0088] According to a particularly preferred embodiment, the liver microtissue comprises 20-60% (based on number) of liver cells that express cytokeratin 19.

[0089] Cells that express cytokeratin 19 are preferentially biliary epithelial cells.

[0090] In the context of the present invention, liver cells are preferably selected from mature hepatocytes, immature hepatocytes, hepatoblasts, biliary epithelial cells and mixtures thereof.

[0091] The liver microtissue may also contain cells expressing CD73 and CD90. Cells expressing CD73 and CD90 are preferentially mesenchymal stem cells.

[0092] Mesenchymal stem cells are a cell population well known for their properties in tissue repair and regeneration, especially in the liver.Advantageously, the presence of mesenchymal stem cells derived from induced pluripotent stem cells in the treated patient can ensure the effectiveness of tissue repair in livers that exhibit liver failure.

[0093] Thus, in certain embodiments, the microtissue according to the invention comprises at least: - hepatic lineage cells, preferentially at least immature hepatocytes, mature hepatocytes and bile duct epithelial cells; - cells expressing CD73 and CD90, preferentially at least mesenchymal stem cells, Includes.

[0094] Preferably, the liver microtissue comprises at least: - liver cells, of which 20% to 60% (by number) of the cells express cytokeratin 19; - cells expressing CD73 and CD90; Includes.

[0095] Liver microtissues are preferentially - 50-99% of liver cells, of which preferentially 20-60% (by number) of cells are cells expressing cytokeratin 19; - 1-20% of cells expressing CD73 and CD90; Includes (percentage by number).

[0096] The liver microtissue according to the invention may also contain other cells, such as, inter alia, Ito cells (stellate cells), Kupffer cells, endothelial cells, hepatoblasts. Thus, in another particular embodiment, the liver microtissue comprises: -Mature hepatocytes, immature hepatocytes, hepatoblasts, biliary epithelial cells, Ito cells and / or Kupffer cells and / or endothelial cells, and optionally mesenchymal stem cells, Includes.

[0097] According to one variant, the liver microtissue according to the invention may also comprise smooth muscle cells and / or fibroblasts.

[0098] The cell phenotype contained in the liver microtissue is preferably compatible with the liver microenvironment.

[0099] The cellular diversity provided by the liver microtissue according to the invention makes it possible to repair and regenerate diseased livers, restoring affected functions.

[0100] The enrichment of mature hepatocytes, biliary epithelial cells and mesenchymal stem cells ensures a sustained effect during cell therapy. It is particularly important to obtain a content of mature and / or immature hepatocytes of more than 50% in culture in order to obtain a sufficient effect as soon as possible after microtissue transplantation.

[0101] If the concentration of mature and / or immature hepatocytes is less than 50%, the efficiency of the graft is limited, and the low concentration (based on number) of hepatocytes requires a larger graft volume.In fact, it is estimated that functional hepatocytes are required at least 5% of the liver mass to treat, for example, acute liver failure, and liver metabolic defects such as diseases related to the secretion of factor VIII, factor IX and VWF, Wilson's disease and hereditary hemochromatosis require a similar amount.Mature hepatocytes express albumin but do not express alpha-fetoprotein.These markers can be easily identified and quantified by detection methods well known to those skilled in the art, such as flow cytometry.

[0102] Particularly preferably, the liver microtissue comprises at least one lumen, at least one cell of the microtissue in contact with both the lumen and the medium outside the microtissue, and at least one cell that is surrounded only by cells.

[0103] In the context of the present invention, such tissue structures are characteristic of functional microtissues that are ready for use in cell therapy.

[0104] The particular organization of the microtissues is possible in particular due to the presence of polarized liver cells which make it possible to structure and organize the microtissues.

[0105] Thus, the microtissues may contain polarized liver cells. Polarization of liver cells in the microcompartments may be an indication of functional microtissue formation.

[0106] The liver microtissues according to the present invention do not comprise human embryonic stem cells.

[0107] The liver microtissue according to the present invention may be in the form of a spheroid.

[0108] Advantageously, the elliptical shape of the microtissue makes it possible to promote the survival and integration of the liver microtissue. When the liver microtissue is injected into the systemic circulation, its elliptical shape makes it possible to facilitate its flow into the blood vessels in case of administration by injection via the vascular route (in some embodiments via the portal vein), but also in the cannula in case of administration by intracellular implantation. Improved injection of the liver microtissue improves the integration of the liver microtissue by the liver of the treated patient.

[0109] The liver microtissues according to the invention preferably have a diameter or smaller dimension of 100-300 μm, preferably 150-280 μm. The maximum dimension of the liver microtissues is preferably less than 1 mm, very preferably 500-700 μm.

[0110] Advantageously, the size of the liver microtissue is compatible with administration via the portal vein.

[0111] A liver microtissue according to the present invention may comprise 300 to 14,000 cells, preferably 500 to 8,000 cells, even more preferably 900 to 5,000 cells, especially 4,500 cells.

[0112] Preferably, the liver microtissue comprises at least one bile duct. The bile duct collects and transports bile produced by liver cells to the gallbladder. The presence of at least one bile duct in the liver microtissue facilitates the microtissue to function properly in the liver and to reconstruct defective bile ducts.

[0113] According to another embodiment, the liver microtissue comprises at least one glycogen granule. Glycogen serves as a storage form of carbohydrate in the body and is mainly stored in the liver. Under the action of insulin, liver cells store glucose in the form of glycogen. Under the action of glucagon, liver cells hydrolyze glycogen and release glucose into the blood. The presence of glycogen granules in liver cells is a physiological element of the metabolic function of liver cells and thus of the function of liver cells. Thus, the presence of at least one glycogen granule in the liver microtissue ensures favorable conditions for the liver microtissue to function in order to obtain optimal therapeutic effects. Preferably, the liver microtissue comprises at least one bile duct and at least one glycogen granule.

[0114] According to one embodiment, the liver microtissue according to the invention is a three-dimensional liver microtissue having a maximum dimension of 500-700 μm. Preferentially, the liver microtissue according to the invention expresses CYP3A4 monooxygenase with an activity of at least 75,000 RLU per million cells and / or produces at least 18 μg urea per million cells per 24 hours.

[0115] Cytochrome P450 is a hemoprotein involved in the oxidative metabolism of many molecules. Cytochrome P450 is an enzyme involved in the biotransformation of exogenous compounds, both in the phenomenon of detoxification and intoxication through the formation of reactive substances. The most abundant human hepatic form (CYP3A4) is responsible for the metabolism of more than 60% of drugs. Its presence in the microcompartments is a guarantee of its functionality. Urea is a nitrogenous product resulting from the catabolism of proteins. It is synthesized exclusively in the liver via the urea cycle, and the amount of urea formed depends on the amount of ingested protein, protein catabolism and the state of liver function. A CYP3A4 activity of at least 75,000 RLU per million cells associated with a production of at least 18 μg of urea per million cells per 24 hours ensures sufficient metabolic activity to guarantee the presence of mature hepatocytes and a significant effect on impaired liver function.

[0116] Preferably, the activity of CYP3A4 is at least 80,000 RLU, and even more preferably at least 100,000 RLU.

[0117] According to a preferred embodiment, the liver microtissue produces at least 40 μg urea per million cells per 24 hours, even more preferably at least 60 μg urea, especially at least 80 μg urea.

[0118] Advantageously, liver microtissues according to the present invention exhibit metabolic activity approaching that of a healthy liver, and are therefore particularly effective in restoring the function of treated livers.

[0119] Preferably, the liver microtissue according to the invention comprises 50-99% liver cells. The liver microtissue comprises liver cells which preferentially secrete at least 75 μg albumin per million cells per 24 hours, even more preferentially at least 150 μg albumin, in particular at least 250 μg albumin.

[0120] According to certain embodiments, the liver microtissue comprises liver cells obtained from induced pluripotent stem cells and secreting at least 75 μg of albumin per million cells per 24 hours at least 20 days after initiation of differentiation.

[0121] Albumin is the most abundant protein in blood. Produced by the liver, it is responsible, among other things, for stabilizing blood pressure and transporting many substances.

[0122] Advantageously, a production of at least 75 μg of albumin per million cells per 24 hours is indicative of a properly functioning liver microtissue.

[0123] The liver microtissue according to the present invention can be obtained by differentiation of induced pluripotent stem cells at least 20 days, preferably at least 30 days, after encapsulation of 1 to 200, preferably 5 to 150, in particular 15 to 80 induced pluripotent stem cells in a three-dimensional closed microcompartment.

[0124] Preferably, the liver microtissue is obtainable from the encapsulation of 1-200, preferably 5-150, in particular 15-80 induced pluripotent stem cells in a three-dimensional closed microcompartment.

[0125] Preferably, the induced pluripotent lines form cysts in the microcompartments prior to differentiation into cells of the liver microtissue according to the invention.

[0126] The subject of the present invention is also a set of liver microtissues, which comprises at least one liver microtissue according to the present invention.Preferably, the set of liver microtissues is a set of several three-dimensional liver microtissues in culture medium, of which at least one liver microtissue is the liver microtissue according to the present invention.According to a preferred variant, at least 50% (based on number) of the liver microtissues of the set of liver microtissues is the liver microtissue according to the present invention.

[0127] The set of liver microtissues according to the present invention is adapted to be administered to a patient with liver failure. The administration can be by injection with a biocompatible solution. The injection can be in the portal vein so as to reach the liver without dispersing into the systemic circulation, directly in the liver, or ectopically. If the injection is ectopically, it can be intraperitoneally or under the kidney capsule.

[0128] Preferably, the effective amount of the set of microtissues injected into the patient corresponds to a mass of 1-20%, preferably 2-10%, in particular 5% of the liver mass of the treated patient.

[0129] According to another aspect, the object of the invention is a microcompartment comprising at least one liver microtissue according to the invention.

[0130] The microcompartments according to the invention comprise an external hydrogel layer. Preferentially, the hydrogel used is biocompatible, i.e. non-toxic to the cells. The external hydrogel layer must allow the diffusion of oxygen and nutrients to supply the cells contained within the microcompartments and allow them to survive. According to one embodiment, the external hydrogel layer comprises at least alginate. It may consist exclusively of alginate. The alginate may in particular be sodium alginate composed of 80% α-L-guluronate and 20% β-D-mannuronate, with an average molecular weight of 100 to 400 kDa and a total concentration of 0.5 to 5% by weight. The external hydrogel layer is cell-free.

[0131] The external hydrogel layer makes it possible in particular to protect the cells from the mechanical stresses of the bioreactor and to limit potentially toxic molecules if they accumulate in the culture medium.

[0132] The average thickness of the outer layer may vary. It is preferably between 20 and 60 μm, more preferably between 30 and 40 μm. The ratio of the minimum radius of the inner part to this thickness is preferably between 2 and 10 μm.

[0133] The microcompartments according to the invention advantageously exhibit at least a 15-fold increase in proliferation rate of induced pluripotent stem cells 20 days after the initiation of differentiation.

[0134] Thus, the present invention promotes expansion with high proliferation rates, resulting in shorter culture times to obtain functional microtissues.

[0135] The microcompartments according to the invention can be obtained after encapsulation of induced pluripotent stem cells with or without the addition of an extracellular matrix, natural or synthetic.

[0136] According to one variant, the microcompartments according to the invention comprise in their internal part an extracellular matrix such as Matrigel® and / or Geltrex®, and / or a hydrogel-type matrix of plant or synthetic origin, such as modified alginates, or a copolymer of poly(N-isopropylacrylamide) and poly(ethylene glycol) (PNIPAAm-PEG) of the Mebiol® type.

[0137] According to one variant, the microcompartments according to the invention can be obtained after encapsulation of the induced pluripotent stem cells without the addition of extracellular matrix, which can be peptide or peptidomimetic sequences, mixtures of proteins, extracellular compounds or structural proteins, such as collagens, laminins, entactins, vitronectins, and growth factors or cytokines.

[0138] Within the microcompartments, the induced pluripotent stem cells are differentiated into liver tissue for at least 20 days, preferably at least 30 days.

[0139] Unexpectedly, microcompartments provide a favorable microenvironment for the development of liver microtissues.Indeed, during differentiation in microcompartments, cells organize to form structures similar to liver buds.These liver buds are found during in vivo liver organogenesis, and these particular structures give rise to hepatocytes and bile duct epithelial cells.The presence of structures similar to liver buds during the formation of liver microtissues is an indication of good tissue quality.

[0140] Thus, liver microtissues according to the invention are preferably obtained following the formation of liver bud-like structures in a single three-dimensional closed microcompartment.

[0141] The microcompartments according to the invention make it possible to isolate induced pluripotent stem cells from the mechanical stresses present in a bioreactor, allowing the microtissues to establish and maintain a topology that closely resembles the spatial and structural organization of liver organogenesis present in vivo.

[0142] Unexpectedly, after differentiation in the microcompartments for at least 20 days, and preferentially 30 days, the cells retain their conformation and thus are better able to proliferate while maintaining their functional phenotype, which in turn allows for a reduced number of passages and shorter culture times required to reach the required final number of liver cells.

[0143] The cells present in the microcompartments according to the invention are preferentially obtained after encapsulation of 1 to 200, preferably 5 to 150, in particular 15 to 80 induced pluripotent stem cells in the outer hydrogel layer and after at least two cell division cycles.

[0144] Preferably, the cells present in the microcompartments according to the invention are obtained after at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 28, 30, preferably at least 5, even more preferably at least 6 cell division cycles after encapsulation in the outer hydrogel layer, resulting in up to 8,000 mature hepatocytes per microcompartment. For example, the mature hepatocytes present in the microcompartments are obtained after at least 6 cell division cycles after encapsulation of the cells in the outer hydrogel layer.

[0145] Preferentially, the number of cell divisions for the implementation of the method according to the invention is less than 100, and even more preferentially less than 30.

[0146] Preferably, the microcompartments are obtained after at least 2 passages after encapsulation, more preferably after at least 3, 4 or 5 passages, each passage lasting for example 2 to 10 days, in particular 2 to 4 days.

[0147] Preferably, the microcompartments are obtained after at least one resealing, more preferably after 1 to 14 resealings, in particular after 2 to 7 resealings. Very preferentially, the resealing corresponds to a new passage, each encapsulation cycle corresponding to a passage.

[0148] Preferably, the microcompartments according to the invention are obtained less than 30 days, even more preferably less than 20 days after encapsulation of at least 1, preferably 5, 20 and up to 100 induced pluripotent stem cells in the internal portion defined by the outer hydrogel layer.

[0149] A microcompartment according to the invention may contain 100-14,000 cells, preferably 300-10,000 cells, even more preferably 300-5,000, more particularly at least 50 mature hepatocytes and at least 20 bile duct epithelial cells.

[0150] Advantageously, the microcompartments according to the invention protect the induced pluripotent stem cells from mechanical stresses, thereby making it possible to obtain proliferation rates that are particularly suitable for large-scale culture.

[0151] The microcompartments according to the invention can be of any three-dimensional form, i.e. they can have the shape of any object in space. They can have any form that is suitable for cell encapsulation. Preferably, the microcompartments according to the invention are spherical or elongated or ellipsoidal, or substantially spherical or elongated in shape. The microcompartments according to the invention can have an ovoid, cylindrical, spheroidal, or spherical shape, or substantially this shape.

[0152] The outer layer of the microcompartment, ie the hydrogel layer, gives the microcompartment according to the invention its size and shape.Preferably, the smallest dimension of the microcompartment according to the invention is between 150 μm and 500 μm, preferably between 200 μm and 450 μm.

[0153] Its maximum dimension is preferably greater than 350 μm, more preferably between 350 μm and 600 μm.

[0154] The micro-compartments according to the invention may optionally be frozen for storage. The micro-compartments according to the invention must then preferentially be thawed before use.

[0155] The invention also relates to several microcompartments according to the invention used together.

[0156] The present invention also relates to an assembly of microcompartments or a series of microcompartments comprising at least two three-dimensional cellular microcompartments, characterized in that at least one microcompartment is a microcompartment according to the invention.

[0157] Preferably, the series of microcompartments according to the invention are in a culture medium, in particular in a culture medium that is at least partially convected. Any culture medium suitable for culturing induced pluripotent stem cells can be used, for example the medium "HCM Hepatocyte Culture Medium BulletKit (Lonza)" or "Medium E from William (Thermofisher Scientific)", since the concentration of dissolved salts is compatible with maintaining alginate cross-linking by divalent cations.

[0158] According to a particularly preferred embodiment, the subject of the invention is a series of cell microcompartments as described above in a closed chamber such as a bioreactor, preferentially in a culture medium in a closed chamber such as a bioreactor. Preferentially, the microcompartments are therefore arranged in the culture medium in a closed bioreactor.

[0159] A set or series of microcompartments according to the invention preferably comprises 2 to 10 16 It contains micro compartments.

[0160] Thus, the microcompartments according to the invention are suitable for large-scale culture by providing liver microtissues that exhibit a functional phenotype, in particular with regard to their detoxification capacity (in particular the activity of CYP3A) and secretory capacity (in particular the secretion of albumin), which ensures optimal efficacy in vivo.

[0161] Uses of liver microtissues according to the present invention The liver microtissues or microcompartments according to the invention can be used for any application, in particular as a medicine, in particular in cell therapy in humans.

[0162] An object of the present invention is therefore a liver microtissue according to the invention or a set of liver microtissues comprising at least one liver microtissue according to the invention, or a microcompartment according to the invention or a set of microcompartments comprising at least one microcompartment according to the invention, for use as a medicament, in particular in cell therapy.

[0163] The present invention is also directed to a method of therapeutic treatment, in particular as a cell therapy treatment, which consists of implanting and / or injecting the microtissue according to the invention into a human.

[0164] Preferably, liver microtissues or microcompartments according to the invention may be used for the prevention or treatment of symptoms associated with liver failure, in particular acute liver failure, chronic liver failure, or acute exacerbation of chronic liver failure.

[0165] Preferably, the liver microtissues or microcompartments according to the invention can be used for the prevention or treatment of diseases such as fibrosis, cirrhosis, steatosis, non-alcoholic fatty liver, hepatitis, metabolic diseases of the liver, such as diseases associated with the secretion of factors VIII and IX and VWF, Wilson's disease, and hereditary hemochromatosis, etc. In fact, all these indications can be treated by liver transplantation, and transplantation of a set of microtissues according to the invention that synthesizes liver functions is exactly what is needed to solve this.

[0166] An object of the present invention is therefore a liver microtissue according to the invention or a set of liver microtissues comprising at least one liver microtissue according to the invention, or a microcompartment according to the invention or a set of microcompartments comprising at least one microcompartment according to the invention, for use in the prevention or treatment of symptoms associated with liver failure, in particular acute liver failure, chronic liver failure or acute exacerbation of chronic liver failure, in particular liver diseases such as fibrosis, cirrhosis, steatosis, non-alcoholic fatty liver, hepatitis, metabolic diseases of the liver, such as diseases associated with the secretion of factors VIII and IX and VWF.

[0167] The present invention also relates to a liver microtissue according to the invention or a set of liver microtissues comprising at least one liver microtissue according to the invention, or a microcompartment according to the invention or a set of microcompartments comprising at least one microcompartment according to the invention, for use in molecular assessment or modelling of liver disease.

[0168] Methods for preparing microcompartments and microtissues according to the present invention The present invention also relates to a method for preparing the microcompartments according to the invention.

[0169] The method for preparing a microcompartment or an assembly of microcompartments according to the invention comprises the following steps: -(a) preparing, inside an external hydrogel layer, three-dimensional closed cellular microcompartments comprising induced pluripotent stem cells and, optionally, extracellular matrix elements or natural or synthetic extracellular matrices, -(b) inducing cell differentiation within the cell microcompartments to obtain liver cells of at least three different phenotypes; may include.

[0170] The preparation of the microcompartments in step (a) can be carried out in any culture medium suitable for culturing induced pluripotent stem cells, such as mTeSR™1 or mTeSRPlus medium from Stemcell technologies, StemMACS™ iPS-Brew XF (StemFlex from Miltenyi Biotec or thermofisher Scientific).

[0171] The microcompartments of step (a) may be obtained by encapsulation of between 1 and 150, preferably at least 50, in particular at least 100 induced pluripotent stem cells.

[0172] Preferably, the encapsulation is carried out according to techniques known to those skilled in the art. In fact, any method of producing a cellular microcompartment containing an outer hydrogel layer and cells inside can be used to carry out the preparation method according to the present invention. In particular, it is possible to prepare the microcompartment by adapting the method and microfluidic device described in Alessandri et al., 2016 ("A 3D printed microfluidic device for production of functionalized hydrogel microcapsules for culture and differentiation of human Neuronal Stem cells (hNSC)", Lab on a Chip, 2016, vol. 16, no. 9, pp. 1593-1604) according to the steps described below.

[0173] Preferably, step (a) is carried out by adding extracellular matrix elements or extracellular matrices, natural or synthetic. According to one variant, step (a) is carried out without adding extracellular matrix elements or extracellular matrices, natural or synthetic.

[0174] In the context of the present invention, step (a) is preferably carried out in a device capable of producing hydrogel capsules using a microfluidic chip. For example, the device may comprise syringe pumps for several solutions that are concentrically injected by a microfluidic injector, which allows the formation of a jet that breaks into droplets, which are then collected in a calcium bath. According to a particularly preferred embodiment, the following two or three solutions are loaded into two or three syringe pumps: - hydrogel solutions, e.g. alginate, Optionally, an isotonic intermediate solution, preferentially without divalent cations such as Ca2+, Mg2+, such as a sorbitol solution, to avoid excessively premature crosslinking of the hydrogel in the syringe; A solution comprising induced pluripotent stem cells and a culture medium.

[0175] These three solutions are co-injected concentrically using a microfluidic injector or microfluidic chip, which allows the formation of a jet that breaks into droplets with an outer layer of the hydrogel solution and a core of the solution of step (a) containing the induced pluripotent stem cells; these droplets are collected in a calcium reservoir that crosslinks and / or gels the alginate solution to form a shell.

[0176] To improve the monodispersity of the cell microcompartments, preferentially the hydrogel solution is charged with direct current (1-10 kV). A ring to ground may be optionally placed at a distance of 1 mm to 20 cm from the tip, preferentially 3 mm to 10 cm, even more preferentially 1 cm to 5 cm from the tip, in a plane perpendicular to the axis of the jet emerging from the microfluidic injector (coextrusion tip) to generate an electric field.

[0177] According to the invention, it is necessary to produce capsules whose inner part has an average radius of at least 100 μm or less. To produce capsules with such dimensions using a coextrusion tip (microfluidic injector or microfluidic chip), the invention proposes in particular to modify the flow rate of the coextrusion solution and the final opening of the coextrusion tip. "Flow rate" means the flow rate of each solution that reaches the injector. "Final opening of the coextrusion tip" means the inner opening of the exit channel of the tip.

[0178] the flow rate will preferentially go from a value in the range of 20 to 40 mL / h for each coextrusion solution for standard capsule sizes known from the prior art, to a value in the range of 45 to 150 mL / h in one variant of the invention, preferentially from 45 to 110 mL / h.

[0179] the final opening diameter of the coextrusion tip (microfluidic injector) preferentially goes from values ​​in the range of 50-120 μm for standard capsule sizes known from the prior art to diameter values ​​in the range of 150-300 μm, preferentially to diameter values ​​in the range of 180-240 μm.

[0180] Thus, according to a particular embodiment, the encapsulation in step (a) is carried out using a microfluidic injector, the final opening diameter of which is between 150 and 300 μm, preferably between 180 and 240 μm, and the flow rate of each of the three solutions is comprised in the range of 45 to 150 mL / h, preferably between 45 to 110 mL / h.

[0181] According to one variant, step (a) of preparing the microcompartments can be carried out using stem cells, progenitor stem cells or cells capable of differentiating into hepatic cells.

[0182] The cell differentiation step (b) is preferably carried out for at least 20 days, even more preferably for at least 30 days.

[0183] During differentiation in step (b), the cells organize into tissues resembling liver buds, which are typically characterized by structuring in the form of two cell subpopulations that display two distinct tissues, one epithelial (i.e., apical-basal polarized cell base morphology and tight junctions) and the other mesenchymal (i.e., lacking apical-basal organization and displaying focal adhesions).

[0184] The differentiation of the induced pluripotent stem cells into liver microtissues in step (b) can be carried out by any known differentiation method, such as those described by Raggi et al., Stem Cell Report 2022 or Mallanna and Duncan, Curr Protoc Cell Bil, 2014.

[0185] According to one embodiment, steps (a) and / or (b) are carried out with permanent or sequential agitation, which is important to maintain the homogeneity of the culture environment and to avoid the formation of any diffusion gradients.

[0186] Preferably, step (b) is carried out under hypoxic conditions, more preferably the first 5 days of differentiation are carried out under hypoxic conditions.

[0187] Unexpectedly, differentiation under hypoxic conditions makes it possible to obtain better proliferation rates.

[0188] By carrying out the method according to the invention it is possible to obtain microcompartments which contain at least 100, preferably at least 500, at least 800, at least 1,000, in particular at least 3,000 cells.

[0189] The method according to the invention is preferentially carried out in a closed chamber, such as a closed bioreactor.

[0190] The present invention also provides a method for preparing liver microtissues, comprising the steps of: -(a) preparing, inside an external hydrogel layer, three-dimensional closed cellular microcompartments comprising induced pluripotent stem cells and, optionally, extracellular matrix elements or natural or synthetic extracellular matrices, -(b) inducing cell differentiation within the cell microcompartments to obtain liver cells of at least three different phenotypes; - (c) removing the outer hydrogel layer to recover liver microtissues of ovoid, cylindrical, spheroidal or spherical shape, or substantially ovoid, cylindrical, spheroidal or spherical or elliptical shape, comprising at least 300 cells corresponding to at least three different liver cell phenotypes; The present invention relates to a method comprising:

[0191] Step (c) consists of dissociating the microcompartments to obtain liver microtissues, the removal of the outer hydrogel layer can be carried out in particular by hydrolysis, dissolution, perforation and / or rupture by any means that is biocompatible, i.e. non-toxic to the cells. For example, the removal can be achieved using phosphate buffered saline, divalent ion chelators, enzymes, such as alginate lyase if the hydrogel comprises alginate, and / or laser microdissection.

[0192] Step (c) of removing the outer hydrogel layer allows for the recovery of liver microtissues of ovoid, cylindrical, spheroidal or spherical shape, or substantially ovoid, cylindrical, spheroidal or spherical or elliptical shape, comprising at least 300 cells corresponding to at least three different liver cell phenotypes.

[0193] Preferably, the liver microtissues harvested in step (c) have an oval shape.

[0194] The liver microtissue from step (c) may comprise at least 300, preferably at least 500, especially between 300 and 14,000, even more preferably between 900 and 5,000 cells.

[0195] The liver microtissue from step (c) comprises at least one lumen, at least one cell in contact with both the lumen and the medium outside the microtissue, and at least one cell that is surrounded only by cells.

[0196] Advantageously, the method according to the invention makes it possible to produce liver microtissues according to the invention on a large scale, so as to form a set of liver microtissues capable of forming up to 20% of the liver mass of the treated patient in less than 50 days, preferably less than 40 days, in particular less than 35 days.

[0197] In a preferred variant, the method according to the present invention comprises at least one resealing of liver microtissue after step (c), i.e. at least two encapsulation cycles.Preferably, each encapsulation cycle corresponds to a passage.In this variant of the method (at least one resealing of cells after step (c)), the number of cell divisions in the whole method (for all passages) is at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30 cell division cycles.

[0198] In the method according to the invention there may be several resealings, preferentially between 1 and 100, in particular between 1 and 10 resealings.

[0199] Each repackage may include: - a step consisting of dissociating the microcompartment or a series of microcompartments to obtain liver microtissues; the removal of the outer hydrogel layer can be carried out in particular by hydrolysis, dissolution, perforation and / or rupture by any means that is biocompatible, i.e. non-toxic to the cells. For example, the removal can be achieved using phosphate buffered saline, divalent ion chelators, enzymes, such as alginate lyase if the hydrogel comprises alginate, and / or laser microdissection, and - reencapsulation of all or part of the liver microtissue in a hydrogel capsule, which is a suitable means to increase cell expansion arising from the pluripotent stage and to reduce the risk of mutations.

[0200] Resealing consists of removing the outer hydrogel layer, resuspending the cells, which were preferentially in the form of cysts within the microcompartments, in a partially or completely dissociated manner, and reperforming the steps of the method.

[0201] According to one embodiment, the reencapsulation comprises the following steps: a hydrogel defining an interior portion, the interior portion having a minimum or average radius of at least 300 μm; -(iv) culturing the obtained microcompartments in a culture medium, -(vii) optionally recovering the resulting cell micro-compartments; Includes.

[0202] Compartmentalization into microcompartments allows the elimination of microcompartments that contain more mutated cells than other capsules. Even if the mutated cells grow rapidly, they reach intracapsular confluence and their increase is limited. Compartmentalization also allows not to contaminate the entire cell population and allows the elimination of capsules that contain mutated cells at any time, in particular before the resealing step. This sorting can be done, for example, by in-line analysis or by removing the filled capsules more quickly than the other capsules.

[0203] In one embodiment, at least one of the steps (preferably all steps) is carried out at a temperature suitable for cell survival, comprised in the range of 4-42° C. The temperature during cell growth should preferably be between 32-37° C. to avoid inducing mutations by reducing the performance of repair enzymes. Similarly, the temperature should preferably be low (ideally around 4° C.) to manage stress on the cells in step (c).

[0204] At any point, the method according to the invention may include a step consisting of verifying the phenotype of the cells contained in the microcompartments. This verification can be carried out by identifying the expression by at least a portion of the cells contained in the microcompartments of specific markers of the desired phenotype, selected from the following: - expression of alpha-fetoprotein, albumin and cytokeratin 19, which characterize hepatoblasts; - absence of expression of alpha-fetoprotein and albumin, and of cytokeratin 19, which characterize immature hepatocytes; - the absence of expression of albumin and of alpha-fetoprotein and cytokeratin 19, which characterize mature hepatocytes; the absence of expression of cytokeratins and of albumin and alpha-fetoprotein, which characterize bile duct epithelial cells; - expression of CD31, which characterizes endothelial cells; -Expression of CD90 and CD73, which characterize mesenchymal stem cells.

[0205] The cell microcompartments obtained according to the method of the invention can then be frozen before any use. Freezing is preferentially carried out at a temperature between -190°C and -80°C. Thawing can be carried out by immersing the sealed freezing container (screw ampoule or plastic pouch) in a warm water bath (preferentially at 37°C) so that the cells are thawed very quickly. The microcompartments according to the invention before use may be kept at a temperature higher than 4°C, preferentially between 4°C and 38°C, for a limited time before use.

[0206] The present invention specifically promotes expansion at high proliferation rates, resulting in short culture times and very large numbers of functional lymphocytes. EXAMPLES

[0207] Example 1: Differentiation Protocol Liver microtissues according to the present invention can be obtained from any known differentiation method.

[0208] To demonstrate this, we adapted two protocols known from the prior art: Protocol A described in Raggi et al., Stem cell Reports 2022, and Protocol B, which has been modified from Mallanna and Duncan, Curr Protoc Cell Bil, 2014 and Raggi et al., Stem cell Reports, 2022.

[0209] Materials and Methods iPSCs were cultured on vitronectin-coated T75 flasks and passaged periodically using the dissociation reagent ReLeSR (Stem Cell Technologies). All experiments were performed with iPSCs at passages 20–26.

[0210] Encapsulation of iPSCs was performed in alginate microcapsules of similar size between the two protocols A and B, and in the presence of extracellular matrix.

[0211] Regarding Protocol A: Minimum major axis diameter = 220 μm Maximum diameter of major axis = 550 μm Mean diameter of major axis = 415 μm

[0212] Regarding Protocol B: Minor axis minimum diameter = 200 μm Maximum minor axis diameter = 385 μm Mean minor axis diameter = 295 μm

[0213] First, iPSCs were detached into small groups of 3–5 cells with Accutase, resuspended at a concentration of 0.8 E6 cells / mL in a mix consisting of 50% Matrigel and 50% mTESR1 medium containing 10 μM Rhock inhibitor (Y-27632), and encapsulated by adapting the method and microfluidic device described in Alessandri et al., 2016 ("A 3D printed microfluidic device for production of functionalized hydrogel microcapsules for culture and differentiation of human Neuronal Stem cells (hNSC)", Lab on a Chip, 2016, vol. 16, no. 9, pp. 1593-1604). The encapsulated cells were resuspended in mTESR1 medium containing 10 μM Rhock inhibitor at a ratio of 0.2 mL capsules per 1 mL total medium. Cells were allowed to form cysts within the capsules using a 30 mL ABLE bioreactor (Reprocell) in mTESR1 medium at 37°C, 5% CO2, under stirred conditions with medium changes every 24 h for 96 h.

[0214] Differentiation was initiated 96 hours after encapsulation by shifting to RPMI medium containing 1 mM Ca2+, 1% KnockOut serum replacement (KOSR), insulin-free B27, 100 ng / mL activin A and 3 μM CHIR-99021 for 2 days.

[0215] For the next 3 days, the medium was replaced with RPMI medium containing 1 mM Ca2+, 1% KOSR, insulin-free B27 and 100 ng / mL activin A.

[0216] Then, for the next 5 days, the medium was replaced with RPMI medium containing 1 mM Ca2+, 1% KOSR, insulin-free B27 supplemented with 20 ng / mL BMP-4, 5 ng / mL bFGF, 1 μM A83-01 (TGFβ pathway inhibitor) and 4 μM IWP-2 (Wnt channel inhibitor) for protocol A.

[0217] In protocol B, 1 mM Ca2+, 1% KOSR, insulin-free B27 supplemented with 20 ng / mL BMP-4 and 5 ng / mL bFGF. From day 11 to day 15, medium consisted of RPMI medium containing 1 mM Ca2+, 2% KOSR, insulin-containing B27 supplemented with 20 ng / mL HGF, 3 μM CHIR-99021, 5 ng / mL bFGF and 20 ng / mL BMP-4.

[0218] On day 16, the medium was changed to EGF-free HCM medium (Lonza) supplemented with 20 ng / mL HGF, 3 μM CHIR-99021, 5 ng / mL bFGF, 20 ng / mL BMP-4, 20 ng / mL oncostatin M, 10 μM dexamethasone and 1% KOSR.

[0219] From day 20 onwards for the next 5 days, medium consisted of EGF-containing HCM medium supplemented with 20 ng / mL oncostatin M, 10 μM dexamethasone and 1% KOSR.

[0220] From day 25 onwards, the medium consisted of HCM medium containing EGF, 10 μM dexamethasone and 1% KOSR.

[0221] Gene expression analysis Gene expression analysis was performed by RT-Q-PCR.

[0222] qPCR was performed on a Roche LightCycler® 480 instrument. Expression of each gene was normalized by the expression of the reference genes YWHAZ, NONO and VCP. Data are expressed as 2^delta Ct, where delta Ct = Ct of gene - mean Ct of reference genes.

[0223] The results are shown in Figures 1a, 1b and 1c. These results demonstrate that liver microtissues according to the invention can be obtained by any suitable differentiation method.

[0224] Example 2: Comparison of hepatocyte differentiation in microcompartments according to the invention with differentiation in two dimensions. iPSCs were encapsulated in alginate microcapsules with the following characteristics: Minimum major axis diameter = 450 μm Maximum diameter of major axis = 685 μm Mean diameter of major axis = 575 μm Minor axis minimum diameter = 313 μm Maximum minor axis diameter = 546 μm Mean minor axis diameter = 423 μm

[0225] When encapsulating iPSCs in matrix-free alginate microcapsules with an average diameter of approximately 575 μm, iPSCs were first detached into small groups of 3-5 cells with Accutase, resuspended in mTESR1 medium containing 10 μM Rhock inhibitor (Y-27632) at a concentration of 10 E6 cells / mL, and encapsulated by adapting the method and microfluidic device described in Alessandri et al., 2016 ("A 3D printed microfluidic device for production of functionalized hydrogel microcapsules for culture and differentiation of human Neuronal Stem cells (hNSC)", Lab on a Chip, 2016, vol. 16, no. 9, pp. 1593-1604). The encapsulated cells were resuspended at 0.5 E6 cells / mL in mTESR1 medium containing 10 μM Rhock inhibitor, with the capsule to medium ratio not exceeding 20%. Cells were allowed to aggregate within the capsules for 24 h under stirring conditions at 37°C and 5% CO2 using a 30 mL ABLE bioreactor (Reprocell).

[0226] Differentiation was initiated 24 hours after encapsulation by transfer to RPMI medium containing 1 mM Ca2+, 1% KnockOut Serum Replacement (KOSR), insulin-free B27, 100 ng / mL activin A and 3 μM CHIR-99021 for 2 days.

[0227] For the next 3 days, the medium was replaced with RPMI medium containing 1 mM Ca2+, 1% KOSR, insulin-free B27 and 100 ng / mL activin A.

[0228] The medium was then replaced with RPMI medium containing 1 mM Ca2+, 1% KOSR, insulin-free B27 supplemented with 20 ng / mL BMP-4 and 5 ng / mL bFGF for the next 5 days. From day 11 to day 15, the medium consisted of RPMI medium containing 1 mM Ca2+, 2% KOSR, insulin-containing B27 supplemented with 20 ng / mL HGF, 3 μM CHIR-99021, 5 ng / mL bFGF and 20 ng / mL BMP-4.

[0229] On day 16, the medium was changed to EGF-free HCM medium (Lonza) supplemented with 20 ng / mL HGF, 3 μM CHIR-99021, 5 ng / mL bFGF, 20 ng / mL BMP-4, 20 ng / mL oncostatin M, 10 μM dexamethasone and 1% KOSR.

[0230] From day 20 onwards for the next 5 days, medium consisted of EGF-containing HCM medium supplemented with 20 ng / mL oncostatin M, 10 μM dexamethasone and 1% KOSR.

[0231] From day 25 onwards, the medium consisted of HCM medium containing EGF, 10 μM dexamethasone and 1% KOSR.

[0232] The medium was changed every day from day 1 to day 25, and every other day from day 25 onwards.

[0233] 2D hepatocyte differentiation iPSCs were dissociated into individual cells using Accutase and plated onto Matrigel-coated 6-well plates at a density of 1x105 cells / cm2 in mTESR1 medium containing 10μM Rhock inhibitor. Differentiation was initiated 24 hours after seeding using the same differentiation protocol and medium composition as for the 3D capsules.

[0234] Gene expression analysis Gene expression analysis was performed by RT-Q-PCR.

[0235] qPCR was performed on a Roche LightCycler® 480 instrument. Expression of each gene was normalized by the expression of the reference genes YWHAZ, NONO and VCP. Data are expressed as 2^delta Ct, where delta Ct = Ct of gene - mean Ct of reference genes.

[0236] To verify the induction of endoderm during differentiation, a comparison of gene expression between hepatocytes obtained in 2D and 3D is shown in Figure 2. The expression of EOMES, CXCR4, FOXA2 and SOX17 genes indicates that the differentiation indeed proceeds through a transient stage of endodermal differentiation towards definitive endoderm, a process expected to generate hepatocytes from pluripotent cells.

[0237] Analysis of protein expression of endoderm markers was performed on day 5 and is shown in Figure 3. These results indicate that differentiation proceeds in the order expected by one of skill in the art, i.e., via definitive endoderm towards the liver bud.

[0238] Analysis of gene expression during the differentiation protocol is shown in Figures 4a and 4b. These results indicate that differentiation progresses towards a composition consistent with the goal of the cellular composition of the liver, specifically hepatocytes.

[0239] Proliferation measurements: The proliferation rate is measured by taking the ratio of the number of cells counted on day X of culture divided by the number of cells at the start of culture (day of encapsulation or day of placement in culture medium). The proliferation rate or amplification factor was measured during differentiation. Tracking of the proliferation rate during the differentiation process is shown in Figure 5. At least a 15-fold proliferation rate is observed 20 days after the start of differentiation.

[0240] Assessment of albumin and urea production To evaluate albumin and urea production, conditioned medium was sampled periodically after 24 h (± 2 h) of medium change and stored at -80°C. Levels of albumin and urea secreted into the medium were measured using an ELISA kit for human albumin (Invitrogen) and a Quantichrom urea assay kit (Gentaur), respectively, according to the manufacturer's instructions. The amount of molecules secreted at 24 h was then normalized to the cell number by counting performed after dissociation at each time point.

[0241] The results are shown in Figures 6 and 7. It can be seen that albumin secretion and urea production are greater in the microcompartments according to the invention.

[0242] Measurement of CYP3A4 activity: Cyp3A4 activity was performed using P450-Glo™ CYP3A4 assay (Promega) with luciferin-IPA according to the manufacturer's instructions. Briefly, a sample of 3D capsules containing microtissues was removed from the capsules and the number of cells in a given volume of capsules containing microtissues was determined. Microtissues removed from capsules corresponding to 1E5 cells or 1E5 cells differentiated in 2D were mixed with 50 μl of substrate proluciferin P450-Glo 3 μM in a 96-well plate with round bottom and incubated at 37° C. and 5% CO2 for 3 hours. Five replicates were performed for each condition, and cell culture medium alone and iPSCs were used as negative controls. Subsequently, 25 μl of culture medium from each well was transferred to an opaque white 96-well luminometer plate and mixed with 25 μl of luciferin detection reagent. The plate was incubated at room temperature for 20 min and luminescence was read using a Spectramax i3x microplate reader (Molecular devices) with an integration time of 1 s per well. Net signal was calculated by subtracting background luminescence values ​​from control wells without cells.

[0243] The results are shown in Figure 8. It is observed that the activity of CYP3A4 in cells encapsulated in microcompartments according to the invention is significantly higher.

[0244] Example 3: Morphological examination of microcompartments according to the invention The goal of this example is to characterize the morphology of cells within differentiating microcompartments.

[0245] histology Liver microtissue samples were fixed in AFA fixative for 24 h at room temperature, washed with PBS, and pre-embedded in histogel. After dehydration in successive baths of ethanol, acetone, and xylene, samples were embedded in paraffin and sectioned at 5 μm thickness with a microtome.

[0246] Hematoxylin-Eosin-Saffron (HES) staining After removing the paraffin, the sections were stained with Harris hematoxylin and eosin G. After dehydration, the sections were stained with saffron and mounted with Entellan. The cytoplasm is stained pink, the nucleus is stained blue-purple, and the extracellular matrix is ​​stained yellow-pink.

[0247] PAS (Periodic Acid-Schiff) staining After removing the paraffin, the sections were pretreated with 1% periodic acid and then stained sequentially with Schiff's reagent and Mayer's hematoxylin. Glycogen is stained pink and nuclei are stained blue-purple.

[0248] The images obtained from the staining are shown in Figure 9. The characteristic cuboidal shape of the hepatocytes and the presence of glycogen granules within the microcompartments can be clearly seen.

[0249] Immunofluorescence in 3D microtissues Microtissues were fixed in capsules in a 4% solution of paraformaldehyde in calcium-containing PBS for 1 h at room temperature. After rinsing with Ca2+-free PBS to remove the capsules, they were permeabilized in 1% Triton X-100 for 30-60 min at room temperature. Microtissues were incubated with primary antibody solution for 72 h at 4 °C with agitation. After washing with PBS, they were incubated with a solution of labeled secondary antibody (Alexa Fluor, Life Technologies) and DAPI overnight at 4 °C with agitation and protected from light.

[0250] For structural imaging with phalloidin-DAPI, staining was performed with the capsules still attached (omitting the step of removing the capsules from the permeabilized tissue and washing with PBS containing calcium). Microtissues were incubated with DAPI and phalloidin for 72 h at 4°C with agitation.

[0251] After washing with PBS, cells were mounted on slides with a gap of 0.5 mm. Images were acquired under a confocal microscope (SP5, Leica).

[0252] [Table 1]

[0253] The evolution of the morphology of the microcompartments was carried out over 30 days during differentiation. These results are shown in Figures 10a to 10e. It is possible to observe the formation of structures close to those of liver buds on day 7 and the presence of specific structures such as cellular organelles characteristic of liver microtissues according to the invention. Indeed, it is possible to distinguish at least one lumen, at least one cell in contact with both the lumen and the medium outside the microtissue, and at least one cell surrounded only by cells. In addition to examining the morphology, the inventors were able to characterize the presence of at least three different phenotypes of liver cells in the microcompartments 30 days after the start of differentiation (Figure 11).

[0254] Evaluation of cell survival after transplantation in mice compared to transplantation of microtissues according to the invention and single-cell hepatocytes obtained in 2D The aim of this study is to inoculate immune competent mice with stem cell-derived hepatocytes prepared as a cell suspension or microtissues according to the invention to assess their survival and function in vivo.

[0255] The test conditions are described below.

[0256] animal: The study was performed in 10-week-old female Balb / cByJ (Mus musculus) mice. Seven (7) animals were housed in an animal room. Temperature was controlled at approximately 21-22°C with frequent ventilation and air handling. Humidity was maintained at approximately 50%. Artificial light was maintained for 12 hours per day. Food (pellets) and drinking (tap water) amounts and access were monitored daily. Cages were changed weekly and the environment was enriched to minimize anxiety.

[0257] Injection method: On day 42 of hepatic differentiation, cells generated in 2D (according to Example 2 "2D hepatocyte differentiation") were dissociated into single cells and liver microtissues generated according to the invention (according to Example 1) were removed from the capsule. - Microtissue suspension: microtissues according to the invention obtained according to example 1, suspended in culture medium, were used: approximately 3 x 106 cells per tube were diluted in 170 μL of basal medium. Before inoculation, 170 μL of Matrigel® was added to each tube. To allow for the subcutaneous inoculation of such a volume, i.e. 340 μL, a skin incision was made on the right flank of the mouse to create a subcutaneous pocket, into which the microtissue suspension was then injected using a pipette, after which the wound was closed with surgical adhesive to prevent the suspension from spreading. - Cell suspension: Cells obtained according to the protocol of Example 2 "2D hepatocyte differentiation" suspended in culture medium were used: approximately 3 x 106 cells per tube in 120 μL of basal medium. Before seeding, 120 μL of Matrigel® was added to each tube. A skin incision was made on the right flank of the mouse to create a subcutaneous pocket to allow for the subcutaneous inoculation of a volume of 240 μL, the cell suspension was then injected into the pocket using a pipette, and the wound was then closed with surgical adhesive to prevent the suspension from spreading. - Control solution: 100 μL of basal medium solution (vehicle) was mixed with 100 μL of Matrigel®. This solution was injected subcutaneously using the same method as the microtissue and cell suspensions.

[0258] sampling: Blood samples were taken from all available mice by retro-orbital bleeding for serum preparation (approximately 80 μL): - Before cell inoculation, day 0, n=7 - 2 days after vaccination, day 2, n=7 - 6 days after vaccination, day 6, n=3

[0259] The grafts, including the surrounding skin to maintain structure, were harvested as follows: - 2 days after vaccination, day 2, n=4 Cell suspension n=2 For microtissues, n=2 - 6 days after vaccination, day 6, n=3 n=1 for cell suspension For microtissues, n=1 n=1 for vehicle

[0260] The explants were harvested, fixed, paraffin-embedded, sectioned, and stained with HES (hematoxylin, eosin, and saffron) and specific antibodies against the human cell marker Stem121 (detecting a human cytoplasmic protein), the liver cell markers albumin, and CytK19 (cytokeratin 19).

[0261] Prior to implantation, microtissues were also stained for HES, albumin and CytK19.

[0262] sampling: Images obtained by optical microscopy (Leica DM2000 microscope) are shown in Figures 12a-12d. -12a: * Single cell grafts on the second day after transplantation into Balb / c mice (recipients 1 and 2, top row). * Micrografts 2 days after transplantation into Balb / c mice (recipients 4 and 5, lower panel). -12b: * Single cell grafts 6 days after transplantation into Balb / c mice (recipient 3, top row). * Micrografts 6 days after transplantation into Balb / c mice (recipient 6, lower panel). -12c: Tissue from the site of injection of vehicle alone (basal medium containing 50% Matrigel) without grafts in Balb / c mice. -12d: Microtissue according to the invention before implantation.

[0263] Histological analysis of the grafts 2 days after transplantation revealed the presence of round-shaped microtissues positive for the human cell marker Stem121 in mice transplanted with liver microtissues according to the present invention, confirming that the detected microtissues are of human origin. Some of the cells constituting the microtissues were positive for the hepatocyte marker albumin, and some cells, mainly those at the periphery of the microtissues, were positive for the bile duct epithelial cell marker CytK19. The cellular organization and compartmentalization of cells expressing albumin and CytK19 after in vivo transplantation were similar to those observed in the microtissues before transplantation.

[0264] In mice transplanted with 2D-generated single cells, only rare cells positive for the human cell markers Stem121, albumin and cytokeratin 19 were detected 2 days after transplantation.

[0265] The presence of cells expressing Stem121 and CytK19 was still detectable in mice implanted with microtissues at day 6, whereas no Stem121- or CytK19-positive cells were present in mice implanted with single cells. Finally, no Stem121-, albumin- or cytokeratin 19-positive cells were detected in areas of immune cell infiltration in mice implanted with vehicle alone (control).

[0266] Furthermore, the presence of human A1AT was analyzed in the serum of transplanted Balb / c mice (analysis was performed before transplantation (day 0), and on days 2 and 6 after transplantation). The presence of human A1AT (α1-antitrypsin) was analyzed by ELISA. A1AT secretion by cells and microtissues before transplantation was also analyzed. The results obtained are shown in Figures 13a and 13b. -13a: Graph showing A1AT secretion before implantation of 2D (2D) differentiated single cells and microtissues according to the invention (present invention). -13b: Graph showing A1T detection in serum of Balb / c mice before (day 0), 2 and 6 days after transplantation of 2D differentiated single cells (SC), microtissues according to the invention (MT) and vehicle only (control).

[0267] These results show that liver microtissues according to the invention can secrete higher levels of A1AT than 2D differentiated cells. Consistent with histological observations, the concentration of human A1AT is higher in the serum of mice implanted with microtissues according to the invention than in the serum of mice implanted with single cells. Human A1AT concentration is lower or even undetectable on day 0. It is highest on day 2 after implantation and decreases on day 6, but is still significant and higher than 2D differentiated single cells. These data are consistent with histological results and show that microtissues according to the invention persist over time and are more functional after implantation than 2D differentiated single cells, even in immune-competent mouse models.

[0268] Taken together, these results show that liver microtissues transplanted as 3D objects according to the present invention survive post-transplantation in immunocompetent mice and have a better and longer post-transplant survival time than 2D-generated liver cells transplanted as single cell suspensions.

Claims

1. A three-dimensional liver microtissue comprising liver cells of at least three different phenotypes, characterized in that all cells of the microtissue are obtained from induced pluripotent stem cells encapsulated in a single three-dimensional closed microcompartment.

2. The liver microtissue according to claim 1, characterized by comprising immature hepatocytes, mature hepatocytes, and bile duct epithelial cells.

3. at least, - Immature hepatocytes characterized by the absence of α-fetoprotein and albumin expression and cytokeratin 19 expression, - Mature hepatocytes characterized by the absence of albumin expression and the absence of α-fetoprotein and cytokeratin 19 expression, - Bile duct epithelial cells characterized by the absence of cytokeratin expression and the absence of albumin and α-fetoprotein expression, The liver microtissue according to claim 1, characterized by containing the following:

4. The liver microtissue according to claim 1, characterized in that the liver microtissue includes cells expressing CD73 and CD90, preferably mesenchymal stem cells.

5. The liver microtissue according to claim 1, characterized in that the liver microtissue comprises at least liver cells, and 20 to 60% of the liver cells are cells expressing cytokeratin 19.

6. The liver microtissue according to claim 1, characterized in that all cells of the microtissue are obtained from the differentiation of at least one cyst of induced pluripotent stem cells encapsulated in a single three-dimensional closed microcompartment.

7. - At least one lumen, - At least one cell in contact with both the lumen and the culture medium outside the microtissue, - At least one cell surrounded only by other cells, The liver microtissue according to claim 1, including the liver microtissue described in claim 1.

8. The liver microtissue according to claim 1, characterized by having an oval shape.

9. The liver microtissue according to claim 1, characterized in that the liver cells are polarized.

10. The liver microtissue according to claim 1, characterized in that the liver microtissue comprises liver cells that are 50% to 99% liver cells, of which 20% to 60% are bile duct epithelial cells, and 1% to 20% of the cells express CD73 and CD90.

11. The liver microtissue according to claim 1, characterized by having a diameter or minimum dimension of 100 μm to 300 μm, and / or a maximum dimension of 500 μm to 700 μm.

12. The liver microtissue according to claim 1, characterized by containing 300 to 14,000 cells.

13. The liver microtissue according to claim 1, characterized by comprising at least one bile duct and / or at least one glycogen granule.

14. The liver microtissue according to claim 1, characterized in that the liver cells are selected from mature hepatocytes, immature hepatocytes, hepatoblasts, bile duct epithelial cells, and mixtures thereof.

15. The liver microtissue according to claim 1, which expresses CYP3A4 monooxygenase having an activity of at least 75,000 RLU per million cells and / or produces at least 18 μg of urea per million cells per 24 hours.

16. A three-dimensional closed microcompartment comprising an external hydrogel layer defining an internal portion, wherein the internal portion comprises at least one liver microtissue according to claim 1.

17. The cell microcompartment according to claim 16, characterized in that the microcompartment is obtained 20 days after encapsulation of 1 to 200 induced pluripotent stem cells in the internal portion defined by the external hydrogel layer, and / or exhibits a proliferation rate of at least 15 times 20 days after the initiation of differentiation.

18. The cellular microcompartment according to claim 16, characterized in that the thickness of the outer layer is variable, and is between 20 and 60 μm.

19. The cellular microcompartment according to claim 16, characterized in that the outer layer contains alginate.

20. The cellular microcompartment according to claim 16, characterized in that the internal portion between the outer layer and the liver microtissue contains extracellular matrix elements or natural or synthetic extracellular matrix.

21. The cellular microcompartment according to claim 16, characterized by having a diameter or minimum dimension of 300 μm to 400 μm, and / or a maximum dimension of 400 μm to 600 μm.

22. A microcompartment assembly comprising at least two three-dimensional cellular microcompartments, wherein at least one microcompartment is the microcompartment described in claim 16.

23. The assembly of microcompartments according to claim 22, characterized in that the microcompartments are placed in a culture medium within a bioreactor.

24. A microtissue according to claim 1 or a microcompartment according to claim 16 for use as a pharmaceutical agent.

25. A microtissue according to claim 1 or a microcompartment according to claim 16 for use in the prevention or treatment of symptoms associated with hepatic failure, preferably acute hepatic failure, chronic hepatic failure, or acute exacerbation of chronic hepatic failure.

26. A microtissue according to claim 1 or a microcompartment according to claim 16 for use in the treatment or prevention of metabolic diseases of the liver, hepatic fibrosis and cirrhosis, fatty liver, non-alcoholic fatty liver, hepatitis, diseases related to the secretion of factor VIII and factor IX and VWF, Wilson's disease, and hereditary hemochromatosis.

27. A method for preparing a microcompartment according to claim 16, - a) A step of creating a three-dimensional closed cell microcompartment containing induced pluripotent stem cells and optionally extracellular matrix elements or natural or synthetic extracellular matrix inside an outer hydrogel layer. -b) A step of inducing cell differentiation within the cellular microcompartment to obtain liver cells with at least three different phenotypes, Methods that include...

28. The method according to claim 27, characterized in that the cell differentiation method in step b) lasts for at least 20 days, and / or in step a), 40 to 150 induced pluripotent stem cells are present in the microcompartment.

29. A method for preparing liver microtissue according to claim 1, - To carry out the method for preparing the microcompartments described in claim 16, - Removing the outer layer of the hydrogel and recovering the liver microtissue, A method characterized by including

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