How to obtain healthy intestinal organoids

Abstract

The present invention relates to a biofunctional three-dimensional hydrogel suitable for the proliferation of freshly isolated or frozen intestinal cells and the formation of intestinal organoids derived therefrom, which avoids the use of naturally occurring matrices, such as Matrigel®, and results in intestinal organoids suitable for clinical use and produced in a commercially viable manner. The present invention also relates to kit contents comprising said hydrogel in combination with a suitable culture medium, and methods for generating organoids from freshly isolated or frozen intestinal cells and for cell proliferation using said kit contents.

Description

[Technical Field]

[0001] The present invention relates to the field of regenerative medicine and precision medicine, in particular to a method for preparing patient-derived healthy intestinal organoids (PDO).

[0002] The number of people suffering from inflammatory bowel disease (IBD) continues to increase worldwide. Mucosal healing and re-establishment of intestinal barrier function are important therapeutic goals, associated with substantially better prognosis, lower recurrence and hospitalization rates, and reduced risk of surgery and colon cancer. However, current medical treatments are not effective for all IBD patients, and approximately 16–47% of patients require surgery within 10 years of diagnosis (Frolkis et al., Risk of surgery for inflammatory bowel diseases has decreased over time: a systematic review and meta-analysis of population-based studies; Gastroenterology 2013;145:996–1006). This clearly highlights the unresolved challenges facing novel therapeutic approaches based on stem cell-enriched intestinal organoids, which hold great promise as a regenerative material for transplantation and re-establishment of intestinal barrier function.

[0003] Organoids, including cell spheroids or clusters, are three-dimensional structures of stem cells or organ-specific cells derived from stem cells that self-organize (or self-pattern) through cell sorting and spatially restricted lineage constraints in a manner similar to the in vivo situation. Thus, organoids exhibit the natural physiology of cells and have a cellular composition (including residual stem cells and / or specialized cell types at various stages of differentiation) and a biological structure that mimics the natural situation. Stem cells can be isolated from tissues or organoid fragments. The cells from which organoids are derived can differentiate to form organ-like tissues that display multiple cell types that self-organize, forming structures that closely resemble organs in vivo (i.e., cell differentiation). Therefore, organoids are an excellent model for studying human organs and their development in a system that closely resembles in vivo development. Organoids are also used to grow and expand cells for clinical applications. Organoids grown from isolated intestinal crypts or stem cells may also be referred to in the art as "enteroids" or "colonoids."

[0004] Intestinal organoids have been successfully delivered to mice with experimental colitis, demonstrating that the cells adhere to and become an integral part of the epithelium (Yui et al., Functional engraftment of colon epithelium expanded in vitro from a single adult Lgr5+ stem cell, Nature Medicine Vol. 18, no. 4 2012, 618-624; Fordham et al., Transplantation of Expanded Fetal Intestinal Progenitors Contributes to Colon Regeneration after Injury, Cell Stem Cell 13, 734-744, December 5, 2013; and Sugimoto et al., Reconstruction of the Human Colon Epithelium In Vivo, Cell Stem Cell 22, 1-6, February 1, 2018 (https: / / doi.org / 10.1016 / j.stem.2017.11.012)). In these studies, successful growth and transplantation of intestinal organoids relied on the use of animal-derived matrices (e.g., Matrigel®) for PDO establishment and proliferation.

[0005] 3D culture systems for intestinal organoids that maintain basic crypt-villus physiology with stem cell turnover in Matrigel® have been described (Sato et al., Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche, Nature, Vol 459, 14 May 2009, 262-266; Sato et al., Paneth cells constitute the niche for Lgr5 stem cells in intestinal crypts, Nature, Vol 469, 20 January 2011, 415-419; Sato et al., Long-term Expansion of Epithelial Organoids From Human Colon, Adenoma, Adenocarcinoma, and Barrett's Epithelium, GASTROENTEROLOGY 2011;141:1762-1772; WO 2009 / 022907 A2; WO 2010 / 090513 A2; WO 2012 / 168930 A2; WO 2013 / 093812 A2). In these studies, a major challenge was the identification of suitable components for the cell culture medium used to feed cells grown in Matrigel®.

[0006] However, batch-to-batch variability and unclear composition of animal-derived matrices, such as Matrigel®, have hindered regulatory approval for their use in humans. Therefore, to translate intestinal organoid transplantation therapy to human patients, several aspects of intestinal organoid cultures must be modified for clinical use. These include the development of support matrices and media that are well-defined, approved for human use, scalable, and preferably xeno-free (i.e., free of animal-derived components).

[0007] A defined, but not synthetic (i.e., neither allo-free nor xeno-free) fibrin hydrogel supplemented with laminin-111 was shown to support the growth of intestinal organoids in Broguiere et al., Growth of Epithelial Organoids in a Defined Hydrogel, Adv. Mater. 2018, 1801621. The natural presence of RGD domains in fibrin was shown to be essential for organoid growth.

[0008] Gjorevski (Gjorevski et al., Designer matrices for intestinal stem cell and organoid culture, Nature, Vol. 539, 24 November 2016, 560-56; Gjorevski et al., Synthesis and characterization of well-defined hydrogel matrices and their application to intestinal stem cell and organoid culture, Nature protocols, Vol. 12, no. 11, 2017, 2263-2274; WO 2017 / 036533 A1 and WO 2017 / 037295 A1) developed enzymatically (factor XIII) crosslinked 8-arm polyethylene glycol (PEG) hydrogels with functionalized RGD peptides and different degradation rates, including specific enzymatic and autolytic rate control (hydrolysis of PEG-acrylate), for the growth of primary mouse and human small intestinal organoids and human colorectal cancer organoids. The addition of laminin-111 purified from mouse tissue (whole protein) was necessary to support organoid differentiation.

[0009] Although this method has been successful in growing mouse cells and forming organoids, the system has not been shown to be suitable for growing freshly isolated or frozen human cells from human biopsies (and was even questioned in Gjorevski 2017, p. 2265). Furthermore, the only system that has employed enzymatic cross-linking with factor XIII has proven expensive, difficult to scale up and / or automate for commercial purposes, and difficult to reproduce.

[0010] Cruz-Acuna's research (Cruz-Acuna et al., Synthetic hydrogels for human intestinal organoid generation and colonic wound repair, Nature cell biology, advanced online publication published online 23 October 2017; DOI: 10.1038 / ncb3632, 1-23; Cruz-Acuna et al., PEG-4MAL hydrogels for human organoid generation, culture, and in vivo delivery, Nature protocols, Vol. 13, September 2018, 2102-2119; and WO 2018 / 165565 A1) is based on the development of a fully synthetic 4-arm PEG-maleimide hydrogel functionalized with RGD and cross-linked with the protease-degradable peptide GPQ-W for growing intestinal organoids using human embryonic stem cells and induced pluripotent stem cells. Organoids grown in these synthetic gels were then injected into a mouse colon injury model for proof-of-concept, demonstrating the therapeutic potential of intestinal organoid transplantation.

[0011] This system has not been shown to be able to grow freshly isolated or frozen cells from patient biopsies to form organoids, and the cross-linking moieties must be enzymatically degraded in this system.

[0012] Currently, the standard for establishing ex vivo organoid cultures involves first encapsulating freshly isolated cells (derived from tissues) in the "gold standard" Matrigel® (a commercially available basement membrane extract (BME)), followed by several passages to grow the cells (i.e., expand the cell population). BME (e.g., Matrigel®) is a gel derived from mouse sarcoma extracts, and as noted above, poor batch-to-batch consistency and unclear composition preclude its clinical use, making regulatory approval difficult or impossible (Madl et al., Nature 557 (2018), 335-342).

[0013] Eliminating the use of gels with undefined xenogeneic or human components for the establishment of organoids would overcome one of the major obstacles to using organoids in clinical applications, such as regenerative medicine, precision medicine, drug testing, or patient stratification.

[0014] Proof-of-concept studies of freshly isolated cells obtained from biopsies cultured in a fully defined (not fully synthetic) matrix have been presented in Mazzocchi et al., "In vitro patient-derived 3D mesothelioma tumor organoids facilitate patient-centric therapeutic screening," Scientific Reports (2018) 8:2886; Votanopoulos et al., "Appendiceal Cancer Patient-Specific Tumor Organoid Model for Predicting Chemotherapy Efficacy Prior to Initiation of Treatment: A Feasibility Study," Ann Surg Oncol (2019) 26:139-147; and WO 2018 / 027023 A1. Briefly, cells derived from mesothelioma and appendiceal cancer patients were cultured in a hyaluronic acid / collagen-based hydrogel to develop a platform for predicting drug response. However, like Matrigel®, collagen is a naturally derived matrix and faces similar challenges.

[0015] To date, there have been no successful reports of growing freshly isolated or frozen human cells obtained from biopsies or tissue resections (i.e., cells obtained directly from a human and not previously cultured or established in another system) and subsequently forming organoids from said human cells in a fully defined and / or fully synthetic hydrogel matrix that is not a naturally derived matrix, such as Matrigel® or collagen. Despite the clear need for such an approach, as articulated in the prior art discussed above, to date the gold standard remains the use of Matrigel® for at least the initial steps of cell growth. This is a testament to the challenges involved in conducting research in semi-synthetic or fully synthetic three-dimensional hydrogel systems.

[0016] The challenge underlying the present invention was to provide a method for the proliferation of freshly isolated or frozen human cells obtained by biopsy and the subsequent formation of intestinal organoids derived from said human cells, which completely avoids the use of naturally occurring matrices, such as Matrigel®, and results in intestinal organoids that are suitable for clinical use and are produced in a commercially viable manner, i.e., cost-effective, reliable, reproducible, automatable and scalable.

[0017] According to the present invention, the above problem is solved by the hydrogel, the contents of the kit and the method as defined in the independent claims.

[0018] In particular, the present invention relates to a biofunctional three-dimensional hydrogel suitable for the growth and proliferation of freshly isolated or frozen intestinal cells and for the formation of intestinal organoids derived from intestinal cells, said hydrogel comprising: - at least one precursor molecule which is a multi-arm PEG having ethylenically unsaturated groups selected from the group consisting of vinyl sulfone and acrylate; - a cross-linking molecule having at least two, preferably two, nucleophilic groups capable of reacting with the ethylenically unsaturated groups of said multi-arm PEG via a Michael addition reaction; and - at least one biofunctional ligand selected from the group consisting of natural laminins, such as laminin 111, recombinant laminin isoforms, preferably laminin 511, and biofunctional fragments thereof; is the reaction product of The hydrogel is characterized in that the crosslinking molecules contain at least one RGD motif and are preferably not known to be enzymatically degradable.

[0019] Compared with the prior art hydrogels of Gjorevski 2016 and 2017, WO2017 / 036533 A1, and WO2017 / 037295 A1, the hydrogel of the present invention is less expensive. The hydrogel of the present invention can be easily scaled up for commercial purposes because it does not require the presence of enzymes and activators for crosslinking. This is also because the gel precursor described above in the present invention (i.e., the multi-arm PEG having an ethylenically unsaturated group, which is a crosslinking molecule containing two nucleophilic groups and at least one biofunctional ligand) does not require additional steps for the preparation of the gel precursor, as is required for the prior art gels of Gjorevski 2016 and 2017, WO2017 / 036533 A1, and WO2017 / 037295 A1.

[0020] In particular, the gel precursors of the present invention are highly suitable for scale-up of production and automation of their use, i.e., for applications such as methods using pipetting robots. The hydrogels of the present invention are highly reliable and reproducible. Furthermore, in contrast to the enzymatically crosslinked gels of Gjorevski et al., the hydrogels of the present invention are comparable to commercially available media, such as IntestiCult™ Organoid Growth Medium from STEMCELL Technologies, i.e., the hydrogels of the present invention do not degrade rapidly.

[0021] The hydrogels of the present invention are based on a different chemistry that is more suitable for automation, i.e., for application in methods using pipetting robots, etc., and are more easily scalable, compared to the prior art hydrogels of Cruz-Acuna 2017 and 2018 and WO2018 / 165565 A1. This is because all of the gel precursor materials of the present invention (i.e., the multi-arm PEG having an ethylenically unsaturated group, the crosslinking molecule having two nucleophilic groups, and the at least one biofunctional ligand) can be premixed and lyophilized during the manufacturing process without premature reaction, which is not possible under the conditions of the Cruz-Acuna et al. system due to the precursors reacting.

[0022] An important aspect of the hydrogels of the present invention is the fact that, compared to prior art hydrogels in which the RGD motif is present in the ligand attached ("pendant") to the crosslinked hydrogel, at least one RGD motif is present in the crosslinking molecule. The approach of the present invention makes it possible to significantly increase the amount of RGD motif in the hydrogel. Surprisingly, it has been found that this leads to a significant improvement in the properties of the hydrogel.

[0023] According to the present invention, it has been surprisingly found that the hydrogel described herein, when used in combination with a suitable culture medium, is suitable for the de novo formation of organoids derived from freshly isolated or frozen intestinal cells (single cells and / or clusters) obtained from human biopsies or tissue resections, the growth, subculture and proliferation of these cells, and the subsequent differentiation of organoids derived therefrom.Therefore, the present invention completely avoids the need to use naturally derived matrices, such as Matrigel®.

[0024] In the present invention, the term "freshly isolated or frozen human cells obtained by biopsy or tissue resection" refers to cells obtained directly from a human by the methods described above and not previously cultured or established in another system before use in the method for forming organoids. Typically, such fresh cells are used in the methods of the present invention immediately after collection or within a maximum of 3-4 days. If the cells are not used immediately after collection, they may be frozen under conventional conditions for storage. The collected cells may be single cells and / or "clusters" of cells, including dissociated cells, crypts, and small pieces of tissue. In a preferred embodiment of the present invention, epithelial cells are used.

[0025] In the present invention, the term "de novo formation of organoids" refers to the first time that freshly isolated or frozen human cells (e.g., human biopsy or excised tissue) are grown ex vivo (i.e., outside the original organism). The terms "initial ex vivo cell growth" or "passage 0 (P0)" are used interchangeably.

[0026] As used herein, the term "pre-established organoids" refers to cells, single cells and / or cell clusters (e.g., cell aggregates, organoids, etc.) that have been grown in other systems (e.g., Matrigel®, 2D or 3D systems, in vivo patient-derived xenografts (PDX)) prior to application to the hydrogel of the present invention.

[0027] In the present invention, the term "cell growth" refers to the successful growth of cells derived from pre-established organoids or organoids formed de novo.

[0028] In the present invention, the term "passaging cells" or "passage" or "cell division" refers to the process of removing cells from one gel and seeding and growing said cells in another gel having the same or different characteristics as the previous gel.

[0029] In the present invention, the term "cell expansion" refers to the process by which cells grow and increase in cell number (e.g., within the same passage or from one passage to another).

[0030] In the present invention, the term "organoid differentiation" refers to the successful induction of cell differentiation in organoids.

[0031] The present invention also provides kit contents for the growth and proliferation of freshly isolated or frozen intestinal cells and the formation of intestinal organoids derived therefrom, comprising: - at least one precursor molecule which is a multi-arm PEG having ethylenically unsaturated groups selected from the group consisting of vinyl sulfone and acrylate; - a cross-linking molecule comprising at least one RGD motif, having at least two, preferably two, nucleophilic groups capable of reacting with the ethylenically unsaturated groups of said multi-arm PEG via a Michael addition reaction; and - at least one biofunctional ligand selected from the group consisting of natural laminins, such as laminin 111, recombinant laminin isoforms, preferably laminin 511, and biofunctional fragments thereof, and - a medium comprising R-spondin1, preferably an R-spondin1 conditioned medium, and Wnt3a, preferably a Wnt3a conditioned medium The present invention relates to the contents of the kit, including:

[0032] The present invention also provides a method for the growth and proliferation of freshly isolated or frozen intestinal cells, comprising the steps of: a) forming organoids de novo from freshly isolated or frozen human intestinal cells by incubating the freshly isolated or frozen intestinal cells with the hydrogel of the kit in the presence of the culture medium of the kit using the contents of the kit as described herein; b) growing, optionally passage and expanding the cells derived from the intestinal organoids of step a) using the contents of the kit as described in the specification; c) optionally differentiating the organoids of step a) in the presence of a modified medium that induces cell differentiation. Including, The method is characterized in that only fully synthetic hydrogels or fully defined semi-synthetic hydrogels are used.

[0033] In the present invention, the term "fully synthetic hydrogel" refers to a hydrogel formed exclusively from synthetic precursors, i.e., in the absence of any naturally occurring precursors, such as native laminin-111.

[0034] In the present invention, the term "fully defined semi-synthetic hydrogel" refers to a hydrogel that contains at least one naturally occurring precursor, such as native laminin 111, but whose structure and / or composition are fully defined because the nature of the precursor molecule used in its synthesis is known. Thus, fully defined semi-synthetic hydrogels differ from naturally occurring hydrogels, such as Matrigel®, whose structure and / or composition are unknown.

[0035] The hydrogels of the present invention are based, as precursor molecules, on multi-arm PEG (polyethylene glycol) with ethylenically unsaturated groups selected from the group consisting of vinyl sulfones or acrylates.

[0036] A hydrogel (gel) is a matrix containing a network of hydrophilic polymer chains. Biofunctional hydrogels are hydrogels that contain bioadhesive (or bioactive) molecules and / or cell signaling molecules that interact with viable cells to promote cell viability and desired cell phenotypes.

[0037] According to a preferred embodiment of the present invention, the multi-armed PEG is selected from the group consisting of PEGs having 2 to 12 arms, preferably 4-armed or 8-armed, i.e., preferably 4-armed or 8-armed PEG. The molecular weight of PEG may be 1,000 to 1,000,000, 1,000 to 500,000, 1,000 to 250,000, 1,000 to 150,000, 1,000 to 100,000, 1,000 to 50,000, 5,000 to 100,000, 5,000 to 50,000, 10,000 to 100,000, 10,000 to 50,000, 20,000 to 100,000, 20,000 to 80,000, 20,000 to 60,000, 20,000 to 40,000, or 40,000 to 60,000. The molecular weight is an average molecular weight expressed in Da, determined by a method such as GPC or MALDI.

[0038] Such PEGs are known in the art and commercially available, and consist of a core that may be pentaerythritol in the case of 4-arm PEGs and tripentaerythritol or hexaglycerol in the case of 8-arm PEGs:

[0039] [ka]

[0040] In the case of 4-arm PEG-VS or 8-arm PEG-VS, the free OH group at the end of the 4-arm PEG or 8-arm PEG is converted to a vinyl sulfone group under conditions known in the art, and R in the above formula is, for example,

[0041] [ka]

[0042] In the case of 4-arm PEG-Acr or 8-arm PEG-Acr, the free OH group at the end of the 4-arm PEG or 8-arm PEG is converted to an acrylate group under conditions known in the art, and in the above formula, R is, for example,

[0043] [ka]

[0044] Preferably, all of the free OH groups at the ends of the 4-arm PEG or 8-arm PEG are converted to vinyl sulfone or acrylate.

[0045] Vinyl sulfones or acrylates are suitable ethylenically unsaturated groups for crosslinking PEG precursor molecules via the Michael addition reaction. The Michael addition reaction is a well-known chemical reaction involving the reaction of a suitable nucleophilic moiety with a suitable electrophilic moiety. For example, acrylates or vinyl sulfones are known to be suitable Michael donors (i.e., nucleophiles) and suitable Michael acceptors (i.e., electrophiles) that react with, for example, thiols.

[0046] Thus, to obtain a hydrogel according to the present invention, the PEG precursor molecule is reacted with a crosslinking molecule having at least two, preferably two, nucleophilic groups capable of reacting with the ethylenically unsaturated groups of the multi-arm PEG via a Michael addition reaction.

[0047] A bridge molecule is a molecule formed by linking together at least two of the above-described PEG precursor molecules. In this application, the bridge molecule must have at least two, preferably two, nucleophilic groups, one of which reacts with a first PEG precursor molecule and the other nucleophilic group with a second PEG precursor molecule.

[0048] Although the crosslinking molecules used in the present invention may have three or more nucleophilic groups, it is not necessary for the formation of a three-dimensional network because each PEG precursor molecule can react with two or more crosslinking molecules.

[0049] An important aspect of the hydrogels of the present invention is the fact that, compared to prior art hydrogels in which the RGD motif is present in the ligand attached ("pendant") to the crosslinked hydrogel, at least one RGD motif is present in the crosslinking molecule. The approach of the present invention makes it possible to significantly increase the amount of RGD motif in the hydrogel. Surprisingly, it has been found that this leads to a significant improvement in the properties of the hydrogel.

[0050] For the hydrogels of the present invention, the presence of a significant amount of RGD motifs in the hydrogel is necessary. The results described in WO2017 / 037295 A1 that the RGD motif is not essential when laminin-111 ligands are present in the hydrogel could not be confirmed for the hydrogels of the present invention.

[0051] Thus, the cross-linking molecule used in the present invention is a peptide containing at least one RGD motif, preferably at least two RGD motifs, more preferably 2 to 8 RGD motifs, even more preferably 2 to 5 RGD motifs, and especially preferably 2, 3 or 4 RGD motifs.

[0052] The term RGD or RGD sequence refers to the minimal biologically active RGD sequence, which is the arginine-glycine-aspartic acid (RGD) sequence, a minimal (minimal) fibronectin-derived amino acid sequence sufficient to mimic cell binding to fibronectin and / or promote anchorage-dependent cell adhesion. In addition, amino acid sequences containing lysine or arginine, such as RGD, are suitable substrates for proteases, such as trypsin-like enzymes, used, for example, for gel dissociation.

[0053] Examples of suitable RGD motifs are RGD, RGDS, RGDSP, RGDSPG, RGDSPK, RGDTP or RGDSPASSKP, but primarily any RGD sequence that is known and has been used successfully in the field of hydrogels and cell culture can be used.

[0054] According to a preferred embodiment of the present invention, the bridging molecule is a peptide containing at least two RGD motifs and at least two cysteines, which are amino acids containing a thiol group, i.e., a Michael donor.

[0055] According to a particularly preferred embodiment of the present invention, the cross-linking molecule is Ac-GCREGRGDSPGGRGDSPGERCG-NH2.

[0056] According to the present invention, the amount of RGD motif in the hydrogel is higher than that in the prior art hydrogels mentioned above (WO 2017 / 037295 A1, page 31, Example 5, argues that increasing RGD in the hydrogel above 0.5 mM does not result in an improvement; in Cruz-Acuna gels (Cruz-Acuna 2017), 2 mM RGD was consistently used). According to the present invention, the amount of RGD motif in the hydrogel is 1 to 15 mM, preferably 2.5 to 5.5 mM, and particularly preferably 2.5 to 5 mM.

[0057] Crosslinking of hydrogel precursor molecules is typically carried out in the presence of cells cultured within the hydrogel, with single cells and / or "clusters" of cells, including dissociated cells, crypts, and small pieces of tissue, being encapsulated in the forming hydrogel matrix, i.e., in separate cell culture microenvironments.

[0058] According to a particularly preferred embodiment of the present invention, the hydrogel comprises: - a reaction product which is a 4-arm or 8-arm PEG vinyl sulfone crosslinked with a peptide comprising at least two, preferably two thiol groups and at least two, preferably two RGD motifs, or - a reaction product which is a 4-arm or 8-arm PEG vinyl sulfone cross-linked with a peptide comprising at least two, preferably two thiol groups and at least two, preferably two RGD motifs, and / or a reaction product which is a 4-arm or 8-arm PEG acrylate cross-linked with a peptide comprising at least two, preferably two thiol groups and at least two, preferably two RGD motifs.

[0059] In each embodiment, a biofunctional ligand selected from the group consisting of a native laminin, e.g., laminin 111, a recombinant laminin isoform, and a biofunctional fragment thereof, is further present in the hydrogel. Examples of suitable recombinant laminin isoforms are laminin 111, laminin 211, laminin 332, laminin 411, laminin 511, or laminin 521, with laminin 511 being preferred.

[0060] The first of these embodiments uses only 4-arm or 8-arm PEG vinyl sulfone as the precursor molecule, and the hydrogels according to this embodiment are mechanically stable, i.e., the shear modulus of the hydrogel does not decrease and the hydrogel does not soften over time.

[0061] The second of these embodiments uses 4-arm or 8-arm PEG vinyl sulfone and 4-arm or 8-arm PEG acrylate, or 4-arm or 8-arm PEG acrylate alone, as precursor molecules. As described below, the presence of 4-arm or 8-arm PEG acrylate in the hydrogel makes the hydrogel mechanically dynamic. That is, the shear modulus of the hydrogel decreases, and the hydrogel softens over time. The degree of softening of the hydrogel can be controlled by the ratio of 4-arm or 8-arm PEG vinyl sulfone to 4-arm or 8-arm PEG acrylate present in the hydrogel. According to a particularly preferred embodiment of the present invention, the ratio of 4-arm or 8-arm PEG vinyl sulfone to 4-arm or 8-arm PEG acrylate present in the hydrogel is 5:1 to 1:5, preferably 3:1 to 1:3, and particularly preferably 1:1.

[0062] Mechanically dynamic hydrogels, preferably those containing a 1:1 ratio of 4-arm or 8-arm PEG vinyl sulfone to 4-arm or 8-arm PEG acrylate, have been found to be favorable for their suitability for the growth and passage of freshly isolated or frozen intestinal cells (efficient gel dissociation and reduced cell loss compared to non-degradable gels of similar structure). Furthermore, the hydrogels of the present invention have surprisingly been found to exhibit highly advantageous dynamic degradation behavior. In contrast to prior art hydrogels (Gjorevski 2016 & 2017, WO 2017 / 036533 A1, and WO 2017 / 037295 A1), which degrade prematurely under the culture conditions preferred for the present invention (i.e., in the presence of the most preferred culture medium of the present invention) and are therefore unsuitable for cell passaging and growth, the hydrogels of the present invention remain viable (i.e., do not degrade) even after the typically required 8-11 days, making them particularly suitable for cell passaging and further growth.

[0063] The hydrogel according to the present invention further comprises, as an essential component, at least one biofunctional ligand selected from the group consisting of natural laminin, such as laminin 111, recombinant laminin isoforms and biofunctional fragments thereof. Examples of suitable recombinant laminin isoforms are laminin 111, laminin 211, laminin 332, laminin 411, laminin 511, or laminin 521.

[0064] Preferably, at least one biofunctional ligand selected from the group consisting of native laminin, e.g., laminin 111, recombinant laminin isoforms, and biofunctional fragments thereof, is present in the hydrogel at a concentration of 0.01 g / L to 3 g / L, more preferably 0.05 g / L to 0.5 g / L.

[0065] Laminin is an extracellular matrix glycoprotein with a heterotrimeric structure consisting of α, β, and γ chains. For example, "111" indicates the chain composition of the α1β1γ1 isoform. Laminin 111 is synonymous with laminin 1. In cell adhesion, laminin 111 and other isoforms are extracellular matrix Important for anchoring cells to the extracellular matrix (ECM) protein The binding between cells and ECM is cell surface receptors They are formed by binding a protein to one end of the laminin α chain and an ECM component to another region of the laminin. Spherical domains (G-domain) Integrin , glycoproteins , sulfated glycolipids and Dystroglycan This is the region of laminin 111 that allows the binding of . In addition to anchoring cells to the ECM, laminin is also involved in signal transduction between cells and other components of the ECM.

[0066] In one embodiment, a natural laminin is used, such as laminin 111, preferably mouse laminin 111. In this case, the hydrogel of the present invention is semi-synthetic, since the natural laminin 111 is of biological origin. However, since the structure of natural laminin 111, preferably mouse laminin 111, is known, the hydrogel of the present invention is completely defined and therefore reliably reproducible.

[0067] In another embodiment, recombinant laminin isoforms are used. Examples of suitable recombinant laminin isoforms are laminin-111, laminin-211, laminin-332, laminin-411, laminin-511, or laminin-521, preferably laminin-511. Again, the laminin isoforms are of synthetic origin, and therefore the hydrogels of the present invention are completely synthetic. Methods for producing recombinant laminin isoforms are known in the art.

[0068] According to another embodiment, a biofunctional fragment of laminin 511 is used. A biofunctional fragment of laminin 511 is a molecule that contains a portion of laminin 511 that confers the required biological function. In other words, the fragment must contain one of the portions of laminin 511 that can interact with other molecules (e.g., integrins, cell surface receptor proteins, components of the ECM).

[0069] Embodiments that avoid the use of native laminin 111 are preferred as they result in the use of a completely synthetic three-dimensional matrix, and therefore offer advantages with regard to regulatory approval for clinical use.

[0070] In another embodiment, the hydrogel of the present invention may further comprise at least one other biofunctional ligand in addition to the biofunctional ligand selected from the group consisting of native laminin 111, recombinant laminin isoforms, and biofunctional fragments thereof. For example, a ligand containing at least one RGD motif, as described above, may be attached to the hydrogel.

[0071] At least one biofunctional ligand may be attached to the crosslinked hydrogel by methods known in the art, preferably by reaction of a cysteine thiol group in the biofunctional ligand with a Michael acceptor group (vinyl sulfone or acrylate) in the hydrogel polymer.

[0072] Another aspect of the present invention relates to a method for producing the three-dimensional hydrogel of the present invention, in particular the method comprising: a1) dispensing onto the surface of the substrate or into separate spaces of the substrate, preferably a multi-well plate, one or more different hydrogel precursor molecules, which are multi-arm PEGs with ethylenically unsaturated groups selected from the group consisting of vinyl sulfones and acrylates; The hydrogel precursor molecules - a cross-linking molecule comprising at least one RGD motif, having at least two, preferably two, nucleophilic groups capable of reacting with the ethylenically unsaturated groups of said multi-arm PEG via a Michael addition reaction; and - at least one biofunctional ligand selected from the group consisting of natural laminins, such as laminin 111, recombinant laminin isoforms, preferably laminin 511, and biofunctional fragments thereof; or a2) dispensing onto the surface of the substrate or into discrete spaces of the substrate, preferably in a multi-well plate, a resuspension of unreacted powder, preferably lyophilized unreacted powder, The unreacted powder is - one or more different hydrogel precursor molecules that are multi-arm PEGs with ethylenically unsaturated groups selected from the group consisting of vinyl sulfones and acrylates; - a cross-linking molecule comprising at least one RGD motif, having at least two, preferably two, nucleophilic groups capable of reacting with the ethylenically unsaturated groups of said multi-arm PEG via a Michael addition reaction; and - at least one biofunctional ligand selected from the group consisting of natural laminins, such as laminin 111, recombinant laminin isoforms, preferably laminin 511, and biofunctional fragments thereof; Process including b) adding human biopsy cells, preferably freshly isolated or frozen intestinal cells, to the surface of the substrate or into the separate spaces of the substrate, or to the hydrogel precursor formulation of a1) or a2) before adding them to the surface of the substrate or into the separate spaces of the substrate; and c) crosslinking the hydrogel precursor molecules with the crosslinking molecules to form a hydrogel. Includes:

[0073] According to a preferred embodiment, the hydrogel precursor formulation in the form of an unreacted powder is resuspended and dispensed into discrete spaces on or within a substrate, preferably a multi-well plate, containing all of the components necessary for the formation of a hydrogel according to the present invention, i.e., one or more different hydrogel precursor molecules as described above, one or more cross-linking molecules, and at least one biofunctional ligand, and preferably also cells, preferably freshly isolated or frozen intestinal cells obtained from a human biopsy.

[0074] The provision of the unreacted powder of the hydrogel precursor formulation is known, for example, from WO2011 / 131642 A1.

[0075] According to another embodiment, the hydrogel precursor formulation itself can be dispensed onto or into discrete spaces of a substrate without prior lyophilization and resuspension.

[0076] The hydrogel of the present invention is a so-called soft hydrogel, i.e., the shear modulus (rigidity) of the three-dimensional hydrogel of the present invention is usually 50 to 1000 Pa, preferably 200 to 500 Pa. The desired range of rigidity is achieved by fixing the total content of the polymer (PEG) and the crosslinker in the hydrogel, preferably at 1.0 to 10% w / v.

[0077] In one embodiment of the present invention, the hydrogel is autodegradable. This is achieved by the presence of acrylate moieties in the hydrogel precursor molecules, as known in the art (Gjorevski 2016, p. 563, Fig. 4a), which undergo hydrolysis (i.e., their bonds are broken down in the presence of water). Thus, in this embodiment, the three-dimensional hydrogel of the present invention has an initial shear modulus (stiffness) of 50 to 1000 Pa, preferably 200 to 500 Pa, and a final shear modulus (stiffness) of 0 to 50 Pa, preferably 0 Pa. The final shear modulus (stiffness) is typically reached 7 to 18 days after hydrogel formation.

[0078] The shear modulus of a hydrogel is equal to the modulus of stiffness, G, or elasticity or elasticity of the hydrogel. Shear modulus is defined as the ratio of shear stress to shear strain. The shear modulus of a hydrogel can be measured using a rheometer. Briefly, preformed hydrogel disks, 1-1.4 mm thick, are allowed to swell in complete cell culture medium for at least 3 hours and then sandwiched between the parallel plates of the rheometer. The mechanical response of the gel is recorded by performing frequency sweep (0.1-10 Hz) measurements in constant strain (0.05) mode at room temperature. The shear modulus (G') is reported as a measure of the gel's mechanical properties.

[0079] The technical properties of 3D hydrogels can be tuned by varying the content of hydrophilic polymer in the hydrogel, as well as the molecular weight and / or functionality (number of sites available for cross-linking) of the polymeric hydrogel precursors.

[0080] The sum of the polymer (i.e., PEG molecules) and crosslinker contents of the hydrogel swollen to equilibrium in the buffer solution can be 0.3-10% w / v, with preferred ranges being 1.1-4.0% w / v and 1.5-3.5% w / v.

[0081] Contrary to the teachings of Cruz-Acuna 2017 and 2018 and WO2018 / 165565 A1, the present invention has found that enzymatic degradation sites in the hydrogel are undesirable with respect to the efficacy of the hydrogel. Thus, the hydrogels of the present invention are preferably not susceptible to degradation by enzymes that require specific peptide sequences for cleavage, such as degradation by MMPs or cathepsins, i.e., the hydrogels of the present invention do not contain moieties that are susceptible to cleavage by proteases secreted by cells.

[0082] The microenvironment provided by the hydrogels of the present invention provides the biochemical, biophysical, and biological complexity necessary for organoid formation from fresh, isolated, or frozen human cells or tissues.

[0083] According to the present invention, it has been found that the hydrogels described herein, when used in combination with an appropriate culture medium, are suitable for the growth of freshly isolated or frozen intestinal cells and the formation of intestinal organoids derived therefrom.

[0084] It has been found that in order to grow freshly isolated or frozen intestinal cells and to form intestinal organoids derived therefrom, a medium must be used that contains as essential components a Wnt agonist, such as R-spondin1, preferably R-spondin1 conditioned medium, and Wnt3a, preferably Wnt3a conditioned medium.

[0085] R-spondin1 can be used in the form of a conditioned medium, for example, the supernatant of cells stably transfected to secrete R-spondin1. Alternatively, recombinant R-spondin1, preferably purified recombinant R-spondin1, can be used.

[0086] Wnt3a is preferably used in the form of a conditioned medium, e.g., the supernatant of cells stably transfected to secrete Wnt3a. However, afamin has been shown to stabilize Wnt proteins (Mihara et al., Active and water-soluble form of lipidated Wnt protein is maintained by a serum glycoprotein afamin / a-albumin eLife 2016; 5:e11621). Afamin / recombinant Wnt3a complexes or other similar complexes that stabilize Wnt proteins can also be used instead of conditioned medium (Holmberg et al., Culturing human intestinal stem cells for regenerative applications in the treatment of inflammatory bowel disease. EMBO Mol Med 2017 9: 558-57). Similarly, Wnt surrogate proteins described in Janda, Surrogate Wnt agonists that phenocopy canonical Wnt / β-catenin signaling, Nature 2017 May 11; 545(7653): 234-237; and WO 2016 / 040895 A1 can be used.

[0087] According to a preferred embodiment of the present invention, the medium further contains FBS (fetal bovine serum).

[0088] Optionally, one or more of the following components may be present in the medium: components of basal medium, such as adDMEM / F12, amino acids, such as glutamine, proteins, such as transferrin, noggin, such as recombinant mouse noggin and epidermal growth factor (EGF), such as recombinant mouse EGF, antibiotics, such as penicillin-streptomycin, antioxidants, such as glutathione, N-acetyl-l-cysteine (NAC), catalase and superoxide dismutase, vitamins, such as biotin, niacin, niacin, niacin, niacin, niacin-containing compounds, niacin-containing ferricyanide, niacin-containing cellulose ... In some embodiments, the compound may be selected from the group consisting of thyroid hormones, thyroid hormones, thyroid hormones, thyroid hormones (T3), thyroid hormones (T4), thyroid hormones (T5), thyroid hormones (T6), thyroid hormones (T7), thyroid hormones (T8), thyroid hormones (T9), thyroid hormones (T10), thyroid hormones (T11), thyroid hormones (T12), thyroid hormones (T13), thyroid hormones (T14), thyroid hormones (T15), thyroid hormones (T16), thyroid hormones (T16), thyroid hormones (T18 ...

[0089] Table 1 below lists preferred embodiments of media (standard medium (SCM) and differentiation medium (DM) compositions) suitable for the present invention:

[0090] [Table 1]

[0091] A commercially available medium suitable for the present invention is IntestiCult™ (available from STEMCELL Technologies).

[0092] According to the present invention, it has been found that the above-mentioned commercially available culture medium (IntestiCult™) cannot be used with the hydrogels of the prior art, e.g., Gjorevski 2016 and 2017, WO2017 / 036533 A1, and WO2017 / 037295 A1. Prior art hydrogels typically degrade within the culture medium within one day. Prior art hydrogels cannot be maintained in these culture media for a period sufficient for cell growth and proliferation.

[0093] The present invention also provides a method for the growth and proliferation of freshly isolated or frozen intestinal cells, comprising the steps of: a) forming organoids de novo from freshly isolated or frozen human intestinal cells by incubating the freshly isolated or frozen intestinal cells with the hydrogel of the kit in the presence of the culture medium of the kit using the contents of the kit as described herein; b) growing, optionally passage and expanding the cells derived from the intestinal organoids of step a) using the contents of the kit as described in the specification; c) optionally differentiating the organoids of step a) in the presence of a modified medium that induces cell differentiation. Including, The method is characterized in that only fully synthetic hydrogels or fully defined semi-synthetic hydrogels are used.

[0094] Thus, the method of the present invention completely avoids the need to have to use naturally derived matrices such as Matrigel®.

[0095] Cells for use in the methods of the present invention can be obtained by any suitable method, for example, standard sigmoidoscopy, obtaining one to several, for example five, standard punch biopsies.

[0096] The cells thus obtained may be isolated by standard procedures known in the art, for example, by chelation (using a chelating agent such as EDTA), or single cells or "clusters" of cells, including dissociated cells, crypts, and small pieces of tissue, may be prepared, for example, by exposure to trypsin-like enzymes.

[0097] The single cells or "clusters" of cells thus obtained, including dissociated cells, crypts and small pieces of tissue, can then be cultured using the contents of the kit of the invention, i.e., in the hydrogel of the invention in combination with the culture medium defined above, until a sufficient number of cells for the desired clinical application (e.g., for regenerative medicine, a minimum of 10 7The cells are then grown in vitro for 2-3 passages (e.g., 2 passages) to obtain a total of 1000 cells (e.g., 1000 cells). During cell growth, the medium is periodically replaced, e.g., every 2-4 days.

[0098] The resulting organoids can be implanted into a patient using standard procedures known in the art, for example, using a standard colonoscopy catheter.

[0099] In a further aspect, the present invention provides a method for regenerating intestinal tissue, comprising: a) encapsulating freshly isolated or frozen intestinal cells obtained from a human biopsy in the three-dimensional hydrogel of the present invention and allowing them to grow and form organoids; and b) transplanting the formed organoids or cells into a patient.

[0100] The method of the present invention allows for the maintenance of freshly isolated or frozen intestinal cells for at least 10 passages, with organoid formation capacity comparable to that of Matrigel® and with a similar number of proliferating cells as in Matrigel®.The present invention allows for the production of organoids that comply with relevant regulatory requirements for use in human and / or clinical applications.

[0101] The invention will now be explained in more detail with reference to non-limiting figures and embodiments. [Brief explanation of the drawings]

[0102] [Figure 1] Figure 1 shows organoid formation from freshly isolated intestinal cells obtained from human biopsies at various culture passages (P, days shown in parentheses) cultured in the hydrogel of Example 1 (fully defined semi-synthetic), the hydrogel of Example 2 (synthetic), prior art gels (PEG-RGD and PEG-RGD LAM), or Matrigel® using the commercially available medium IntestiCult™. Scale bar: 500 μm. [Figure 2]Figure 2 shows the organoid formation efficiency (calculated on the day in parentheses after seeding the cells in the three-dimensional matrix) of human intestinal organoids formed from fresh human biopsy samples and cultured in vitro for five passages (P). [Figure 3] Figure 3 shows organoid formation from intestinal cells pre-cultured in Matrigel® and grown for 18 days in the hydrogel of Example 1 (fully defined semi-synthetic), prior art gels (PEG-RGD and PEG-RGD LAM), or Matrigel® using the commercially available medium IntestiCult™. Scale bar: 500 μm. [Example]

[0103] Materials and Methods a) Isolation and primary encapsulation of cells from human colonic biopsies. Colon punch biopsies were obtained from healthy subjects after they signed informed consent. Enterocytes were isolated by chelation (using EDTA) and enzymatically dissociated into single cells using TrypLE Express (Thermo Fisher Scientific). Single cells from freshly dissociated biopsies were resuspended in an appropriate volume of basal medium (DMEM / F12, GlutaMAX (1x), Pen-Strep (100 U / ml) (Thermo Fisher Scientific), and HEPES (10 mM) (Thermo Fisher Scientific)) to a concentration of 2500 cells / μl (5x final concentration) and prepared for initial encapsulation (passage 0) with Matrigel®, fully defined semi-synthetic hydrogel (Example 1), synthetic hydrogel (Example 2), and prior art gels (PEG-RGD and PEG-RGD-LAM).

[0104] The prior art hydrogels, PEG-RGD and PEG-RGD LAM, were prepared as described in Gjorevski et al., "Synthesis and characterization of well-defined hydrogel matrices and their application to intestinal stem cell and organoid culture," Nature Protocols, Vol. 12, No. 11, 2017, pp. 2263-2274. Briefly, to generate the hydrogel precursors, the termini of 8-arm PEG-VS macromers and 8-arm PEG-Acr macromers were functionalized with lysine- and glutamine-bearing peptides, which act as substrates for the activated transglutaminase factor XIIIa (FXIIIa). FXIIIa crosslinked the macromers to form gels via ε-(α-glutamyl)lysine isopeptide side chain crosslinking between the two peptide substrates. The same crosslinking mechanism was used to attach the RGD residues contained in the peptide sequences to the hydrogel backbone. In the case of PEG-RGD LAM, native mouse laminin 111 was added to the hydrogel.

[0105] The hydrogel used in Example 1 was derived from a 1:1 ratio of 8-arm PEG-VS and 8-arm PEG-Acr as precursor molecules, a peptide crosslinker molecule containing two cysteines and two RGD motifs and not enzymatically degradable, and native mouse laminin 111 as the biofunctional molecule.

[0106] The hydrogel used in Example 2 was derived from a 1:1 ratio of 8-arm PEG-VS and 8-arm PEG-Acr as precursor molecules, a peptide crosslinker molecule containing two cysteines and two RGD motifs and not enzymatically degradable, and recombinant laminin 511 as the biofunctional molecule.

[0107] The mixture of gel and cells (approximately 10,000 cells) was cast in 20 μl droplets in a 48-well plate and incubated at 37°C. The assay plate was inverted every minute until gelation occurred to prevent cell settling. After 20 minutes, 300–330 μl of IntestiCult™ medium supplemented with ROCK inhibitor (10 μmol / L) was added on top of the gel droplet. The medium was changed every 2–4 days, and the ROCK inhibitor was maintained in the medium until day 7.

[0108] b) Organoid passaging and cell expansion Depending on cell growth, organoid passage was performed every 8-11 days by enzymatic (TrypLE) and mechanical dissociation of organoids encapsulated in Matrigel® and the hydrogel of the present invention into single cells. After dissociation of the gel and organoids, cells were counted and replated in the hydrogel of the present invention or Matrigel® at a cell density of approximately 10,000 cells per 20 μl droplet.

[0109] c) Experiments using pre-established organoids In experiments performed with pre-established organoids, organoids grown in Matrigel® for several passages were dissociated and replated onto prior art gels, hydrogels of the present invention, and b) Matrigel®.

[0110] d) Organoid formation efficiency and planned total number of cells Colony or organoid formation efficiency (OFE) was assessed by image analysis and was defined as the ratio of formed organoids to the total number of objects (i.e., the sum of single cells and organoids) in each image at a particular time point.

[0111] 1-3, it can be seen that the hydrogels of the present invention exhibit an organoid formation efficiency (OFE) comparable to that of Matrigel®. Thus, the hydrogels of the present invention are at least as suitable for organoid formation as the previously used standard Matrigel®, but with the advantages mentioned above, in particular the advantage of a completely defined composition, especially a fully synthetic composition, which provides a clear advantage in terms of regulatory approval.

[0112] From Figures 1 to 3, it can be seen that under the conditions shown in a) or b), the hydrogels of the present invention were stable and had very good organoid formation efficiency (OFE), whereas the hydrogels of Gjorevski 2017 ("PEG RGD" and "PEG RGD LAM" in the figures) immediately decomposed and had inadequate organoid formation efficiency (OFE). The following claims as originally filed in this application are appended as embodiments. [1] A biofunctional three-dimensional hydrogel suitable for the growth and proliferation of freshly isolated or frozen intestinal cells and the formation of intestinal organoids derived therefrom, said hydrogel comprising: - at least one precursor molecule which is a multi-arm PEG having ethylenically unsaturated groups selected from the group consisting of vinyl sulfone and acrylate; - a cross-linking molecule having at least two, preferably two, nucleophilic groups capable of reacting with the ethylenically unsaturated groups of said multi-arm PEG via a Michael addition reaction; and - at least one biofunctional ligand selected from the group consisting of natural laminins, such as laminin 111, recombinant laminin isoforms, preferably laminin 511, and biofunctional fragments thereof; is the reaction product of the cross-linking molecule comprises at least one RGD motif; Biofunctional three-dimensional hydrogels. [2] The hydrogel described in [1], wherein the multi-arm PEG is selected from the group consisting of 4-arm and 8-arm PEG. [3] The hydrogel according to [1] or [2], wherein the hydrogel has a shear modulus of 50 to 1000 Pa. [4] The hydrogel i) a reaction product that is an 8-arm PEG vinyl sulfone crosslinked with a peptide containing at least two thiol groups and at least two RGD motifs, or ii) comprising a reaction product which is an 8-arm PEG vinyl sulfone crosslinked with a peptide comprising at least two thiol groups and at least two RGD motifs, and / or a reaction product which is an 8-arm PEG acrylate crosslinked with a peptide comprising at least two thiol groups and at least two RGD motifs; The hydrogel according to any one of [1] to [3]. [5] The hydrogel according to any one of [1] to [4], wherein the shear modulus of the hydrogel decreases over time. [6] The hydrogel according to any one of [1] to [5], wherein the hydrogel is not susceptible to enzymatic degradation. [7] The hydrogel described in any one of [1] to [6], wherein the crosslinking molecule is a peptide containing at least two RGD motifs and at least two cysteines. [8] The cross-linking molecule is Ac-GCREGRGDSPGGRGDSPGERCG-NH 2 [7] The hydrogel according to [7], [9] Contents of a kit for the growth and proliferation of freshly isolated or frozen intestinal cells and the formation of intestinal organoids derived therefrom, comprising: - at least one precursor molecule which is a multi-arm PEG having ethylenically unsaturated groups selected from the group consisting of vinyl sulfone and acrylate; - a cross-linking molecule comprising at least one RGD motif, having at least two, preferably two, nucleophilic groups capable of reacting with the ethylenically unsaturated groups of said multi-arm PEG via a Michael addition reaction; - at least one biofunctional ligand selected from the group consisting of natural laminins, such as laminin 111, recombinant laminin isoforms, preferably laminin 511, and biofunctional fragments thereof, and - a medium comprising R-spondin1, preferably an R-spondin1 conditioned medium, and Wnt3a, preferably a Wnt3a conditioned medium The contents of the kit include:

[10] The contents of the kit described in [9], wherein the cross-linking molecule is a peptide containing at least two RGD motifs and at least two cysteines.

[11] The contents of the kit according to [9] or

[10] , wherein the precursor molecule is 8-arm PEG vinyl sulfone or a combination of 8-arm PEG vinyl sulfone and 8-arm PEG acrylate.

[12] A method for producing a three-dimensional hydrogel according to any one of [1] to [8], the method comprising: a1) dispensing onto the surface of the substrate or into separate spaces of the substrate, preferably a multi-well plate, one or more different hydrogel precursor molecules, which are multi-arm PEGs with ethylenically unsaturated groups selected from the group consisting of vinyl sulfones and acrylates; The hydrogel precursor molecules - a cross-linking molecule comprising at least one RGD motif, having at least two, preferably two, nucleophilic groups capable of reacting with the ethylenically unsaturated groups of said multi-arm PEG via a Michael addition reaction; and - at least one biofunctional ligand selected from the group consisting of natural laminins, such as laminin 111, recombinant laminin isoforms, preferably laminin 511, and biofunctional fragments thereof; or a2) dispensing onto the surface of the substrate or into discrete spaces of the substrate, preferably in a multi-well plate, a resuspension of unreacted powder, preferably lyophilized unreacted powder, The unreacted powder is - one or more different hydrogel precursor molecules that are multi-arm PEGs with ethylenically unsaturated groups selected from the group consisting of vinyl sulfones and acrylates; - a cross-linking molecule comprising at least one RGD motif, having at least two, preferably two, nucleophilic groups capable of reacting with the ethylenically unsaturated groups of said multi-arm PEG via a Michael addition reaction; and - at least one biofunctional ligand selected from the group consisting of natural laminins, such as laminin 111, recombinant laminin isoforms, preferably laminin 511, and biofunctional fragments thereof; a process comprising: b) adding human biopsy cells, preferably freshly isolated or frozen intestinal cells, to the surface of the substrate or into the separate spaces of the substrate, or to the hydrogel precursor formulation of a1) or a2) before adding them to the surface of the substrate or into the separate spaces of the substrate; and c) crosslinking said hydrogel precursor molecules and said crosslinking molecules to form a hydrogel.

[13] A method for the growth and proliferation of freshly isolated or frozen intestinal cells, comprising the steps of: a) forming organoids de novo from freshly isolated or frozen human intestinal cells by incubating the freshly isolated or frozen intestinal cells with the hydrogel of the kit in the presence of the culture medium of the kit using the contents of the kit as described herein; b) growing, optionally passage, and expanding the cells derived from the intestinal organoids of step a) using the contents of the kit according to any one of [9] to

[11] ; c) optionally differentiating the organoids of step a) in the presence of a modified medium that induces cell differentiation. Including, A method characterized by using only fully synthetic hydrogels or fully defined semi-synthetic hydrogels.

[14] The method according to

[13] , wherein only fully defined semi-synthetic hydrogels that are self-degrading are used.

[15] The method according to

[13] , wherein only fully synthetic hydrogels that are self-degrading are used.

Claims

1. A biofunctional three-dimensional hydrogel suitable for the growth and proliferation of freshly isolated or frozen intestinal cells and the formation of intestinal organoids derived therefrom, said hydrogel comprising: at least one precursor molecule which is a multi-arm PEG having ethylenically unsaturated groups selected from the group consisting of vinyl sulfone and acrylate; a bridging molecule having at least two nucleophilic groups capable of reacting with the ethylenically unsaturated groups of said multi-arm PEG via a Michael addition reaction, and - at least one biofunctional ligand selected from the group consisting of native laminin, recombinant laminin isoforms and biofunctional fragments thereof; is the reaction product of The bridging molecule comprises at least one RGD motif. Biofunctional three-dimensional hydrogels.

2. 2. The hydrogel of claim 1, wherein the multi-arm PEG is selected from the group consisting of 4-arm and 8-arm PEG.

3. 3. The hydrogel of claim 1, wherein the hydrogel has a shear modulus of 50 to 1000 Pa.

4. The hydrogel is i) a reaction product which is an 8-arm PEG vinyl sulfone crosslinked with a peptide containing at least two thiol groups and at least two RGD motifs, or ii) a reaction product which is an 8-arm PEG vinyl sulfone cross-linked with a peptide comprising at least two thiol groups and at least two RGD motifs, and / or a reaction product which is an 8-arm PEG acrylate cross-linked with a peptide comprising at least two thiol groups and at least two RGD motifs; The hydrogel according to any one of claims 1 to 3.

5. 5. The hydrogel of any one of claims 1 to 4, wherein the shear modulus of the hydrogel decreases over time.

6. The hydrogel according to any one of claims 1 to 5, wherein the cross-linking molecule is a peptide containing at least two RGD motifs and at least two cysteines.

7. The cross-linking molecule is Ac-GCREGRGDSPGGRGDSPGERCG-NH ２ 7. The hydrogel of claim 6, wherein:

8. 1. The contents of a kit for the growth and proliferation of freshly isolated or frozen intestinal cells and the formation of intestinal organoids derived therefrom, comprising: at least one precursor molecule which is a multi-arm PEG having ethylenically unsaturated groups selected from the group consisting of vinyl sulfone and acrylate; a cross-linking molecule containing at least one RGD motif and having at least two nucleophilic groups capable of reacting with the ethylenically unsaturated groups of said multi-arm PEG by a Michael addition reaction; - at least one biofunctional ligand selected from the group consisting of native laminin, recombinant laminin isoforms and biofunctional fragments thereof, and - medium containing R-spondin1 conditioned medium and Wnt3a conditioned medium The contents of the kit include:

9. 9. The contents of the kit of claim 8, wherein the bridging molecule is a peptide containing at least two RGD motifs and at least two cysteines.

10. The contents of the kit according to claim 8 or 9, wherein the precursor molecule is 8-arm PEG vinyl sulfone or a combination of 8-arm PEG vinyl sulfone and 8-arm PEG acrylate.

11. A method for producing a three-dimensional hydrogel according to any one of claims 1 to 7, said method comprising: a1) dispensing onto the surface of the substrate or into discrete spaces of the substrate one or more different hydrogel precursor molecules, wherein the multi-arm PEG has an ethylenically unsaturated group selected from the group consisting of vinyl sulfone and acrylate; The hydrogel precursor molecules a cross-linking molecule containing at least one RGD motif and having at least two nucleophilic groups capable of reacting with the ethylenically unsaturated groups of said multi-arm PEG by a Michael addition reaction; and - at least one biofunctional ligand selected from the group consisting of native laminin, recombinant laminin isoforms and biofunctional fragments thereof; or a2) dispensing a resuspension of unreacted powder onto the surface of the substrate or into discrete spaces of the substrate, The unreacted powder is one or more different hydrogel precursor molecules that are multi-arm PEGs with ethylenically unsaturated groups selected from the group consisting of vinyl sulfones and acrylates; a cross-linking molecule containing at least one RGD motif and having at least two nucleophilic groups capable of reacting with the ethylenically unsaturated groups of said multi-arm PEG by a Michael addition reaction; and - at least one biofunctional ligand selected from the group consisting of native laminin, recombinant laminin isoforms and biofunctional fragments thereof; a process comprising: b) adding cells obtained from a human biopsy to the surface of the substrate or into the discrete spaces of the substrate, or to the hydrogel precursor formulation of a1) or a2) before adding cells to the surface of the substrate or into the discrete spaces of the substrate; and c) crosslinking said hydrogel precursor molecules and said crosslinking molecules to form a hydrogel.

12. 1. A method for the growth and proliferation of freshly isolated or frozen intestinal cells, comprising the steps of: a) forming organoids de novo from freshly isolated or frozen human intestinal cells by incubating the freshly isolated or frozen intestinal cells with the hydrogel of the kit in the presence of the culture medium of the kit using the contents of the kit as described herein; b) growing and expanding cells derived from the intestinal organoids of step a) using the contents of the kit according to any one of claims 8 to 10. Including, A method characterized by using only fully synthetic hydrogels or fully defined semi-synthetic hydrogels.

13. 13. The method of claim 12, wherein the method uses only fully defined semi-synthetic hydrogels that are self-degrading.

14. 13. The method of claim 12, wherein the method uses only fully synthetic hydrogels that are self-degrading.

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