Specific three-dimensional liver microtissue and uses in the treatment of liver failure

Abstract

The invention relates to a specific liver microtissue, the largest dimension of which is between 500 and 700 µm, which expresses monooxygenase CYP3A4 with an activity of at least 75,000 RLU per million cells and produces at least 18 µg of urea per million cells in 24 hours. The invention also relates to a method for preparing a liver microtissue of this kind and to the uses thereof in the treatment or prevention of liver failure.

Description

[0001] SPECIAL THREE-DIMENSIONAL LIVER MICROTISSUE AND USES IN THE TREATMENT OF LIVER FAILURE

[0002] Technical field

[0003] The present invention relates to the treatment of liver failure by using a liver microtissue obtained from particular cellular microcompartments. The invention relates in particular to a particular liver microtissue, its preparation method and its uses.

[0004] Prior art

[0005] The liver is one of the most complex organs in the human body. An integral part of the digestive system, the liver is constantly supplied with nutrients and toxic substances resulting from digestion. The liver's processing of these substances is essential for the body and serves the following purposes:

[0006] - the storage and distribution of nutrients from digestion

[0007] - the degradation of toxic substances

[0008] - the synthesis of most blood proteins, and

[0009] -the production of bile.

[0010] To carry out these functions, the liver is composed of a wide variety of cells, such as hepatocytes, bile duct cells (cholangiocytes), stellate cells (Ito cells), Kupffer cells, mesenchymal stem cells and endothelial cells.

[0011] Hepatocytes are the most represented liver cells and are responsible for the majority of liver functions, from fatty acid synthesis to urea production and plasma protein synthesis.

[0012] Cholangiocytes are the polarized epithelial cells that form the walls of the bile ducts. They play a role in regulating bile secretion and collecting bile for transport from hepatocytes to the intestine.

[0013] Other cells ensure proper vascularization as well as signaling and interaction functions with other liver cells and with cells of the immune system. When the liver can no longer perform its functions, we speak of liver failure. The main causes of liver failure are viral infections, drug overdoses, immunological disorders, hereditary diseases, or blood circulation disorders. When the damage to liver function is irreversible, the recommended treatment is liver transplantation.

[0014] Thus, liver transplantation represents the standard treatment for people with end-stage liver disease.

[0015] Today, there are great difficulties in finding organ donors who can provide a liver of sufficient quality for a transplant.

[0016] Like any organ transplant, liver transplantation can only be performed if the liver is of good quality in order to avoid the risks associated with the transplant such as infections, cancers, prolonged immunosuppression and major surgery.

[0017] Today, only two-thirds of patients can benefit from a transplant, and only when their quality of life has significantly deteriorated. Furthermore, the expected cost of a liver transplant is around €1 million (or $1 million) in the United States.

[0018] Faced with these problems, several innovative solutions have emerged.

[0019] Isolated hepatocyte transplantation has emerged as an attractive approach, however clinical trials have remained few and inconclusive, limited by the poor survival, integration and expansion of isolated hepatocytes following in vivo transplantation, factors consequently limiting short- and long-term therapeutic effects. Indeed, isolated hepatocytes from donors have a very limited capacity for proliferation in vivo and in vitro. In addition, isolated hepatocytes placed in culture tend to enter a process of dedifferentiation, thus reducing the chances of obtaining a sufficient number of mature hepatocytes.

[0020] Although this technique allows for the treatment of a large number of patients, the dose administered is often insufficient and presents a risk when injecting cells into the general circulation.

[0021] This is mainly due to the difficulty for single cells to integrate within the liver, as well as poor quality induced by primary cell preparation and in vitro culture and rejection of some hepatocytes despite immunosuppression. Therefore, the low number of hepatocyte donors, stability and limited functionality of these hepatocytes constitute a barrier to their use.

[0022] The development of protocols for guided differentiation of pluripotent stem cells, i.e. embryonic stem cells and induced pluripotent stem cells, has, however, made it possible to obtain an almost inexhaustible source of hepatocytes.

[0023] Although promising, hepatocytes derived from pluripotent cells are very difficult to cultivate on a large scale and generate excessive production costs. For example, the expected cost for the production of autologous liver grafts from induced pluripotent stem cells is around 9.7 million dollars.

[0024] To date, guided differentiation protocols do not allow the production of a diversity of functional cell phenotypes and in particular sufficient mature hepatocytes; the cells obtained retain characteristics of fetal liver hepatocytes, notably with persistent expression of alpha-fetoprotein as well as low albumin production.

[0025] However, some protocols allow for the production of liver cells with better functional characteristics. These protocols remain complex and require dissociation, reaggregation, or co-culture steps, particularly with mesenchymal stem cells and endothelial cells. These additional steps add additional risks, increasing the overall cost and reducing control over the finished product.

[0026] For application in cell therapy, it is necessary to be able to adapt existing processes to: i) obtain production with a limited number of steps to be effective on a large scale, ii) improve the integration of grafted cells. The techniques developed today do not allow the production of liver microtissue obtained from induced pluripotent stem cells suitable for large-scale culture and presenting functional hepatocytes.

[0027] Current liver microtissue production processes are still too complex, involve multiple steps and therefore a very high cost, and are difficult to scale up.

[0028] There is therefore a significant need for a solution enabling large-scale production of liver microtissue comprising multiple liver cell phenotypes, producible on a large scale and directly transplantable, to meet an essential demand for liver grafts.

[0029] The objective of the invention is therefore to meet all of these needs and to overcome the drawbacks and limitations of the prior art.

[0030] Summary of the invention

[0031] To meet this objective, the invention proposes a particular hepatic microtissue, suitable for use in cell therapy and in particular in the fight against hepatic insufficiency.

[0032] For this purpose, the invention relates to a three-dimensional hepatic microtissue whose largest dimension is between 500 and 700 pm, expressing the monooxygenase CYP3A4 with an activity of at least 75,000 RLU per million cells and producing at least 18 pg of urea per million cells per 24 hours.

[0033] Advantageously, the activity of CYP3A4 associated with the production of urea makes it possible to guarantee a hepatic microtissue comprising at least functional hepatic cells.

[0034] Preferably, liver cells secrete at least 75pg of albumin per million cells per 24 hours.

[0035] Thus, liver microtissue exhibits metabolic activity similar to a healthy liver.

[0036] According to another subject, the invention relates to a hepatic microtissue comprising at least 3 different phenotypes of hepatic cells, said cells of the microtissue having all been obtained from induced pluripotent stem cells encapsulated in a single three-dimensional closed microcompartment.

[0037] Advantageously, hepatic microtissue has sufficient cellular diversity to restore and / or improve liver functions sustainably.

[0038] Preferably, the hepatic microtissue comprises at least immature hepatocytes, mature hepatocytes and cholangiocytes.

[0039] According to a preferred object of the invention, the hepatic microtissue comprises at least:

[0040] - liver cells, of which at least 40% of the liver cells are cells expressing cytokeratin 19 (CK19), and

[0041] - cells expressing CD73 and CD90. Preferably, cells expressing CD73 and CD90 are mesenchymal stem cells and 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 hepatic microtissue according to the invention comprises:

[0044] - at least one lumen,

[0045] - at least one cell of the microtissue in contact with both a lumen and the environment external to the microtissue, and

[0046] - at least one cell surrounded only by cells.

[0047] Advantageously, the organization of the hepatic microtissue is close to the organization of the hepatic tissue during development, which allows it in particular to promote its integration into the liver and the proper execution of metabolic functions in the treated liver. According to a particularly preferred embodiment, the hepatic microtissue is in an ovoid, cylindrical, spheroid or spherical or substantially ovoid, cylindrical, spheroid or spherical or ellipsoidal shape. Preferably, the hepatic microtissue is in an ellipsoidal shape.

[0048] Advantageously, the ellipsoidal shape of the microtissue helps promote the survival of the hepatic microtissue. Thus, a greater proportion of the hepatic microtissues are integrated by the treated liver.

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

[0050] According to a preferred object of the invention, the hepatic microtissue comprises:

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

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

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

[0054] According to another aspect, the invention relates to a three-dimensional closed cellular microcompartment comprising an external hydrogel layer defining an internal part, said internal part comprising at least one hepatic microtissue according to the invention.

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

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

[0057] According to another aspect, the invention relates to a method for preparing a microcompartment or a set of microcompartments comprising at least the implementation of the following steps, the production of a microcompartment comprising induced pluripotent stem cells, the induction of cellular differentiation within the microcompartment so as to obtain at least 3 different phenotypes of hepatic cells.

[0058] Finally, the invention relates to a hepatic microtissue according to the invention or a microcompartment containing it, or a set of hepatic microtissues according to the invention or a set of microcompartments containing them, for its use as a medicament, preferably in the prevention or treatment of hepatic insufficiency caused by diseases such as hepatic fibrosis and cirrhosis, steatosis, non-alcoholic steatosis, hepatitis, metabolic liver diseases, diseases linked to secretion of factors VIII, alpha 1 antitrypsin, IX and / or VWF, Wilson's disease and hereditary hemochromatosis.

[0059] Other features and advantages will emerge from the detailed description of the invention and the examples which follow.

[0060] Brief description of the figures

[0061] Figure 1a is a comparative representation of the expression of genes of interest (EÛMES, CXCR4, HHEX, PROXI, AFP, ASGR1) during differentiation depending on the differentiation protocol.

[0062] Figure 1b is a comparative representation of the expression of genes of interest (SOX17, FOXA2, TBX, HNF4A, ALB, KT18) during differentiation depending on the differentiation protocol. Figure 1c is a comparative representation of the expression of genes of interest (GATA4, HNF1B, SOX9, KT19, TAT) during differentiation depending on the differentiation protocol.

[0063] Figure 2 is a comparative representation of the expression of genes of interest (EÛMES, CXCR4, FOXA2, SOX17) as a function of culture conditions.

[0064] Figure 3 is a comparative representation of the expression of proteins of interest (SOX17, FOXA2) as a function of 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) as a function of culture conditions.

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

[0067] Figure 5 is a comparative representation of the expansion factor over 30 days as a function of culture conditions.

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

[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] Figure 9 is a set of images obtained by confocal microscopy of a microcompartment showing hepatocytes.

[0072] Figure 10a is a set of images obtained by confocal microscopy of a microcompartment from D-0 to D-9 after the start of differentiation.

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

[0074] Figure 10c is a set of images obtained by confocal microscopy of a microcompartment of J-15 after the start of differentiation.

[0075] Figure 10d is a set of images obtained by confocal microscopy of a microcompartment of J-20 after the start of differentiation.

[0076] Figure 10e is a set of confocal microscopy images of a microcompartment of J-30 after the start of differentiation. Figure 11 is a set of confocal microscopy images of a microcompartment identifying some liver cell phenotypes.

[0077] Figure 12a is a set of images obtained by confocal microscopy:

[0078] - single-cell grafts on day 2 (recipients 1 and 2, top row) after transplantation into Balb / c mice,

[0079] - of microtissue grafts on day 2 (recipients 4 and 5, bottom row) after transplantation into Balb / c mice.

[0080] Figure 12b is a set of images obtained by confocal microscopy:

[0081] - single-cell grafts on day 6 (recipients, top row) after transplantation into Balb / c mice,

[0082] - of microtissue grafts on day 6 (recipient 6, bottom row) after transplantation into Balb / c mice.

[0083] Figure 12c is a set of images obtained by confocal microscopy of the tissue at the injection site with vehicle alone (basal medium containing 50% matrigel) without graft in Balb / c mice.

[0084] Figure 12d is a set of images obtained by confocal microscopy of microtissues according to the invention before transplantation.

[0085] Figure 13a is a graphical representation of AlAT (alpha-1 antitrypsin) secretion prior to transplantation of 2D differentiated single cells (2D) and microtissues according to the invention (Invention).

[0086] Figure 13b is a graphical representation of AlT (alpha-1 antitrypsin) detection in serum of Balb / c mice before (day 0), at day 2 and at day 6 after transplantation with 2D differentiated single cells (SC), with microtissues of the invention (MT) and with vehicle alone (control).

[0087] Detailed description of the invention

[0088] Definitions

[0089] For the purposes of the invention, the term "alginate" means linear polysaccharides formed from pD-mannuronate and aL-guluronate, salts and derivatives thereof.

[0090] For the purposes of the invention, the term “hydrogel capsule” or “hydrogel microcompartment” means a three-dimensional structure formed from a matrix of polymer chains, swollen by a liquid and preferably water.

[0091] For the purposes of the invention, the term "human cells" means human cells or immunologically humanized non-human mammalian cells. Even when not specified, the cells, stem cells, progenitor cells and tissues according to the invention consist of or are obtained from human cells or from immunologically humanized non-human mammalian cells.

[0092] For the purposes of the invention, the term "embryonic stem cell" means a pluripotent stem cell derived from the inner cell mass of the blastocyst. The pluripotency of embryonic stem cells can be assessed by the presence of markers such as the transcription factors OCT4, NANOG and SOX2 and surface markers such as SSEA3 / 4, Tra-1-60 and Tra-1-81. The embryonic stem cells used in the context of the invention are obtained without destroying the embryo from which they originate, for example using the technique described in Chang et al. (Cell Stem Cell, 2008, 2(2)): 113-117). Alternatively, embryonic stem cells from humans can be excluded.

[0093] By "a cell surrounded only by cells" in a microtissue within the meaning of the invention is meant a cell which is neither in contact with a lumen nor in contact with the exterior of the microtissue.

[0094] For the purposes of the invention, the term “mutant cell” means a cell carrying at least one mutation.

[0095] For the purposes of the invention, the term "pluripotent stem cell" or "pluripotent cell" means a cell that has the capacity to form all the tissues present in the entire original organism, without being able to form an entire organism as such. Human pluripotent stem cells may be referred to as hPSCs in the present application. In particular, they may be induced pluripotent stem cells (iPSCs or hiPSCs for human induced pluripotent stem cells), embryonic stem cells or MUSE cells (for "Multilineage-differentiating Stress Enduring").

[0096] For the purposes of the invention, the term "induced pluripotent stem cell" means a pluripotent stem cell induced to pluripotency by genetic reprogramming of differentiated somatic cells. These cells are in particular positive for pluripotency markers, such as alkaline phosphatase staining and expression of the proteins NANOG, SOX2, OCT4 and SSEA3 / 4. Examples of methods for obtaining induced pluripotent stem cells are described in the articles Yu et al. (Science 2007, 318 (5858): 1917-1920), Takahashi et al (Cell, 207, 131(5): 861-872) and Nakagawa et al (Nat Biotechnol, 2008, 26(1): 101-106).

[0097] For the purposes of the invention, the term “progenitor cell” means a stem cell already engaged in differentiation into liver cells but not yet differentiated.

[0098] By "Féret diameter" of a microcompartment (or of a part of a microcompartment) or of a microtissue according to the invention, is meant the distance "d" between two tangents to said microcompartment (or to said part) or of the microtissue, these two tangents being parallel, such that the entire projection of said microcompartment (or of said part) or of the microtissue is between these two parallel tangents. A Féret diameter of the internal part of the microcompartment is measured between two interfaces of the internal part and the external layer of the microcompartment, i.e. the distance "d" between two tangents to said internal part, these two tangents being parallel, such that the entire projection of said internal part is between these two parallel tangents.

[0099] For the purposes of the invention, the term "variable thickness" of a layer means that the layer for the same microcompartment or the same microfabric does not have the same thickness everywhere.

[0100] For the purposes of the invention, the term “microcompartment” or “capsule” means a partially or totally closed three-dimensional structure containing several cells.

[0101] For the purposes of the invention, the term “microtissue” or “liver microtissue” means a three-dimensional human tissue comprising at least liver cells and the largest dimension of which is less than 1 mm.

[0102] For the purposes of the invention, the term "medium" means an aqueous solution containing cells or microtissues, compatible with the survival, development and / or metabolism of the cells. It may be a culture medium.

[0103] For the purposes of the invention, the term “convective culture medium” means a culture medium driven by internal movements.

[0104] For the purposes of the invention, the term "mutation" means a genetic or epigenetic mutation, preferably a functional mutation. In particular, it may be a specific modification of the genetic sequence, a structural variant, an epigenetic modification, or a modification of mitochondrial DNA.

[0105] For the purposes of the invention, the term "functional mutation" means a transmissible genetic or epigenetic modification which confers a gain or loss of function or potential loss of function to the mutant cell concerned. This is preferably a mutation resulting in a modification of the phenotype of the mutant cell concerned. Very preferably, it is a change in the sequence of the genome and / or the epigenome which alters the therapeutic potential of a population of cells, either by increasing the risk associated with the therapy produced or by reducing the benefit provided by the therapy produced.

[0106] By "smallest dimension" of a microcompartment or a layer of cells within the meaning of the invention, we mean the value of the smallest Feret diameter of said microcompartment. By "light" or "lumen" within the meaning of the invention, we mean a volume of aqueous solution topologically surrounded by cells. Preferably, its contents are not in diffusive equilibrium with the volume of convective liquid present outside the microcompartment.

[0107] By "smallest radius" of the internal part of a microcompartment according to the invention, we mean half the value of the smallest Féret diameter of the internal part of the microcompartment.

[0108] By "average radius" of the internal part of a microcompartment according to the invention, we mean the average of the radii of the smallest compartment, each radius corresponding to half the value of a Féret diameter of the internal part of the microcompartment.

[0109] By "expansion rate at X days" according to the invention, we mean 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 the culture divided by the number of cells at the start of the culture (day of encapsulation or day of culturing).

[0110] By "large-scale culture" according to the invention, we mean a cell culture method suitable for a production batch of hepatic microtissue making it possible to treat at least 1 patient, preferably 10 patients, more preferably 100 patients, even more preferably more than 1000 patients.

[0111] By "functional phenotype" of a microtissue according to the invention is meant the presence of mature hepatocytes characterized by the expression of albumin and the absence of expression of alphafetoprotein and cytokeratin 19. By "liver bud" according to the invention is meant a cellular organization characterized by a cellular extension of the endoderm of the embryonic foregut which gives rise to the parenchyma of the liver and the bile duct. This is a particular conformation giving rise to hepatocytes and cholangiocytes.

[0112] According to the invention, the term "URL" means the relative unit of light. It corresponds to the measurement of the quantity of light produced by a bioluminescence reaction. This measurement can be carried out using a luminometer.

[0113] Liver microtissue

[0114] The invention therefore relates to a three-dimensional hepatic microtissue whose largest dimension is between 500 and 700 pm, expressing the monooxygenase CYP3A4 with an activity of at least 75,000 URLs per million cells and producing at least 18 pg of urea per million cells per 24 hours.

[0115] Cytochromes P450 are hemoproteins involved in the oxidative metabolism of many molecules. Cytochrome P450 are enzymes involved in the biotransformation of exogenous compounds, both in detoxification and intoxication phenomena through the formation of reactive entities. The most abundant human hepatic form (CYP3A4) is responsible for the metabolism of more than 60% of drugs. Its presence within the microcompartment is a functional guarantee. Urea is a nitrogenous product resulting from protein catabolism. It is exclusively synthesized in the liver via the urea cycle and the amount of urea formed depends on the amount of ingested proteins, protein catabolism and the state of liver function.A CYP3A4 activity of at least 75,000 RLU per million cells combined with a urea production of at least 18 pg per million cells per 24 hours ensures the presence of mature hepatocytes and sufficient metabolic activity to ensure a significant effect on affected liver functions.

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

[0117] According to a preferred embodiment, the hepatic microtissue produces at least 40pg of urea per million cells per 24 hours, even more preferably at least 60pg, in particular at least 80pg. Advantageously, the hepatic microtissue according to the invention has a metabolic activity close to that of a healthy liver. Thus, the hepatic microtissue is particularly effective in restoring the functions of the treated liver.

[0118] Preferably, the hepatic microtissue according to the invention comprises between 50 and 99% of hepatic cells.

[0119] The liver microtissue according to the invention can be obtained from induced pluripotent stem cells.

[0120] Alternatively, 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 enclosed microcompartment.

[0121] Hepatic microtissue preferentially comprises hepatic cells secreting at least 75pg of albumin per million cells per 24 hours, even more preferentially at least 150pg, in particular at least 250pg.

[0122] According to a particular embodiment, the hepatic microtissue is obtained from induced pluripotent stem cells and comprises hepatic cells secreting at least 75 pg of albumin per million cells per 24 hours at least 20 days after the start of differentiation.

[0123] Albumin is the most abundant protein in the blood. Produced by the liver, albumin is responsible for stabilizing blood pressure and transporting many substances.

[0124] Advantageously, an albumin production of at least 75pg per million cells per 24 hours is an indicator of the proper functioning of the hepatic microtissue.

[0125] According to another preferred embodiment, the liver microtissue comprises at least 3 different phenotypes of liver cells, all of the cells of the microtissue having been obtained from induced pluripotent stem cells encapsulated in a single three-dimensional enclosed microcompartment.

[0126] Preferably, the hepatic microtissue comprises at least 4 different phenotypes of hepatic cells, even more preferably at least 5.

[0127] Advantageously, the hepatic microtissue according to the invention has significant cellular diversity making it possible to reproduce the hepatic microenvironment. The diversity and their proximity of the cells present in the hepatic microtissue allows a significant number of cellular interactions thus cooperating in the performance of numerous metabolic and transport functions.

[0128] The cellular diversity found in the liver microtissue according to the invention is obtained from induced pluripotent stem cells in a single, three-dimensional, closed microcompartment. In this way, the different cell types can organize themselves within the microcompartment reproducing the hepatic microenvironment.

[0129] According to a preferred embodiment, all the cells of the microtissue have all been obtained by differentiation of induced pluripotent stem cells encapsulated in a single three-dimensional closed microcompartment, preferably said induced pluripotent stem cells all being of the same lineage.

[0130] Very preferably all the cells of the microtissue were all obtained from induced pluripotent stem cells encapsulated in a single three-dimensional closed microcompartment, by a single differentiation process implemented in said microcompartment.

[0131] Preferably, the induced pluripotent stem cells before differentiation into microtissue according to the invention form a cyst in the microcompartment. Also, preferably, all the cells of the microtissue have all been obtained from at least one cyst of induced pluripotent stem cells encapsulated in a single microcompartment closed in three dimensions, preferably by differentiation, in particular by a single differentiation method implemented in said microcompartment.

[0132] Advantageously, the microenvironment of the microcompartment from which the microtissue according to the invention is obtained reproduces the conditions of hepatic organogenesis. Indeed, the microcompartment according to the invention makes it possible to limit the various physical constraints and / or stress and promotes interactions between the different cell types within the microcompartment.

[0133] According to a particularly suitable embodiment, the hepatic microtissue according to the invention comprises at least immature hepatocytes, mature hepatocytes and cholangiocytes. Preferably, it comprises at least:

[0134] - immature hepatocytes, characterized by the expression of alphafetoprotein (AFP) and albumin (ALB) and the absence of expression of cytokeratin 19 (CK19)

[0135] - mature hepatocytes characterized by the expression of albumin and the absence of expression of alphafetoprotein and cytokeratin 19

[0136] - cholangiocytes characterized by the expression of cytokeratin 19 and the absence of expression of albumin and alphafetoprotein.

[0137] Alternatively, the liver microtissue of the invention comprises hepatoblasts. The hepatoblasts may be characterized by the expression of alpha-fetoprotein, albumin, and cytokeratin 19.

[0138] The presence of these hepatic cell phenotypes guarantees a multifactorial effect on liver functions. This effect can help restore or optimize certain liver functions.

[0139] In a particular embodiment, the hepatic microtissue comprises at least 50% mature and / or immature hepatocytes.

[0140] According to a particularly suitable embodiment, the hepatic microtissue comprises within the hepatic cells between 20 and 60% (in number) of cells expressing cytokeratin 19.

[0141] Cells expressing cytokeratin 19 are preferentially cholangiocytes.

[0142] In the context of the invention, the hepatic cells are preferentially chosen from mature hepatocytes, immature hepatocytes, hepatoblasts, cholangiocytes and mixtures thereof.

[0143] Liver microtissue may also include cells expressing CD73 and CD90. Cells expressing CD73 and CD90 are preferentially mesenchymal stem cells.

[0144] Mesenchymal stem cells are a cell population well known for their properties on tissue repair and regeneration, particularly in the liver. Advantageously, the presence of mesenchymal stem cells derived from induced pluripotent stem cells of the patient to be treated ensures effective tissue repair in livers with hepatic insufficiency.

[0145] Thus, in a particular embodiment, the microfabric according to the invention comprises at least:

[0146] - hepatic cells, preferably at least immature hepatocytes, mature hepatocytes, cholangiocytes, and

[0147] - cells expressing CD73 and CD90, preferably at least mesenchymal stem cells.

[0148] Preferably, the hepatic microtissue comprises at least:

[0149] - liver cells of which between 20 and 60% (in number) of the cells are cells expressing cytokeratin 19, and

[0150] - cells expressing CD73 and CD90.

[0151] Hepatic microtissue preferentially comprises (number percentages):

[0152] - between 50 and 99% of liver cells, of which preferably between 20 and 60% (in number) of the cells are cells expressing cytokeratin 19, and

[0153] - between 1 and 20% of cells expressing CD73 and CD90.

[0154] The hepatic microtissue according to the invention may also comprise other cells, such as in particular Ito cells (stellate cells), Kupffer cells, endothelial cells, hepatoblasts. Thus, in another particular embodiment, the hepatic microtissue comprises:

[0155] - mature hepatocytes, immature hepatocytes, hepatoblasts, cholangiocytes,

[0156] - Ito cells and / or Kupffer cells and / or endothelial cells,

[0157] - and possibly mesenchymal stem cells.

[0158] According to one variant, the hepatic microtissue according to the invention may also comprise smooth muscle cells and / or fibroblasts.

[0159] The phenotypes of cells included in the liver microtissue are preferentially compatible with the hepatic microenvironment.

[0160] The cellular diversity provided by the hepatic microtissue according to the invention makes it possible to repair and regenerate the diseased liver in order to restore the affected functions.

[0161] The concentration of mature hepatocytes, cholangiocytes and mesenchymal stem cells ensures a long-lasting effect during cell therapy. It is particularly important during culture to achieve a mature and / or immature hepatocyte content above 50% in order to achieve a sufficient effect as soon as possible after microtissue transplantation.

[0162] A concentration of mature and / or immature hepatocytes below 50% would limit the effectiveness of the graft and would require a graft volume that is all the greater as the concentration (in number) of hepatocytes is low. Indeed, it is estimated that at least 5% of the liver mass in functional hepatocytes is necessary for the treatment of acute liver failure, for example, and metabolic liver failures such as diseases linked to secretion of factor VIII and factor IX and VWF, Wilson's disease and hereditary hemochromatosis require the same order of magnitude.

[0163] Mature hepatocytes express albumin but do not express alpha-fetoprotein. These markers are easily identifiable and quantifiable by detection methods well known to those skilled in the art such as flow cytometry.

[0164] Particularly preferably, the hepatic microtissue comprises at least one lumen, at least one cell in contact with both a lumen and the environment external to the microtissue and at least one cell surrounded only by cells.

[0165] In the context of the invention, such an organization is characteristic of a functional microtissue ready to be used in cell therapy.

[0166] The particular organization of the microtissue is possible in particular thanks to the presence of polarized hepatic cells which make it possible to structure and organize the microtissue.

[0167] 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.

[0168] The liver microtissue according to the invention does not comprise human embryonic stem cells.

[0169] The hepatic microtissue according to the invention can be in ellipsoidal form.

[0170] Advantageously, the ellipsoidal shape of the microtissue allows for the survival and integration of the hepatic microtissue to be promoted. When the hepatic microtissue is injected into the general circulation, its ellipsoidal shape allows it to facilitate its flow in the blood vessels in the case of administration by vascular injection (in a realization via the portal vein) but also their flow within a cannula if the administration is done by intratissue graft. The improvement in the injection of the hepatic microtissue leads to an improvement in the integration of the hepatic microtissue by the liver of the treated patient.

[0171] The hepatic microtissue according to the invention preferably has a diameter or a smaller dimension of between 100 and 300 pm, preferably between 150 and 280 pm. The largest dimension of the hepatic microtissue is preferably less than 1 mm, and is very preferably between 500 and 700 pm.

[0172] Advantageously, the size of the hepatic microtissue is suitable for administration via the portal vein.

[0173] The hepatic microtissue according to the invention may comprise between 300 and 14,000 cells, preferably between 500 and 8,000, even more preferably between 900 and 5,000, in particular 4,500 cells.

[0174] Preferably, the liver microtissue comprises at least one bile duct. Bile ducts collect bile produced by liver cells and transport it to the gallbladder. The presence of at least one bile duct within the liver microtissue promotes proper functioning of the microtissue within the liver and the reconstruction of defective bile ducts.

[0175] According to another embodiment, the hepatic microtissue comprises at least one glycogen granule. Glycogen serves as a storage for carbohydrates in the body and is mainly stored in the liver. Under the action of insulin, liver cells store glucose in the form of glycogen. Under the action of glucagon, the liver cells hydrolyze the glycogen and release the glucose into the blood. The presence of glycogen granules in the liver cell is a physiological element of the functioning of its metabolism and therefore of its function. Thus, the presence of at least one glycogen granule in the hepatic microtissue guarantees favorable conditions for the functioning of the hepatic microtissues for an optimal therapeutic effect.

[0176] Preferably, the hepatic microtissue comprises at least one bile duct and at least one glycogen granule.

[0177] The hepatic microtissue according to the invention can be obtained by differentiation of induced pluripotent stem cells at least 20 days, preferably at least 30 days after encapsulation in a three-dimensional closed microcompartment of 1 to 200 induced pluripotent stem cells, preferably between 5 and 150, in particular between 15 and 80. Preferably, the hepatic microtissue can be obtained from encapsulation in a three-dimensional closed microcompartment of 1 to 200 induced pluripotent stem cells, preferably between 5 and 150, in particular between 15 and 80. Preferably, the induced pluripotent stem cells form a cyst in the microcompartment before differentiation into cells of the hepatic microtissue according to the invention.

[0178] The invention also relates to a set of hepatic microtissues comprising at least one hepatic microtissue according to the invention. Preferably, it is a set of several hepatic microtissues in three dimensions in a medium, of which at least one hepatic microtissue is a hepatic microtissue according to the invention.

[0179] According to a variant, at least 50% (in number) of the hepatic microtissues of the set of hepatic microtissues are hepatic microtissues according to the invention.

[0180] The set of hepatic microtissues according to the invention is suitable for administration to patients with hepatic insufficiency. Administration can be carried out by injection in a biocompatible solution. The injection can be carried out in the portal vein so as to reach the liver without dispersion in the general circulation, directly in the liver or ectopically. When the injection is ectopic it can be carried out in the abdomen or under the renal capsule.

[0181] Preferably, the effective quantity of the microtissue set injected into the patient corresponds to a mass between 1 and 20% of the mass of the liver of the patient treated, preferably between 2 and 10%, in particular 5%.

[0182] According to another aspect, the invention relates to a microcompartment comprising at least one hepatic microtissue according to the invention.

[0183] The microcompartment according to the invention comprises an outer hydrogel layer. Preferably, the hydrogel used is biocompatible, i.e., it is not toxic to the cells. The outer hydrogel layer must allow the diffusion of oxygen and nutrients to feed the cells contained in the microcompartment and allow their survival. According to one embodiment, the outer hydrogel layer comprises at least alginate. It may consist exclusively of alginate. The alginate may in particular be a sodium alginate, composed of 80% α-L-guluronate and 20% β-D-mannuronate, with an average molecular mass of 100 to 400 kDa and a total concentration of between 0.5 and 5% by mass. The outer hydrogel layer is devoid of cells.

[0184] The external hydrogel layer helps protect cells from the mechanical stress of bioreactors and limits potentially toxic molecules when they accumulate in the environment.

[0185] The average thickness of the outer layer can be variable. It is preferably between 20 and 60 pm, more preferably between 30 and 40 pm. The ratio between the smallest radius of the inner part and this thickness is preferably between 2 and 10 pm.

[0186] The microcompartment according to the invention advantageously has an expansion rate of induced pluripotent stem cells of at least 15 times, 20 days after the start of differentiation.

[0187] The invention thus promotes amplification with a high expansion rate, which consequently reduces the culture time to obtain a functional microtissue.

[0188] The microcompartment according to the invention can be obtained after encapsulation of induced pluripotent stem cells with or without the addition of extracellular matrix, natural or synthetic.

[0189] According to a variant, the microcompartment according to the invention comprises in its internal part extracellular matrix such as Matrigel® and / or Geltrex® and / or a hydrogel type matrix of plant origin such as modified alginates or of synthetic origin or copolymer of poly(N-isopropylacrylamide) and poly(ethylene glycol) (PNIPAAm-PEG) type Mebiol®.

[0190] Alternatively, the microcompartment of the invention may be obtained after encapsulation of induced pluripotent stem cells without the addition of any extracellular matrix element. The extracellular matrix elements may be peptide or peptidomimetic sequences, protein mixtures, extracellular compounds or structural proteins, such as collagen, laminins, entactin, vitronectin, as well as growth factors or cytokines.

[0191] Within the microcompartment, induced pluripotent stem cells differentiate into liver tissue for a period of at least 20 days, preferably at least 30 days.

[0192] Unexpectedly, the microcompartment provides a microenvironment favorable to the development of hepatic microtissue. Indeed, during differentiation within the microcompartment, cells organize to form a structure similar to a liver bud. This liver bud is found during liver organogenesis in vivo; it is this particular structure that gives rise to hepatocytes and cholangiocytes. The presence of a structure similar to a liver bud during the formation of hepatic microtissue is an indicator of good tissue quality.

[0193] Thus, the hepatic microtissue according to the invention is preferentially obtained after the formation of a structure similar to a hepatic bud in a single closed three-dimensional microcompartment.

[0194] The microcompartment according to the invention makes it possible to isolate the induced pluripotent stem cells from the mechanical constraints present within the bioreactor. This mechanical isolation allows the microtissue to establish and maintain a topology that approximates the spatial and structural organization of the hepatic organogenesis existing in vivo.

[0195] Unexpectedly, after at least 20 days, preferably 30 days, of differentiation within the microcompartment, the cells retain their conformation, thus allowing for better proliferation while maintaining a functional phenotype. Consequently, this makes it possible to reduce the number of passages and reduce the culture time required to reach the final number of liver cells required.

[0196] The cells present in the microcompartment according to the invention were preferably obtained after at least two cycles of cell division after encapsulation in an external layer of hydrogel of 1 to 200 induced pluripotent stem cells, preferably between 5 and 150, in particular between 15 and 80.

[0197] Preferably, the cells present in the microcompartment according to the invention have been obtained after at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 28, 30 cycles of cell division after encapsulation in an external hydrogel layer, preferably at least 5, even more preferably at least 6, to obtain up to 8000 mature hepatocytes per microcompartment. For example, the mature hepatocytes present in the microcompartment have been obtained after at least six cycles of cell division after encapsulation of the cells in the external hydrogel layer.

[0198] Preferably the number of cell divisions for implementing the method according to the invention is less than 100, even more preferably less than 30.

[0199] Preferably the microcompartment is obtained after at least 2 passes after encapsulation, more preferably at least 3, 4 or 5 passes. Each pass can last for example between 2 and 10 days, in particular between 2 and 4 days.

[0200] Preferably, the microcompartment is obtained after at least one re-encapsulation, more preferably between 1 and 14 re-encapsulations, in particular between 2 and 7 re-encapsulations. Very preferably, one re-encapsulation corresponds to a new passage and each encapsulation cycle corresponds to one passage.

[0201] Preferably, the microcompartment according to the invention was obtained in less than 30 days after encapsulation, even more preferably in less than 20 days after encapsulation of at least 1 induced pluripotent stem cell in the internal part defined by the external hydrogel layer, preferably 5, 20 and up to 100.

[0202] The microcompartment according to the invention can contain between 100 and 14,000 cells, preferably between 300 and 10,000 cells, even more preferably between 300 and 5,000, more particularly at least 50 mature hepatocytes and at least 20 cholangiocytes. Advantageously, the microcompartment according to the invention protects the induced pluripotent stem cells from mechanical stress, thus making it possible to obtain an expansion rate particularly suited to large-scale culture.

[0203] The microcompartment according to the invention can be in any three-dimensional form, that is to say it can have the shape of any object in space. The microcompartment can have any shape compatible with the encapsulation of cells. Preferably, the microcompartment according to the invention is in a spherical or elongated or ellipsoidal or substantially spherical or elongated form. It can have the shape of an ovoid, a cylinder, a spheroid or a sphere or substantially this shape.

[0204] It is the outer layer of the microcompartment, i.e. the hydrogel layer, which gives the microcompartment according to the invention its size and shape. Preferably, the smallest dimension of the microcompartment according to the invention is between 150 μm and 500 μm, preferably between 200 μm and 450 μm.

[0205] Its largest dimension is preferably greater than 350 pm, more preferably between 350 pm and 600 pm.

[0206] The microcompartment according to the invention can optionally be frozen for storage. It should then preferably be defrosted before use.

[0207] The invention also relates to several microcompartments according to the invention used together.

[0208] The invention also relates to a set or series of microcompartments comprising at least two three-dimensional cellular microcompartments, characterized in that at least one microcompartment is a microcompartment according to the invention.

[0209] Preferably, the series of microcompartments according to the invention is in a culture medium, in particular in an at least partially convective culture medium. Any culture medium suitable for the culture of induced pluripotent stem cells can be used, such as for example the medium “HCM Hepatocyte Culture Medium BulletKit (Lonza)” or “William’s Medium E (Thermofisher Scientific)” provided that the concentration of dissolved salts is compatible with the maintenance of the crosslinking of the alginate by the divalent cations.

[0210] According to a particularly suitable embodiment, the invention relates to a series of cellular microcompartments in a closed enclosure, such as a bioreactor, preferably in a culture medium in a closed enclosure, such as a bioreactor. Thus, preferably, the microcompartments are arranged in a culture medium in a closed bioreactor.

[0211] The set or series of microcompartments according to the invention preferably comprises between 2 and 1016 microcompartments.

[0212] Thus, the microcompartment according to the invention is suitable for large-scale culture by providing hepatic microtissues exhibiting a functional phenotype, particularly with regard to their detoxifying (particularly CYP3A activity) and secretory (particularly albumin secretion) capacities, guaranteeing optimal efficacy in vivo.

[0213] Use of the hepatic microtissue according to the invention

[0214] The hepatic microtissue or microcompartment according to the invention can be used for all applications, in particular as a medicine, in particular in cell therapy in humans.

[0215] Thus, the subject of the invention is a hepatic microtissue according to the invention or a set of hepatic microtissues comprising at least one hepatic microtissue according to the invention, or a microcompartment according to the invention or a set of microcompartments comprising at least one microcompartment according to the invention, for its use as a medicament, in particular in cell therapy. The invention also relates to a method of therapeutic treatment which consists of grafting and / or injecting microtissues according to the invention into humans, in particular as a treatment in cell therapy.

[0216] Preferably, the hepatic microtissue or microcompartment according to the invention can be used in the prevention or treatment of symptoms associated with liver failure, in particular acute, chronic or acute-on-chronic liver failure. Preferably, the hepatic microtissue or microcompartment according to the invention can be used in the prevention or treatment of diseases such as fibrosis, cirrhosis, steatosis, non-alcoholic steatosis, hepatitis, metabolic liver diseases such as diseases linked to secretion of factor VIII and factor IX and VWF, Wilson's disease and hereditary hemochromatosis. Indeed, all these indications can be treated by liver transplantation and this is precisely what the transplantation of a set of microtissues according to the invention restoring liver functions is intended to solve.

[0217] Thus, the subject of the invention is a hepatic microtissue according to the invention or a set of hepatic microtissues comprising at least one hepatic microtissue according to the invention, or a microcompartment according to the invention or a set of microcompartments comprising at least one microcompartment according to the invention, for its use in the prevention or treatment of symptoms associated with hepatic insufficiency, in particular acute, chronic or acute-on-chronic hepatic insufficiency, in particular in the prevention or treatment of hepatic diseases such as fibrosis, cirrhosis, steatosis, non-alcoholic steatosis, hepatitis, metabolic diseases of the liver such as diseases linked to secretion of factor VIII and factor IX and VWF.

[0218] The invention also relates to a hepatic microtissue according to the invention or a set of hepatic microtissues comprising at least one hepatic microtissue according to the invention, or a microcompartment according to the invention or a set of microcompartments comprising at least one microcompartment according to the invention, for its use in the evaluation of molecules or in the modeling of hepatic diseases.

[0219] Method for preparing microcompartments and microtissues according to the invention The invention also relates to a method for preparing microcompartments according to the invention. The method for preparing a microcompartment or a set of microcompartments according to the invention may comprise the following steps:

[0220] -(a) preparation of a closed three-dimensional cellular microcompartment comprising, inside an external hydrogel layer, induced pluripotent stem cells, and optionally extracellular matrix elements or a natural or synthetic extracellular matrix,

[0221] -(b) inducing cellular differentiation within the cellular microcompartment, so as to obtain a microcompartment comprising hepatic cells.

[0222] The preparation of the microcompartment of step (a) can be carried out in any culture medium suitable for the culture of induced pluripotent stem cells such as mTeSR™l or mTeSRPIus media from Stemcell technologies, StemMACS™ iPS-Brew XF (Miltenyi Biotec or StemFlex from Thermofisher Scientific).

[0223] The microcompartment of step (a) can be obtained by encapsulation of 1 to 150 induced pluripotent stem cells, preferably at least 50, in particular at least 100.

[0224] Preferably, the encapsulation is carried out according to techniques known to those skilled in the art. Indeed, any method for producing cellular microcompartments containing cells and an external layer of hydrogel inside can be used for implementing the preparation method according to the invention. In particular, it is possible to prepare microcompartments by adapting the method and the 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, p. 1593-1604), in accordance with the steps described below.

[0225] Preferably, step (a) is implemented by adding extracellular matrix elements or a natural or synthetic extracellular matrix. According to a variant, step (a) is implemented without adding extracellular matrix elements or extracellular matrices or a natural or synthetic extracellular matrix.

[0226] In the context of the invention, step (a) is preferably implemented in a device capable of generating hydrogel capsules using a microfluidic chip. For example, the device may comprise syringe pumps for several solutions injected concentrically using a microfluidic injector which makes it possible to form a jet which splits into drops then collected in a calcium bath. According to a particularly suitable embodiment, two or three solutions loaded onto two or three syringe pumps:

[0227] - a hydrogel solution, for example alginate,

[0228] - optionally an isotonic intermediate solution, preferably an isotonic solution not containing a divalent cation such as Ca2+ Mg2+ to avoid crosslinking of the hydrogel too early in the injector, such as for example a sorbitol solution,

[0229] - a solution comprising induced pluripotent stem cells and culture medium The three solutions are co-injected (injected simultaneously) concentrically using a microfluidic injector or microfluidic chip which makes it possible to form a jet which splits into drops whose outer layer is the hydrogel solution and the core the solution from step (a) comprising the induced pluripotent stem cells; These drops are collected in a calcium bath which crosslinks and / or gels the alginate solution to form the shell.

[0230] To improve the mono-dispersity of the cellular microcompartments, the hydrogel solution is preferably charged with a direct current at (between 1 and 10 kV). A grounded ring can optionally be placed at a distance from the tip of between 1 mm and 20 cm, preferably 3 mm to 10 cm, even more preferably 1 cm to 5 cm, from the tip in the plane perpendicular to the axis of the jet exiting the microfluidic injector (coextrusion chip) to generate the electric field.

[0231] According to the invention, it is necessary to generate capsules whose internal part has a mean radius or smaller radius of at least 100 pm. To generate capsules with such dimensions with a coextrusion chip (microfluidic injector or microfluidic chip) the invention proposes in particular to modify the flow rate of the coextruded solutions and the final opening of the coextrusion chip. By flow rate is meant the flow rate of each solution that arrives at the injector. By final opening of the coextrusion chip is meant the internal opening of the outlet channel of the chip.

[0232] - for the flow rate: we preferentially go from a value between 20 and 40 ml / h for each coextruded solution for the standard sizes of capsules known from the prior art, to a value between 45 and 150 mL / h, preferentially between 45 and 110 mL per hour for a variant of the invention, - for the diameter of the final opening of the coextrusion chip (microfluidic injector) which goes from a value preferentially between 50 and 120 pm for the standard sizes of capsules known from the prior art, to a diameter value between 150 and 300 pm, preferentially between 180 and 240 pm.

[0233] Thus, according to a particular embodiment, the encapsulation of step (a) is carried out using a microfluidic injector whose final opening diameter is between 150 and 300 pm, preferably between 180 and 240 pm, and with the flow rate of each of the 3 solutions between 45 and 150 mL / h, preferably between 45 and 110 mL / h.

[0234] Alternatively, step (a) of preparing a microcompartment may be carried out with stem cells, progenitor stem cells or cells capable of differentiating into liver cells.

[0235] Step (b) of cell differentiation is preferably carried out for at least 20 days, even more preferably at least 30 days.

[0236] During the differentiation of stage (b) the cells organize themselves in an organization similar to that of a liver bud. This organization is typically marked by the structuring in the form of two cell subpopulations presenting two different organizations, one of the epithelial type (i.e. in the form of a cell base polarized apico-basally and presenting tight junctions) and the other of the mesenchymal type, i.e. without apicobasal organization and presenting focal junctions.

[0237] Differentiation of induced pluripotent stem cells from step (b) into liver microtissue can be achieved by any known differentiation method as described in Raggi, et al, Stem cell Report 2022 or Mallanna and Duncan, Curr Protoc Cell Bil, 2014.

[0238] According to one embodiment, steps (a) and / or (b) are carried out with continuous or sequential stirring. This stirring is important because it maintains the homogeneity of the culture environment and avoids the formation of any diffusive gradient.

[0239] Preferably, step (b) is carried out under hypoxia conditions, more preferably the first 5 days of differentiation are carried out under hypoxia conditions.

[0240] Unexpectedly, differentiation under hypoxia conditions allows for a better expansion rate.

[0241] The implementation of the method according to the invention makes it possible to obtain microcompartments comprising at least 100, preferably at least 500, at least 800, at least 1000, in particular at least 3000 cells.

[0242] The method according to the invention is preferably implemented in a closed enclosure such as a closed bioreactor.

[0243] The invention also relates to a method for preparing a hepatic microtissue comprising the implementation of the steps:

[0244] -(a) preparation of a closed three-dimensional cellular microcompartment comprising, inside an external hydrogel layer, induced pluripotent stem cells, and optionally extracellular matrix elements or a natural or synthetic extracellular matrix,

[0245] -(b) induce cellular differentiation within the cellular microcompartment so as to differentiate the induced pluripotent stem cells into hepatic cells

[0246] -(c) removing the outer hydrogel layer to recover ovoid, cylindrical, spheroid or spherical or substantially ovoid, cylindrical, spheroid or spherical or ellipsoidal shaped liver microtissue comprising at least 300 cells expressing CYP3A4 monooxygenase with an activity of at least 75,000 RLU per million cells and producing at least 18pg of urea per million cells per 24 hours.

[0247] Step (c) consists of dissociating the microcompartment to obtain a hepatic microtissue; the removal of the external hydrogel layer can be carried out in particular by hydrolysis, dissolution, piercing and / or rupture by any biocompatible means, i.e. non-toxic to the cells. For example, the removal can be carried out using a phosphate buffered saline, a divalent ion chelator, an enzyme such as alginate lyase if the hydrogel comprises alginate and / or laser microdissection.

[0248] Step (c) of removing the outer hydrogel layer makes it possible to recover the hepatic microtissue of ovoid, cylindrical, spheroid or spherical or substantially ovoid, cylindrical, spheroid or spherical or ellipsoidal shape comprising at least 300 cells corresponding to at least 3 different phenotypes of hepatic cells.

[0249] Preferably, the hepatic microtissue recovered in step (c) is ellipsoidal in shape.

[0250] The hepatic microtissue from step (c) may comprise at least 300, in particular between 300 and 14,000, even more preferably between 900 and 5,000 cells. The hepatic microtissue from step (c) comprises at least one lumen, at least one cell in contact both with a lumen and with the environment external to the microtissue, and at least one cell surrounded only by cells.

[0251] Advantageously, the method according to the invention makes it possible to produce on a large scale the hepatic microtissue according to the invention so as to form a set of hepatic microtissues which can form up to 20% of the mass of the liver of the patient to be treated in less than 50 days, preferably in less than 40 days, in particular in less than 35 days.

[0252] In a preferred variant, the method according to the invention comprises at least one re-encapsulation of the liver microtissue after step (c), i.e. at least two encapsulation cycles. Preferably, each encapsulation cycle corresponds to one passage. In this variant of the method (at least one re-encapsulation of the cells after step (c)) the number of cell divisions of the entire method (for all the passages) is at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30 cell division cycles. In a method according to the invention, there may be several re-encapsulations, preferably between 1 and 100, in particular between 1 and 10 re-encapsulation(s).

[0253] Each re-encapsulation may include:

[0254] - a step of dissociating the microcompartment or series of microcompartments to obtain a hepatic microtissue; the removal of the external hydrogel layer can be carried out in particular by hydrolysis, dissolution, piercing and / or rupture by any biocompatible means, i.e. non-toxic to the cells. For example, the removal can be carried out using a saline phosphate buffer, a divalent ion chelator, an enzyme such as alginate lyase if the hydrogel comprises alginate and / or laser microdissection, and - a step of re-encapsulation of all or part of the hepatic microtissue in a hydrogel capsule. Re-encapsulation is a suitable means for increasing the cell amplification obtained from the pluripotent stage, and reducing the risks of mutation.

[0255] Re-encapsulation consists of removing the outer layer of hydrogel, preferably resuspending the cells that were in the form of cysts in the microcompartments in a partially or totally dissociated manner and repeating the process steps.

[0256] According to one embodiment, the re-encapsulation comprises the following steps: - (i) removing the external hydrogel layer,

[0257] - (ii) resuspend the liver microtissue that was contained in the microcompartment

[0258] - (iii) encapsulating the liver microtissue in a hydrogel layer so as to form a microcompartment of ovoid, cylindrical, spheroidal or spherical or substantially ovoid, cylindrical, spheroidal or spherical or ellipsoidal shape, comprising an outer hydrogel layer defining an inner portion, the smallest radius or the average radius of said inner portion being at least 300 pm;

[0259] - (iv) cultivate the microcompartments obtained in a culture medium

[0260] - (vii) optionally recover the cellular microcompartments obtained.

[0261] Compartmentalization into microcompartments allows the elimination of microcompartments containing more mutated cells than other capsules. Even if the mutated cells grow rapidly, they will reach capsular confluence, which will contain their multiplication. Compartmentalization also allows the entire cell population to be avoided and capsules containing mutant cells to be eliminated at any time, particularly before a reencapsulation step. This sorting can be done either by online analysis or by eliminating capsules that fill more quickly than the others, for example.

[0262] In one embodiment, at least one of the steps (preferably all steps) is carried out at a temperature suitable for cell survival, between 4 and 42°C. The temperature during cell proliferation should preferably be between 32 and 37°C to avoid triggering mutations by lowering the performance of the repair enzymes. Similarly, preferably, the temperature should be low (ideally around 4°C) to manage cell stress in step (c).

[0263] 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 carried out by identifying the expression by at least part of the cells contained in the microcompartment, of markers specific to the desired phenotype chosen from:

[0264] - the expression of alpha-fetoprotein, albumin and cytokeratin 19 to characterize hepatoblasts the expression of alpha-fetoprotein and albumin and the absence of expression of cytokeratin 19 to characterize immature hepatocytes

[0265] - albumin expression and absence of alphafetoprotein or cytokeratin 19 expression to characterize mature hepatocytes cytokeratin expression and absence of albumin and alphafetoprotein expression to characterize cholangiocytes

[0266] -CD31 expression to characterize endothelial cells

[0267] -the expression of CD90 and CD73 to characterize mesenchymal stem cells. The cellular microcompartments obtained according to the methods of the invention can then be frozen before 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 vehicle (screw-on ampoule or plastic bag) in a warm water bath (preferably 37 degrees) so that the cells thaw fairly quickly. The microcompartments according to the invention, before use, can be kept at more than 4°C for a limited period of time before use, preferably between 4°C and 38°C.

[0268] The invention mainly promotes amplification with a high expansion rate, which consequently reduces the culture time and the number of divisions to obtain a very large number of functional lymphocytes.

[0269] Example :

[0270] Example 1: Differentiation protocol

[0271] The hepatic microtissue according to the invention can be obtained from any known differentiation method.

[0272] To demonstrate this, the inventors adapted two known prior art protocols. Protocol A is described in 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.

[0273] Materials and methods

[0274] iPSCs were cultured on vitronectin-coated T75 flasks and were routinely passaged using ReLeSR dissociation reagent (Stem Cell Technologies). All experiments were performed with iPSCs between passages 20 and 26.

[0275] Encapsulation of iPSCs was performed in alginate microcapsules of similar size between the two protocols A and B and in the presence of extracellular matrix.

[0276] For protocol A:

[0277] Minimum diameter on the long axis = 220 pm

[0278] Maximum diameter on the long axis = 550 pm

[0279] Average diameter on the long axis = 415 pm

[0280] For protocol B:

[0281] Minimum diameter on the short axis = 200 pm

[0282] Maximum diameter on the short axis = 385 pm

[0283] Average diameter on the short axis = 295 pm

[0284] iPSCs were first detached with accutase in small groups of 3 to 5 cells and resuspended at a concentration of 0.8E6 cells / ml in a mix composed of 50% Matrigel and 50% mTESRl medium containing 100M of Rhock inhibitor (Y-27632) and encapsulated 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 mTESRl medium containing 100M of Rhock inhibitor in a proportion of 0.2 ml of capsule to 1 ml of total medium.Cells were allowed to form cysts in the capsules for 96 h under shaking conditions, using a 30 ml ABLE bioreactor (Reprocell), at 37°C and 5% CO2 with a medium change every 24 h in mTESRl medium.

[0285] Differentiation was initiated 96h after encapsulation by switching to RPMI medium, containing 1 / 100M Ca2+, 1% KnockOut Serum Replacement (KOSR), insulin-free B27, 100ng / ml Activin A and 3pM CHIR-99021 for 2 days.

[0286] For the next 3 days, the medium was replaced with RPMI medium, containing 1 / 100M Ca2+, 1% KOSR, insulin-free B27, and 100ng / ml Activin A.

[0287] Then, for the next 5 days, the medium was replaced with RPMI medium, containing for protocol A: 1mM Ca2+, 1% KOSR, B27 without insulin, supplemented with 20ng / ml BMP-4, 5ng / ml bFGF, 1pM A83-01 (TGF beta pathway inhibitor) and 4pM IWP-2 (Wnt pathway inhibitor).

[0288] For protocol B: ImM Ca2+, 1% KOSR, B27 without insulin, supplemented with 20ng / ml BMP-4 and 5ng / ml bFGF. From day 11 to day 15, the medium consisted of RPMI medium, containing ImM Ca2+, 2% KOSR, B27 with insulin, supplemented with 20ng / ml HGF, 3pM CHIR-99021, 5ng / ml bFGF and 20ng / ml BMP-4.

[0289] On day 16, the medium was changed to HCM medium without EGF (Lonza), supplemented with 20ng / ml HGF, 3pM CHIR-99021, 5ng / ml bFGF, 20ng / ml BMP-4, 20ng / ml Oncostatin M, 10pM Dexamethasone and 1% KOSR.

[0290] From day 20 and for the following 5 days, the medium consisted of HCM plus EGF medium, supplemented with 20ng / ml Oncostatin M, 10M Dexamethasone and 1% KOSR.

[0291] From day 25, the medium consisted of HCM medium plus EGF, 1% Dexamethasone and 1% KOSR.

[0292] Gene expression analysis

[0293] Gene expression analysis was performed by RT-Q-PCR.

[0294] qPCR was performed on a Roche LightCycler® 480 instrument. Expression of each gene was normalized to the expression of the reference genes YWHAZ, NONO, and VCP. Data are expressed as 2 A delta Ct, where delta Ct=Ct of the gene - average Ct of the reference genes.

[0295] The results are presented in Figure 1a, 1b and 1c. These results show that the liver microtissue according to the invention can be obtained with any suitable differentiation method.

[0296] Example 2: Differentiation into hepatocytes within the microcompartment according to the invention versus in two dimensions.

[0297] Encapsulation of iPSCs was performed in alginate microcapsules with the following characteristics:

[0298] Minimum diameter on the long axis = 450pm

[0299] Maximum diameter on the long axis = 685pm

[0300] Average diameter on the long axis = 575 pm

[0301] Minimum diameter on the short axis = 313pm

[0302] Maximum diameter on the short axis = 546pm

[0303] Average diameter on the short axis = 423 pm

[0304] For encapsulation of iPSCs in alginate microcapsules of approximately 575 pm average diameter and in the absence of matrix, iPSCs were first detached with accutase in small groups of 3 to 5 cells and resuspended at a concentration of 10E6 cells / ml in mTESRl medium containing 100M of Rhock inhibitor (Y-27632) and encapsulated 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 mTESRl medium containing 100M Rhock inhibitor at 0.5E6 cells / ml, with the proportion of capsules to medium not exceeding 20%.Cells were allowed to form agglutinates in the capsules for 24 h under shaking conditions, using a 30 ml ABLE bioreactor (Reprocell), at 37°C and 5% CO2.

[0305] Differentiation was initiated 24h after encapsulation by switching to RPMI medium, containing 1 / 100M Ca2+, 1% KnockOut Serum Replacement (KOSR), insulin-free B27, 100ng / ml Activin A and 3pM CHIR-99021 for 2 days.

[0306] For the next 3 days, the medium was replaced with RPMI medium, containing 1 / 100M Ca2+, 1% KOSR, insulin-free B27, and 100ng / ml Activin A.

[0307] Then, for the next 5 days, the medium was replaced with RPMI medium, containing 1 / 100 μM 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 / 100 μM 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.

[0308] On day 16, the medium was changed to HCM medium without EGF (Lonza), supplemented with 20ng / ml HGF, 3pM CHIR-99021, 5ng / ml bFGF, 20ng / ml BMP-4, 20ng / ml Oncostatin M, 10pM Dexamethasone and 1% KOSR.

[0309] From day 20 and for the following 5 days, the medium consisted of HCM plus EGF medium, supplemented with 20ng / ml Oncostatin M, 10M Dexamethasone and 1% KOSR.

[0310] From day 25, the medium consisted of HCM medium plus EGF, 1% Dexamethasone and 1% KOSR.

[0311] The medium was changed daily from day 1 to day 25, and every other day from day 25.

[0312] iPSCs were dissociated into single cells using accutase and plated on Matrigel-coated 6-well plates in mTESR1 medium containing 100M Rhock inhibitor at a density of 1x105 cells / cm2. Differentiation was initiated 24h after plating using the same differentiation protocol and medium composition as for the 3D capsules.

[0313] Gene expression analysis

[0314] Gene expression analysis was performed by RT-Q-PCR.

[0315] qPCR was performed on a Roche LightCycler® 480 instrument. Expression of each gene was normalized to the expression of the reference genes YWHAZ, NONO, and VCP. Data are expressed as 2 A delta Ct, where delta Ct=Ct of the gene - average Ct of the reference genes.

[0316] Comparison of gene expression between hepatocytes obtained in two dimensions versus three dimensions is illustrated in Figure 2 to verify the induction of endoderm during differentiation. The expression of the genes EÛMES, CXCR4, FOXA2 and SOX17 shows that differentiation does indeed go through a transitional stage of endodermal differentiation towards the definitive endoderm, a passage expected to generate hepatic cells from pluripotent cells.

[0317] Analysis of protein expression of endoderm markers was performed on day 5 and is shown in Figure 3. These results show that differentiation progresses in a sequence consistent with the expectations of those skilled in the art, i.e. via the definitive endoderm and towards the liver bud.

[0318] An analysis of gene expression during the differentiation protocols is shown in Figures 4a and 4b. These results show that differentiation progresses towards a composition consistent with the target cellular composition of the liver, particularly hepatocytes.

[0319] Measurement of the expansion rate:

[0320] 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 the culture (day of encapsulation or day of culturing). The expansion rate or amplification factor was measured during differentiation. Monitoring of the expansion rate during the differentiation process is shown in Figure 5. An expansion rate of at least times 15, 20 days after the start of differentiation is found. Evaluation of albumin and urea production

[0321] To assess albumin and urea production, the conditioned medium was sampled periodically 24 hours (±2 h) after medium change and stored at -80°C. The levels of albumin and urea secreted into the medium were measured using the Human Albumin ELISA Kit (Invitrogen) and the Quantichrom Urea Assay Kit (Gentaur), respectively, according to the manufacturer's instructions. The amount of molecules secreted in 24 h was then normalized by the number of cells according to the count performed after dissociation for each time point.

[0322] The results are presented in Figures 6 and 7. It can be seen that albumin secretion and urea production are greater in the microcompartments according to the invention.

[0323] Measurement of CYP3A4 activity:

[0324] Cyp3A4 activity was performed using the P450-Glo™ CYP3A4 assay with Luciferin-IPA (Promega) according to the manufacturer's instructions. Briefly, a sample of the 3D capsules containing the microtissues was decapsulated and the number of cells in a given volume of capsules containing the microtissues was determined. The decapsulated microtissues corresponding to 1E5 cells, or 1E5 cells that were differentiated in 2D, were mixed with 50 μl of the proluciferin substrate P450-Glo 3 μM in a 96-well round-bottom plate and incubated for 3 h at 37°C, 5% CO2. For each condition, 5 replicates were performed, cell culture medium alone and iPSCs were used as negative controls. Subsequently, 25 μl of culture medium from each well was transferred into a 96-well opaque white luminometer plate and mixed with 25 μl of luciferin detection reagent.The plate was incubated for 20 minutes at room temperature and the luminescence was read using the Spectramax i3x microplate reader (Molecular devices) with an integration time of 1 second per well. The net signal was calculated by subtracting the background luminescence values ​​from the control wells without cells.

[0325] The results are presented in Figure 8. A significantly higher CYP3A4 activity is observed in the cells encapsulated in the microcompartment according to the invention. according to the invention

[0326] The objective of this example is to characterize the morphology of cells within the microcompartment during differentiation.

[0327] Histology

[0328] Liver microtissue samples were fixed with AFA fixative solution for 24 hours 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 at a thickness of 5 μm. Hematoxylin-Eosin-Saffron (HES) staining

[0329] After removal of paraffin, sections were stained with Harris hematoxylin and eosin G. After dehydration, sections were stained with Safran and mounted with Entellan. Cell cytoplasm is stained pink, nuclei blue-violet, and extracellular matrix pinkish-yellow.

[0330] PAS (Periodic-Acid-Schiff) staining

[0331] After removal of paraffin, sections were pretreated with 1% perodic acid, followed by successive staining with Schiff's reagent and Mayer's hematoxylin. Glycogen is stained pink and nuclei blue-violet.

[0332] The images resulting from the staining are shown in Figure 9. The characteristic cubic shape of the hepatocytes is clearly visible, as well as the presence of glycogen granules within the microcompartment.

[0333] Immunofluorescence on 3D microtissues

[0334] Microtissues were fixed inside the capsules in a 4% paraformaldehyde solution in PBS with calcium for 1 hour at room temperature. After being rinsed with PBS without Ca2+ to remove the capsules, they were permeabilized in 1% Triton X-100 for 30-60 minutes at room temperature. Microtissues were incubated with the primary antibody solution for 72 hours at 4°C with shaking. After washing with PBS, they were incubated with a labeled secondary antibody solution (Alexa Fluor, Life Technologies) and DAPI overnight at 4°C with shaking and protected from light.

[0335] For structural imaging by phalloidin-DAPI, staining was performed while preserving the capsules (the decapsulation step before permeabilization was skipped, washes were done with PBS containing calcium). Microtissues were incubated with DAPI and Phalloidin for 72 h at 4 °C with shaking. After washing with PBS, cells were mounted on a slide with a spacing of 0.5 mm. Image acquisition was performed on a confocal microscope (SP5, Leica).

[0336] Table 1:

[0337] The evolution of the morphology of the microcompartment was carried out over 30 days during differentiation. These results are shown in Figures 10a to 10e. The presence of particular structures can be observed, such as the formation of a structure similar to a liver bud on day 7 and a cellular organization characteristic of the liver microtissue according to the invention. Indeed, at least one lumen, at least one cell in contact with both a lumen and the environment outside the microtissue, and at least one cell surrounded only by cells can be distinguished. In addition to the study of the morphology, the inventors were able to characterize the presence of at least 3 different phenotypes of liver cells within the microcompartment, 30 days after the start of differentiation (Figure 11).

[0338] Microtissue grafting according to the invention and evaluation of cell survival after transplantation in mice compared to transplantation of hepatocytes in the form of single cells obtained in 2D

[0339] The objective of this study is to inoculate immunocompetent mice with stem cell-derived hepatocytes, prepared in the form of cell suspension or microtissues according to the invention, with a view to evaluating their survival and function in vivo.

[0340] The conditions of the study are described below:

[0341] Animals :

[0342] The study was carried out on 10-week-old female Balb / cByJ mice (Mus musculus).

[0343] Seven (7) animals were housed in the animal facility. Ventilation and air treatment were achieved through frequent renewals and the temperature was controlled around 21-22°C. Humidity was maintained around 50%. Artificial lighting was maintained for 12 hours per day. The quantity and access to food (pellets) and drink (tap water) were controlled daily. Cages were renewed once a week and environmental enrichment was done to minimize anxiety.

[0344] Injection process:

[0345] On day 42 of hepatic differentiation, the cells generated in 2D (according to Example 2 “2D Hepatocyte Differentiation”) were dissociated into single cells and the liver microtissues generated according to the invention (according to Example 1) were decapsulated.

[0346] Microtissue suspension: microtissues according to the invention obtained according to Example 1, suspended in a culture medium, were used: approximately 3x106 cells per tube diluted in 170pL of basal medium. Before inoculation, 170pL of Matrigel® was added to each tube.

[0347] To perform subcutaneous inoculation of such a volume, i.e. 340pL, a skin incision in the right flank of the mouse was made to prepare a subcutaneous pocket. The microtissue suspension was then injected into this pocket using a pipette before closing the wound with surgical glue to prevent diffusion of the suspension.

[0348] Cell suspension: Cells obtained according to the protocol of Example 2 “2D Hepatocyte Differentiation”, suspended in a culture medium, were used: approximately 3x106 cells per tube in 120pL of basal medium. Before inoculation, 120pL of Matrigel® was added to each tube.

[0349] To perform the subcutaneous inoculation of this 240pL volume, a skin incision was made on the right flank of the mouse to prepare a subcutaneous pocket. The cell suspension was then injected into this pocket using a pipette before closing the wound with surgical glue to prevent diffusion of the suspension.

[0350] Control solution: A 100pL basal medium (vehicle) solution was mixed with 100pL of Matrigel®. This solution was injected subcutaneously following the same procedure as that used for microtissues and cell suspension. Sample collection:

[0351] Blood samples were collected by retro-orbital bleeding for serum preparation (approximately 80 pL) from all available mice:

[0352] - Before cell inoculation, day 0, n=7

[0353] - Two days after inoculation, day 2, n=7

[0354] - Six days after inoculation, day 6, n=3

[0355] The grafts, including the surrounding skin to preserve the structure, were harvested as follows:

[0356] - Two days after inoculation, day 2, n=4 on=2 for cell suspension on=2 for microtissues

[0357] - Six days after inoculation, day 6, n=3 on=l for cell suspension on=l for microtissues on=l for vehicle

[0358] The grafts were harvested, fixed, embedded in paraffin, sectioned and stained with HES (hematoxylin eosin saffron) and with antibodies specific for the human cell marker Steml21 (detects human cytoplasmic proteins), the liver cell markers albumin and CytK19 (cytokeratin 19).

[0359] Before transplantation, microtissues were also stained with HES, albumin and

[0360] CytK19.

[0361] Sample collection:

[0362] Images obtained by optical microscopy (Leica DM2000 microscope) are shown in Figures 12a to 12d:

[0363] - 12a:

[0364] * single-cell grafts at day 2 (recipients 1 and 2, top row) after transplantation into Balb / c mice,

[0365] * Microtissue grafts on day 2 (recipients 4 and 5, bottom row) after transplantation into Balb / c mice.

[0366] - 12b:

[0367] * single-cell grafts at day 6 (recipients, top row) after transplantation into Balb / c mice,

[0368] * Microtissue grafts at day 6 (recipient 6, bottom row) after transplantation into Balb / c mice.

[0369] - 12c: tissue at the injection site with vehicle alone (basal medium containing 50% matrigel) without graft in Balb / c mice.

[0370] - 12d: microtissues according to the invention before transplantation.

[0371] Histological analysis of the grafts on day 2 after transplantation revealed the presence of rounded-shaped microtissues positive for the human cell marker Steml21 in mice transplanted with the liver microtissues according to the invention, confirming that the detected microtissues are of human origin. Some of the cells composing the microtissues were positive for the hepatocyte marker albumin and some cells, mainly those located at the periphery of the microtissues, were positive for the collangiocyte marker CytK19. The cellular organization and compartmentalization of the cells expressing albumin and CytK19 after transplantation in vivo were similar to those observed in the microtissues before transplantation.

[0372] In mice transplanted with 2D-generated single cells, only rare cells positive for the human cell marker Steml21, albumin, and cytokeratin 19 were detected at day 2 after transplantation.

[0373] The presence of cells expressing Steml21 and CytK19 was still detectable at day 6 in mice transplanted with microtissues, no cells positive for Steml21 or CytK19 were present in mice transplanted with single cells. Finally, in the area of ​​immune cell infiltration in mice transplanted with vehicle alone (control), no cells positive for Steml21, albumin or cytokeratin 19 were detected. Furthermore, the presence of human AlAT was analyzed in sera from transplanted Balb / c mice (analyses before transplantation (day 0), at day 2 and at day 6 after transplantation). The presence of human AlAT (alpha-1 antitrypsin) was analyzed by ELISA. The secretion of AlAT by cells and microtissues before transplantation was also analyzed. The results obtained are presented in Figures 13a and 13b:

[0374] 13a: Graphical representation of AlAT secretion before transplantation of 2D differentiated single cells (2D) and microtissues according to the invention (Invention).

[0375] 13b: Graphical representation of AlT detection in Balb / c mouse serum before (day 0), at day 2 and at day 6 after transplantation with 2D differentiated single cells (SC), with the invention microtissues (MT) and with vehicle alone (control).

[0376] These results show that the liver microtissues according to the invention are capable of secreting a higher level of AlAT than the 2D-differentiated cells. In accordance with the histological observation, the concentration of human AlAT is higher in the serum of mice transplanted with the microtissues according to the invention than in that of mice transplanted with single cells. The concentration of human AlAT is lower, or even undetectable, at day 0. It is higher at day 2 after transplantation and decreases at day 6 while still being significant and higher than that of the 2D-differentiated single cells. These data are consistent with the histological results and show that the microtissues according to the invention persist over time and are more functional after transplantation than the 2D-differentiated single cells, even in an immunocompetent mouse model.Overall, these results show that the liver microtissues according to the invention and grafted as 3D objects survive after transplantation in immunocompetent mice, and have better and longer survival after transplantation than liver cells generated in 2D and grafted as a single cell suspension.

Claims

DEMANDS

1. Three-dimensional liver microtissue, the largest dimension of which is between 500 and 700 pm, expressing CYP3A4 monooxygenase with an activity of at least 75,000 RLU per million cells and producing at least 18 pg of urea per million cells per 24 hours.

2. Liver microtissue according to the preceding claim, characterized in that it comprises between 50 and 99% liver cells.

3. Liver microtissue according to any one of the preceding claims, characterized in that it is obtained from induced pluripotent stem cells

4. Liver microtissue according to any one of the preceding claims, characterized in that it is obtained from induced pluripotent stem cells encapsulated in a single three-dimensional closed microcompartment.

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

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

7. Three-dimensional liver microtissue according to any one of the preceding claims, comprising at least 3 different liver cell phenotypes, characterized in that all cells of the microtissue were all obtained from induced pluripotent stem cells encapsulated in a single closed three-dimensional microcompartment.

8. Liver microtissue according to any one of the preceding claims, characterized in that the liver microtissue comprises immature hepatocytes, mature hepatocytes and cholangiocytes.

9. Liver microtissue according to any 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 expression 19, - mature hepatocytes characterized by albumin expression and the absence of alpha-fetoprotein and cytokeratin expression 19, and -cholangiocytes characterized by the expression of cytokeratin and the absence of expression of albumin and alpha-fetoprotein.

10. Microtissue according to any 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 any one of the preceding claims, characterized in that the liver microtissue comprises at least liver cells of which between 20 and 60% of the liver cells are cells expressing cytokeratin 19.

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

14. Liver microtissue according to any one of the preceding claims, comprising: - at least one lumen, - at least one cell in contact with both a lumen and the environment external to the microtissue, and - at least one cell surrounded only by other cells.

15. Liver microtissue according to any one of the preceding claims, characterized in that it has an ellipsoidal shape.

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

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

18. Liver microtissue according to any one of the preceding claims, characterized in that it has a diameter or smaller dimension between 100 pm and 300 pm.

19. Liver microtissue according to any one of the preceding claims, characterized in that it has a larger dimension of between 500 µm and 700 µm.

20. Liver microtissue according to any one of the preceding claims, characterized in that it comprises between 300 and 14,000 cells.

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

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

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

24. Three-dimensional closed microcompartment, comprising an outer hydrogel layer defining an inner part, said inner part comprising at least one liver microtissue according to any one of claims 1 to 23.

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

26. A cell microcompartment according to claim 24 or 25, characterized in that it exhibits an expansion rate of at least 15 times, 20 days after the start of differentiation.

27. ​​Cell microcompartment according to any one of claims 24 to 26, characterized in that the thickness of the outer layer is variable and between 20 and 60µm.

28. Cell microcompartment according to any one of claims 24 to 27, characterized in that the outer layer comprises alginate.

29. Cell microcompartment according to any one of claims 24 to 28, characterized in that it comprises extracellular matrix elements or a natural or synthetic extracellular matrix in the inner part between the outer layer and the liver microtissue.

30. Cell microcompartment according to any one of claims 24 to 29, characterized in that it has a diameter or smaller dimension between 300 pm and 400 pm.

31. A cell microcompartment according to any one of claims 24 to 30, characterized in that it has a largest dimension of between 400 µm and 600 µm.

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

33. A set of microcompartments according to the preceding claim, characterized in that the microcompartments are arranged in a culture medium in a bioreactor.

34. Microtissue according to any one of claims 1 to 23 or microcompartment according to any one of claims 24 to 31 for its use as a medicinal product.

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

36. Microtissue or microcompartment for use according to claim 35, wherein the liver failure is acute, chronic, or acute-over-chronic liver failure.

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

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

39. A method for preparing a microcompartment according to any one of claims 24 to 31, comprising the steps of: - a) produce a closed, three-dimensional cellular microcompartment comprising, within an outer hydrogel layer, induced pluripotent stem cells, and optionally extracellular matrix elements or a natural or synthetic extracellular matrix, - b) induce cell differentiation within the cell microcompartment, so as to obtain a microcompartment comprising liver cells.

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

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