Specific three-dimensional liver microtissue and use in treatment of hepatic failure

Abstract

The present invention relates to a specific liver micro-tissue having a maximum size of between 500 [mu] m and 700 [mu] m, expressing the monooxygenase CYP3A4 with an activity of at least 75,000 RLU per million cells, and producing at least 18 [mu] g urea per million cells within 24 hours. The invention also relates to a method for preparing the liver micro-tissue and application of the liver micro-tissue in treating or preventing liver failure.

Description

Technical Field

[0001] The present invention relates to the treatment of liver failure by using liver microtissues obtained from specific cell microcompartments. In particular, the present invention relates to specific liver microtissues, methods for their preparation and uses. Prior Art

[0002] The liver is one of the most complex organs in the human body. As an integral part of the digestive system, the liver is constantly supplied with nutrients or toxic substances resulting from digestion. The processing of these substances by the liver is essential for the organism and has the following purposes:

[0003] -Stores and distributes nutrients from digestion

[0004] -Degradation of toxic substances

[0005] -Synthesizes most blood proteins, and

[0006] - Produces bile.

[0007] To perform these functions, the liver is composed of various cells such as hepatocytes, bile duct cells (cholangiocytes), stellate cells (Ito cells), Kupffer cells, mesenchymal stem cells, and endothelial cells.

[0008] Hepatocytes are the most representative liver cells and are responsible for most liver functions, from fatty acid synthesis to urea production to plasma protein synthesis.

[0009] Cholangiocytes are polarized epithelial cells that form the walls of the bile ducts. They have a role in regulating bile secretion and collecting bile for transport from the hepatocytes to the intestine.

[0010] Other cells provide fine vascularization as well as signaling functions and provide interactions with other liver cells and with cells of the immune system.

[0011] When the liver can no longer perform its functions, it is called liver failure. The main causes of liver failure are viral infections, drug overdose, immune disorders, genetic diseases or blood circulation disorders. When damage to liver function is irreversible, the recommended treatment is a liver transplant.

[0012] Therefore, liver transplantation represents the standard of care for people with advanced liver disease.

[0013] Today, there is great difficulty in finding organ donors who can provide livers of sufficient quality for transplantation.

[0014] As with any organ transplant, liver transplantation can only be performed if the liver is of good quality to avoid the risks associated with transplantation, such as infection, cancer, long-term immunosuppression, and major surgery.

[0015] Today, only 2 / 3 of patients can benefit from a transplant, and then only when their quality of life has greatly deteriorated. In addition, the expected cost of a liver transplant in the United States is about 1 million euros (or 1 million US dollars).

[0016] Several innovative solutions have emerged to address these problems.

[0017] Transplantation of isolated hepatocytes has emerged as an attractive approach, but clinical trials remain rare and inconclusive, limited by poor survival, integration, and expansion of isolated hepatocytes after in vivo transplantation, which limits both short-term and long-term therapeutic efficacy. In fact, hepatocytes isolated from donors have very limited in vivo and in vitro proliferation capacity. In addition, isolated hepatocytes subjected to culture tend to enter a dedifferentiation process, reducing the chances of obtaining sufficient numbers of mature hepatocytes.

[0018] Although this technology allows the treatment of a large number of patients, the doses administered are often insufficient and present a risk of escape into the systemic circulation during injection of the cells.

[0019] This is mainly due to the difficulty of single cells integrating into the liver, as well as the poor quality induced by the preparation of primary cells and their in vitro culture, and the rejection of some hepatocytes despite immunosuppression.

[0020] Therefore, the low number of hepatocyte donors, the stability and limited functionality of these hepatocytes constitute obstacles to their use.

[0021] However, the development of protocols for directed differentiation of pluripotent stem cells (ie, embryonic stem cells and induced pluripotent stem cells) has made available a virtually inexhaustible source of hepatocytes.

[0022] Despite their promise, hepatocytes derived from pluripotent cells are extremely difficult to grow on a large scale and incur disproportionate production costs. For example, the expected cost of producing an autologous liver transplant from induced pluripotent stem cells is approximately $9.7 million.

[0023] To date, directed differentiation protocols have failed to generate multiple functional cell phenotypes, especially adequate mature hepatocytes; the cells obtained retain features of fetal hepatocytes, in particular with persistent expression of alpha-fetoprotein and low albumin production.

[0024] However, some protocols make it possible to obtain liver cells with better functional properties. These protocols are still complex and require steps of dissociation, reaggregation or co-cultivation with, in particular, mesenchymal stem cells and endothelial cells. These additional steps add additional risks, increase the overall cost and reduce the control of the finished product.

[0025] For application in cell therapy, existing methods must be adaptable in order to: i) obtain production with a limited number of steps in order to be effective on a large scale, ii) improve integration of transplanted cells. Currently developed technologies do not allow the generation of liver microtissues derived from induced pluripotent stem cells that are suitable for large-scale culture and present functional hepatocytes.

[0026] Current methods for generating liver microtissues are still too complex, have multiple steps, are therefore very costly, and difficult to scale up.

[0027] Therefore, there is a strong need for solutions that can generate liver microtissues containing a variety of hepatocyte phenotypes on a large scale.

[0028] Liver microtissues can be produced on a large scale and directly transplanted to meet the important need for liver transplantation.

[0029] It is therefore an object of the present invention to meet all of these needs and to overcome the shortcomings and limitations of the prior art. Summary of the invention

[0030] To achieve this object, the present invention proposes a specific liver microtissue suitable for use in cell therapy and in particular in combating liver failure.

[0031] To this end, the subject of the invention is a three-dimensional liver microtissue having a maximum dimension between 500 μm and 700 μm, expressing CYP3A4 monooxygenase with an activity of at least 75,000 RLU per million cells and producing at least 18 μg urea per million cells per 24 hours.

[0032] Advantageously, the activity of CYP3A4 associated with urea production makes it possible to ensure that the liver microtissue contains at least functional hepatocytes.

[0033] Preferably, the liver cells secrete at least 75 μg of albumin per million cells per 24 hours.

[0034] Therefore, liver microtissues exhibit metabolic activity similar to that of a healthy liver.

[0035] According to another object, the present invention relates to a liver microtissue comprising at least 3 different phenotypes of liver cells, said cells of the microtissue having all been obtained from induced pluripotent stem cells encapsulated in a single three-dimensional closed microcompartment.

[0036] Advantageously, the liver microtissue has sufficient cellular diversity to sustainably restore and / or improve liver function.

[0037] Preferably, the liver microtissue comprises at least immature hepatocytes, mature hepatocytes and bile duct cells.

[0038] According to a preferred purpose of the present invention, the liver microtissue at least comprises:

[0039] - liver cells, wherein at least 40% of the liver cells express cytokeratin 19 (CK19), and

[0040] -Cells expressing CD73 and CD90.

[0041] Preferably, the cells expressing CD73 and CD90 are mesenchymal stem cells, and the cells expressing CK19 are cholangiocytes.

[0042] Advantageously, the phenotypic composition of the liver microtissue is close to that of a healthy human liver.

[0043] Preferably, the liver microtissue according to the present invention comprises:

[0044] - at least one inner cavity,

[0045] - at least one cell of the microtissue in contact with the lumen and the medium outside the microtissue, and

[0046] - At least one cell that is surrounded only by cells.

[0047] Advantageously, the architecture of the liver microtissue is close to that of developing liver tissue, which makes the liver microtissue particularly capable of promoting its integration into the liver and the proper performance of metabolic functions in the treated liver. According to a particularly preferred embodiment, the liver microtissue is oval, cylindrical, spheroid or spherical or substantially oval, cylindrical, spheroid or spherical or ellipsoidal in shape. Preferably, the liver microtissue is ellipsoidal in shape.

[0048] Advantageously, the oval shape of the microtissue makes it possible to promote the survival of the liver microtissue. Thus, most of the liver microtissue is integrated into the treated liver.

[0049] Preferably, the liver microtissue according to the present invention comprises at least one bile duct and / or at least one glycogen granule.

[0050] According to a preferred purpose of the present invention, the liver microtissue comprises:

[0051] -50% to 99% of liver cells, including 20% ​​to 60% of cells expressing CK19, and

[0052] -1% to 20% of cells expressing CD73 and CD90.

[0053] An object of the invention is also a set of several three-dimensional liver microtissues in a medium, wherein at least one liver microtissue is a liver microtissue according to the invention. Preferably, at least 50% (by number) of the liver microtissues in the set are liver microtissues according to the invention.

[0054] According to another aspect, the present invention relates to a three-dimensional closed cell microcompartment comprising an outer hydrogel layer defining an inner part, said inner part comprising at least one liver microtissue according to the present invention.

[0055] The microcompartments according to the invention make it possible to ensure a microenvironment suitable for the culture of pluripotent stem cells and for their differentiation into the cells constituting the microtissue according to the invention. Indeed, such microcompartments make it possible to reproduce the in vivo conditions of the cellular microenvironment during liver organogenesis.

[0056] The invention also relates to a set of cell microcompartments according to the invention.

[0057] According to another aspect, the present invention relates to a method for preparing a microcompartment or a group of microcompartments, the method comprising at least the following steps: generating a microcompartment containing induced pluripotent stem cells, inducing cell differentiation in the microcompartment to obtain at least 3 different phenotypes of liver cells.

[0058] Finally, the present invention relates to a liver microtissue according to the invention or a microcompartment containing the liver microtissue, or a set of liver microtissues according to the invention or a set of microcompartments containing liver microtissues, which are used as a medicament, preferably in the prevention or treatment of liver failure caused by the following diseases, such as liver fibrosis and cirrhosis, steatosis, non-alcoholic steatosis, hepatitis, liver metabolic diseases, diseases related to the secretion of factor VIII, α1 antitrypsin, IX and / or VWF, Wilson's disease and hereditary hemochromatosis.

[0059] Other features and advantages will become apparent from the detailed description and the examples that follow. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Figure 1a is a comparative representation of the expression of genes of interest (EOMES, CXCR4, HHEX, PROX1, AFP, ASGR1) during differentiation according to the differentiation protocol.

[0061] Figure 1b is a comparative representation of the expression of genes of interest (SOX17, FOXA2, TBX, HNF4A, ALB, KT18) during differentiation according to the differentiation protocol.

[0062] Figure 1cis a comparative representation of the expression of genes of interest (GATA4, HNF1B, SOX9, KT19, TAT) during differentiation according to the differentiation protocol.

[0063] Figure 2 It is a comparative representation of the expression of genes of interest (EOMES, CXCR4, FOXA2, SOX17) according to culture conditions.

[0064] Figure 3 Comparative expression of the proteins of interest (SOX17, FOXA2) according to culture conditions 5 days after the start of differentiation.

[0065] Figure 4a is a comparative representation of the expression of genes of interest (PROX1, TBX, AFP, KT18, KT19, HNF4A) according to culture conditions.

[0066] Figure 4b is a comparative representation of the expression of genes of interest (HNF1B, SOX9, ASGR1, ALB, TAT) according to culture conditions.

[0067] Figure 5 It is a comparative representation of the 30-day expansion factor as the culture conditions change.

[0068] Figure 6 is a graphical representation of albumin secretion as a function of culture conditions during differentiation.

[0069] Figure 7 is a graphical representation of urea production during differentiation as a function of culture conditions.

[0070] Figure 8 is a graphical representation of CYP3A4 activity as a function of culture conditions.

[0071] Fig. 9 is a set of images of microcompartments presenting hepatocytes obtained by confocal microscopy.

[0072] Fig.10a is a set of images of microcompartments obtained by confocal microscopy from D-0 to D-9 after the initiation of differentiation.

[0073] Fig.10b is a set of images of microcompartments obtained by confocal microscopy from D-12 to D-30 after the initiation of differentiation.

[0074] Fig.10c is a set of images of microcompartments obtained by confocal microscopy at D-15 after the initiation of differentiation.

[0075] Fig.10dis a set of images of microcompartments obtained by confocal microscopy at D-20 after the initiation of differentiation.

[0076] Fig.10e is a set of images of microcompartments obtained by confocal microscopy at D-30 after the initiation of differentiation.

[0077] Fig.11 is a set of images of microcompartments obtained by confocal microscopy, identifying certain liver cell phenotypes.

[0078] Fig.12a A set of images obtained by confocal microscopy are shown of:

[0079] - Single cell grafts at day 2 after transplantation into Balb / c mice (recipients 1 and 2, top row),

[0080] - Microtissue grafts at day 2 after transplantation into Balb / c mice (recipients 4 and 5, bottom row).

[0081] Figure 12b A set of images obtained by confocal microscopy are shown of:

[0082] - Single cell grafts at day 6 after transplantation into Balb / c mice (recipients, top row),

[0083] - Microtissue grafts at day 6 after transplantation into Balb / c mice (recipient 6, bottom row).

[0084] Fig.12c is a set of images of injection site tissue obtained by confocal microscopy in Balb / c mice with vehicle alone (basal medium containing 50% Matrigel) without grafts.

[0085] Fig.12d is a set of images of microtissues according to the invention obtained by confocal microscopy before transplantation.

[0086] Fig.13a is a graphical representation of A1AT (alpha-1 antitrypsin) secretion before transplantation of 2D differentiated single cells (2D) and microtissues according to the invention (invention).

[0087] Fig.13b is a graphical representation of A1T (alpha-1 antitrypsin) detection in Balb / c mouse serum before (day 0), 2 days and 6 days after transplantation of 2D differentiated single cells (SC), microtissues according to the present invention (MT) and vehicle alone (control). DETAILED DESCRIPTION

[0088] definition

[0089] For the purposes of the present invention, "alginate" refers to a linear polysaccharide formed from β-D-mannuronic acid and α-L-guluronic acid, their salts and derivatives.

[0090] For the purposes of the present invention, "hydrogel capsule" or "hydrogel microcompartment" means a three-dimensional structure formed by a matrix of polymer chains swollen with a liquid, preferably water.

[0091] For the purposes of the present invention, "human cell" means a human cell or an immunohumanized non-human mammal cell. Even when this is not specified, the cells, stem cells, progenitor cells and tissues according to the present invention consist of or are obtained from human cells or immunohumanized non-human mammal cells.

[0092] For the purposes of the present invention, "embryonic stem cells" means pluripotent stem cells derived from the cells of the inner cell mass of the blastocyst. The pluripotency of embryonic stem cells can be evaluated 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. The embryonic stem cells used in the context of the present invention are obtained without destroying the embryos from which they originate, for example using the techniques described by Chang et al. (Cell Stem Cell, 2008, 2 (2): 113-117). Alternatively, human embryonic stem cells can be excluded.

[0093] Within the meaning of the present invention, "only cells surrounded by cells" in a microtissue means cells that are in contact neither with the lumen nor with the exterior of the microtissue.

[0094] For the purposes of the present invention, the term "mutant cell" refers to a cell that carries at least one mutation.

[0095] For the purposes of the present invention, "pluripotent stem cells" or "multipotent cells" means cells that have the ability to form all tissues present in the entire origin organism, but cannot form the entire organism itself. Human pluripotent stem cells may be referred to as hPSCs in this application. These cells may especially be induced pluripotent stem cells (iPSCs or human induced pluripotent stem cells hiPSCs), embryonic stem cells, or MUSE cells (for "tolerance to multilineage differentiation stress").

[0096] For the purposes of the present invention, "induced pluripotent stem cells" means pluripotent stem cells that are induced to become pluripotent by genetic reprogramming of differentiated somatic cells. These cells are particularly positive for pluripotency markers, such as staining with alkaline phosphatase and the expression of proteins NANOG, SOX2, OCT4 and SSEA3 / 4. Examples of methods for obtaining induced pluripotent stem cells are described in 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).

[0097] Within the meaning of the present invention, "progenitor cells" are intended to mean stem cells which have participated in differentiation into liver cells but have not yet differentiated.

[0098] Within the meaning of the present invention, the "Féret diameter" of a microcompartment (or a part of a microcompartment) or a microtissue according to the present invention means the distance "d" between two tangents to said microcompartment (or said part) or to said microtissue, which are parallel so that the entire projection of said microcompartment (or said part) or to said microtissue lies between these two parallel tangents. The Feret diameter of the inner part of a microcompartment is measured between the two interfaces of the inner part of the microcompartment and the outer layer, i.e. the distance "d" between two tangents to said inner part, which are parallel so that the entire projection of said inner part lies between these two parallel tangents.

[0099] Within the meaning of the present invention, "variable thickness" of a layer refers to the fact that a layer of the same microcompartment or the same microtissue does not have the same thickness everywhere.

[0100] For the purposes of the present invention, a "microcompartment" or "vesicle" means a partially or completely enclosed three-dimensional structure containing several cells.

[0101] Within the meaning of the present invention, "microtissue" or "liver microtissue" is intended to mean a three-dimensional human tissue comprising at least liver cells and having a maximum dimension of less than 1 mm.

[0102] Within the meaning of the present invention, "medium" means an aqueous solution comprising cells or microtissues that is compatible with the survival, development and / or metabolism of cells. It can be a culture medium.

[0103] For the purposes of the present invention, "convective medium" means a medium that is agitated by internal motion.

[0104] For the purposes of the present invention, the term "mutation" means a genetic or epigenetic mutation, preferably a functional mutation. In particular, it may include point modifications of genetic sequences, structural variants, epigenetic modifications or modifications of mitochondrial DNA.

[0105] The term "functional mutation" within the meaning of the present invention refers to a transmissible genetic or epigenetic modification that confers a potential gain of function or loss of function or potential loss of function to the relevant mutant cell. It is preferentially related to mutations that cause a phenotypic change in the affected mutant cell. Very preferentially, it is related to changes in the genomic and / or epigenomic sequence that alter the therapeutic potential of the cell population, or increase the risks associated with the resulting therapy, or reduce the benefits provided by the resulting therapy.

[0106] For the purposes of the present invention, the "minimum dimension" of a microcompartment or cell layer means the value of the minimum Feret diameter of the microcompartment. For the purposes of the present invention, "lumen" means a volume of aqueous solution surrounded by a cell topology. Preferably, the contents of the lumen are not in diffusion equilibrium with the volume of convective liquid present outside the microcompartment.

[0107] The "smallest radius" of the inner part of a microcell according to the present invention means half the value of the smallest Feret's diameter of the inner part of the microcell.

[0108] The "average radius" of the inner part of a microcompartment according to the invention means the average value of the radii of the smallest compartments, each radius corresponding to half the value of the Feret's diameter of the inner part of a microcompartment.

[0109] "Expansion rate at day X" according to the present invention means a measurement of cell proliferation at time t = X. The expansion 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 beginning of culture (the day of packaging or the day of placing in culture).

[0110] According to the present invention, "large-scale culture" means a cell culture method suitable for the production batches of liver microtissues, so that at least 1 patient, preferably 10 patients, more preferably 100 patients, even more preferably more than 1,000 patients can be treated.

[0111] According to the present invention, the "functional phenotype" of the microtissue means the presence of mature hepatocytes, which are characterized by the expression of albumin and the absence of alpha-fetoprotein and cytokeratin 19.

[0112] According to the present invention, "liver bud" means a cell structure characterized by the cell expansion of the endoderm of the embryonic foregut, which gives rise to the parenchyma of the liver and bile duct. This is a specific conformation that gives rise to hepatocytes and bile duct cells.

[0113] According to the present invention, "RLU" means relative light unit. It corresponds to the measurement of the amount of light produced by a bioluminescent reaction. The measurement can be performed using a luminometer.

[0114] Liver microtissue

[0115] The object of the present invention is therefore a three-dimensional liver microtissue having a maximum dimension between 500 μm and 700 μm, expressing CYP3A4 monooxygenase with an activity of at least 75,000 RLU per million cells and producing at least 18 μg urea per million cells per 24 hours.

[0116] Cytochrome P450 is a heme protein involved in the oxidative metabolism of many molecules. Cytochrome P450 is an enzyme involved in the biotransformation of exogenous compounds in detoxification phenomena and intoxication phenomena produced by forming reaction entities. The most abundant human liver form (CYP3A4) is responsible for more than 60% of drug metabolism. Its presence in the microcompartment is a functional guarantee. Urea is a nitrogenous product produced by the catabolism of proteins. It is synthesized only in the liver via the urea cycle, and the amount of urea formed depends on the amount of protein ingested, the catabolism of proteins and the state of liver function. CYP3A4 activity of at least 75,000 RLU per million cells together with a urea production of at least 18 μg per million cells per 24 hours ensures the presence of mature hepatocytes and sufficient metabolic activity to ensure a significant effect on impaired liver function.

[0117] Preferably, the activity of CYP3A4 is at least 80,000 RLU, even more preferably at least 100,000 RLU.

[0118] According to a preferred embodiment, the liver microtissue produces at least 40 μg urea per million cells per 24 hours, even more preferentially at least 60 μg, in particular at least 80 μg.

[0119] Advantageously, the liver microtissues according to the present invention exhibit metabolic activity close to that of a healthy liver. Therefore, the liver microtissues are particularly effective in restoring the function of the treated liver.

[0120] Preferably, the liver microtissue according to the present invention comprises 50% to 99% liver cells.

[0121] The liver microtissue according to the present invention can be obtained from induced pluripotent stem cells.

[0122] According to a variant, the liver microtissue may be obtained from stem cells, progenitor cells and / or cells capable of differentiating into liver cells. Preferably, the liver microtissue is obtained from induced pluripotent stem cells encapsulated in a single three-dimensional closed microcompartment.

[0123] The liver microtissue preferably comprises liver cells which secrete at least 75 μg albumin per million cells per 24 hours, even more preferably at least 150 μg, in particular at least 250 μg albumin.

[0124] According to a specific embodiment, the liver microtissue is obtained from induced pluripotent stem cells and comprises liver cells that secrete at least 75 μg of albumin per million cells per 24 hours at least 20 days after the start of differentiation.

[0125] Albumin is the most abundant protein in the blood. Produced by the liver, albumin is responsible, among other things, for stabilizing blood pressure and transporting many substances.

[0126] Advantageously, an albumin production of at least 75 μg per million cells per 24 hours is an indicator of a correct functioning of the liver microtissue.

[0127] According to another preferred embodiment, the liver microtissue comprises at least 3 different phenotypes of liver cells, and all cells of the microtissue have been obtained from induced pluripotent stem cells encapsulated in a single three-dimensional closed microcompartment.

[0128] Preferably, the liver microtissue comprises at least 4 different phenotypes of liver cells, even more preferably at least 5.

[0129] Advantageously, the liver microtissue according to the present invention has a significant cellular diversity, making it possible to reproduce the liver microenvironment. The diversity of cells present in the liver microtissue and their proximity allow a large number of cells to interact, thereby collaborating in the execution of many metabolic and transport functions.

[0130] The cellular diversity found in the liver microtissues according to the present invention is obtained from induced pluripotent stem cells in a single three-dimensional closed microcompartment. In this way, different cell types can self-organize in microcompartments that reproduce the liver microenvironment.

[0131] According to a preferred embodiment, all cells of the microtissue have been obtained by differentiation of induced pluripotent stem cells encapsulated in a single three-dimensional closed microcompartment, said induced pluripotent stem cells being preferentially all of the same lineage.

[0132] Very preferentially, all cells of the microtissue have been obtained from induced pluripotent stem cells encapsulated in a single three-dimensional closed microcompartment by a single differentiation process carried out in said microcompartment.

[0133] Preferably, the induced pluripotent stem cells form cysts in the microcompartment before differentiation in the microtissue according to the invention. Therefore, preferably, all cells of the microtissue have been obtained from at least one cyst of induced pluripotent stem cells encapsulated in the microcompartment by differentiation, in particular by a single differentiation method implemented in a single three-dimensional closed microcompartment.

[0134] Advantageously, the microenvironment of the microcompartment from which the microtissue according to the invention is obtained reproduces the conditions of liver organogenesis.In fact, the microcompartment according to the invention makes it possible to limit various physical and / or stress constraints and promotes the interaction between the various cell types within the microcompartment.

[0135] According to a particularly suitable embodiment, the liver microtissue according to the present invention comprises at least immature hepatocytes, mature hepatocytes and bile duct cells. Preferably, the liver microtissue comprises at least:

[0136] - Immature hepatocytes, characterized by the expression of alpha-fetoprotein (AFP) and albumin (ALB) and the absence of cytokeratin 19 (CK19) expression

[0137] -Mature hepatocytes characterized by albumin expression and the absence of alpha-fetoprotein and cytokeratin 19 expression

[0138] - Cholangiocytes, characterized by the expression of cytokeratin 19 and the absence of albumin and alpha-fetoprotein expression.

[0139] According to one variant, the liver microtissue according to the invention comprises hepatoblasts. Hepatoblasts may be characterized by the expression of alpha-fetoprotein, albumin and cytokeratin 19.

[0140] The presence of these hepatocyte phenotypes ensures a multifactorial effect on liver function that may restore or optimize certain liver functions.

[0141] In a specific embodiment, the liver microtissue comprises at least 50% mature hepatocytes and / or immature hepatocytes.

[0142] According to a particularly suitable embodiment, the liver microtissue comprises between 20% and 60% (by number) of cells expressing cytokeratin 19 within the liver cells.

[0143] Cells expressing cytokeratin 19 are preferentially cholangiocytes.

[0144] In the context of the present invention, liver cells are preferably selected from the group consisting of mature hepatocytes, immature hepatocytes, hepatoblasts, cholangiocytes and mixtures thereof.

[0145] The liver microtissue may also contain cells expressing CD73 and CD90. The cells expressing CD73 and CD90 are preferentially mesenchymal stem cells.

[0146] Mesenchymal stem cells are a cell population well known for their properties for tissue repair and regeneration, in particular of the liver. Advantageously, the presence of mesenchymal stem cells derived from induced pluripotent stem cells of the patient to be treated makes it possible to ensure a tissue repair effect in a liver exhibiting liver failure.

[0147] Thus, in a particular embodiment, the microtissue according to the invention comprises at least:

[0148] - liver cells, preferably at least immature hepatocytes, mature hepatocytes and cholangiocytes, and

[0149] - Cells expressing CD73 and CD90, preferably at least mesenchymal stem cells.

[0150] Preferably, the liver microtissue comprises at least:

[0151] - liver cells, of which 20% to 60% (by number) are cells expressing cytokeratin 19, and

[0152] -Cells expressing CD73 and CD90.

[0153] The liver microtissue preferably includes (number percentage):

[0154] - 50% to 99% of the liver cells, of which preferably 20% to 60% (by number) of the liver cells are cells expressing cytokeratin 19, and

[0155] -1% to 20% of cells expressing CD73 and CD90.

[0156] The liver microtissue according to the present invention may also comprise other cells, such as, in particular, Ito cells (stellate cells), Kupffer cells, endothelial cells, hepatoblasts. Therefore, in another specific embodiment, the liver microtissue comprises:

[0157] -Mature hepatocytes, immature hepatocytes, hepatoblasts, cholangiocytes,

[0158] - Ito cells and / or Kupffer cells and / or endothelial cells,

[0159] - and optionally mesenchymal stem cells.

[0160] According to a variant, the liver microtissue according to the invention may also contain smooth muscle cells and / or fibroblasts.

[0161] The liver microtissues contain cells with a phenotype that is preferentially compatible with the stem cell microenvironment.

[0162] The cellular diversity provided by the liver microtissue according to the invention makes it possible to repair and regenerate a diseased liver in order to restore the affected functions.

[0163] The concentration of mature hepatocytes, cholangiocytes and mesenchymal stem cells ensures a lasting effect during cell therapy. In order to achieve a sufficient effect as quickly as possible after microtissue transplantation, it is particularly important to obtain a mature hepatocyte and / or immature hepatocyte content of more than 50% in culture.

[0164] A concentration of mature and / or immature hepatocytes less than 50% will limit the efficiency of the graft and will require a larger graft volume due to the low concentration of hepatocytes (by number). In fact, it is estimated that at least 5% of the liver mass in functional hepatocytes is necessary to treat, for example, acute liver failure, and metabolic defects of the liver such as diseases associated with the secretion of Factor VIII, Factor IX and VWF, Wilson's disease and hereditary hemochromatosis require a similar order of magnitude.

[0165] Mature hepatocytes express albumin, but not 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.

[0166] Particularly preferably, the liver microtissue comprises at least one lumen, at least one cell of the microtissue in contact with the lumen and a medium outside the microtissue, and at least one cell surrounded solely by cells.

[0167] In the context of the present invention, such architectures are characteristic of functional microtissues ready for cell therapy.

[0168] The specific architecture of the microtissues is possible, in particular, due to the presence of polarized liver cells, which make the structure and architecture of the microtissues possible.

[0169] Thus, the microtissue may contain polarized liver cells. Polarization of liver cells in the microcompartment may be an indicator of the formation of functional microtissue.

[0170] The liver microtissue according to the present invention does not contain human embryonic stem cells.

[0171] The liver microtissue according to the present invention may be in the form of an ellipsoid.

[0172] 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 allows it to promote its flow into the blood vessels in the case of administration by injection via the vascular route (in one embodiment, via the portal vein), and also promotes its flow within the cannula if it is administered by intratissue transplantation. The improvement of the liver microtissue injection leads to improved integration of the liver microtissue by the liver of the treated patient.

[0173] The liver microtissue according to the present invention preferably has a diameter of 100 μm to 300 μm, preferably 150 μm to 280 μm or less. The maximum dimension of the liver microtissue is preferably less than 1 mm, and very preferably between 500 μm and 700 μm.

[0174] Advantageously, the liver microtissue is of a size suitable for administration via the portal vein.

[0175] The liver microtissue according to the present invention may contain 300 to 14,000 cells, preferably 500 to 8,000 cells, even more preferably 900 to 5,000 cells, in particular 4,500 cells.

[0176] Preferably, the liver microtissue comprises at least one bile duct. The bile duct collects bile produced by liver cells to transport it to the gallbladder. The presence of at least one bile duct in the liver microtissue promotes the correct function of the microtissue in the liver and the reconstruction of defective bile ducts.

[0177] According to another embodiment, the liver microtissue comprises at least one glycogen granule. Glycogen serves as a reservoir of carbohydrates in the body; it is primarily 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 will hydrolyze glycogen and release glucose into the blood. The presence of glycogen granules in liver cells is a physiological element for their metabolic function and, therefore, for their function. Therefore, 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 the best therapeutic effect.

[0178] Preferably, the liver microtissue comprises at least one bile duct and at least one glycogen granule.

[0179] The liver microtissue according to the present invention can be obtained by inducing pluripotent stem cell differentiation after 1 to 200, preferably 5 to 150, and especially 15 to 80 induced pluripotent stem cells are encapsulated in a three-dimensional closed microcompartment for at least 20 days, preferably at least 30 days. Preferably, the liver microtissue can be obtained from 1 to 200 induced pluripotent stem cells, preferably 5 to 150, and especially 15 to 80 induced pluripotent stem cells encapsulated in a three-dimensional closed microcompartment.

[0180] Preferably, the induced pluripotent line forms cysts in the microcompartment and then differentiates into cells of the liver microtissue according to the invention.

[0181] An object of the present invention is also a set of liver microtissues comprising at least one liver microtissue according to the present invention. Preferably, it is a set of several three-dimensional liver microtissues in a medium, wherein at least one liver microtissue is a liver microtissue according to the present invention.

[0182] According to one variant, at least 50% (by number) of the liver microtissues in the set of liver microtissues are liver microtissues according to the invention.

[0183] The set of liver microtissues according to the invention is suitable for administration to patients suffering from liver failure. It can be administered by injection in a biocompatible solution. The injection can be performed in the portal vein so as to reach the liver without being dispersed in the systemic circulation, directly in the liver or ectopically. When ectopically injected, the injection can be performed in the abdomen or under the kidney capsule.

[0184] Preferably, the effective amount of the set of microtissues injected into the patient corresponds to between 1% and 20% by mass, preferably between 2% and 10%, in particular 5% by mass of the liver of the patient to be treated.

[0185] According to another aspect, the invention has for its object a microcompartment comprising at least one liver microtissue according to the invention.

[0186] The microcompartment according to the present invention comprises an outer hydrogel layer. Preferentially, the hydrogel used is biocompatible, that is to say it is non-toxic to cells. The outer hydrogel layer must allow diffusion of oxygen and nutrients in order to supply and enable the cells contained in the microcompartment to survive. According to one embodiment, the outer hydrogel layer comprises at least alginate. It can consist only of alginate. Alginate can especially be sodium alginate, consisting of 80% α-L-guluronate and 20% β-D-mannuronate, with an average molecular weight of 100 kDa to 400 kDa and a total concentration of 0.5 wt % to 5 wt %. The outer hydrogel layer has no cells.

[0187] This external hydrogel layer makes it possible to specifically protect the cells from the mechanical stresses of the bioreactor, limiting potentially toxic molecules when they accumulate in the medium.

[0188] The average thickness of the outer layer may be variable, preferably between 20 μm and 60 μm, more preferably between 30 μm and 40 μm. The ratio between the smallest radius of the inner part and this thickness is preferably between 2 μm and 10 μm.

[0189] The microcompartments according to the invention advantageously exhibit an at least 15-fold expansion rate of induced pluripotent stem cells 20 days after the start of differentiation.

[0190] The present invention thus facilitates scale-up with high expansion rates, which therefore reduce the culture time to obtain functional microtissues.

[0191] The microcompartments according to the present invention can be obtained after encapsulation of induced pluripotent stem cells, with or without the addition of an extracellular matrix, a natural matrix or a synthetic matrix.

[0192] According to a variant, the microcompartment according to the invention contains in its internal part an extracellular matrix such as and / or and / or a hydrogel-type matrix of plant origin such as modified alginates or a hydrogel-type matrix of synthetic origin or Type of copolymer of poly(N-isopropylacrylamide) and poly(ethylene glycol) (PNIPAAm-PEG).

[0193] According to a variant, the microcompartments according to the invention can be obtained after encapsulation of induced pluripotent stem cells without the addition of an extracellular matrix. The extracellular matrix components can be peptide or peptidomimetic sequences, mixtures of proteins, extracellular compounds or structural proteins, such as collagen, laminin, entactin, vitronectin and growth factors or cytokines.

[0194] In the microcompartments, induced pluripotent stem cells are differentiated into liver tissue for at least 20 days, preferably at least 30 days.

[0195] Unexpectedly, this microcompartment provides a favorable microenvironment for the development of liver microtissues. In fact, during differentiation within this microcompartment, the cells organize themselves to form a structure similar to a liver bud. This liver bud is found during liver organogenesis in vivo; it is this special structure that gives rise to hepatocytes and bile duct cells. The presence of a structure similar to a liver bud during the formation of liver microtissues is an indicator of good tissue quality.

[0196] Therefore, the liver microtissue according to the present invention is preferentially obtained after forming a liver bud-like structure in a single three-dimensional closed microcompartment.

[0197] The microcompartments according to the invention make it possible to isolate induced pluripotent stem cells from the mechanical stresses present in a bioreactor. This mechanical isolation allows the microtissue to establish and maintain a topology that approximates the spatial and structural organization of liver organogenesis present in vivo.

[0198] Unexpectedly, after at least 20 days of differentiation in microcompartments, preferably 30 days, the cells maintained their conformation, allowing them to have better proliferation while maintaining a functional phenotype. This therefore makes it possible to reduce the number of passages and the culture time necessary to reach the final desired number of liver cells.

[0199] The cells present in the microcompartments according to the invention are preferentially obtained after at least two cell division cycles following encapsulation of 1 to 200, preferentially 5 to 150, in particular 15 to 80 induced pluripotent stem cells in the outer hydrogel layer.

[0200] 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 cell division cycles after preferentially at least 5, even more preferentially at least 6, encapsulation in the outer hydrogel layer, to obtain up to 8,000 mature hepatocytes per microcompartment. For example, the mature hepatocytes present in the microcompartment are obtained after at least six cell division cycles after encapsulation of the cells in the outer hydrogel layer.

[0201] Preferably, the number of cell divisions used to carry out the method according to the invention is less than 100, even more preferentially less than 30.

[0202] Preferably, the microcompartment is obtained after at least 2 passages, more preferably at least 3, 4 or 5 passages after encapsulation. Each passage may, for example, last 2 to 10 days, especially 2 to 4 days.

[0203] Preferably, the microcompartments are obtained after at least one recapsulation, more preferably 1 to 14 recapsulations, especially 2 to 7 recapsulations. Very preferably, the recapsulations correspond to a new passage and each encapsulation cycle corresponds to a passage.

[0204] Preferably, the microcompartment according to the invention is obtained less than 30 days after encapsulation of at least 1 induced pluripotent stem cell, preferentially 5, 20 and up to 100 induced pluripotent stem cells, in the inner part defined by the outer hydrogel layer, even more preferentially less than 20 days after encapsulation.

[0205] The microcompartment according to the invention may contain 100 to 14,000 cells, preferably 300 to 10,000 cells, even more preferably 300 to 5,000 cells, more particularly at least 50 mature hepatocytes and at least 20 bile duct cells. Advantageously, the microcompartment according to the invention protects induced pluripotent stem cells from mechanical stress, thereby enabling them to obtain an expansion rate particularly suitable for large-scale culture.

[0206] The microcompartment according to the present invention can be in any three-dimensional form, i.e., it can have the shape of any object in space. The microcompartment can have any form compatible with cell encapsulation. Preferably, the microcompartment according to the present invention is spherical or elongated or ellipsoidal or substantially spherical or elongated in shape. It can have the shape of an oval, a cylinder, a spheroid or a sphere or substantially such a shape.

[0207] It is the outer layer of the microcompartments, ie the hydrogel layer, which gives the microcompartments according to the invention their size and shape. Preferably, the smallest dimension of the microcompartments according to the invention is between 150 μm and 500 μm, preferentially between 200 μm and 450 μm.

[0208] The maximum dimension thereof is preferably greater than 350 μm, more preferably between 350 μm and 600 μm.

[0209] The microcompartments according to the invention can optionally be stored frozen. They should then preferably be thawed before use.

[0210] The invention also relates to several microcompartments according to the invention used together.

[0211] The invention also relates to a microcompartment assembly or series comprising at least two three-dimensional cellular microcompartments, characterized in that at least one microcompartment is a microcompartment according to the invention.

[0212] Preferably, the microcompartment series according to the present invention is in a culture medium, especially in a culture medium that is at least partially convective. Any culture medium suitable for culturing induced pluripotent stem cells can be used, such as the culture medium "HCM Hepatocyte Culture Medium Bullet Kit (Lonza)" or "Culture Medium E from William (Thermofisher Scientific)", because the concentration of dissolved salts is compatible with maintaining alginate crosslinking by divalent cations.

[0213] According to a particularly suitable embodiment, the object of the present invention is a series of cell microcompartments as described above in a closed chamber such as a bioreactor, preferably in a closed chamber such as a culture medium in a bioreactor. Therefore, preferably, the microcompartment is arranged in the culture medium in a closed bioreactor.

[0214] The set of microcompartments or the series of microcompartments according to the invention preferably comprises from 2 to 1,016 microcompartments.

[0215] Therefore, the microcompartments according to the invention are suitable for large-scale cultivation by providing liver microtissues that exhibit a functional phenotype, especially with regard to their detoxification (particularly the activity of CYP3A) and secretory (particularly the secretion of albumin) capacities, thereby ensuring optimal efficacy in vivo.

[0216] Use of liver microtissue according to the invention

[0217] The liver microtissue or microcompartment according to the invention can be used for all applications, in particular as a medicament, in particular in human cell therapy.

[0218] Therefore, the object of the present invention is a liver microtissue according to the invention or a group of liver microtissues comprising at least one liver microtissue according to the invention, or a microcompartment according to the invention or a group of microcompartments comprising at least one microcompartment according to the invention, which is used as a medicament, in particular in cell therapy.

[0219] The invention also relates to a method of therapeutic treatment consisting of transplanting and / or injecting the microtissue according to the invention into a human body, in particular as a cell therapy treatment.

[0220] Preferably, the liver microtissue or microcompartment according to the present invention can be used to prevent or treat symptoms associated with liver failure, especially acute liver failure, chronic liver failure or chronic acute liver failure. Preferably, the liver microtissue or microcompartment according to the present invention can be used to prevent or treat diseases such as fibrosis, cirrhosis, steatosis, non-alcoholic steatosis, hepatitis, liver metabolic diseases such as diseases associated with factor VIII and factor IX and VWF secretion, Wilson's disease and hereditary hemochromatosis. In fact, all of these indications can be treated by liver transplantation, and this is exactly the problem to be solved by transplanting a group of microtissues according to the present invention to restore liver function.

[0221] Therefore, the object of the present invention is a liver microtissue according to the present invention or a group of liver microtissues comprising at least one liver microtissue according to the present invention, or a microcompartment according to the present invention or a group of microcompartments comprising at least one microcompartment according to the present invention, for use in the prevention or treatment of symptoms associated with liver failure, especially acute liver failure, chronic liver failure or acute-on-chronic liver failure, in particular in the prevention or treatment of liver diseases such as fibrosis, cirrhosis, steatosis, non-alcoholic steatosis, hepatitis, liver metabolic diseases such as diseases associated with the secretion of factor VIII, factor IX and VWF.

[0222] The present invention also relates to a liver microtissue according to the invention or a set of liver microtissue molecules comprising at least one liver microtissue according to the invention or a microcompartment according to the invention or a set of microcompartment sets comprising at least one microcompartment according to the invention, which are used in molecular evaluation or in the modeling of liver diseases.

[0223] Method for preparing microcompartments and microtissues according to the invention

[0224] The invention also relates to a method for producing the microcompartments according to the invention.

[0225] The method for preparing a microcompartment or a microcompartment assembly according to the present invention may comprise the following steps:

[0226] - (a) preparing a closed three-dimensional cell microcompartment comprising induced pluripotent stem cells and optionally an extracellular matrix component or a natural or synthetic extracellular matrix within an outer hydrogel layer,

[0227] - (b) inducing cell differentiation within the cell microcompartments to obtain microcompartments containing liver cells.

[0228] The preparation of the microcompartments of step (a) can be carried out in any medium suitable for culturing induced pluripotent stem cells, such as mTeSR from Stemcell technologies. TM 1 or mTeSRPlus medium, StemMACS TM iPS-Brew XF (Miltenyi Biotec or StemFlex from thermofisher Scientific).

[0229] The microcompartment of step (a) can be obtained by encapsulating 1 to 150 induced pluripotent stem cells, preferably at least 50, especially at least 100 induced pluripotent stem cells.

[0230] Preferably, encapsulation is implemented according to techniques known to those skilled in the art. In fact, any method for producing a cell microcompartment containing an external hydrogel layer and cells inside can be used to implement the preparation method according to the present invention. Specifically, according to the following steps, the microcompartment can be prepared by adapting the method and microfluidic device described in Alessandri et al. 2016 ("A3D 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).

[0231] Preferably, step (a) is performed by adding extracellular matrix components or a natural or synthetic extracellular matrix. According to a variant, step (a) is performed without adding extracellular matrix components or an extracellular matrix or a natural or synthetic extracellular matrix.

[0232] In the context of the present invention, step (a) is preferably implemented in a device capable of producing hydrogel capsules using a microfluidic chip. For example, the device may comprise a syringe pump for concentrically injecting several solutions by means of a microfluidic syringe, which makes it possible to form a jet that breaks up into droplets that are then collected in a calcium bath. According to a particularly suitable embodiment, two or three solutions are loaded on two or three syringe pumps:

[0233] - hydrogel solutions, such as alginate,

[0234] - optionally an isotonic intermediate solution, preferably an isotonic solution free of divalent cations such as Ca2+ and Mg2+ to avoid premature crosslinking of the hydrogel in the syringe, for example a sorbitol solution,

[0235] - A solution containing induced pluripotent stem cells and a culture medium. The three solutions are co-injected (simultaneously injected) concentrically using a microfluidic syringe or a microfluidic chip so that a jet can be formed, the jet splits into droplets, the outer layer of the droplets is the hydrogel solution and the core of the droplets is the solution containing induced pluripotent stem cells of step (a); These droplets are collected in a calcium bath, which crosslinks and / or gels the alginate solution to form a shell.

[0236] To improve the monodispersity of the cell microcompartments, the hydrogel solution is preferably charged with a direct current of (1 kV to 10 kV). A grounding ring can be optionally arranged in a plane perpendicular to the axis of the jet leaving the microfluidic injector (coextrusion chip) at a distance of 1 mm to 20 cm from the tip, preferably 3 mm to 10 cm from the tip, and even more preferably 1 cm to 5 cm, to generate an electric field.

[0237] According to the present invention, capsules must be produced, the inner part of which has an average radius of at least 100 μm or less. In order to produce capsules of such size with a co-extruded chip (microfluidic syringe or microfluidic chip), the present invention proposes, in particular, to change the flow rate of the co-extruded solution and the final opening of the co-extruded chip. "Flow rate" means the flow rate of each solution reaching the syringe. "Final opening of the co-extruded chip" means the inner opening of the outlet channel of the chip.

[0238] - For the flow rate: the process preferentially varies from a value of 20 to 40 mL / h to a value of 45 to 150 mL / h for each coextruded solution of a standard capsule size known from the prior art, preferentially 45 to 110 mL per hour for the variant of the invention.

[0239] - The final opening diameter for the coextruded chip (microfluidic syringe) changes from a value preferably between 50 μm and 120 μm for standard capsule sizes known from the prior art to a diameter value between 150 μm and 300 μm, preferably between 180 μm and 240 μm.

[0240] Therefore, according to a specific embodiment, the encapsulation of step (a) is carried out using a microfluidic syringe with a final opening diameter between 150 μm and 300 μm, preferably between 180 μm and 240 μm, and the flow rate of each of the three solutions is between 45 mL / h and 150 mL / h, preferably between 45 mL / h and 110 mL / h.

[0241] According to one variant, step (a) of preparing the microcompartments can be carried out with stem cells, progenitor stem cells or cells capable of differentiating into hepatocytes.

[0242] Step (b) of cell differentiation is preferably performed for at least 20 days, even more preferably for at least 30 days.

[0243] During the differentiation of step (b), the cells organize themselves in a structure similar to a liver bud. This structure is typically marked by structuring in the form of two cell subpopulations presenting two different structures, one cell subpopulation being of epithelial type (i.e. in the form of a cell matrix with apical-basal polarization and presenting tight junctions, and the other cell subpopulation being of mesenchymal type, that is to say without apical-basal structure and presenting focal junctions.

[0244] The differentiation of induced pluripotent stem cells into liver microtissues in step (b) can be performed by any known differentiation method, such as described in Raggi et al., Stem cell Report 2022 or Mallanna and Duncan, Curr Protoc Cell Bil, 2014.

[0245] According to one embodiment, steps (a) and / or (b) are carried out under permanent or continuous stirring. Such stirring is important because it maintains the homogeneity of the culture environment and avoids the formation of any diffusion gradients.

[0246] Preferably, step (b) is performed under hypoxic conditions; more preferably, the first 5 days of differentiation are performed under hypoxic conditions.

[0247] Unexpectedly, differentiation under hypoxic conditions allowed for better expansion rates.

[0248] Implementing the method according to the invention makes it possible to obtain microcompartments comprising at least 100, preferentially at least 500, at least 800, at least 1,000 and in particular at least 3,000 cells.

[0249] The method according to the invention is preferably performed in a closed chamber, such as a closed bioreactor.

[0250] The present invention also relates to a method for preparing liver microtissue, the method comprising the steps of:

[0251] - (a) preparing a closed three-dimensional cell microcompartment comprising induced pluripotent stem cells and optionally an extracellular matrix component or a natural or synthetic extracellular matrix within an outer hydrogel layer,

[0252] - (b) inducing cell differentiation in the cell microcompartment to differentiate the induced pluripotent stem cells into liver cells

[0253] (c) removing the outer hydrogel layer to recover an ovoid, cylindrical, spheroid or spherical or substantially ovoid, cylindrical, spheroid or spherical or ellipsoidal liver microtissue comprising at least 300 cells expressing CYP3A4 monooxygenase at an activity of at least 75,000 RLU per million cells and producing at least 18 μg urea per million cells per 24 hours.

[0254] Step (c) consists in dissociating the microcompartments to obtain liver microtissues; the removal of the outer hydrogel layer can be carried out in particular by any biocompatible, i.e. cell-free, manner, by hydrolysis, dissolution, puncture and / or rupture. For example, elimination can be accomplished using phosphate buffered saline, divalent ion chelators, enzymes (such as alginate lyase, if the hydrogel comprises alginate) and / or laser microdissection.

[0255] Step (c) of removing the outer hydrogel layer allows recovery of liver microtissues of oval, cylindrical, spheroid or spherical or substantially oval, cylindrical, spheroid or spherical or ellipsoidal shape, comprising at least 300 cells corresponding to at least 3 different liver cell phenotypes.

[0256] Preferably, the liver microtissue recovered in step (c) has an oval shape.

[0257] The liver microtissue from step (c) may contain at least 300, in particular 300 to 14,000, even more preferably 900 to 5,000 cells.

[0258] The liver microtissue from step (c) comprises at least one lumen, at least one cell in contact with the lumen and the medium outside the microtissue, and at least one cell surrounded solely by cells.

[0259] 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 that can form up to 20% of the liver mass of the patient to be treated in less than 50 days, preferentially less than 40 days and in particular less than 35 days.

[0260] In a preferred variant, the method according to the invention comprises at least one repackaging of the liver microtissue after step (c), that is to say, at least two packaging cycles. Preferably, each packaging cycle corresponds to one passage. In this variant of the method (at least one repackaging of the cells after step (e)), the number of cell divisions of the entire 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. In the method according to the invention, there can be several repackagings, preferably 1 to 100, in particular 1 to 10 repackagings.

[0261] Each repackaging may include:

[0262] - a step of dissociating the microcompartment or the series of microcompartments to obtain liver microtissue; the removal of the outer hydrogel layer can be carried out in particular by any biocompatible, i.e. cell-free manner, by hydrolysis, dissolution, piercing and / or rupture. For example, elimination can be accomplished using phosphate buffered saline, divalent ion chelators, enzymes (such as alginate lyases, if the hydrogel contains alginate) and / or laser microdissection, and - a step of re-encapsulating all or some cells or cell clusters in a hydrogel capsule. Re-encapsulation is a means suitable for increasing the cell expansion produced by the pluripotent step and reducing the risk of mutation.

[0263] Reencapsulation consists in removing the outer hydrogel layer, in resuspending the cells in the form of cysts, preferably in a partially or completely dissociated manner, in the microcompartments, and in re-performing the steps of the method.

[0264] According to one embodiment, repackaging comprises the following steps:

[0265] - (i) removing the outer hydrogel layer,

[0266] - (ii) resuspending the liver microtissue contained in the microcompartment

[0267] - (iii) encapsulating the liver microtissue in a hydrogel layer so as to form a microcompartment of oval, cylindrical, spheroid or spherical shape, or a substantially oval, cylindrical, spheroid or spherical or ellipsoidal shape, the microcompartment comprising an outer hydrogel layer defining an inner portion, the inner portion having a minimum radius or an average radius of at least 300 μm

[0268] - (iv) culturing the resulting microcompartments in culture medium

[0269] - (vii) optionally recovering the resulting cell microcompartments.

[0270] Compartmentalization in microcompartments makes it possible to eliminate microcompartments containing more mutant cells than other capsules. Even if the mutant cells have a rapid growth, they will reach the capsule confluence, which will limit their multiplication. Compartmentalization also makes it possible not to contaminate the entire cell population at any time, especially before the recapsulation step, and also to eliminate capsules containing mutant cells. For example, this sorting can be done by online analysis, or by eliminating capsules that are filled faster than others.

[0271] In one embodiment, at least one step (preferably all steps) is performed at a temperature suitable for cell survival (4-42°C, inclusive). The temperature during cell proliferation must preferably be between 32 and 37°C to avoid triggering mutations by reducing the performance of repair enzymes. Likewise, preferably, the temperature must be low (ideally about 4°C) to control the stress on the cells in step (c).

[0272] At any time, the method according to the invention may comprise a step consisting of verifying the phenotype of the cells contained in the microcompartment. This verification may be performed by identifying the expression of markers characteristic of the sought phenotype by at least a portion of the cells contained in the microcompartment, these markers being selected from:

[0273] - Expression of alpha-fetoprotein, albumin, and cytokeratin 19 to characterize hepatoblasts

[0274] - Expression of alpha-fetoprotein and albumin and absence of cytokeratin 19 expression to characterize immature hepatocytes

[0275] - Albumin expression and absence of alpha-fetoprotein and cytokeratin 19 expression to characterize mature hepatocytes

[0276] - Cytokeratin expression and absence of albumin and alpha-fetoprotein expression to characterize cholangiocytes

[0277] -CD31 expression, used to characterize endothelial cells

[0278] - Expression of CD90 and CD73, used to characterize mesenchymal stem cells.

[0279] The cell microcompartments obtained according to the method of the present invention can then be frozen before any use. Freezing is preferably 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 bag) in a warm water bath (preferably 37 degrees) so that the cells are thawed quite quickly. The microcompartments according to the present invention can be maintained at more than 4°C for a limited time before use, preferably between 4°C and 38°C.

[0280] The present invention particularly facilitates expansion with a high expansion rate, thereby reducing the culture time and the number of divisions to obtain very large numbers of functional lymphocytes.

[0281] Example :

[0282] Example 1: Differentiation Protocol

[0283] The liver microtissues according to the present invention can be obtained from any known differentiation method.

[0284] To demonstrate this, the inventors have adapted 2 protocols known from the prior art. Protocol A is described by Raggi et al., Stem cell Report 2022, and protocol B is adapted from Mallanna and Duncan, Curr Protoc Cell Bil, 2014 and Raggi et al., Stem Cell Reports, 2022.

[0285] Materials and methods

[0286] iPSCs were cultured on vitronectin-coated T75 flasks and passaged regularly using the dissociation agent ReLeSR (Stem Cell Technologies). All experiments were performed with iPSCs between passage 20 and 26.

[0287] Encapsulation of iPSCs was performed in alginate capsules of similar size between the two protocols A and B and in the presence of an extracellular matrix.

[0288] For scenario A:

[0289] Minimum diameter of major axis = 220 μm

[0290] Maximum diameter of long axis = 550 μm

[0291] Average diameter of major axis = 415 μm

[0292] For plan B:

[0293] Minimum minor axis diameter = 200 μm

[0294] Maximum minor axis diameter = 385 μm

[0295] Average minor axis diameter = 295 μm

[0296] First, iPSCs were separated into groups of 3 to 5 cells using accutase and resuspended in a mixture of 50% Matrigel and 50% mTESR1 medium containing 10 μM Rhock inhibitor (Y-27632) at a concentration of 0.8E6 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 in mTESR1 medium containing 10 μM Rhock inhibitor at a ratio of 1 mL total medium to 0.2 mL capsule. The cells were allowed to form cysts in mTESR1 medium at 37° C. and 5% CO 2 for 96 hours under agitation conditions using a 30 mL ABLE bioreactor (Reprocell), with medium changes every 24 hours.

[0297] At 96 hours after encapsulation, differentiation was initiated by passage of the cells for 2 days in RPMI medium containing 1 mM Ca2+, 1% KnockOut Replacement Serum (KOSR), insulin-free B27, 100 ng / mL Activin A, and 3 μM CHIR-99021.

[0298] During the next 3 days, the medium was replaced with RPMI medium containing 1 mM Ca2+, 1% KOSR, B27 without insulin, and 100 ng / mL Activin A.

[0299] Then, during the next 5 days, the medium was replaced with RPMI medium containing for protocol A: 1 mM Ca2+, 1% KOSR, B27 without insulin, 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).

[0300] For regimen B, it contained: 1 mM Ca2+, 1% KOSR, B27 without insulin, supplemented with 20 ng / mL BMP-4 and 5 ng / mL bFGF. From day 11 to day 15, the medium consisted of RPMI medium containing 1 mM Ca2+, 2% KOSR, B27 with insulin, supplemented with 20 ng / mL HGF, 3 μM CHIR-99021, 5 ng / mL bFGF and 20 ng / mL BMP-4.

[0301] On day 16, the medium was changed to HCM medium without EGF (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.

[0302] From day 20 and for the following 5 days, the culture medium consisted of HCM medium plus EGF, supplemented with 20 ng / mL Oncostatin M, 10 μM Dexamethasone and 1% KOSR.

[0303] From day 25 onwards, the culture medium consisted of HCM medium plus EGF, 10 μM dexamethasone and 1% KOSR.

[0304] Gene expression analysis

[0305] Gene expression analysis was performed by RT-Q-PCR.

[0306] qPCR at Roche The expression of each gene was normalized by the expression of the reference genes YWHAZ, NONO and VCP. The data are expressed as 2^ΔCt, where ΔCt = gene Ct - average Ct of the reference gene.

[0307] The results are shown in Figure 1a , Figure 1b and Figure 1c These results show that liver microtissues according to the present invention can be obtained using any suitable differentiation method.

[0308] Example 2: Differentiation into hepatocytes in two dimensions in microcompartments according to the invention .

[0309] IPSCs were encapsulated in alginate microcapsules with the following features:

[0310] Minimum diameter of major axis = 450 μm

[0311] Maximum diameter of long axis = 685 μm

[0312] Average diameter of major axis = 575 μm

[0313] Minimum short axis diameter = 313 μm

[0314] Maximum minor axis diameter = 546 μm

[0315] Average minor axis diameter = 423 μm

[0316] In order to encapsulate iPSC in alginate microcapsules with an average diameter of about 575 μm and without a matrix, iPSCs were first separated in groups of 3 to 5 cells with accutase and resuspended in mTESR1 medium containing 10 μM Rhock inhibitor (Y-27632) at a concentration of 10E6 cells / mL, and encapsulated by adapting the method and microfluidic device described in Alessandri et al. 2016 ("A 3Dprinted 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 0.5E6 cells / mL, and the proportion of capsule relative to the medium did not exceed 20%. The cells were allowed to aggregate in the capsules for 24 hours at 37°C and 5% CO2 under stirring conditions using a 30 mL ABLE bioreactor (Reprocell).

[0317] 24 hours after encapsulation, differentiation was initiated by passage of the cells through RPMI medium containing 1 mM Ca2+, 1% KnockOut Replacement Serum (KOSR), insulin-free B27, 100 ng / mL Activin A, and 3 μM CHIR-99021 for 2 days.

[0318] During the next 3 days, the medium was replaced with RPMI medium containing 1 mM Ca2+, 1% KOSR, B27 without insulin, and 100 ng / mL Activin A.

[0319] Then, for the next 5 days, the medium was replaced with RPMI medium containing 1 mM Ca2+, 1% KOSR, B27 without insulin, supplemented with 20 ng / mL BMP-4 and 5 ng / mL bFGF. From day 11 to day 15, the medium consisted of RPMI medium containing 1 mM Ca2+, 2% KOSR, B27 with insulin, supplemented with 20 ng / mL HGF, 3 μM CHIR-99021, 5 ng / mL bFGF and 20 ng / mL BMP-4.

[0320] On day 16, the medium was changed to HCM medium without EGF (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.

[0321] From day 20 and for the following 5 days, the culture medium consisted of HCM medium plus EGF, supplemented with 20 ng / mL Oncostatin M, 10 μM Dexamethasone and 1% KOSR.

[0322] From day 25 onwards, the culture medium consisted of HCM medium plus EGF, 10 μM dexamethasone and 1% KOSR.

[0323] The medium was changed every day from day 1 to day 25, and from day 25 onwards the medium was changed every other day.

[0324] 2D Hepatocyte Differentiation

[0325] iPSCs were dissociated into single cells using accutase and plated at a density of 1x105 cells / cm2 in Matrigel-coated 6-well plates in mTESR1 medium containing 10 μM Rhock inhibitor. Differentiation was initiated 24 hours after plating using the same differentiation protocol and medium composition as for 3D cysts.

[0326] Gene expression analysis

[0327] Gene expression analysis was performed by RT-Q-PCR.

[0328] qPCR at Roche The expression of each gene was normalized by the expression of the reference genes YWHAZ, NONO and VCP. The data are expressed as 2^ΔCt, where ΔCt = gene Ct - average Ct of the reference gene.

[0329] exist Figure 2A comparison of gene expression between hepatocytes obtained in two and three dimensions is shown in , in order to verify the induction of endoderm during differentiation. The expression of EOMES, CXCR4, FOXA2 and SOX17 genes shows that the differentiation does pass through a transient phase of endoderm differentiation towards definitive endoderm, which is expected to generate hepatocytes from pluripotent cells.

[0330] Analysis of protein expression of endoderm markers was performed on day 5 and is shown in Figure 3 These results show that differentiation proceeds in an order consistent with that expected by a person skilled in the art, that is, via definitive endoderm and towards liver buds.

[0331] Analysis of gene expression during the differentiation protocol is shown in Figure 4a and Figure 4b These results show that differentiation progresses toward a composition consistent with the goal of the cellular composition of the liver, particularly hepatocytes.

[0332] Measurement of amplification rate:

[0333] The expansion 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). The expansion rate or expansion factor is measured during differentiation. Tracking of the expansion rate during the differentiation process is shown in Figure 5 At 20 days after the start of differentiation, an expansion rate of at least 15-fold was found.

[0334] Evaluation of albumin and urea production

[0335] To assess albumin and urea production, conditioned medium was sampled regularly 24 hours (± 2 hours) after medium replacement and stored at -80°C. The 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 secreted molecules within 24 hours was then normalized by the number of cells based on the counts performed after dissociation at each time point.

[0336] The results are shown in Figure 6 and Figure 7 It was found that albumin secretion and urea production were higher in the microcompartments according to the present invention.

[0337] Measurement of CYP3A4 activity :

[0338] Using P450-Glo TMCYP3A4 assay, Cyp3A4 activity was measured with luciferin-IPA (Promega) according to the manufacturer's instructions. In brief, samples of 3D capsules containing microtissues were decapsulated and the number of cells in a capsule containing microtissues of a given volume was determined. The decapsulated microtissues corresponding to 1E5 cells or corresponding to 1E5 cells differentiated in 2D were mixed with 50 μl of substrate proluciferin P450-Glo 3 μM in a 96-well plate with a round bottom and incubated for 3 hours at 37 ° C, 5% CO2. For each condition, 5 replicates were performed, and cell culture medium and iPSC alone 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 minutes, and the luminescence was read using a Spectramax i3x microplate reader (Molecular devices) with an integration time of 1 second per well. Net signal was calculated by subtracting background luminescence from control wells without cells.

[0339] The results are shown in Figure 8 A significantly higher activity of CYP3A4 was observed for cells encapsulated in the microcompartments according to the invention.

[0340] Example 3: Morphological study of microcompartments according to the present invention

[0341] The purpose of this example was to characterize the morphology of cells within microcompartments during differentiation.

[0342] Histology

[0343] Liver microtissue samples were fixed with 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, the samples were embedded in paraffin and sectioned with a microtome to a thickness of 5 μm.

[0344] Hematoxylin-eosin-saffron (HES) staining

[0345] After deparaffinization, the sections were stained with Harris hematoxylin and eosin G. After deparaffinization, the sections were stained with saffron and mounted with Entellan. The cytoplasm was stained pink, the nucleus was stained purple-blue, and the extracellular matrix was stained yellow-pink.

[0346] PAS (Periodic Acid-Schiff) staining

[0347] After removal of paraffin, sections were pretreated with 1% periodic acid and subsequently stained with Schiff's reagent and Mayer's hematoxylin in succession. Glycogen was stained pink and cell nuclei were stained blue-purple.

[0348] The images produced by staining are shown in Fig. 9 Middle. The characteristic cubic shape of the hepatocytes can be clearly seen, as well as the presence of glycogen granules within the microcompartments.

[0349] Immunofluorescence on 3D microtissues

[0350] The microtissues were fixed in 4% paraformaldehyde in PBS containing calcium inside the capsule for 1 hour at room temperature. After rinsing with PBS without Ca2+ to remove the capsule, they were permeabilized in 1% Triton X-100 for 30 to 60 minutes at room temperature. The microtissues were incubated with primary antibody solutions at 4°C with stirring for 72 hours. After washing with PBS, they were incubated with labeled secondary antibodies (Alexa Fluor, Life Technologies) and DAPI solutions at 4°C with stirring overnight and protected from light.

[0351] For structural imaging via phalloidin-DAPI, staining was performed while the capsules were preserved (skipping the decapsulation step prior to permeabilization and washing with PBS containing calcium). Microtissues were incubated with DAPI and phalloidin at 4°C for 72 hours with agitation.

[0352] After washing with PBS, cells were mounted on glass slides at a spacing of 0.5 mm. Images were acquired on a confocal microscope (SP5, Leica).

[0353] Table 1:

[0354] Antibody company Ig types Dilution AFP DAKO rabbit 1:2000 ALB Bethyl goat 1:400 CK19 DAKO Mouse 1:100

[0355] The evolution of microcompartment morphology was performed during differentiation for 30 days. These results are shown in Figures 10a to 10e The presence of specific structures can be observed, such as the formation of a structure similar to a liver bud on day 7, as well as the cell architecture unique to the liver microtissue according to the invention. In fact, at least one lumen, at least one cell in contact with the lumen and the medium outside the microtissue, and at least one cell surrounded only by cells can be seen. In addition to morphological studies, the inventors were also able to characterize the presence of at least 3 different phenotypes of liver cells within the microcompartment 30 days after the start of differentiation ( Fig.11 ).

[0356] Compared with the transplantation of single-cell hepatocytes obtained in 2D, the transplantation of microtissues according to the present invention and in mice Assessment of cell survival after transplantation

[0357] The goal of this study was to inoculate immunocompetent mice with stem cell-derived hepatocytes prepared as cell suspensions or microtissues according to the invention in order to evaluate their survival and functionality in vivo.

[0358] The conditions of the study are described below:

[0359] animal :

[0360] The study was performed on 10 week old female Balb / cByJ mice (Mus musculus).

[0361] Seven (7) animals were housed in an animal room. Ventilation and air handling were performed with frequent changes and the temperature was controlled at approximately 21-22°C. Humidity was maintained at approximately 50%. Artificial lighting was maintained for 12 hours per day. The amount and access to food (pellets) and water (tap water) were monitored daily. Cages were changed weekly and the environment was enriched to minimize anxiety.

[0362] Injection process :

[0363] - On day 42 of hepatic differentiation, cells generated in 2D ("2D hepatocyte differentiation" according to Example 2) were dissociated into single cells and liver microtissues generated according to the invention (according to Example 1) were decapsulated.

[0364] - Microtissue suspension: Use the microtissues according to the invention obtained in Example 1 suspended in culture medium: approximately 3 x 106 cells per tube diluted in 170 μL of basal culture medium. Before inoculation, add 170 μL of

[0365] - To enable subcutaneous inoculation of such a volume, ie 340 μL, a skin incision was made in the right flank of the mouse to prepare a subcutaneous pocket. The microtissue suspension was then injected into this pocket using a pipette, and the wound was then closed with surgical glue to prevent the suspension from spreading.

[0366] - Cell suspension: Use cells obtained according to the protocol "2D hepatocyte differentiation" in Example 2 suspended in culture medium: approximately 3x106 cells per tube in 120 μL basal medium. Before inoculation, add 120 μL of

[0367] To enable subcutaneous inoculation of this 240 μL volume, a skin incision was made in the right flank of the mouse to prepare a subcutaneous pocket. The cell suspension was then injected into this pocket using a pipette, and the wound was then closed with surgical glue to prevent the suspension from spreading.

[0368] -Control solution: 100 μL of basal medium solution (vehicle) and 100 μL Mix. Inject the solution subcutaneously using the same procedure as for the microtissue and cell suspensions.

[0369] sampling :

[0370] Blood samples were collected from all available mice by retro-orbital bleeding for serum preparation (approximately 80 μL):

[0371] - Before cell seeding, day 0, n=7

[0372] - Two days after inoculation, day 2, n=7

[0373] - Six days after inoculation, day 6, n=3

[0374] The graft is harvested as follows, including the surrounding skin to maintain structure:

[0375] - Two days after inoculation, day 2, n=4

[0376] ○ For cell suspension, n = 2

[0377] ○For microtissue, n=2

[0378] - Six days after inoculation, day 6, n=3

[0379] ○For cell suspension, n=1

[0380] ○For microtissue, n=1

[0381] ○For vehicle, n=1

[0382] The grafts were harvested, fixed, paraffin-embedded, sectioned and stained with specific antibodies for HES (hematoxylin-eosin-saffron) and the human cell marker Stem121 (detecting human cytoplasmic protein), the hepatocyte marker albumin and CytK19 (cytokeratin 19).

[0383] Microtissues were also stained with HES, albumin, and CytK19 before transplantation.

[0384] sampling :

[0385] Images obtained by optical microscopy (Leica DM2000 microscope) are shown in Figures 12a to 12d middle:

[0386] -12a:

[0387] * Single cell grafts at day 2 after transplantation into Balb / c mice (recipients 1 and 2, top row),

[0388] *Microtissue grafts at day 2 after transplantation into Balb / c mice (recipients 4 and 5, bottom row).

[0389] -12b:

[0390] * Single cell grafts at day 6 after transplantation into Balb / c mice (recipients, top row),

[0391] *Microtissue grafts at day 6 after transplantation into Balb / c mice (recipient 6, bottom row).

[0392] -12c: Tissue at the injection site in Balb / c mice with vehicle alone (basal medium containing 50% Matrigel) without grafts.

[0393] -12d: Microtissue according to the present invention before transplantation.

[0394] Histological analysis of the grafts on day 2 after transplantation revealed the presence of round microtissues positive for the human cell marker Stem121 in mice transplanted with the liver microtissues according to the present invention, confirming that the detected microtissues were of human origin. Some 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. After in vivo transplantation, the cell architecture and compartmentalization of cells expressing albumin and CytK19 were similar to those observed in the microtissues before transplantation.

[0395] In mice transplanted with 2D-generated single cells, only rare cells positive for human cell markers Stem121, albumin, and cytokeratin 19 were detected on day 2 after transplantation.

[0396] On day 6, the presence of cells expressing Stem121 and CytK19 could still be detected in mice transplanted with microtissues, while no Stem121 or CytK19 positive cells were present in mice transplanted with single cells. Finally, in the areas of immune cell infiltration of mice transplanted with vehicle alone (control), no cells positive for Stem121, albumin or cytokeratin 19 were detected. Sera from transplanted Balb / c mice were analyzed for the presence of human A1AT (before transplantation (day 0), day 2 and day 6 after transplantation). The presence of human A1AT (alpha-1 antitrypsin) was analyzed by ELISA. A1AT secretion by cells and microtissues before transplantation was also analyzed. The results obtained are shown in Fig.13a and Fig.13b middle:

[0397] -13a: Graphical representation of A1AT secretion before transplantation of 2D differentiated single cells and microtissues according to the invention.

[0398] -13b: Graphical representation of A1T detection in Balb / c mouse serum before (day 0), 2 days and 6 days after transplantation of 2D differentiated single cells (SC), microtissues according to the invention (MT) and vehicle alone (control).

[0399] These results show that the liver microtissues according to the present invention are able to secrete higher levels of A1AT than 2D differentiated cells. Consistent with the histological observations, the concentration of human A1AT in the serum of mice transplanted with the microtissues according to the present invention was higher than that of mice transplanted with single cells. On day 0, the concentration of human A1AT was low, or even undetectable. It was highest on day 2 after transplantation and decreased on day 6, but still significant and higher than 2D differentiated single cells. These data are consistent with the histological results and show that the microtissues according to the present invention are more functional than 2D differentiated single cells over time and after transplantation, even in an immunocompetent mouse model.

[0400] Overall, these results show that liver microtissues according to the present invention and transplanted as 3D objects survive transplantation in immunocompetent mice and have better and longer survival after transplantation than 2D-generated liver cells transplanted as single cell suspensions.

Claims

1. A three-dimensional liver microtissue having a maximum dimension between 500 μm and 700 μm, expressing CYP3A4 monooxygenase at an activity of at least 75,000 RLU per million cells and producing at least 18 μg urea per million cells per 24 hours.

2. Liver microtissue according to the preceding claim, characterized in that The liver microtissues contain 50% to 99% liver cells.

3. Liver microtissue according to one of the preceding claims, characterized in that The liver microtissues are obtained from induced pluripotent stem cells.

4. Liver microtissue according to one of the preceding claims, characterized in that The liver microtissues were obtained from induced pluripotent stem cells encapsulated in individual three-dimensional closed microcompartments.

5. Liver microtissue according to one of the preceding claims, characterized in that The liver cells secrete at least 75 μg of albumin per million cells per 24 hours.

6. Liver microtissue according to one of the preceding claims, characterized in that The liver microtissue is obtained from induced pluripotent stem cells and the liver cells secrete at least 75 μg of albumin per million cells per 24 hours for at least 20 days after the start of differentiation.

7. The liver microtissue according to claim 1, comprising at least 3 different phenotypes of liver cells, characterized in that All cells of the microtissue have been obtained from induced pluripotent stem cells encapsulated in a single three-dimensional closed microcompartment.

8. Liver microtissue according to one of the preceding claims, characterized in that The liver microtissue comprises immature hepatocytes, mature hepatocytes and bile duct cells.

9. Liver microtissue according to one of the preceding claims, characterized in that The liver microtissue comprises at least: - Immature hepatocytes characterized by the expression of alpha-fetoprotein and albumin and the absence of cytokeratin 19 expression - mature hepatocytes characterized by albumin expression and the absence of alpha-fetoprotein and cytokeratin 19 expression, and - Cholangiocytes, characterized by the expression of cytokeratins and the absence of albumin and alpha-fetoprotein expression.

10. Liver microtissue according to one of the preceding claims, characterized in that The liver microtissue comprises cells expressing CD73 and CD90.

11. Liver microtissue according to the preceding claim, characterized in that The cells expressing CD73 and CD90 are mesenchymal stem cells.

12. Liver microtissue according to one of the preceding claims, characterized in that The liver microtissue at least comprises liver cells, wherein 20% to 60% of the liver cells are cytokeratin 19 expressing cells.

13. Liver microtissue according to one of the preceding claims, characterized in that All cells of the microtissue have been obtained from the differentiation of at least one cyst of induced pluripotent stem cells encapsulated in a single three-dimensional closed microcompartment.

14. The liver microtissue according to one of the preceding claims, comprising: - at least one inner cavity, - at least one cell in contact with the lumen and the medium outside of said microtissue, and - At least one cell that is surrounded only by cells.

15. Liver microtissue according to one of the preceding claims, characterized in that The liver microtissue has an oval shape.

16. Liver microtissue according to one of the preceding claims, characterized in that The liver cells are polarized.

17. Liver microtissue according to one of the preceding claims, characterized in that The liver microtissue comprises 50% to 99% of liver cells, wherein 20% to 60% of the liver cells are cells expressing cytokeratin 19, and 1% to 20% of the liver cells are cells expressing CD73 and CD90.

18. Liver microtissue according to one of the preceding claims, characterized in that The liver microtissue has a diameter or smallest dimension between 100 μm and 300 μm.

19. Liver microtissue according to one of the preceding claims, characterized in that The liver microtissue has a maximum dimension between 500 μm and 700 μm.

20. Liver microtissue according to one of the preceding claims, characterized in that The liver microtissues contained between 300 and 14,000 cells.

21. Liver microtissue according to one of the preceding claims, characterized in that The liver microtissue comprises at least one bile duct.

22. Liver microtissue according to one of the preceding claims, characterized in that The liver microtissue comprises at least one glycogen granule.

23. Liver microtissue according to one of the preceding claims, characterized in that The liver cells are selected from the group consisting of mature hepatocytes, immature hepatocytes, hepatoblasts, cholangiocytes and mixtures thereof.

24. A three-dimensional closed microcompartment comprising an outer hydrogel layer defining an inner portion, the inner portion comprising at least one liver microtissue according to one of claims 1 to 23.

25. The cell microcompartment according to claim 24, characterized in that The microcompartment is obtained 20 days after encapsulating 1 to 200 induced pluripotent stem cells in the inner portion defined by the outer hydrogel layer.

26. The cell microcompartment according to claim 24 or 25, characterized in that The cell microcompartments exhibited an expansion rate of at least 15-fold 20 days after the start of differentiation.

27. The cell microcompartment according to claim 24, characterized in that The thickness of the outer layer is variable and is between 20 μm and 60 μm.

28. The cell microcompartment according to claim 24, characterized in that The outer layer comprises alginate.

29. The cell microcompartment according to claim 24, characterized in that The cell microcompartment comprises extracellular matrix components or natural or synthetic extracellular matrix in the inner portion between the outer layer and the liver microtissue.

30. The cell microcompartment according to claim 24, characterized in that The cell microcompartments have a diameter or smallest dimension between 300 μm and 400 μm.

31. The cell microcompartment according to claim 24, characterized in that The cell microcompartments have a maximum dimension between 400 μm and 600 μm.

32. A microcompartment assembly comprising at least two three-dimensional cell microcompartments, characterized in that At least one microcompartment is a microcompartment according to one of claims 24 to 31 .

33. Microcompartment assembly according to the preceding claim, characterized in that The microcompartments are arranged in a culture medium in a bioreactor.

34. The microtissue according to one of claims 1 to 23 or the microcompartment according to one of claims 24 to 31 for use as a medicament.

35. The microtissue according to one of claims 1 to 23 or the microcompartment according to one of claims 24 to 31 for use in the prevention or treatment of symptoms associated with liver failure.

36. The microtissue or microcompartment for use according to claim 35, wherein the liver failure is acute liver failure, chronic liver failure or acute-on-chronic liver failure.

37. Microtissue according to one of claims 1 to 23 or microcompartment according to one of claims 24 to 31 for use in the treatment or prevention of metabolic diseases of the liver.

38. The microtissue according to one of claims 1 to 23 or the microcompartment according to one of claims 24 to 31 for use in the treatment or prevention of liver fibrosis and cirrhosis, steatosis, non-alcoholic steatosis, hepatitis, diseases associated with the secretion of factor VIII and factor IX and VWF, Wilson's disease, hereditary hemochromatosis.

39. A method for preparing a microcompartment according to one of claims 24 to 31, comprising the following steps: a) preparing a closed three-dimensional cell microcompartment comprising induced pluripotent stem cells and optionally an extracellular matrix component or a natural or synthetic extracellular matrix within an outer hydrogel layer, -b) inducing cell differentiation in the cell microcompartments to obtain microcompartments containing liver cells.

40. The method according to the preceding claim, characterized in that The cell differentiation method of step b) lasts for at least 20 days The method according to one of claims 39 to 40, characterized in that In step a), 40 to 150 induced pluripotent stem cells are present in the microcompartment The method for preparing liver microtissue according to one of claims 1 to 23, characterized in that The method comprises: - implementing a method for producing microcompartments according to one of claims 24 to 31, - Removing the outer hydrogel layer to recover the liver microtissue.

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