Cellular microcompartments containing cells with maintained genomic integrity after amplification and preparation methods - Patents.com

Abstract

Solutions are needed to maintain the genetic integrity of cells in culture, particularly for large-scale production of cell therapies. [Solution] The present invention relates to a three-dimensional cellular microcompartment or a three-dimensional cellular microcompartment assembly, comprising at least one outer hydrogel layer and, inside said outer layer, at least one layer of cells and / or at least one cell-based layer, wherein less than 20% of the total population of cells present in the microcompartment or microcompartment assembly are cells having at least one mutation. The present invention also relates to a method for producing such a microcompartment or a cellular microcompartment assembly.

Description

[Technical field]

[0001] The present invention relates to maintaining the genomic integrity of cells during ex vivo division over several cell division cycles, particularly in the context of three-dimensional cell culture. [Background technology]

[0002] Ex vivo cell culture is a field that is gaining increasing interest. The cultured cells can be any type of cell. The cultured cells can include both differentiated cells, progenitor cells and stem cells with different phenotypes. A significant advance in cell culture technology is the introduction of three-dimensional culture systems. Three-dimensional cultures are actually closer to the in vivo natural system and can be used for many applications, especially for the development of therapeutics.

[0003] However, cell therapy and tissue engineering are conditioned by the availability of industrial quantities of cells, which requires resorting to a large increase in cell number and therefore a large number of divisions in a short time. In modern cell culture systems, this increase causes the emergence and selection of mutations, in particular deleterious genomic and / or epigenetic functional mutations, at each division over a large number of cells during the growth of the culture, thus compromising their use, in particular in therapy.

[0004] The mutations can be point mutations in gene sequences (coding or non-coding, silent or not with respect to peptide sequences), structural variants, epigenetic modifications, or even mitochondrial DNA modifications. Only mutant cells with one or more functional or potentially functional mutations are of concern for the use of cells in therapy, i.e. any transmissible genetic or epigenetic modification that confers a gain or loss of function, or a potential loss of function, to the cultured cells. It can be, in particular, a growth advantage, a reduced susceptibility to cell death, the modification of genes involved in tumorigenesis, or the suppression of tumorigenesis. The most impactful mutations are those that allow the clonal expansion of cells that become dominant in the culture.

[0005] Examples of particularly recurrent genetic mutations are described, inter alia, in Y. Avior, K. Eggan, N. Benvenisty, Cancer-Related Mutations Identified in Primed and Naive Human Pluripotent Stem Cells. Cell Stem Cell. 25, 456-461 (2019). Among the best known are mutations in the P53 gene (FT Merkle, S. Ghosh, N. Kamitaki, J. Mitchell, Y. Avior, C. Mello, S. Kashin, S. Mekhoubad, D. Ilic, M. Charlton, G. Saphier, RE Handsaker, G. Genovese, S. Bar, N. Benvenisty, SAMc Carroll, K. Eggan, Human pluripotent stem cells recurrently acquire and expand dominant negative P53 mutations. Nature. 545, 229-233 (2017)) and mutations due to amplification of the 20q11 chromosomal region (N. Lefort, M. Feyeux, C. Bas, O. Feraud, A. Bennaceur-Griscelli, G. Tachdjian, M. Peschanski, AL Perrier, Human embryonic stem cells reveal recurrent genomic instability at 20q11.21. Nature Biotechnology. 26, 1364-1366 (2008)).

[0006] The problem of genetic stability and integrity of cultured cells is known and has been widely studied, especially for pluripotent stem cells, see, for example, S. Attwood, M. Edel, iPS-Cell Technology and the Problem of Genetic Instability-Can It Ever Be Safe for Clinical Use? Journal of Clinical Medicine.8,288(2019) or P. Andrews, Human pluripotent stem cells: genetic instability;or stability;Regenerative medicine,vol.16,No 2,March 2,2021. It is also known that mutagenesis is currently very problematic for culturing stem cells as soon as they are programmed as described in Ji, S. Ng, V. Sharma, D. Necut, S. Human, M. Sam, Q. Trinh, GMChurch, JD McPherson, A. Nagy, N.N. Batada, Elevated coding mutation rate during the reprogramming of human somatic cells into induced pluripotent stem cells. Stem Cells. 30, 435-440 (2012), and V. Turinetto, L. Orlando, C. Giachino, Induced pluripotent stem cells: Advances in the quest for genetic stability during reprogramming process. International Journal of Molecular Sciences. 18 (2017), doi:10.3390 / ijms18091952.

[0007] This genetic instability is extremely detrimental to the development of cell therapy, especially for the clinical use of stem cells (Yamanaka, Pluripotent Stem Cell-Based Cell Therapy-Promise and Challenges. Cell stem cell. 27, 523-531 (2020); SE Peterson, JF Lawing, Genomic instability in pluripotent stem cells: Implications for clinical applications. Journal of Biological Chemistry. 289, 4578-4584 (2014); K. Garber, RIKEN suspends first clinical trial involving induced pluripotent stem cells. Nature biotechnology. 33, 890-891 (2015)).

[0008] Thus, there is a great need for solutions to maintain the genetic integrity of cells in culture, particularly for large-scale production of cell therapies.

[0009] Therefore, it is an object of the present invention to fulfill all of these needs and overcome the disadvantages and limitations of the prior art. Summary of the Invention

[0010] By working on the development of cellular microcompartments for cell culture in 3D, the inventors have developed a system that allows for the culture of mass cells while maintaining their genomic integrity.

[0011] To this end, the subject of the present invention is a three-dimensional cellular microcompartment comprising at least one outer hydrogel layer and, inside the outer layer, at least one layer of cells and / or at least one cell-based layer, in which less than 20% of the total population of cells present in the microcompartment, even after several cell divisions, preferentially 0-10%, more preferentially 0-5%, preferentially 0-3%, are cells carrying at least one mutation.

[0012] According to another subject, the invention relates to an assembly of at least two three-dimensional cellular microcompartments, preferably in liquid suspension, each microcompartment comprising at least one outer hydrogel layer and, inside said outer layer, at least one layer of cells and / or at least one cell-based layer, wherein less than 20%, preferentially 0-10%, even more preferentially 0-5%, in particular 0-2%, of the total population of cells present in all microcompartments are cells carrying at least one mutation.

[0013] Advantageously, this level of mutant cells is lower than that of existing cell culture systems. For example, certain studies suggest that inactivating mutations in the P53 gene confer up to 1.9-fold selection advantage per passage in traditional 2D stem cell culture systems (iPS-Cell Technology and the Problem of Genetic Instability-Can It Ever Be Safe for Clinical Use? Attwood & Edel)+Merkle,FT;Ghosh,S.;Kamitaki,N.;Mitchell,J.;Avior,Y.;Mello,C.;Kashin,S.;Mekhoubad,S.;Ilic,D.;Charlton,M.;et al.Human pluripotent stem cells recurrently acquire and expand dominant negative P53 mutations.Nature 2017,545,229-233). This implies a 97% probability of attachment following the occurrence of this mutation (Haldane, JBSA Mathematical Theory of Natural and Artificial Selection, Part V: Selection and Mutation. Math. Proc. Camb. Phils. Soc. 1927, 23, 838-844).

[0014] Maintaining the genomic integrity of the cells allows the use of microcompartments with the cell cultures according to the invention for different applications, in particular in the prevention and / or treatment of pathologies.

[0015] The cell microcompartments according to the invention are in particular -(a) preparing a suspension of cells comprising single cells and / or at least one cluster of cells in an isotonic medium, preferentially a culture medium containing an apoptosis inhibitor, -(b) encapsulating the cell suspension within a hydrogel layer; - (c) culturing the obtained microcompartments in an isotonic solution, preferably in a medium containing an apoptosis inhibitor; - (d) preferentially rinsing the microcompartments to remove apoptosis inhibitors; - (e) culturing the microcompartments for at least two cell division cycles (amplification); -(f) optionally, recovering the obtained cell micro-compartments, The method is characterized in that (upon encapsulation) all of the cells initially encapsulated in step (b) occupy a volume that is less than 50% of the volume of the microcompartment in which they are encapsulated.

[0016] This method makes it possible to obtain microcompartments according to the invention with a stabilized population of cells whose genomic integrity is maintained.

[0017] The present invention is also directed to the use of cell culture compartments and / or such methods for maintaining the genomic integrity of cells during their expansion.

[0018] Other features and advantages will become apparent from the detailed description of the invention and the examples that follow. [Brief description of the drawings]

[0019] [Figure 1] Overview of the three experimental arms "2D culture", "bioreactor aggregates" and "invention" as well as the number and timeline of the passages performed (indicated by rectangles: D4 = day 4, D8 = day 8, etc.). For detailed genetic comparisons, all cultures were stopped on the final day, D28. [Figure 2a] Phase contrast microscopy images showing results from the experimental arm "2D culture" on day 28, the final day before the final sampling. Scale bar, 500 μm. [Figure 2b]Phase contrast microscopy images showing results from the experimental arm "Bioreactor aggregates" on day 28, the final day before the final sampling. The indicated aggregates were removed from culture in suspension and temporarily placed in a Petri dish for microscopic observation. Scale bar, 500 μm. [Figure 2c] Phase contrast microscopy images showing results from the experimental arm "Invention" on the last day before the final sampling, day 28. The indicated microcompartments were removed from the culture in suspension and temporarily placed in a Petri dish for microscopic observation. Scale bar 500 μm. [Diagram 3] It is an indication of the visible growth of cells during the culture time, calculated by counting the cells before and after each passage. The cumulative theoretical amplification factor is expressed as a function of time. The vertical axis (amplification) is shown on a logarithmic scale. The data points shown correspond to all counts performed at the time of passage. [Figure 4] Figure 1 shows the results of phenotypic evaluation of stem cells by flow cytometry. Dissociated cells are fixed and labeled for OCT4 and NANOG pluripotency markers. The percentage of double positive cells for these two markers during serial passage over 28 days is shown here (mean and standard deviation). [Diagram 5] High-resolution karyotypes by Cytoscan HD Array SNP chips for the first day 0 sample and comparative analysis of the analysis of the three experimental arms "2D culture", "bioreactor aggregates" and "invention" at day 28. The CytoScan® HD Array Affymetrix, sold by ThermoFisher, quantifies the average copy number per cell for 2.67 million probes distributed throughout the genome. The boxed region is centered on chromosome 20. [Figure 6]Figure 1 shows the results of the assessment by digital PCR of the average copy number of the 20q11 chromosomal region during 28 days of culture for the three experimental arms (the analysis was carried out with the iPS ddPCR 24 probe test from Stemogenomics). On the left is the count of 20q11 copies as a function of the days of culture. On the right is the ratio of the count of 20q11 copies to the cumulative theoretical amplification over time. The data points correspond to the samplings carried out during the various passages. Squares = "bioreactor aggregates", circles = "2D culture", and triangles = "invention". The associated curves correspond to the corresponding regressions. It should be noted that the standard deviation of these measurements is on average 0.12 (count of 20q11 copies) and the asterisks indicate measurements that are significantly increased. [Figure 7] 1 shows an overview of the percentage of mutant cells during 28 days of culture for the "2D culture", "bioreactor aggregates" and "invention" arms of Example 1. [Figure 8] 1 shows the karyotypes obtained by digital PCR for the two cell lines (GHE and AAVS1_GFP) used in Example 2. [Figure 9] Overview of the two experimental arms "bioreactor aggregates" and "invention" as well as the number and timeline of the passages carried out (indicated by rectangles: D4=day 4, D8=day 8 etc.). [Figure 10] 10A and 10B are phase contrast microscopy images showing the results of experimental arms A: "Bioreactor aggregates" and B: "Invention" on day 19 for "Bioreactor aggregates" and day 21 for "Invention". Aggregates and indicated microcompartments were removed from culture in suspension and temporarily placed in petri dishes for microscopic observation. Scale bar 500 μm. [Figure 11] It is an indication of the visible growth of cells during the culture time, calculated by counting the cells before and after each passage. The cumulative theoretical amplification factor is expressed as a function of time. The vertical axis (amplification) is shown on a logarithmic scale. The data points shown correspond to all counts performed at the time of passage. [Figure 12]Figure 1 shows the results of phenotypic evaluation of stem cells by flow cytometry. Dissociated cells are fixed and labeled for OCT4 and NANOG pluripotency markers. The percentage of double positive cells for these two markers during serial passage over 28 days is shown here (mean and standard deviation). [Figure 13] Figure 1 shows the results of evaluation by digital PCR of the percentage of GFP- (iPSC-GHE) and GFP+ (iPSC-AAVS1) cells during 21 days of culture for the two experimental arms. Left: percentage of GFP- and GFP+ cells as a function of days of culture. Right: percentage of GFP- and GFP+ cells relative to the cumulative theoretical amplification over time. Data points correspond to samplings performed during the various passages. Squares = "bioreactor aggregates" and triangles = "invention". [Figure 14] Figure 2 shows the results of the evaluation by digital PCR of the average copy counts of the 7q and 20q chromosomal regions during 28 days of culture for the two experimental arms (analysis was carried out with the iPS ddPCR 24-probe test from Stemogenomics). On the left are the 7q and 20q copy counts as a function of the number of days in culture. On the right are the 7q and 20q copy counts as a ratio to the cumulative theoretical amplification over time. The data points correspond to samplings carried out during the various passages. Squares = "bioreactor aggregates" and triangles = "invention". [Figure 15] 1 shows a summary of the percentage of mutant cells during 28 days of culture for the "bioreactor aggregates" and "invention" arms of Example 2. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] definition For purposes of the present invention, "alginate" refers to a linear polysaccharide formed from β-D-mannuronate and α-L-guluronate, their salts, and derivatives.

[0021] For the purposes of the present invention, "hydrogel capsule" means a three-dimensional structure formed from a matrix of polymer chains swollen with a liquid, preferentially water.

[0022] For the purposes of the present invention, a "cell expressing a gene" means a cell that contains at least 5-fold more copies, preferentially 10-fold more copies, preferentially 20-fold more copies, preferentially 100-fold more copies of RNA transcribed from the DNA sequence of the gene in question compared to a pluripotent cell.

[0023] For purposes of the present invention, a "differentiated" cell refers to a cell that has a particular phenotype, in contrast to an undifferentiated pluripotent stem cell, or a progenitor cell that has undergone differentiation.

[0024] For the purposes of the present invention, "human cells" refers to human cells or immunologically humanized non-human mammalian cells. Even if this is not specified, the cells, stem cells, progenitor cells, and tissues according to the invention consist of or are derived from human cells or from immunologically humanized non-human mammalian cells.

[0025] For purposes of the present invention, the term "mutated cell" refers to a cell that has at least one mutation.

[0026] For the purposes of the present invention, "progenitor cells" refers to stem cells that are already committed to cell differentiation but have not yet differentiated.

[0027] For the purposes of the present invention, "embryonic stem cells" refers to pluripotent stem cells derived from the inner cell mass of a blastocyst. The pluripotency of embryonic stem cells can be assessed by the presence of markers such as transcription factors OCT4, NANOG, and SOX2, and surface markers such as SSEA3 / 4, Tra-1-60, and Tra-1-81. Embryonic stem cells used in the context of the present invention can be obtained without destroying the embryo from which they are derived, for example, using the techniques described in Chang et al. (Cell Stem Cell, 2008, 2(2):113-117). Optionally, human embryonic stem cells can be excluded from the present invention, in which case the subject matter of the present invention excludes human embryonic stem cells.

[0028] For the purposes of the present invention, "pluripotent stem cells" or "pluripotent cells" refers to cells that have the potential to form all tissues present in the whole organism of origin, but are unable to form the whole organism itself. Human pluripotent stem cells may be referred to as hPSCs in this application. These may in particular be induced pluripotent stem cells (iPSCs or hiPSCs for human induced pluripotent stem cells), embryonic stem cells, or MUSE cells (for "multi-lineage differentiation stress resistant").

[0029] For the purposes of the present invention, "induced pluripotent stem cells" refers to pluripotent stem cells that have been induced to become pluripotent by genetic reprogramming of differentiated somatic cells. These cells are positive for pluripotency markers, such as, inter alia, staining with alkaline phosphatase and expression of the proteins NANOG, SOX2, OCT4, and SSEA3 / 4. Examples of methods for obtaining induced pluripotent stem cells are described in the articles by Yu et al. (Science 2007,318(5858):1917-1920), Takahashi et al. (Cell,207,131(5):861-872), and Nakagawa et al. (Nat Biotechnol,2008,26(1):101-106).

[0030] The "Ferret diameter" of a microcompartment according to the present invention means the distance "d" between two tangents to said microcompartment, said two tangents being parallel, such that the entire projection of said microcompartment is contained between said two parallel tangents.

[0031] For the purposes of the present invention, "variable thickness" of the lining of human cells undergoing cell differentiation means that in the same microcompartment, the lining does not have the same thickness throughout.

[0032] For the purposes of the present invention, "microcompartment" or "capsule" means a partially or completely enclosed three-dimensional structure that contains several cells.

[0033] For purposes of the present invention, "convective culture medium" means a culture medium that is agitated by internal movement.

[0034] For the purposes of the present invention, the term "mutation" refers to a genetic or epigenetic mutation, preferentially a functional mutation. It may in particular involve point modifications of the gene sequence, structural variants, epigenetic modifications or modifications of mitochondrial DNA. It may also involve mutations due to amplification of chromosomal regions, for example mutations due to amplification of the 20q chromosomal region, in particular 20q11, or 7q.

[0035] The term "functional mutation" in the sense of the present invention refers to a transmissible genetic or epigenetic modification that confers a potential gain or loss of function or a potential loss of function to the relevant mutant cells. It preferably involves a mutation that causes a modification of the phenotype of the affected mutant cells. Very preferentially, it involves a change in the genomic and / or epigenetic sequence that alters the therapeutic potential of the collection of cells or by increasing the risk associated with the therapy produced or by reducing the benefit provided by the therapy produced.

[0036] The "maximum dimension" of a microcompartment or cell cluster, or of a layer of cells or of a cell-based layer in the sense of the present invention is understood to mean the value of the maximum Feret's diameter of the aforementioned microcompartments.

[0037] The "smallest dimension" of a microcompartment or cell cluster, or of a layer of cells or of a cell-based layer in the sense of the present invention is understood to mean the value of the smallest Feret's diameter of the abovementioned microcompartments.

[0038] For the purposes of the present invention, "tissue" or "biological tissue" has the general meaning of tissue in biology, i.e., an intermediate level of organization between cells and organs. Tissues are assemblies of similar cells and of the same origin (most commonly derived from a common cell line, although they can result from the association of different cell lines) grouped into clusters, networks, or bundles (fibers). Tissues form functional assemblies, i.e., their cells contribute to the same function. Biological tissues regenerate regularly and assemble together to form organs.

[0039] For purposes of the present invention, "lumen" refers to the volume of aqueous solution that is topographically surrounded by the cell, the contents of which are preferentially not in diffusion equilibrium with the volume of convective liquid present outside the microcompartment.

[0040] Cellular microcompartments The subject of the present invention is a three-dimensional cellular microcompartment comprising at least one outer hydrogel layer and, inside the outer layer, at least one layer of cells and / or at least one cell-based layer, in which less than 20% of the total population of cells present are cells having at least one mutation.

[0041] The microcompartments comprise an external hydrogel layer. Preferentially, the hydrogel used is biocompatible, i.e. non-toxic to the cells. The hydrogel layer must allow the diffusion of oxygen and nutrients to supply the cells contained in the microcompartments and allow them to survive. According to one embodiment, the external hydrogel layer comprises at least alginate. It may consist exclusively of alginate. The alginate may in particular be sodium alginate, composed of 80% α-L-guluronate and 20% β-D-mannuronate, with an average molecular weight of 100 to 400 kDa and a total concentration of 0.5 to 5% by weight. The hydrogel layer is cell-free.

[0042] The hydrogel layer makes it possible in particular to protect the cells from the external environment and to limit uncontrolled proliferation of the cells.

[0043] A microcompartment according to the invention comprises at least one layer of cells and / or at least one cell-based layer, which or these layers of cells and / or cell-based layers are three-dimensionally organized within the microcompartment.

[0044] The microcompartments are in particular - one or more layers of cells and / or one or more cell-based layers that are three-dimensionally organized, or - may comprise one or more layers of cells and / or one or more cell-based layers, three-dimensionally organized, as well as cells in suspension in microcompartments.

[0045] The cells present in the microcompartments can be of any cell type. Preferably, the cells are human or animal cells.

[0046] In certain embodiments, the microcompartments contain pluripotent stem cells. Pluripotent stem cells or pluripotent cells refer to cells that have the ability to form all tissues present in the whole organism of origin, but cannot form the whole organism itself. Pluripotent stem cells can be, in particular, induced pluripotent stem cells (iPSCs), MUSE cells ("Multilineage-differentiating Stress Enduring") cells found in the skin and bone marrow of adult mammals, or embryonic stem cells (ES).

[0047] According to a particularly preferred variant of the invention, the microcompartments according to the invention contain human or animal induced pluripotent stem cells.

[0048] In another particular embodiment, the microcompartments according to the invention comprise human or animal multipotent cells and / or human or animal progenitor cells derived from these multipotent cells. The multipotent and / or progenitor cells are preferentially obtained from pluripotent stem cells, in particular human pluripotent stem cells, or optionally from non-pluripotent human cells artificially modified so that the transcriptional profile matches that of a particular multipotent and / or progenitor cell, typically by forcing the expression of transcription factors specific for the target cell phenotype. Preferentially, the multipotent and / or progenitor cells are obtained from pluripotent stem cells after contact with a solution capable of initiating the differentiation of said stem cells.

[0049] According to another variant, the microcompartments according to the invention contain differentiated cells of a human or animal. The differentiated cells are preferably obtained from pluripotent stem or progenitor cells, in particular human pluripotent stem or progenitor cells, or optionally from non-pluripotent human cells, whose transcriptional profile has been artificially modified to connect with that of a particular differentiated cell, typically by forcing the expression of transcription factors specific for the target cell phenotype. Preferentially, the differentiated cells are obtained from pluripotent or multipotent, or progenitor stem cells, after contact with a solution capable of initiating the differentiation of said stem cells. According to one variant, the cellular content of the microcompartments comprises homogenous or mixed cellular identity.

[0050] The differentiated cells may be in the form of a three-dimensional tissue or microtissue, or in the form of multiple webs or microtissues within a microcompartment, among others. The differentiated cells may be in the form of a compacted tissue or microtissue.

[0051] The microcompartments according to the invention may contain several types of cells, in particular the microcompartments according to the invention may contain, for example, induced pluripotent stem cells and / or multipotent cells and / or progenitor cells and / or differentiated cells.

[0052] If the cells encapsulated in the microcompartments are intended to be used for cell therapy in humans, the cells may be immunocompatible with the humans intended to receive them, to avoid any risk of rejection.

[0053] The cells present in the microcompartment have few or no functional mutations. According to the invention, less than 20% of the total population of cells present are cells that have at least one mutation, in particular at least one functional, genetic or epigenetic mutation.

[0054] The present invention is particularly directed to microcompartments in which less than 20% of the total population of cells present are cells carrying at least one functional mutation, preferentially in which less than 20% of the total population of cells present are cells carrying at least one mutation that results in an altered phenotype of the associated mutant cell.

[0055] The present invention is also directed to microcompartments in which less than 20% of the total population of cells present are cells that have at least one mutation that allows for the clonal expansion of the cells to become predominant in the culture.

[0056] According to a particularly preferred variant, the invention relates to a microcompartment in which less than 20% of the total cell population present are cells carrying at least one mutation selected from oncogenic mutations, wherein the at least one mutation is an oncogenic mutation.

[0057] According to one embodiment, the invention relates to a microcompartment in which less than 20% of the total cell population present are cells carrying at least one mutation in a gene and / or a mutation due to amplification of a chromosomal region.

[0058] In one embodiment of the invention, less than 20% of the total cell population present in the microcompartment are cells carrying at least one mutation in the P53 gene and / or an amplification of the 20q and / or 7q chromosomal region (a mutation resulting from an amplification of the 20q and / or 7q chromosomal region), in particular an amplification of the 20q11 chromosomal region (a mutation resulting from an amplification of the 20q11 chromosomal region).

[0059] Preferably, cells carrying at least one mutation according to one of the embodiments of the present invention represent 0-15%, in particular 0-14%, 0-12%, in particular 0-10%, even more preferentially 0-8%, 0-5%, 0-2% of the total cell population present in the microcompartment.

[0060] The percentage of mutant cells in cell populations can be measured by various methods known to those skilled in the art.For the detection of point mutations, sequencing methods with high reading depth are preferred (whole genome sequencing, exome sequencing, amplitude, etc.).For the detection of structural variants, high resolution methods are preferred (high resolution SNP array, Bionano optical genome mapping, digital PCR, etc.).For the detection of epigenetic variants, several tools can be envisaged (RRBS methylation array, bisulfite sequencing / pyrosequencing, etc.).

[0061] Advantageously, the microcompartments according to the invention have a very low level of mutant cells, which occurs after several cell division cycles. The cells according to the invention are in fact cells obtained by amplification from at least one cell.

[0062] Indeed, the cells present in the microcompartments according to the invention were obtained after at least two cell division cycles following encapsulation of at least one cell in the outer hydrogel layer.

[0063] Preferably, the cells present in the microcompartments 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 cell division cycles following encapsulation in the outer hydrogel layer of at least one cell, preferentially 1-5, 1-10, 1-15, 1-20, 1-30, 1-40, 1-50, 1-60, 1-100 cells. For example, the cells present in the microcompartments have been obtained after at least 6 cell division cycles following encapsulation in the outer hydrogel layer of at least one cell, preferentially 1-50 cells.

[0064] Preferably, the microcompartments are obtained after at least 2 passages after encapsulation, more preferably after at least 3, 4, 5, 6, 7, 8, 9 or 10 passages, each passage may last for example from 2 to 15 days, in particular from 3 to 10 days.

[0065] Preferably, the microcompartments are obtained after at least one reencapsulation, more preferably after 1 to 14 reencapsulations, in particular after 2 to 7 reencapsulations. Very preferentially, the reencapsulation corresponds to a new passage, each encapsulation cycle corresponding to a passage.

[0066] Preferably, all of the cells initially encapsulated in a microcompartment prior to the first cell division cycle occupy less than 50% of the volume of the microcompartment in which they are encapsulated, more preferably less than 40%, 30%, 20%, 10% of the volume of the microcompartment in which they are encapsulated.

[0067] Thus, according to one embodiment, the cells present in the microcompartments 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 cell division cycles following encapsulation in the outer hydrogel layer of cells occupying a volume of less than 50% of the volume of the microcompartment in which they are encapsulated, more preferably less than 40%, 30%, 20%, 10% of the volume of the microcompartment in which they are encapsulated.

[0068] Preferably, in a microcompartment according to the invention, the cells occupy more than 50% by volume relative to the volume of the microcompartment, even more preferably more than 60%, 70%, 75%, 80%, 85%, 90% by volume relative to the volume of the microcompartment.

[0069] A microcompartment according to the invention comprises several cells, preferably at least 20 cells, even more preferably at least 100, at least 500, at least 1000, at least 10,000 cells.

[0070] In addition to the outer layer and the cells, the microcompartments according to the invention may contain other elements, in particular: - culture medium, and / or - may comprise at least one intermediate layer of an isotonic aqueous solution and / or extracellular matrix elements.

[0071] The culture medium is a medium suitable for the cells present in the microcompartments according to the knowledge of the person skilled in the art.

[0072] The intermediate layer of isotonic aqueous solution preferentially contains peptides or peptidomimetic sequences capable of binding to extracellular matrix elements, e.g. integrins. "Isotonic aqueous solution" means an aqueous solution having an osmolality of 200-400 mOsm / L. This layer is preferably located between (a) the layer of cells and / or cell base layer and (b) the outer hydrogel layer.

[0073] The intermediate layer may consist of elements that have been added during production of the microcompartment and / or elements that have been added to the microcompartment and / or elements that are secreted or induced by other components of the microcompartment.

[0074] The intermediate layer may in particular comprise or consist of an extracellular matrix and / or a culture medium. If it comprises an extracellular matrix, this may be an extracellular matrix secreted by the cells of the inner layer and / or an extracellular matrix added during the preparation / production of the microcompartments.

[0075] The intermediate layer preferentially comprises a mixture of proteins and extracellular compounds necessary for culturing the cells undergoing differentiation. Preferentially, the intermediate layer comprises structural proteins such as collagen, laminin, entactin, vitronectin, and growth factors such as TGF-beta and / or EGF. According to one variant, the intermediate layer may consist of or comprise a hydrogel type matrix of plant or synthetic origin, such as Matrigel® and / or Geltrex® and / or modified alginates, or a copolymer of poly(N-isopropylacrylamide) and poly(ethylene glycol) (PNIPAAm-PEG) of the Mebiol® type.

[0076] According to one variant, the intermediate layer may form a gel.

[0077] The intermediate layer may optionally contain one or more cells at the surface of the intermediate layer that is in contact with the inner layer of human cells undergoing differentiation.

[0078] Preferably, the intermediate layer has a Young's modulus of 0.05 to 3 kDa. The Young's modulus can be measured by any method known to the person skilled in the art, in particular by measuring the rheology of a gel of the same composition as the intermediate layer, or by AFM (atomic force microscopy).

[0079] An intermediate layer of an isotonic aqueous solution and / or comprising extracellular matrix elements, preferentially an intermediate layer of extracellular matrix, having such a Young's modulus value makes it possible to improve the maintenance of the cellular phenotype and / or genomic integrity of the cells contained in this intermediate layer during cell division.

[0080] According to a particular embodiment of the invention, the microcompartment also comprises at least one opening or lumen. Preferably, the microcompartment comprises an internal lumen. The microcompartment according to the invention may also comprise several lumens. The lumen may contain liquids, in particular culture medium and / or liquids secreted by the cells. Advantageously, the presence of this hollow part allows the cells to have a small diffusion volume whose composition can be controlled, facilitating cell communication.

[0081] In one variation of the invention, the microcompartment comprises the following in sequence organized around the lumen: at least one layer of cells and / or at least one cell-based layer, preferentially epithelial cells, in particular stem cells, in particular human or animal induced pluripotent stem cells, an intermediate layer of an isotonic aqueous solution and / or extracellular matrix elements, preferentially an extracellular matrix layer, -Outer hydrogel layer.

[0082] In this variation, the inner layer of cells within the microcompartment according to the invention is hollow. This three-dimensional arrangement of a single layer or globular epithelial base surrounding a central lumen may also be called a cyst. The lumen is preferably generated by cells growing and developing on the extracellular matrix layer during the formation of the cyst.

[0083] The cyst-shaped conformation allows to reduce the pressure that the stem cells are subjected to, compared to 2D cultures or aggregates. This conformation reduces cell mortality and increases the culture amplification factor. This in turn allows to reduce the number of passaging and dissociation required, shortening the culture time required to reach the required final cell number. Taken together, these improvements are also responsible for maintaining the genetic integrity of the stem cells in the microcompartments.

[0084] The cell microcompartments according to the invention are closed or partially closed, i.e. the outer layer is closed or partially closed. Preferentially, the microcompartments are closed.

[0085] The microcompartments according to the invention may be of any three-dimensional form, i.e. they may have the shape of any object in space. They may have any form suitable for cell encapsulation. Preferentially, the microcompartments according to the invention are of spherical or elongated form. They may have an ovoid, cylindrical, spheroidal or spherical form. They may in particular have the form of a hollow spheroid, a hollow ovoid, a hollow cylinder or a hollow sphere.

[0086] It is the outer layer of the microcompartment, i.e. the hydrogel layer, that gives the microcompartment according to the invention its size and shape. Preferentially, the smallest dimension of the microcompartment according to the invention is between 10 μm and 1 mm, preferentially between 100 μm and 700 μm. The smallest dimension of the microcompartment according to the invention may be between 10 μm and 600 μm, in particular between 10 μm and 500 μm.

[0087] Its largest dimension is preferentially greater than 10 μm, more preferentially between 10 μm and 1 m, even more preferentially between 10 μm and 50 cm.

[0088] The microcompartments according to the invention contain cells whose genomic integrity is preserved and / or maintained, and a very small percentage of the cells present in the microcompartment are carriers of the mutation, which can be used for any application, in particular as drugs in cell therapy in humans or animals.

[0089] Microcompartments according to the invention may optionally be frozen for storage and must then be thawed prior to use.

[0090] The present invention also relates to the assembly of multiple microcompartments.

[0091] Therefore, the present invention also relates to an assembly of cellular microcompartments or a series of cellular microcompartments as described above comprising at least two cellular microcompartments according to the present invention.

[0092] The present invention also relates to a microcompartment assembly or a series of at least two three-dimensional cellular microcompartments, each microcompartment comprising at least one outer hydrogel layer and, inside said outer layer, at least one layer of cells and / or at least one cell-based layer, wherein at least one microcompartment is a microcompartment according to the present invention.

[0093] Another particular object of the present invention relates to an assembly of at least two three-dimensional cellular microcompartments or a series of at least two three-dimensional cellular microcompartments, each microcompartment comprising at least one outer hydrogel layer and, inside said outer layer, at least one layer of cells and / or at least one cell-based layer, in which less than 20% of the total population of cells present in all microcompartments of the assembly are cells with at least one mutation. Preferably, the cells with at least one mutation represent 0-15%, in particular 0-14%, 0-12%, in particular 0-10%, even more preferentially 0-8%, 0-5%, 0-2% of the total population of cells present in all microcompartments. Preferably, at least one microcompartment is a microcompartment according to the invention.

[0094] Thus, although a series of one or more microcompartments may contain more than 20% of mutant cells relative to the number of cells present in said microcompartments, for all microcompartments forming a microcompartment assembly according to the invention, less than 20% of the total population of cells present in all microcompartments of the assembly are cells carrying at least one mutation, in particular at least one functional, genetic or epigenetic mutation. Preferably, at least one microcompartment is a microcompartment according to the invention.

[0095] The present invention particularly relates to microcompartment assemblies in which less than 20% of the total population of cells present in the assembly are cells having at least one functional mutation, preferably microcompartments in which less than 20% of the total population of cells present in the assembly are cells having at least one mutation that results in an altered phenotype of the associated mutant cell.

[0096] The present invention also relates to a microcompartment assembly in which less than 20% of the total population of cells present in the assembly are cells that have at least one mutation that allows for the clonal expansion of the cells that become predominant in the culture.

[0097] According to a particularly preferred variant, the present invention relates to a microcompartment assembly in which less than 20% of the total population of cells present in the assembly are cells carrying at least one mutation selected from oncogenic mutations, wherein the at least one mutation is an oncogenic mutation.

[0098] According to one embodiment, the present invention relates to a microcompartment assembly, in which less than 20% of the total population of cells present in the assembly are cells carrying at least one mutation in a gene and / or a mutation due to amplification of a chromosomal region.

[0099] In one embodiment of the invention, less than 20% of the total cell population present in the microcompartment assembly are cells having at least one mutation in the P53 gene and / or an amplification of the 20q and / or 7q chromosomal region (a mutation due to an amplification of the 20q and / or 7q chromosomal region), in particular an amplification of the 20q11 chromosomal region (a mutation due to an amplification of the 20q11 chromosomal region).

[0100] Preferably, cells carrying at least one mutation according to one of the embodiments of the present invention represent 0-15%, in particular 0-14%, 0-12%, in particular 0-10%, even more preferentially 0-8%, 0-5%, 0-2% of the total cell population present in the microcompartment assembly.

[0101] Preferably, the cells present in the microcompartments of the microcompartment assembly 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 cell division cycles following encapsulation in an outer hydrogel layer of at least one cell per microcompartment. The microcompartments present in this microcompartment assembly may have one or more characteristics (size, shape, cell count, cell volume, middle layer, lumen, etc.) of the microcompartments according to the invention.

[0102] The microcompartment assembly according to the present invention preferably comprises 2 to 10 16 It contains micro compartments.

[0103] Preferably, the series of microcompartments according to the invention are in a culture medium, in particular in an at least partially convected culture medium.

[0104] According to a particularly preferred embodiment, the subject of the present invention is a series of cell microcompartments as described above in a closed chamber, e.g. a bioreactor, preferentially in a culture medium in a closed chamber, e.g. a bioreactor.

[0105] The presence of an outer hydrogel layer, and possibly an intermediate layer of isotonic aqueous solution, allows for a uniform distribution of cells between the microcompartments. Moreover, this hydrogel layer makes it possible to prevent the microcompartments from integrating, these integration events being a major source of undesirable variability for the homogeneity of the phenotype of the cells.

[0106] Method for obtaining microcompartments according to the invention The present invention also relates to a method for preparing the microcompartments according to the invention.

[0107] The method for preparing a microcompartment or a microcompartment assembly according to the invention comprises the steps of: -(a) preparing a suspension of cells comprising single cells and / or at least one assembly of cells in an isotonic medium, preferentially a culture medium containing an apoptosis inhibitor, -(b) encapsulating the cell suspension within a hydrogel layer; - (c) preferentially culturing the microcompartments obtained in an isotonic solution, preferably in a medium containing an apoptosis inhibitor; - (d) preferentially rinsing the microcompartments to remove apoptosis inhibitors; - (e) culturing the microcompartments in an isotonic solution, preferentially in a culture medium, for at least two cell division cycles; - (f) optionally, recovering the obtained cellular micro-compartments.

[0108] The present invention also contemplates using this method to maintain the genomic integrity of encapsulated cells.

[0109] In the method according to the invention, all of the cells initially encapsulated in step (b) occupy less than 50% of the volume of the microcompartment in which they are encapsulated, more preferably less than 40%, 30%, 20%, 10% of the volume of the microcompartment in which they are encapsulated.

[0110] The apoptosis inhibitor may be, for example, one or more inhibitors of the RHO / ROCK (RHO-associated protein kinase) pathway, or any other apoptosis inhibitor known to the person skilled in the art. The apoptosis inhibitor must be capable of promoting cell survival and, if present, of promoting the attachment of cells to the extracellular matrix at the time the outer hydrogel layer is formed around said extracellular matrix.

[0111] The method according to the invention may comprise, prior to or simultaneously with step (a), a step of dissociation of the cells by chemical, enzymatic or mechanical dissociation. This step is particularly important in the case of adherent cells.

[0112] The encapsulated cells are suspended in the form of single cells and / or clusters or assemblies of at least two cells ("clusters"). Preferably, single cells represent less than 50% of the total number of cells initially encapsulated in step (b). Indeed, encapsulating clusters of cells is preferred, as this reduces chromosomal segregation and, therefore, the appearance of new mutations, which is involved in maintaining the genomic integrity of the cells.

[0113] Preferably, each cell cluster initially encapsulated in step (b) has a larger dimension that is less than 20% and even more preferentially less than 10% of the maximum dimension of the microcompartment in which it is encapsulated. In fact, the cell clusters must not have a size that is too large compared to the size of the microcompartment, because the dimensions of these initial cell clusters that are too large may lead to premature cell confluence in the capsule during cell division. This premature confluence of all or part of the capsule may cause an increase in intracellular pressure, resulting in cell stress and in particular affecting chromosome segregation.

[0114] According to one variant, the method according to the invention may comprise a step of mixing the cells with an extracellular matrix, either between steps (a) and (b) or simultaneously with the encapsulation in step (b).

[0115] Highly preferably, steps (c), (d) and (e) are carried out under continuous or sequential stirring. This stirring is important because it maintains the homogeneity of the culture environment and avoids the formation of diffusion gradients. For example, it allows uniform control of cellular oxygenation levels, thus avoiding necrosis associated with hypoxia or oxidative stress phenomena associated with hyperoxia. By avoiding cell mortality and / or increased oxidative stress, stirring is responsible for maintaining genetic integrity.

[0116] The method according to the invention is preferably carried out in a closed chamber, such as a sealed bioreactor.

[0117] The number of cell divisions in step (e) is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 cell division cycles.

[0118] Preferably, the microcompartments are obtained after at least two passages (wherein a passage corresponds to a complete cycle of steps (a), (b) and (e), optionally (c) and (d)), more preferably after at least 3, 4, 5, 6, 7, 8, 9 or 10 passages. Each passage may last for example from 2 to 15 days, in particular from 3 to 8 days.

[0119] In a preferred variant, the method according to the invention comprises at least one re-encapsulation of the cells after step (e), i.e. at least two encapsulation cycles.Preferably, each encapsulation cycle corresponds to a passage.In this variant of the method (at least one re-encapsulation of the cells after step (e)), the number of cell divisions throughout the method (for all passages) is at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30 cell division cycles.

[0120] In the method according to the invention there may be several reencapsulations, preferably 1 to 100, in particular 1 to 10 reencapsulations.

[0121] Each reencapsulation may include: - a step consisting of dissociating the microcompartment or a series of microcompartments to obtain a suspension of cells or a suspension of cell clusters. The outer hydrogel layer can be removed in particular by hydrolysis, dissolution, perforation and / or destruction by any biocompatible means, i.e. means that are not toxic to cells. For example, removal can be achieved using phosphate buffered saline, divalent ion chelators, enzymes such as alginate lyase if the hydrogel comprises alginate, and / or laser microdissection. - Reencapsulating all or part of the cells or cell clusters into a hydrogel capsule.

[0122] Reencapsulation is a suitable means to increase cell expansion resulting from the pluripotency process and to reduce the risk of mutations.

[0123] According to one embodiment, the reencapsulation comprises: -(i) removing the outer hydrogel layer; -(ii) resuspending the cells contained in the microcompartments so as to obtain single cells and / or at least one assembly or cluster of cells in an isotonic medium, preferentially a culture medium containing an apoptosis inhibitor; -(iii) encapsulating the cell suspension within a hydrogel layer; - (iv) preferentially culturing the microcompartments obtained in an isotonic solution containing an apoptosis inhibitor, preferably in a culture medium containing an apoptosis inhibitor, - (v) preferentially rinsing the microcompartments to remove apoptosis inhibitors; - (vi) culturing the microcompartments in an isotonic solution, preferentially a culture medium, for at least one cell division cycle; -(vii) optionally, recovering the resulting cell micro-compartments.

[0124] Compartmentalization into microcompartments allows to eliminate microcompartments that contain more mutant cells than other capsules. Even if mutant cells grow rapidly, they reach intracapsular confluence and limit their increase. Compartmentalization also allows not to contaminate the entire cell population and also allows to eliminate capsules that contain mutant cells at any time, in particular before the reencapsulation step. This sorting can be done, for example, by in-line analysis or by eliminating filled capsules more quickly than other capsules. Thus, the method according to the invention can include one or more steps of eliminating microcompartments that contain mutant cells, in particular microcompartments that contain more than 20% mutant cells.

[0125] According to one variant of the invention, the cells are pluripotent stem cells that are organized into cysts directly from pluripotent stem cells or from differentiated cells that are reprogrammed into pluripotent cells within the hydrogel capsule during the formation of the microcompartments.

[0126] The incubation of steps (a) and / or (ii) is preferentially carried out for a period comprised between a few minutes and a few hours, preferentially between 2 minutes and 2 hours, more preferably between 10 minutes and 1 hour.

[0127] Steps (c) and / or (iv) of culturing with the apoptosis inhibitor are carried out for a period comprised between 2 and 72 hours, preferentially comprised between 6 and 48 hours, more preferably comprised between 24 and 48 hours.

[0128] The rinsing step can be carried out by one or more rinsing operations in continuous culture medium free of RHO / ROCK pathway inhibitors for less than 96 hours, preferentially less than 72 hours, more preferably 24 to 48 hours, after the initiation of steps (c) and / or (iv).

[0129] In one embodiment, at least one of the steps (preferably all steps) is carried out at a temperature suitable for cell survival, including between 4 and 42° C. The temperature during cell growth should preferably be between 32 and 37° C. to avoid inducing mutations by reducing the performance of repair enzymes. Similarly, the temperature should preferably be low (ideally around 4° C.) to manage stress on the cells in step (b).

[0130] According to one variant, cell reprogramming agents may be added in steps (a) and / or (b) and / or (c) and / or (ii) and / or (iii) and / or (iv). Preferably, they are cell reprogramming agents that do not permeate the hydrogel layer. The addition of reprogramming agents is particularly relevant when the initially encapsulated cells are differentiated cells that are to be dedifferentiated, especially to a pluripotent stage. The skilled person knows how to reprogram differentiated cells into stem cells by reactivating the expression of genes associated with the embryonic stage by means of certain factors, referred to in the present invention as "reprogramming agents". The methods described below may be cited as examples. Takahashi et al., 2006 ("Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors" Cell, 2006 Vol 126, pages 663-676), Ban et al., 2009 ("Efficient induction of transgene-free human pluripotent stem cells using a vector based on Sendai virus, an RNA virus that does not integrate into the host genome" Proc Jpn Acad Ser B Phys Biol Sci. 2009;85(8):348-62) and International Application No. 2010 / 105311 entitled "Production of reprogrammed pluripotent cells". The reprogramming agent is advantageously encapsulated together with the differentiated cells to concentrate the product and promote contact with all cells. In case of reprogramming agents that permeate the hydrogel layer, it is possible to add the aforementioned agents to the culture medium after the encapsulation step. The reprogramming agent makes it possible to impose a series of phenotypic changes on the cells towards the pluripotent stage. Advantageously, the reprogramming process is carried out using a specific culture medium to promote these phenotypic changes.For example, cells are cultured in a first medium containing 10% human or bovine serum, supplemented with a serine / threonine protein kinase receptor inhibitor (e.g., product SB-431542 (C. 22 H 16 N4O3), one or more RHO / ROCK (RHO-associated protein kinase) pathway inhibitors, such as thiazolines and / or Y-27632, fibroblast growth factors, such as FGF-2, ascorbic acid, and antibiotics, such as Trichstan A (C 17 H 22 The cells are cultured in Eagle's Minimum Essential Medium (DMEM) supplemented with N2O3. The culture medium is then replaced with a medium that promotes the expansion of pluripotent cells, such as mTeSR®1 medium.

[0131] At any point, the method according to the invention may include a step consisting of verifying the phenotype of the cells contained in the microcompartments, which can be carried out by identifying the expression, by at least a portion of the cells contained in the microcompartments, of at least one gene specific for the desired phenotype.

[0132] The cell microcompartments obtained according to the method of the invention can then be frozen before any use. Freezing is preferentially carried out at temperatures comprised between -190°C and -80°C. Thawing can be carried out in a warm water bath (preferentially at 37°C) in order to thaw the cells very quickly. The microcompartments according to the invention prior to use may be kept at temperatures above 4°C, preferentially between 4°C and 38°C, for a limited time before use.

[0133] The method according to the invention, due to its particular features, makes it possible to maintain the genomic integrity of the cells during the culture, the final microcompartments comprising cells carrying few or no mutations.

[0134] In particular, the three-dimensional structure of the cells in the microcompartments and the low or even zero percentage of cells isolated during encapsulation (the majority of the cells are encapsulated in the form of clusters of cells) reduces chromosome segregation and, consequently, the appearance of new mutations.

[0135] The present invention also promotes amplification with a high amplification factor, thereby reducing the culture time and number of divisions to obtain a large number of cells, thus limiting mutagenesis.

[0136] Protection of the cells by the outer layer and, when present, the presence of extracellular matrix elements reduces chromosome segregation and limits mechanical stress on the cells, thereby reducing the appearance of new mutations.

[0137] Control of the culture parameters in the bioreactor also reduces oxidative stress, which is responsible for the reduction of new mutations.

[0138] The subject of the invention is also the use of the method according to the invention for maintaining the genomic integrity of a cell during amplification.

[0139] The subject of the present invention is also the use of three-dimensional microcompartments, preferably closed, preferentially of spherical or elongated shape, comprising at least one external hydrogel layer defining an inner part, for maintaining the genomic integrity of cells during their proliferation.Preferably, the subject of the present invention involves the use of cell microcompartments according to the invention in its different variants as described in the present application.The present invention is also directed to the use of assemblies of these microcompartments, preferentially in closed bioreactors, and even more preferentially of the microcompartment assemblies according to the invention and according to all the variants described in the present application, for maintaining the genomic integrity of cells during their proliferation.

[0140] Next, the present invention will be illustrated by two examples and comparative results.

[0141] These examples relate to the culture of human pluripotent stem cells, more specifically human induced pluripotent stem (iPS) cells.

[0142] Example 1 protocol: The cell line used here, called iPS-IMGINE005, has been previously described in this publication: E. Quelennec, C. Banal, M. Hamlin, D. Clemantine, M. Michael, N. Lefort, Generation of two induced pluripotent stem cell lines IMAGINi004-A and IMAGINi005-A from healthy donors. Stem Cell Research, 101959 (2020).

[0143] iPS lines were generated following the usual standards for iPS culture in two dimensions. Karyotype monitoring was performed regularly (every 5–10 passages) to monitor the virtually inevitable emergence of mutations during long-term culture of the lines.

[0144] The starting point for the experiments carried out here is a frozen iPS cell sample at passage 2D number 23 after reprogramming. At this stage of culture, for this sample, high-resolution karyotype testing was unable to detect the amplification of the 20q11 chromosomal region, but it was observed that short culture of this sample (fewer than 10 2D passages) caused the appearance of mutations due to the amplification of the 20q11 chromosomal region.

[0145] This cell origin is particularly suitable for testing positive selection of mutant clones over time in a population of cells in culture.

[0146] Indeed, the mutation due to the amplification of the 20q11 chromosomal region confers a growth advantage to the mutated clone, and the greater the selective pressure in the culture system, the greater the risk that this clone will be rapidly selected and become dominant.

[0147] The encapsulated culture system in stirred suspension (hereinafter referred to as "the present invention") was compared with two standard culture systems in the field of pluripotent stem cell production: culture in two dimensions (hereinafter referred to as "2D culture") and culture in unprotected stirred suspension in the form of aggregates (hereinafter referred to as "bioreactor aggregates").

[0148] Using the first samples (previously described and cultured in 2D), three experimental arms relating to the three culture systems were started in parallel, which were carried out over a period of 28 days. At each passage and for each experimental arm, cells were sampled to allow the performance of genetic tests (see results section). In particular, the frequency of mutations due to amplification of the 20q11 chromosomal region was evaluated at the beginning and at the end of this long-term culture of 28 days.

[0149] The passaging rate for each culture system closely follows the optimal recommendations for each condition. Thus, 2D cultures are passaged every 4-5 days when the confluence is 70-90%. Aggregate cultures are passaged every 5 days, according to the supplier's recommendations (Minibio, ABLE® Bioreactor Systems). Encapsulated cultures are passaged every 7 days, when the average intracapsular confluence is 50-100%.

[0150] All cultures described below are performed using mTeSR 1 culture medium ("Stemcell Technologies"). 10 μM Rock inhibitor treatment is initiated during the first 24 hours after passaging.

[0151] All cultures (2D, bioreactor aggregates and invention) are maintained in a cell culture incubator at 37° C. and 5% CO 2 .

[0152] The two experimental arms culturing stem cells in suspension, "bioreactor aggregates" and "present invention", use 30 mL bioreactors from Minibio's ABLE® Bioreactor Systems brand. A constant agitation speed of 35 revolutions per minute was set from seeding until cell harvest.

[0153] The two experimental arms culturing stem cells in three-dimensional cell mass form, in "bioreactor aggregates" and "invention" suspension, use enzymatic dissociation for serial passaging. On the one hand, aggregates and on the other hand, encapsulated cysts are dissociated by using a TryplE bath at 37°C for 20 minutes. The cells and small populations (clusters) of cells resulting from this dissociation are then used to seed new cultures.

[0154] For culture using extracellular matrix, Matrigel (Corning) is used. Therefore, for 2D culture, flasks (T-Flak T75) are pre-coated with Matrigel. For encapsulation or reencapsulation, cells are mixed with Matrigel before injection into the central microfluidic channel, and culture in aggregates does not require the use of extracellular matrix.

[0155] "2D cultures" were established in flasks (T-Flask T75) pre-coated / carpeted with Matrigel®, with cell seeding densities of 1 cm 2 The number of cells is 10,000-30,000 per day. Passage is performed by the small aggregate method with short-term (less than 5 min) use of the calcium chelator RelesR (Stem cell technologies). The culture medium is completely replaced on day 1 to remove rock inhibitor (constant volume) treatment, and every day thereafter.

[0156] The "bioreactor aggregate" culture is started with the same cell suspension used to seed the "2D" and "invention" cultures, but with an initial concentration of 175,000 cells per mL of medium for a total of 20 mL of medium. The culture medium is completely replaced on day 1 to remove the rock inhibitor (constant volume) treatment, and then 75% of the medium is refreshed every day (constant volume of 20 mL).

[0157] Cultivation of hiPSCs according to the invention (encapsulation according to the invention): Prior to encapsulation, 2D stem cell colonies were detached using ReLeSR for 1 min and then dissociated using Accutase (StemCell Technologies). HiPSCs were then mixed with Matrigel in a 50 / 50 volume ratio at 4 °C to maintain the suspension in a liquid state. Thus, the final concentration of cells in the cell / matrix solution was 0.4–1.0 × 10 6The encapsulation density was 100 viable cells / ml, which was called the encapsulation density. Ethylene-tetrafluoroethylene (ETFE) tubing is connected to the three inlets of the 3D-printed microfluidic co-laminar flow device. For better flow control, a microcapillary tip made from extruded and polished glass (with a nozzle diameter of about 100 μm for most experiments, or 150 μm) is glued to the nozzle outlet. The cell / matrix suspension is filled into the inner channel of the three-way device, which is kept cooled by an in-line cooling system to avoid premature gelation of Matrigel. Sodium alginate solution (Novamatrix Proonova SLG100, 0.25 g at 2% in distilled water) is injected into the outer channel. To prevent gelation of alginate in the microfluidic device due to calcium release by cells in suspension, a calcium-free solution (sorbitol 300 mM, Sigma-Aldrich) was used in the middle channel of the coextrusion tip to act as a barrier against calcium diffusion. The flow rates for the three solutions were around 120 mL / h for the three channels (alginate solution, sorbitol solution and cell+matrix suspension). At these flow rates, the composite solution forms a liquid jet that fragments into droplets (approximately twice the size of the nozzle) due to spontaneous Rayleigh-Plateau instability. To avoid subsequent coalescence of a series of droplets, the alginate-filled part and the copper ring are connected to a high-voltage generator (2000 V). When the composite droplets come into contact with the calcium collection bath (at 100 mM), the outer layer of alginate gels. Thus, the inner cell / matrix solution remains trapped within a closed spherical permeable microcompartment. A few minutes following encapsulation, the capsules are rinsed with culture medium (DMEM) to reduce the basal calcium concentration, and finally, they are transferred in suspension to culture medium.

[0158] The passages of the "invention" arm of this experiment correspond to reencapsulations. These reencapsulations are carried out by dissolving the alginate capsules using a short rinse with ReleSR, followed by dissociation of the cells with TrypLE (a trypsin-based dissociation enzyme, ThermoFisher) for 20 minutes at 37°C. The cells obtained were then treated according to the encapsulation protocol according to the invention.

[0159] result: Four successive encapsulations were performed, each with a period of 7 days. Six successive passages were performed for the "bioreactor aggregate" arm and seven successive passages for the "2D culture arm". Sampling of cells at each passage and on day 28 allowed a comparative evaluation of the three culture arms over time (Figure 1).

[0160] Evaluation by phase contrast microscopy confirms successful formation of two-dimensional colonies, aggregates and encapsulated cysts of stem cells as expected for the "2D culture", "bioreactor aggregates" and "invention" arms (Figures 2a, 2b, 2c).

[0161] At each passage, the cells are counted using a cell counter (Nucleo Counter NC 3000), which makes it possible to establish the cell amplification factor during the culture (Figure 3). The cumulative theoretical amplifications are 151 million, 71 million and 13,330 for the experimental arms "Invention", "2D culture" and "Bioreactor aggregates", respectively. These cumulative amplification factors correspond to an average visible cell division number of 27.2, 26.2 and 13.7 during the 28 days for the "Invention", "2D culture" and "Bioreactor aggregates" culture arms, respectively. The final cell amplification is observed to be higher in the experimental arm "Invention" compared to the two other experimental arms.

[0162] The three culture systems performed successfully according to best standards, as suggested by the pluripotency markers OCT4 and NANOG being similarly expressed in the three compared culture systems (Figure 4).

[0163] Genetic evaluation was first performed by ultra-high-level SNP chips (CytoScan® HD Array Affymetrix, ThermoFisher) (Figure 5). The appearance of structural mutations is observed near chromosome 20 (chromosomal duplications and deletions encompassing the 20q11 region) that are clearly visible for the final samples (D28) of the experimental arms "2D culture" (about 50% mutant cells) and "bioreactor aggregates" (about 50% mutant cells). The similar profile of the rematch of chromosome 20 for these two samples indicates that it does not involve an independent event and that this mutation is inherited from a common ancestor. Thus, even if this mutation was undetectable during the first sampling, this strongly suggests its presence in a low percentage at the first moments of the experiment. For sample D28 of the "invention" arm, a lower amplitude of copy number is noted, which corresponds to a percentage of mutant cells in the aggregate of less than 10%.

[0164] Digital PCR analysis was also performed at each passage for all experimental arms to detect possible appearance of recurrent gene mutations for pluripotent stem cells (iCS-digital PSC 24 probe, StemGenomics). In particular, the PCR probe of this study allowed to quantify the count of copies of the 20q11 chromosomal region over time (Figure 6). The average count of copies of the 20q11 region increases over time during culture for the cells of the three experimental arms. This increase is greater and faster for the "2D culture" arm and the "bioreactor aggregate" arm compared to the "invention" arm. Considering that the count of copies of the region 20q11 for each mutant cell is 3 (an increase of 1 copy, see Figure 5), an average copy count of less than 2.2 corresponds to a percentage of mutant cells in the aggregate of cells of less than 20%.

[0165] Overall, the digital PCR and SNP chip results were consistent, suggesting that the selection of mutant cells during 28 days of culture was at least 5-fold lower in the "invention" arm compared to the "2D culture" and "bioreactor aggregate" arms (Figure 7). In particular, the encapsulation culture system (invention) allowed an average of 6.8 cell divisions per passage to occur while maintaining a percentage of mutant cells below 20% for each encapsulation or by performing four encapsulations end-to-end.

[0166] Example 2: protocol: In this example, two cell lines are used: a commercially available line called iPSC-GHE (Gibco) and a transgenic line that constitutively expresses the GFP fluorescent protein called iPSC-AAVS1-GFP (Coriell, Allen Institute for Cell Science).

[0167] At the beginning of the experiment, the two lines are independently cultured in 2D. Karyotyping by digital PCR (iCS-digital PSC 24 probes, Semenomics) reveals that the iPSC-GHE line has two karyotypic abnormalities with amplification of chromosomal regions 7q and 20q, while the iPSC-AAVS1-GFP line does not show any abnormalities over the 24 zones tested (Fijack 8).

[0168] The use of iPSC-GHE lines with amplification of chromosomal regions 7q and 20q is particularly relevant to test the positive selection of mutant clones over time in a collection of cells in culture. Indeed, the mutations due to the amplification of chromosomal regions 7q and 20q confer a growth advantage to the mutant clone. The greater the selective pressure of the culture system, the greater the risk that this clone will be selected quickly and become dominant.

[0169] The encapsulated culture system in stirred suspension "the present invention" was compared with a standard culture system in the field of pluripotent stem cell production (unprotected culture in stirred suspension in aggregate form "bioreactor aggregates") over a period of 21 days.

[0170] The samples used to start the two experimental arms related to the two culture systems in parallel correspond to a mixture of iPSC-AAVS1-GFP and iPSC-GHE lines: the mixture corresponds to 80% iPSC-AAVS1-GFP and 20% iPSC-GHE.

[0171] At each passage and for each experimental arm, cells are sampled to allow for cytometric and genetic testing (see Results section). In particular, analyses are aimed at monitoring the occurrence of frequency of iPSC-GHE aggregates containing karyotypic abnormalities within the cultures.

[0172] The passaging rate for each culture system closely follows the optimal recommendations for each condition: thus, cultures in aggregates are passed every 5 days according to the supplier's recommendations (Minibio, ABLE® Bioreactor Systems) and encapsulated cultures are passed every 7 days when the average intracapsular confluence is between 50 and 100%.

[0173] All cultures described below are performed using mTeSR1 plus culture medium (Stemcell Technologies). Treatment with 10 μM Rock inhibitor is initiated during the first 24 hours after passaging.

[0174] All cultures (bioreactor aggregates and invention) are maintained in a cell culture incubator at 37° C. and 5% CO 2 .

[0175] The two experimental arms, "bioreactor aggregate" and "invention", used 30 mL mini-bioreactors from Minibio, an ABLE® Bioreactor Systems brand, with a constant stirring speed of 55 rpm for the "bioreactor aggregate" conditions and 100 rpm for the "invention" conditions.

[0176] The two experimental arms "Bioreactor Aggregates" and "Invention" use enzymatic dissociation for serial passaging. The aggregates on the one hand and the encapsulated cysts on the other hand are dissociated by using a TryplE bath at 37° C. The cells and small populations of cells (clusters) obtained from this dissociation are then used to seed new cultures.

[0177] For encapsulation or reencapsulation, cells are mixed with Matrigel® prior to injection into the central microfluidic channel; culturing in aggregates does not require the use of an extracellular matrix.

[0178] The "bioreactor aggregate" culture is started with the same cell suspension used to seed the "invention" culture, but with an initial concentration of 175,000 cells per mL of medium for a total of 10 mL of medium. The culture medium is completely changed on day 1 to remove the rock inhibitor.

[0179] Cultivation of hiPSCs according to the invention (encapsulation according to the invention): Prior to encapsulation, 2D stem cell colonies of iPSC-GHE and iPSC-AAVS1-GFP were dissociated using Accutase (StemCell Technologies). The two iPSC lines were then mixed according to the ratios previously indicated (80%-20%), and this cell suspension itself was mixed with Matrigel in a 50 / 50 volume ratio at 4 °C to keep the suspension in a liquid state. Thus, the final concentration of cells in the cell / matrix solution was 0.4-1.0 × 10 6The encapsulation density was determined to be 100 viable cells / ml, which was called the encapsulation density. Ethylene-tetrafluoroethylene (ETFE) tubing is connected to the three inlets of the 3D-printed microfluidic co-laminar flow device. For better flow control, a microcapillary tip made of extruded and polished glass (with a nozzle diameter of about 100 μm for most experiments, or 150 μm) is glued to the outlet of the nozzle. The cell / matrix suspension is filled into the inner channel of the three-way device, which is kept cooled by an in-line cooling system to avoid premature gelation of Matrigel. Sodium alginate solution (Novamatrix Proonova SLG100, 0.25 g at 2% in distilled water) is injected into the outer channel. To prevent gelation of alginate in the microfluidic device due to calcium release by the cells in suspension, a calcium-free solution (sorbitol 300 mM, Sigma-Aldrich) is used in the middle channel of the co-extruded tip to act as a barrier against calcium diffusion. The flow rates for the three solutions were around 120 mL / h for the three channels (alginate solution, sorbitol solution and cell + matrix suspension). At these flow rates, the composite solution forms a liquid jet that fragments into droplets (approximately twice the size of the nozzle) due to a spontaneous Rayleigh-Plateau instability. To avoid subsequent coalescence of a series of droplets, the alginate-filled part and the copper ring are connected to a high-voltage generator (2000 V). When the composite droplets come into contact with the calcium collection bath (at 100 mM), the outer layer of alginate gels. Thus, the inner cell / matrix solution remains trapped within the closed spherical permeable microcompartments. In the few minutes following encapsulation, the capsules are rinsed with culture medium (DMEM) to reduce the basal calcium concentration. Finally, they are transferred in suspension to the culture medium.

[0180] The passages in the "invention" arm of this experiment correspond to reencapsulations. These reencapsulations are carried out by dissolving the alginate capsules using a short rinse with ReleSR, followed by dissociation of the cells with Accutase for 20 minutes at 37°C. The cells obtained were then treated according to the encapsulation protocol according to the invention.

[0181] result: Three successive encapsulations were performed on the "bioreactor aggregate" arm, with four successive passages, each over a period of seven days. Sampling of cells at each passage and at day 21 allowed for a comparative evaluation of the two culture arms over time (Figure 9).

[0182] Evaluation by phase contrast microscopy confirms successful formation of stem cell aggregates and encapsulated cysts as expected for the "bioreactor aggregate" and "invention" arms (Figure 10).

[0183] At each passage, the cells are counted using a cell counter (Nucleo Counter NC 3000), which makes it possible to establish the cell amplification factor during the culture (Figure 11). The cumulative theoretical amplification is 55,776,699 million and 40,481 for the experimental arms "Invention" and "Bioreactor aggregates", respectively. These cumulative amplification factors correspond to an average visible cell division number of 25.73 and 15.30 during the 21 days for the "Invention" and "Bioreactor aggregates" culture arms, respectively. The final cell amplification is observed to be higher in the experimental arm "Invention" compared to the "Bioreactor aggregates" experimental arm.

[0184] The two culture systems performed successfully according to best standards, as suggested by the pluripotency markers OCT4 and NANOG being similarly expressed in the two compared culture systems (Figure 12).

[0185] The first flow cytometry analysis was performed to monitor the emergence of iPSC-GHE lineages in cell culture. iPSC-GHE (GFP negative) cells contain amplification of 7q and 20q chromosome regions, which confers a selective advantage during the culture of hiPSCs. iPSC-AAVS1-GFP (GFP positive) cells do not contain chromosomal abnormalities.

[0186] Flow cytometry analysis at each passage for all experimental arms allowed the frequency of iPSC-GHE and iPSC-AAVS1-GFP cells to be quantified over time ( FIG. 13 ), and thus by extrapolation of the copy counts of chromosomal regions 7q and 20q. The frequency of iPSC-GHE (negative GFP) aggregates in cell cultures increases over time in the “bioreactor aggregate” arm, but decreases over time in the “invention” arm.

[0187] The flow cytometry analysis was then confirmed by digital PCR analysis to detect the occurrence of gene mutation rates in the aggregates of pluripotent stem cell aggregates (ICs-digital PSC 24 probes, StemGenomics). In particular, the two PCR probes of this test allowed to quantify the count of copies of the 7q and 20q chromosomal regions over time (Figure 14). The average count of copies of the 7q and 20q regions increases over time during culture for the "bioreactor aggregate" arm. In the "invention" arm, the average count of copies of the 7q region decreases over time, and then the average count of copies of the 20q region decreases over time and then increases very slightly, but much less significantly and less rapidly than in the "bioreactor aggregate" arm. Considering that the count of copies of the 7q and 20q regions of each iPSC-GHE mutant cell is 3 (1 copy increase, see Figure 8), the average copy count equivalent to 2.3 corresponds to a percentage of mutant cells in the cell aggregates of 30%.

[0188] Overall, the flow cytometry and digital PCR results were consistent, suggesting that the selection of mutant cells during the 21-day culture was at least 14.7-fold lower in the "invention" arm compared to the "bioreactor aggregate" arm (Figure 15). In particular, the encapsulation culture system (invention) allowed an average of 8.6 cell divisions to occur per passage, while maintaining a percentage of mutant cells below 20% (below 3%) for each encapsulation, or by performing four encapsulations end-to-end.

Claims

1. 1. A three-dimensional cellular microcompartment comprising at least one outer hydrogel layer and at least one layer of cells and / or at least one cell-based layer inside said outer layer, wherein less than 20% of the total population of cells present in said microcompartment are cells having at least one mutation.

2. The microcompartment of claim 1 , wherein the cells occupy more than 50% by volume of the microcompartment.

3. 3. The method according to claim 1 or 2, characterized in that the mutation is selected from a genetic mutation and an epigenetic mutation.

4. The microcompartment according to claim 1 or 2, characterized in that the mutation is a functional mutation.

5. 3. The microcompartment according to claim 1 or 2, characterized in that at least one mutation is an oncogenic mutation.

6. 3. The microcompartment according to claim 1 or 2, characterized in that less than 20% of the cells are cells carrying at least one mutation in the P53 gene and / or at least one mutation due to amplification of the 20q chromosomal region and / or at least one mutation due to amplification of the 7q chromosomal region.

7. 3. The microcompartment according to claim 1 or 2, characterized in that less than 20% of the cells are cells carrying at least one mutation due to amplification of the 20q11 chromosomal region.

8. A microcompartment according to claim 1 or 2, characterized in that the cells carrying said at least one mutation represent 0-10% of the total population of said cells present in said microcompartment.

9. 3. The method according to claim 1 or 2, characterized in that the cells are organized in the form of a tissue or microtissue.

10. 3. The microcompartment of claim 1 or 2, characterized in that it comprises an internal lumen.

11. 2. The microcompartment according to claim 1, further comprising at least one intermediate layer between (a) said cell layer and / or said cell-based layer and (b) said hydrogel layer, said intermediate layer comprising an isotonic aqueous solution and / or extracellular matrix elements.

12. The microcompartment of claim 11, wherein the intermediate layer of the isotonic aqueous solution is an extracellular matrix layer.

13. 13. Microcompartments according to claim 11 or 12, characterized in that the intermediate layer of isotonic aqueous solution has a Young's modulus of 0.05 to 3 kDa.

14. 3. The microcompartment according to claim 1 or 2, characterized in that the cells are human or animal cells.

15. Organized in a continuous fashion around the lumen at least one layer of cells and / or at least one cell-based layer, an intermediate layer of an isotonic aqueous solution; A microcompartment according to claim 1 or 11, characterized in that it comprises an outer hydrogel layer.

16. 12. The microcompartment according to claim 1 or 11, characterized in that the cells are human or animal induced pluripotent stem cells (iPSCs), and / or human or animal multipotent cells, and / or human or animal progenitor cells, and / or human or animal differentiated cells.

17. 12. Microcompartment according to claim 1 or 11, characterized in that it is closed.

18. 12. The microcompartment of claim 1 or 11, characterized in that the outer layer comprises alginate.

19. 12. Microcompartments according to claim 1 or 11, characterized in that they have an ovoid, cylindrical, spheroidal or spherical shape.

20. 12. Microcompartment according to claim 1 or 11, characterized in that it comprises at least 20 cells, preferentially at least 1000 cells.

21. 12. Microcompartments according to claims 1 or 11, characterized in that the cells present in said microcompartments are obtained after at least two cell division cycles following encapsulation of 1 to 50 cells in an outer hydrogel layer.

22. 12. Microcompartments according to claims 1 or 11, characterized in that the cells present in said microcompartments are obtained after at least 5 cell division cycles following encapsulation of 1 to 50 cells in an outer hydrogel layer.

23. 1. An assembly of at least two three-dimensional cellular microcompartments, each microcompartment comprising at least one outer hydrogel layer and, inside said outer layer, at least one layer of cells and / or at least one cell-based layer, wherein less than 20% of the cells constituting the total population of cells present in all said microcompartments are cells having at least one mutation.

24. Assembly of microcompartments according to claim 23, characterized in that at least one microcompartment is a microcompartment according to claim 1.

25. 24. An assembly of microcompartments according to claim 23, characterized in that said microcompartments are placed in a culture medium in a closed bioreactor.

26. 24. A method for preparing an assembly of cellular micro-compartments according to claim 1 or 23, comprising the steps of: - (a) preparing a suspension of cells comprising single cells and / or at least one cluster of cells in an isotonic culture medium, preferentially containing an apoptosis inhibitor; - (b) encapsulating said cell suspension within a hydrogel layer; - (c) preferentially culturing said microcompartments obtained in an isotonic solution containing an apoptosis inhibitor; - (d) rinsing said microcompartments so as to preferentially remove said apoptosis inhibitor; - (e) culturing said microcompartments in an isotonic solution for at least two cell division cycles; - (f) optionally, recovering the obtained cellular micro-compartments, The method, wherein all of the cells initially encapsulated in step (b) occupy a volume that is less than 50% of the volume of the microcompartment in which they are encapsulated.

27. 27. The method of claim 26, wherein each cell cluster initially encapsulated in step (b) has a larger dimension that is less than 20% of the maximum dimension of the microcompartment in which it is encapsulated.

28. 27. The method of claim 26, further comprising mixing the cells with an extracellular matrix either between steps (a) and (b) or simultaneously with the encapsulation in step (b).

29. 27. The method of claim 26, wherein steps (c), (d) and (e) are carried out under continuous or sequential stirring.

30. 27. The method according to claim 26, characterized in that it is carried out in a closed bioreactor.

31. 27. The method of claim 26, characterized in that the method comprises at least one reencapsulation of the cells after step (e).

32. 32. The method of claim 31, wherein the method comprises 2 to 15 reencapsulations of the cells.

33. 33. The method of claim 32, characterized in that each reencapsulation corresponds to a passage.

34. The reencapsulation step comprises: -(i) removing the outer hydrogel layer; - (ii) resuspending the cells contained within said microcompartments so as to obtain single cells and / or at least one cluster of cells in an isotonic culture medium, preferentially containing an apoptosis inhibitor; - (iii) encapsulating said cell suspension within a hydrogel layer; - (iv) preferentially culturing said microcompartments obtained in an isotonic solution containing an apoptosis inhibitor; - (v) preferentially rinsing said microcompartments to remove said apoptosis inhibitors; - (vi) culturing said microcompartments in an isotonic solution for at least one cell division cycle; - (vii) optionally, recovering the obtained cellular micro-compartments.

35. 27. The method of claim 26, characterized in that for each microcompartment, the single cell represents less than 50% of the total number of cells initially encapsulated in step (b).

36. 27. The method according to claim 26, characterized in that prior to or simultaneously with step (a), the method comprises a step of dissociating the cells by chemical, enzymatic or mechanical dissociation.

37. 27. The method of claim 26, further comprising one or more steps of removing the microcompartments containing mutant cells.

38. 27. Use of the method of claim 26 for maintaining the genomic integrity of a cell during its expansion.

39. 24. Use of a microcompartment according to claim 1 or a microcompartment assembly according to claim 23 for maintaining the genomic integrity of a cell during its amplification.

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