Methods and kits for cell growth

Abstract

Providing a drug screening method that uses a single tissue type. [Solution] A drug screening method performed on a type of tissue combined with other cells, the method comprising: a) providing an array having separate spaces of a completely distinct hydrogel matrix by crosslinking one or more different combinations of one or more different hydrogel precursor molecules, at least one crosslinking agent, and cells of the type of tissue to be tested on the surface of a substrate or in separate spaces of a substrate, in order to create conditions for a completely distinct three-dimensional extracellular matrix having different biological, biophysical and / or biochemical properties from one another; b) enabling the cells to grow and proliferate in the separate spaces of the array of hydrogel matrices in the presence of one or more different culture media; and c) manipulating the cells grown in the separate spaces of the array of hydrogel matrices.

Description

Technical Field

[0001] The present invention relates to a cell proliferation method and a kit for cell proliferation, which provide significantly improved tools not only for drug discovery research and development, but also for basic science research, precision medicine, regenerative medicine, and for delivering cells to be transplanted into mammals, preferably humans.

Background Art

[0002] Hydrogels for cell growth and drug screening In the field of ex vivo assays, progress has been made in recent years. In particular, the development of three-dimensional (3D) hydrogel matrices offers significant advantages over two-dimensional cell culture systems that are not sufficiently similar to in vivo conditions.

[0003] First, naturally-derived 3D cell culture systems, such as Matrigel (registered trademark), were used. However, such systems have a composition that is not sufficiently defined, exhibit batch-to-batch variability, and as such, it is impossible to systematically change their properties and independently control their important matrix parameters. Similarly, for screening purposes using multiwell arrays, such naturally-derived 3D cell culture systems are not suitable because their lack of sufficient definition makes it impossible to accurately attribute changes in cell behavior between arrays to specific modifications of the extracellular matrix conditions provided to those arrays.

[0004] Similarly, due to the batch-to-batch variability and undefined composition of animal-derived matrices, such as Matrigel (registered trademark), regulatory approval for their use in humans has not been forthcoming. There is a need for the development of defined, human-use-approved, scalable, and preferably xenobiotic-free (i.e.,不含 animal-origin components) support matrices and media.

[0005] However, in recent years, completely distinct semi-synthetic or totally synthetic hydrogel systems have been developed that are far more suitable for the above purposes. For example, PEG-based hydrogels are described, which are composed of PEG (polyethylene glycol) precursor molecules that can be crosslinked under physiological conditions via either a crosslinking mechanism detailed in Ehrbar et al. (Ehrbar, M., Rizzi, SC, Schoenmakers, RG, Miguel, BS, Hubbell, JA, Weber, FE, and Lutolf, MP, Biomolecular hydrogels formed and degraded via site-specific enzymatic reactions, Biomacromolecules 8 (2007), 3000-3007) to thrombin-activated factor XIIIa, or via a mild chemical reaction via a crosslinking mechanism detailed in Lutolf et al. (Lutolf, MP, and Hubbell, JA, Synthesis and physicochemical characterization of end-linked poly(ethylene glycol)-co-peptide hydrogels formed by Michael-type addition, Biomacromolecules 4, 713-722 (2003)). These PEG hydrogels are tunable in terms of their properties and are biocompatible.

[0006] Applications of such fully defined semi-synthetic or totally synthetic hydrogel systems include human transplantation, basic research, precision medicine, and drug discovery research and development, such as in cancer research.

[0007] In cancer research and clinical practice, resistance of tumor cells to therapeutic treatments (e.g., chemotherapy with cytotoxic substances, immunotherapy, and / or targeted therapies) is a major challenge.

[0008] Drug resistance in tumor cells to chemotherapy has typically been attributed to genetic alterations and clonal genetic heterogeneity. However, multiple mechanisms contribute to drug resistance in cancer cells (e.g., drug inactivation, inhibition of cell death, DNA damage repair, alteration of drug targets, epithelial-mesenchymal transition, drug efflux, physical barriers, etc.), which can act independently or in combination, and may also depend on epigenetic changes in cancer cells and the influence of the tumor microenvironment (Holohan C, et al., 13, 714-726 (2013)).

[0009] In fact, tumors are generally composed of multiple phenotypic subpopulations that differ in their ability to initiate metastasis and their sensitivity to anticancer therapies (Flavahan et al., Epigenetic plasticity and the hallmarks of cancer, Science 357, 266 (2017); Baylin et al., Nat Rev Cancer, 11(10), 726-734 (2011)). Often, cells exhibit transitions between these subpopulations independently of gene mutations, but instead through reversible changes in signaling, as well as through gene expression programs influenced by the composition of tumor stromal cells, vascular system, immune system, and extracellular matrix (ECM) (Juntilla et al., Nature, 501(7467):346-54 (2013)).

[0010] Resistance to targeted therapies can be classified into intrinsic resistance, adaptive resistance, and acquired resistance. Intrinsic resistance may be due to driver mutations that make the drug insensitive. Adaptive resistance occurs when cancer cells undergo adaptive changes that enable their survival after a partial initial response to the treatment. Acquired resistance may be the result of selection by both pre-existing mutations in heterogeneous subpopulations (i.e., not all cells in the tumor are initially target-dependent) and the acquisition of novel modifications (phenotypic or genetic) due to the selective pressure exerted by the treatment. The resistance mechanism may involve either the primary target of the drug or other signaling events that allow the target to be bypassed by inducing other survival and / or growth pathways (Rotow-Bivona et al., Nature Reviews Cancer, 17(11) 637-658(2017)).

[0011] Therefore, using a single condition for in vitro culture of a specific cancer is insufficient to maintain the heterogeneity necessary to express different genetic and phenotypic tumor cell characteristics ex vivo, which can lead to different drug responses and thus drug resistance.

[0012] Initially considered a passive support structure, the extracellular matrix (ECM) is increasingly recognized as playing a central role in the initiation and progression of malignant diseases, both through its associated bioactive domains and as a reservoir for soluble cytokines, and similarly influencing the sensitivity of cancer cells to chemotherapy, such as chemoresistance (Senthebane et al., Int. J. Mol. Sci. 2017, 18, 1586). The structure and composition of the ECM are regulated by multiple cell types in the stroma, influencing various aspects of tumor cell behavior. While genetic and non-genetic factors are substantially involved in phenotypic diversity within tumors, no single approach can definitively resolve all of their relative contributions.

[0013] The use of scaffolds based on synthetic polymers primarily composed of polyethylene glycol (PEG) modified with bioactive peptides was applied to test a model of lung adenocarcinoma cell lines (Gill et al., Cancer Res; 72(22) November 15, 2012). To provide arrays for exploring and testing differences in epithelial morphogenesis originating from the ECM, modified PEG-RGD and MMP-sensitive hydrogels with diverse elastic and adhesive ligand concentrations were applied as disks on glass substrates.

[0014] Biohybrid in situ-forming hydrogels (starPEG) were used to investigate the potential role of bone-cell distribution factors in the behavior of breast cancer cells (Bray et al., Cancers 2018, 10, 292). To examine cell viability, morphology, and migration within their microenvironment, starPEG was also conjugated with matrix metalloproteinase (MMP)-cleavable peptide linkers, with or without collagen I-derived peptides.

[0015] Regarding the interaction between ECM proteins and drug resistance, it has already been described that ECM compositions modulate drug resistance in hyaluronic acid (HA) hydrogels supplemented with fibronectin, laminin, or cyclic cell adhesion peptides (cRGDs) (Blehm et al., Biomaterials. 2015 July; 56: 129-139). This literature provided the first evidence that the composition and construction of the tumor-ECM environment directly influenced drug efficacy, i.e., that ECM characteristics influenced the sensitivity of cancer cells to different drugs.

[0016] Similarly, using biomimetic hydrogels based on type I collagen of varying stiffness, Lam et al. (Mol. Pharmaceutics 2014, 11, 2016-2021) have already compared the effects of matrix stiffness on the proliferative growth and invasion of metastatic breast tumor cells and on drug therapy outcomes.

[0017] Recently, an approach for screening drug responses in cells cultured in a 3D biomaterial environment has been developed to explore how key biophysical and biochemical features of the extracellular matrix (ECM) mediate drug responses (Schwartz et al., Integr. Biol., 2017, 9, 912-924). Using 3D PEG-maleimide (PEG-MAL) hydrogels containing cRGD, the stiffness, dimensionality (i.e., 2D vs. 3D culture), and cell-cell contact were systematically varied to analyze matrix-mediated adaptive resistance. They identified correlated efficacy of combination therapy with MEK inhibitors and sorafenib, which would not have been achieved without using only a screening environment, i.e., single culture conditions, or without performing systems biology analyses. This literature used a single tissue type from cell lines known to be genetically homogeneous (rather than cells derived from a specific patient's tumor). The need to use a gel selected to capture the heterogeneity of cells from a specific patient's tumor or from tumors of different patients cannot be derived from this literature.

[0018] International Publication No. 2014 / 180970 described arrays and the combination methods performed thereby. Different extracellular matrix conditions in separate spaces of a multiwell plate were provided in an automated manner by varying the type and / or amount of hydrogel precursor molecules, crosslinking agents, and bioactive agents bound to the hydrogel precursor molecules.

[0019] Touati et al. (Poster presentation at the AACR 2018 Annual Meeting, Chicago, April 14-18, 2018) reported on the effects of different ECM compositions on the morphology of A549 lung adenocarcinoma cell lines and correlated different sensitivities to drug exposure with ECM-induced cellular phenotypes.

[0020] To more accurately predict the outcomes of patient drug treatment, a system is needed to establish ex vivo cell culture conditions for drug screening / testing that can capture the diverse disease characteristics of patients. More specifically, there is a need for methods and kits that can be easily used to assist and improve the treatment of patients with a particular disease.

[0021] Organoid manufacturing The present invention relates to any cellular structure, such as organoids, tumor organoids, multicellular tumor spheroids, cell spheroids, cell aggregates, tumor spheres, tissue-derived tumor spheres, or fragments of these cellular structures. Hereafter, the term "cell" refers to any such cellular structure.

[0022] Organoids, including cell spheroids or aggregates, are three-dimensional cellular structures of stem cells that develop and self-organize (or self-pattern) through spatially restricted phylogenetic constraints in a manner similar to cell sorting and in vivo situations, and are organ-specific, tissue-specific, or disease-specific cell types. Therefore, organoids represent the innate physiology of cells and possess cellular composition (including stem cells and / or specific cell or tissue types remaining at different stages of differentiation) and anatomy (e.g., cancer, cystic fibrosis, inflammatory bowel disease) that mimic the conditions of innate organs, tissues, and / or diseased cells and tissues. Normal cells and / or diseased cells (e.g., cancer cells) can be isolated from any tissue or any cellular structure, such as organoids or cancer organoids (also called tumor organoids). The cells from which organoids are generated can grow and / or differentiate, self-organize, and form organ-like or disease-like tissues (e.g., cancer, cystic fibrosis, inflammatory bowel disease) exhibiting multiple types of cells that form structures very similar to organs (i.e., cell differentiation) or disease tissues (e.g., multicellular heterogeneity of tumors) in vivo. Therefore, organoids are excellent models for testing human organs, human organ development, cancer, and other diseases in a system very similar to in vivo conditions. Organoids are also used to grow and proliferate cells for clinical applications, such as regenerative medicine and personalized medicine.

[0023] Another example of clinical application is for personalized medicine, in which organoids representing the disease are cultured ex vivo to test drugs in order to identify personalized treatment options for the patient. Briefly, patient-derived cells taken from a biopsy or excision of diseased tissue are grown and proliferated ex vivo as organoids and / or other cellular structures. These patient organoids can then be tested with potential therapeutic options (e.g., drugs, drug combinations) before actually treating the patient. The results of ex vivo drug testing using patient cells may be used by physicians to assist in their decision-making regarding which treatment to administer to the patient.

[0024] In the prior art concerning the potential clinical applications described above, the success of ex vivo patient cell growth and proliferation as organoids relied on the use of animal-derived matrices (e.g., Matrigel®).

[0025] However, their properties, the inherent batch-to-batch variability of animal-derived matrices, such as Matrigel®, and their unclear composition prevent regulatory approval for their use in humans or for growing cells ex vivo for subsequent transplantation into humans. In addition, these issues can also be major barriers to the standardization of organoid cultures, which may require regulatory approval for the use of organoids in drug testing for clinical diagnostics in precision medicine. Therefore, several aspects of organoid culture need to be modified to bridge the gap between organoid use and clinical applications (precision medicine, regenerative medicine, etc.). These include the development of support matrices and culture media that are defined and approved for human use, scalable, and preferably free of heterogeneous substances (i.e., free of animal-derived components).

[0026] In Broguiere et al., Growth of Epithelial Organoids in a Defined Hydrogel, Adv. Mater. 2018, 1801621, it was shown that a defined but not synthetic (i.e., neither xenogeneic nor xeno-free) fibrin hydrogel supplemented with laminin III supports the growth of organoid lines derived from human small intestine epithelium, liver, pancreas, and pancreatic ductal adenocarcinoma (PDAC).

[0027] 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; International Publication Nos. WO 2017 / 036533 and WO 2017 / 037295 developed hydrogels of 8-arm polyethylene glycol (PEG) crosslinked by an enzyme (factor XIII) with different degradation kinetics, including degradation by a functionalized RGD peptide and a specific enzyme and controlled autodegradation kinetics (hydrolysis of PEG-acrylate), for the growth of primary mouse and human small intestine organoids and human colorectal cancer organoids. Addition of laminin III (complete protein) purified from mouse tissue was necessary to assist organoid differentiation.

[0028] While some success with this approach has been demonstrated for proliferation and organoid formation from mouse cells, it has not been shown that the above system is suitable for proliferation and organoid formation from newly isolated or frozen human cells from human biopsies (rather, this was questioned on page 2265 of Gjorevski 2017). Similarly, the only system tested based on enzymatic cross-linking with factor XIII has proven to be expensive, difficult to scale up and / or automate for commercial purposes, and difficult to reproduce.

[0029] The research by Cruz-Acuna (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 international publication No. 2018 / 165565) is based on the development of a totally synthetic 4-arm PEG-maleimide hydrogel functionalized with RGD and crosslinked 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 injected into a mouse colon injury model as a proof-of-concept test, demonstrating the therapeutic potential of intestinal organoid transplantation.

[0030] This system has not been shown to be able to grow newly isolated or frozen cells from patient biopsies into organoids. In this system, the crosslinking agent component must be enzymatically degradable.

[0031] Currently, the standard for establishing organoid culture ex vivo involves first encapsulating newly isolated cells (from tissue) in Matrigel® (Matrigel® is one of the commercially available products of basement membrane extract (BME)), which is considered the "benchmark", and growing the cells through several passages to expand them (i.e., increase the cell number). BME (e.g., Matrigel®) is a gel derived from mouse sarcoma extract, which, as already noted above, lacks batch-to-batch consistency, has an unclear composition, and thus cannot be used for clinical bridging applications. Therefore, obtaining regulatory approval can be difficult or impossible (Madl et al., Nature 557 (2018), 335 - 342).

[0032] Excluding the use of gels with undefined or xenogeneic components for organoid attachment would overcome one of the major barriers in using organoids in clinical applications, such as regenerative medicine, precision medicine, drug testing, or patient stratification.

[0033] Proof of concept for culturing newly isolated cells obtained by biopsy in a completely defined (not totally synthetic) matrix is ​​provided 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 Pre-dicting Chemotherapy Efficacy Prior to Initiation of Treatment: A Feasibility Study, Ann Surg Oncol (2019) 26:139-147 and International Publication No. 2018 / 027023. 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 presents similar problems.

[0034] To date, there have been no reports of successful proliferation of newly isolated or frozen human cells from biopsies or tissue excisions (i.e., cells obtained directly from humans and not pre-cultured or pre-established in another system), nor have there been any reports of subsequent organoid formation from them in a matrix of completely distinct and / or totally synthetic hydrogels that are not naturally derived matrices, such as Matrigel® or collagen. Despite the clear need for such an approach, as described in the prior art above, to date, the standard for at least the first step of cell proliferation remains the use of Matrigel®. This demonstrates the difficulties associated with the creation of semi-synthetic or totally synthetic three-dimensional hydrogel systems.

[0035] Thus, there is a need to provide a method for the proliferation of newly isolated or frozen human cells obtained by biopsy, and for the subsequent formation of organoids therefrom, wherein the method completely eliminates the use of naturally derived matrices, such as Matrigel®, and provides organoids that are suitable for clinical application, commercially viable, i.e., cost-effective, reliable, reproducible, automatable, and scalable.

[0036] To more accurately predict the outcomes of patient drug therapy, the optimal system for establishing ex vivo cell culture conditions for drug screening / testing that can capture the different tumor characteristics of patients also includes the proliferative capacity of newly isolated or frozen human cells obtained from biopsies or excisions, and the subsequent organoid formation capacity therefrom, and this method does not use any naturally derived matrix, such as Matrigel®.

[0037] More specifically, there is a need for methods and kits that can be easily used to assist and improve the treatment of patients with a certain disease, and which do not use any naturally derived matrix, such as Matrigel®. [Disclosure of the Invention]

[0038] The present invention expands upon the prior art by providing a cell growth kit that includes conditions of an extracellular matrix specifically pre-selected for a particular disease or healthy tissue, thus enabling more accurate and efficient prediction of the outcomes of drug treatment for a particular disease or toxicity to a particular healthy tissue. This potential of three-dimensional, fully defined (including total synthesis) hydrogels is not recognized in the aforementioned prior art.

[0039] Based on previously conducted experiments and / or knowledge, it is possible to estimate preferred conditions for the growth and subsequent testing of specific tissue types, such as cancer cells or normal / healthy cells. However, while this addresses the specific characteristics of each tissue type, it is still insufficient to address multiple phenotypic subpopulations of tissue types that differ in their ability to initiate metastasis and their sensitivity to anti-cancer treatments, for example, in the case of cancer cells. Therefore, performing assays for a particular tissue type, even under a single ex vivo culture condition previously established as favorable for the growth of that particular tissue type, does not provide the desired optimal adjunct and improvement in the treatment of patients with a certain disease.

[0040] The present invention provides an array of extracellular matrix (ex vivo culture) conditions that, while based on the prior selection of extracellular matrix conditions established to be suitable for specific types of tissues, offers an alternative embodiment of the prior selection of extracellular matrix conditions. This approach allows for a more extensive and focused assay. On the other hand, in conventional assays using unselected extracellular matrix conditions (e.g., conventional screening of extracellular matrix conditions), a certain number of extracellular matrix conditions used in the assay are unsuitable. However, in the method of the present invention, which utilizes prior selection of extracellular matrix conditions, all extracellular matrix conditions are essentially suitable for the intended purpose, and in a more extensive and focused manner, it is possible to identify the optimal extracellular matrix conditions for a specific phenotypic subpopulation of a particular type of tissue in a patient being treated. Thus, the present invention provides an improvement in personalized medicine.

[0041] Thus, the present invention provides a method for using a single tissue type by optionally combining it with other cells such as stromal cells or immune cells, a) A step of providing an array having separate spaces of a completely distinct hydrogel matrix by crosslinking cells of the type of tissue to be tested with one or more different combinations of one or more different hydrogel precursor molecules, optionally at least one crosslinking agent, on the surface of a substrate or in separate spaces of a substrate, preferably in a multiwell plate, in the presence of optionally one or more biologically active molecules; b) A process that allows cells to grow and proliferate in separate spaces within an array of hydrogel matrices in the presence of one or more different culture media; c) A step of manipulating cells grown in separate spaces within an array of hydrogel matrices; The present invention relates to a method in which a specific combination of hydrogel characteristics is pre-selected for one type of tissue to be tested.

[0042] According to the present invention, step b) of the above method involves growing and proliferating the cells until a sufficient number of cells are reached. Once a sufficient number of cells are reached, the desired operation (e.g., drug testing or creation / establishment of a cell repository / biobank) can be performed in step c). Preferably, step b) (cell proliferation) is performed manually, and it is important to increase the number of cells after each passage. However, step b) can also be performed automatically and / or in a miniaturized form.

[0043] The aforementioned method may be a combination method, that is, a method for simultaneously investigating multiple combinations of ex vivo conditions (such as extracellular matrix conditions) and drugs.

[0044] In one embodiment, the operation performed using cells grown in separate spaces of a hydrogel matrix array may involve adding one or more drugs to the separate spaces of the hydrogel matrix array. According to this embodiment, the method of the present invention is a drug screening test for identifying one or more drugs suitable for treating cell-related conditions from a type of tissue being tested. This can be used in the field of personalized medicine.

[0045] According to a preferred embodiment of the present invention, the tissue type is derived from a specific patient, for example, from newly isolated or frozen cells obtained from a biopsy or excision of the patient, and the drug screening test is an improvement relating to precision medicine and / or personalized medicine, as it helps to accurately identify the most suitable treatment for the patient.

[0046] According to one aspect of the present invention, the type of tissue in which the method is performed may include both other types of cells, such as stromal cells or immune cells, including cancer-associated fibroblasts (CAFs), and cancer cells.

[0047] According to a preferred embodiment of the present invention, the tissue type is lung cancer, preferably non-small cell lung cancer overexpressing c-Met, the hydrogel matrix is ​​a pre-selected non-autodegradable PEG hydrogel, and the crosslinking agent and any bioactive agents do not contain any RGD motifs. Preferably, the culture medium used in the embodiment contains FBS (serum) or a Wnt agonist, such as R-spongin.

[0048] According to another preferred embodiment of the present invention, the tissue type is pancreatic ductal adenocarcinoma (PDAC) cells, the hydrogel matrix is ​​pre-selected as a non-self-degrading PEG hydrogel having a rigidity of 50-3000 Pa, preferably 50-2000 Pa, most preferably 50-1000 Pa, and at least one of the crosslinking agents and / or any bioactive agents comprises an RGD motif. Preferably, the culture medium used in the embodiment comprises a Wnt agonist, e.g., R-spongin and Wnt3a.

[0049] According to another preferred embodiment of the present invention, the tissue type is colorectal cancer (CRC) cells, and a PEG hydrogel is pre-selected, the matrix of which has an initial stiffness of at least 50 to 2000 Pa, and further comprising one or more bioactive molecules, optionally including laminin, preferably laminin-111 or laminin-511, particularly preferably natural mouse laminin-111 or recombinant human laminin-511, and at least one of the crosslinking agents and / or any bioactive agents comprising an RGD motif. Preferably, the culture medium used in the embodiment comprises a Wnt agonist, e.g., R-spongin and Wnt3a.

[0050] According to another preferred embodiment of the present invention, the tissue type is breast cancer cells, the matrix of the hydrogel is preferably a pre-selected enzymatically degradable PEG hydrogel, at least one of the crosslinking agents preferably comprises an enzymatically degradable motif, preferably an MMP-sensitive motif, and the hydrogel further comprises one or more bioactive molecules, optionally including laminin, preferably laminin-111, particularly preferably natural mouse laminin-111. Preferably, the culture medium used in the embodiment comprises FBS (serum) or a Wnt agonist, such as R-spongin.

[0051] According to another preferred embodiment of the present invention, the tissue type is cancer cells that proliferate more slowly than their healthy / normal corresponding cells (e.g., epithelial and / or stromal cells) ex vivo, preferably prostate cancer cells, the hydrogel matrix is ​​preferably a PEG hydrogel having a rigidity of 50 to 2000 Pa, and the crosslinking agent and any bioactive agents are free from any RGD motifs.

[0052] According to another preferred embodiment of the present invention, the tissue type is cancer cells, preferably pancreatic ductal adenocarcinoma (PDAC) cells, combined with stromal cells, preferably fibroblasts, and the hydrogel matrix is ​​a PEG hydrogel pre-selected having a rigidity of 50-3000 Pa, preferably 50-2000 Pa, most preferably 50-1000 Pa, and at least one of the crosslinking agents comprises an enzymatically degradable motif, preferably an MMP-sensitive motif, and at least one of the crosslinking agents and / or any bioactive agents comprises an RGD motif. Preferably, the culture medium used in the embodiment comprises a Wnt agonist, e.g., R-spongin and Wnt3a, and more preferably also comprises FBS (fetal bovine serum).

[0053] In another embodiment, the procedure performed using cells grown in separate spaces of a hydrogel matrix array may be a drug screening / test on healthy organoid cells, particularly in the field of precision medicine. In a preferred embodiment, healthy / normal organoids (e.g., colon or intestinal organoids, normal / healthy prostate cells, or healthy cells from other organs) may be used as a control condition and / or in a cytotoxicity assay in a drug test on diseased cells of the same organ (e.g., to test the toxicity of a drug). For example, healthy / normal colon or intestinal organoids can be used in a drug test as a control condition when testing a drug on, for example, cancer organoids or organoids from cystic fibrosis tissue of the same patient.

[0054] In another embodiment, the operation performed using cells grown in separate spaces of a hydrogel matrix array may be the isolation of grown cells (referred to as organoids in this specification) for use in basic scientific research with 3D cell structures, or for implantation of cells into humans for purposes of regenerative medicine or personalized medicine.

[0055] A major advantage of the preferred embodiment of the method of the present invention is that it completely avoids the use of naturally derived matrices, such as Matrigel®. This long-desired need in the art has surprisingly been found to be achievable by using specifically pre-selected conditions, which will be described later. Since different drug behaviors can clearly be attributable to specific extracellular matrix conditions, performing the entire method under completely clear extracellular matrix conditions provides accurate results for drug screening. Similarly, performing the entire method under completely clear extracellular matrix conditions meets the requirements of regulatory authorities regarding personalized and regenerative medicine compared to prior art methods.

[0056] The present invention also relates to the contents of a kit for operating on or using one or more tissue types, which include: a) A component for producing an array of perfectly clear hydrogel matrices in order to create conditions for a perfectly clear three-dimensional extracellular matrix in which the biological, biophysical and / or biochemical properties are distinct from each other, wherein the component - One or more different hydrogel precursor molecules, - At least one crosslinking agent as needed, - One or more biologically active molecules as needed, Ingredients, b) One or more different culture media, The kit contents include a specific combination of hydrogel characteristics, pre-selected for the type of tissue being tested.

[0057] According to a preferred embodiment of the present invention, the kit is for testing the effects of a drug on lung cancer cells, preferably non-small cell lung cancer cells, that overexpress c-Met, the hydrogel matrix is ​​a pre-selected non-autodegradable PEG hydrogel, the crosslinking agent and any bioactive agent are free of any RGD motifs, and the culture medium preferably contains FBS (serum) or a Wnt agonist, such as R-spongin.

[0058] According to another preferred embodiment of the present invention, the kit is for testing the effects of a drug on pancreatic ductal adenocarcinoma (PDAC) cells, wherein the hydrogel matrix is ​​a pre-selected non-self-degrading PEG hydrogel having a rigidity of 50-3000 Pa, preferably 50-2000 Pa, most preferably 50-1000 Pa, at least one of the crosslinking agent and / or any bioactive agent comprises an RGD motif, and the culture medium preferably comprises a Wnt agonist, such as R-spongin and Wnt3a.

[0059] According to another preferred embodiment of the present invention, the kit is for testing the effects of a drug on colorectal cancer (CRC) cells, wherein a PEG hydrogel is pre-selected, the matrix of which has an initial stiffness of at least 50 to 2000 Pa, and further comprises one or more bioactive molecules, optionally including laminin, preferably laminin-111 or laminin-511, particularly preferably natural mouse laminin-111 or recombinant human laminin-511, at least one of which is a crosslinking agent and / or any bioactive agent, comprising an RGD motif, and the culture medium preferably comprises a Wnt agonist, e.g., R-spongin and Wnt3a.

[0060] According to another preferred embodiment of the present invention, the kit is for testing the effects of a drug on breast cancer cells, wherein the hydrogel matrix is ​​preferably pre-selected as an enzymatically degradable PEG hydrogel, at least one of the crosslinking agents comprises an enzymatically degradable motif, preferably an MMP-sensitive motif, the hydrogel further comprises one or more bioactive molecules, optionally including laminin, preferably laminin-111, particularly preferably natural mouse laminin-111, and the culture medium preferably comprises FBS (serum) or a Wnt agonist, such as R-spongin.

[0061] According to another preferred embodiment of the present invention, the kit is for growing cancer cells, preferably prostate cancer cells, that proliferate more slowly than their healthy / normal counterpart cells (e.g., epithelial and / or stromal cells) in ex vivo, and for investigating the effects of drugs on cancer cells. The hydrogel matrix is ​​preferably a PEG hydrogel with a rigidity of 50 to 2000 Pa, pre-selected, and the crosslinking agent and any bioactive agents are free from any RGD motifs.

[0062] Kits according to the present invention target cells derived from a specific tissue type and can be easily used to isolate grown cells for operations on or using the said tissue type, for example, testing the effects of drugs on the said tissue type, or for use in basic scientific research, personalized medicine, or for implanting cells in humans for regenerative medicine purposes, or for drug discovery / research, or for creating a cell repository / biobank. Kits according to the present invention are indicated correspondingly by the instructions for use provided with the kit, for example, relating to the specific tissue type in which they are used. [Best Mode for Carrying Out the Invention]

[0063] definition The present invention relates to a three-dimensional cell culture model comprising any cell structure, such as a single cell, organoid, tumor organoid, multicellular tumor spheroid, cell spheroid, cell aggregate, tumor sphere, tissue-derived tumor sphere, or fragments thereof.

[0064] From this point forward, the term "cell" refers to any such cellular structure.

[0065] An array is a set of separate spaces that can be aligned in a particular form, for example, by rows and / or columns. For example, a commonly used well plate (e.g., a 48-well plate) provides 48 separate spaces aligned by 8 columns and 6 rows, where each column in this example consists of 6 separate spaces. Each such column in this example is considered an array in this invention. Alternatively, each row consisting of 8 separate spaces in this example can also be considered an array.

[0066] Organoids, including cell spheroids or aggregates, are three-dimensional cellular structures of stem cells and are organ-specific, tissue-specific, or disease-specific cell types that develop and self-organize (or self-pattern) through cell sorting and partially restricted lineage constraints in a manner similar to in vivo situations. Thus, organoids represent the innate physiology of cells and have cellular composition (including stem cells and / or specific cell or tissue types remaining at different stages of differentiation) and anatomy that mimics innate organs, tissues, and / or diseased cell and tissue conditions (e.g., cancer, cystic fibrosis, inflammatory bowel disease). Normal and / or diseased cells (e.g., cancer cells) can be isolated from any tissue or any cellular structure, such as organoids or cancer organoids (also called tumor organoids). Cells that generate organoids can be grown and / or differentiated to form organoid-like or disease-like tissues (e.g., cancer, cystic fibrosis, inflammatory bowel disease) exhibiting multiple types of cells that self-organize and form structures very similar in vivo to organs (i.e., cell differentiation) or disease tissues (e.g., multicellular heterogeneous tumors). Therefore, organoids are excellent models for testing human organs, human organ development, cancer, and other diseases in a system very similar to in vivo conditions. Organoids are also used to grow and proliferate cells for clinical applications, such as regenerative medicine and personalized medicine.

[0067] In this invention, the term "tissue type" refers to a group of cells that have similar structures and work together to perform specific functions. In animals, there are four different tissue types: connective tissue, muscle tissue, nerve tissue, and epithelial tissue. In this invention, cells from the same cell type are a collection of cells that, when healthy, work together to perform specific functions. More preferably, in this invention, cells of the same tissue type have the same origin in the human body (e.g., mammary gland cells).

[0068] In this invention, the same tissue type is understood to encompass both healthy (also called normal) cells and diseased cells such as cancer cells. Cells from the same tissue type may contain different cell types / subtypes, for example, different cell populations (e.g., multicellular heterogeneous tumors).

[0069] According to a preferred embodiment of the present invention, the type of tissue in which the method of the present invention is performed may include both other types of cells, such as stromal cells or immune cells, including cancer-associated fibroblasts (CAFs), and cancer cells.

[0070] Examples of tissue types used for the purposes of the present invention include lung cancer, preferably non-small cell lung cancer overexpressing c-Met; pancreatic ductal adenocarcinoma (PDAC) cells (preferably combined with stromal cells, preferably with fibroblasts), colorectal cancer (CRC) cells, breast cancer cells, or cancer cells that proliferate more slowly than their healthy / normal counterpart cells (e.g., epithelial and / or stromal cells) in ex vivo, preferably prostate cancer cells.

[0071] In the present invention, the term “newly isolated or frozen human cells obtained from a biopsy or tissue excision” refers to cells obtained directly from a human by any of the procedures mentioned and that have not been pre-cultured or pre-established in another system before being used in a method for forming organoids, spheroids, cell aggregates or any cellular structures. Typically, such fresh cells are collected and used immediately or within a maximum of 3-4 days in the method of the present invention. If the cells are not used immediately after collection, they may be frozen under normal conditions for storage purposes. The collected cells may be single cells and / or “aggregates” of cells including dissociated cells, tissue crypts and fragments. In a preferred embodiment of the present invention, epithelial cells are used.

[0072] In this invention, the term "de novo formation of organoids" refers to newly isolated or frozen human cells (e.g., human biopsy or tissue excision) that are growing for the first time ex vivo (i.e., outside the original organism). The terms "primary ex vivo cell growth" or "passage zero (P0)" may be used synonymously.

[0073] In the present invention, the term "pre-implanted organoid" refers to cells, single cells and / or cell aggregates (e.g., cell aggregates, organoids, etc.) that have grown in other systems (e.g., Matrigel®, 2D or 3D systems, or as patient-derived xenografts (PDX) in vivo) before being applied to the hydrogel of the present invention.

[0074] In this invention, the term "cell growth" refers to the success of cell growth.

[0075] In this invention, the terms "cell passage," "passaging," "cell division," or "organoid passage" refer to the steps of extracting cells from one gel and seeding those cells onto another gel having the same or different properties as the previous gel, and growing them.

[0076] In this invention, the terms "cell proliferation" or "organoid proliferation" refer to the process of cell growth and increase in cell number (for example, within the same passage or from one passage to the next).

[0077] In this invention, the term "organoid differentiation" refers to the successful induction of cell differentiation in organoids.

[0078] In the present invention, the term "completely distinct hydrogel" refers to a hydrogel selected from the group consisting of totally synthesized or semi-synthetic hydrogels, i.e., hydrogels having a completely distinct structure and / or composition due to the known properties of the precursor molecules used in their synthesis and the synthesis pathway.

[0079] In this invention, the term "totally synthetic hydrogel" refers to a hydrogel formed solely from synthetic precursors, i.e., one that does not contain naturally occurring precursors, such as natural laminin-111.

[0080] In this invention, the term "fully defined semi-synthetic hydrogel" refers to a hydrogel that contains at least one naturally occurring precursor, such as natural laminin-111, but has a fully defined structure and / or composition because the properties of the precursor molecule used in its synthesis are known. A fully defined semi-synthetic hydrogel is thus different from a naturally occurring hydrogel with an unknown structure and / or composition, such as Matrigel®.

[0081] In this invention, the term "encapsulated in a cell culture microenvironment" or similar expression means that the cells are completely surrounded by a matrix, thereby being embedded in the matrix to mimic naturally occurring cell growth conditions.

[0082] In this specification, "microenvironment" or "space of the microenvironment" respectively refers to a space suitable for high-throughput testing equipment, particularly multi-well plates. The typical space analyzed in a multi-well plate is about 100 nl to about 500 μl, preferably about 2 μl to about 50 μl.

[0083] The term “separate spaces” refers to spatially isolated spots or regions within an array. These isolated spots or regions may be in contact with each other, or preferably separated from each other by, for example, a plastic barrier. Cells of a desired tissue type can be placed in or on each of these separate spaces, such that they remain isolated from one another. They do not come into contact with each other from the beginning of the experiment and remain so over time, thereby growing independently of adjacent spaces, under the influence of their own cell culture microenvironment (ex vivo culture).

[0084] The term "crosslinking agent" refers to a chemical substance containing at least two functional groups that can react with the functional group moieties of a hydrogel precursor molecule in order to link two or more hydrogel precursor moieties together. Examples include peptides containing at least two functional groups such as cysteine ​​moieties, or polyethylene glycols having at least two functional groups such as thiol groups (e.g., two-arm or multi-arm PEGs with terminal thiol moieties).

[0085] The term "crosslinkable by cytocompatibility reactions" (or similar terms) includes reactions based on both (i) a) enzyme-catalyzed reactions, preferably by activated transglutaminase factor XIIIa; and b) covalent bond formation selected from the group consisting of non-enzyme-catalyzed and / or uncatalyzed reactions, preferably such as Michael addition reactions; and / or ii) non-covalent bond formation (e.g., hydrophobic interactions, H-bonds, van der Waals interactions, or electrostatic interactions; particularly induced by temperature changes or changes in the ionic strength of the buffer). These reactions may occur between two hydrogel precursor molecules containing functional groups that may react with each other, or between at least one hydrogel precursor molecule and a crosslinker containing functional groups that may react with each other.

[0086] In this invention, the term “working with grown cells” includes adding one or more drugs to separate spaces in an array of hydrogel matrix. Thus, the method of this invention may be for drug discovery development or drug screening tests to identify one or more drugs suitable for treating cell-related conditions from a type of tissue under test. It can be used in the field of personalized medicine. The method can also be used for drug discovery development as a cytotoxicity assay or in regenerative medicine. The term “working with grown cells” also includes operations to analyze the cells themselves or by-products from cells, for example by DNA or RNA sequencing methods, such as NGS (next-generation sequencing), and operations to analyze the products of these cells (e.g., analysis of supernatant), or operations to isolate cell-derived products (e.g., products of cell passage or cell proliferation) for further use (e.g., for the establishment of a biobank or cell repository, or for organoid implantation).

[0087] In this invention, the term “pre-selected” means that the extracellular matrix conditions, i.e., the hydrogel precursor molecules, optionally crosslinking agents, any bioactive agents, and preferably the culture medium, preferably at least two of them, most preferably all of them, are selected for the type of tissue being tested, such that a specific combination of hydrogel features is pre-selected. Hydrogel features are those that define the structure and / or function of the hydrogel. Examples include the chemical structure of the hydrogel (governed by the precursors used, optionally crosslinking agents, and any bioactive agents), the rigidity of the hydrogel, or its degradability (e.g., by hydrolysis or enzymatic reactions). Compared to the prior art methods considered, in this invention, the extracellular matrix conditions are not selected randomly. Based on previously obtained or available information, extracellular matrix conditions are selected that are known to be suitable for the growth and presentation of the desired phenotypic characteristics of the particular type of tissue being tested. A method for pre-selecting the extracellular matrix conditions is described below. In the present invention, other embodiments of the pre-selected extracellular matrix conditions used in the method of the present invention are also understood to be "pre-selected" because these embodiments are not random but are based on pre-selected extracellular matrix conditions.

[0088] In the present invention, the term "another aspect of the pre-selected extracellular matrix (ex vivo culture) conditions" encompasses conditions that are similar to the pre-selected conditions but differ in at least one parameter, preferably one to three parameters, such as hydrogel characteristics (e.g., stiffness, degradation), culture medium components, the amount of components in the extracellular matrix conditions, and bioactive agents in the extracellular matrix. Generally, the differing parameters are biological properties (e.g., presence or absence of RGD motifs), biophysical properties (e.g., hydrogel stiffness), and / or biochemical properties (e.g., enzymatic degradation).

[0089] In this invention, the term "autodegradability" means that the hydrogel degrades over time without the influence of degrading enzymes. Preferably, autodegradation occurs by hydrolysis of bonds in the hydrogel that are sensitive to reaction with water. As an example, an ester bond formed by the reaction of acrylate groups in a PEG-Acr precursor molecule (i.e., a precursor molecule containing a PEG molecule having a terminal acrylate group) can be cited.

[0090] In this invention, the term "non-self-degrading" means that the hydrogel does not degrade over time unless affected by degrading enzymes. A non-self-degrading hydrogel does not contain bonds that are easily reacted with water. As an example, a hydrogel formed from a PEG-VS precursor molecule (i.e., a precursor molecule containing a PEG molecule having a terminal vinyl sulfone group) can be cited.

[0091] In this invention, the term "RGD" or "RGD sequence" refers to the minimally biologically active RGD sequence, which is an arginine-glycine-aspartate (RGD) sequence and is the smallest (minimal) fibronectin-derived amino acid sequence sufficient to mimic the binding of cells to fibronectin and / or to promote adhesion-dependent cell adhesion. Furthermore, lysine or arginine-containing amino acid sequences such as RGD are suitable substrates for proteases, such as trypsin-like enzymes used for gel dissociation. Examples of suitable RGD motifs include RGD, RGDS, RGSSP, RGDSPG, RGDSPK, RGDTP, RGDSPASSKP, PHSRNSGSGSGSGSGRGDSPG, or cyclic RGD motifs, such as cyclo(RGDfC), but basically any known and successfully utilized RGD sequence can be used in the fields of hydrogels and cell culture.

[0092] The shear modulus of a hydrogel is equivalent to the coefficient of stiffness of the hydrogel, G, the elastic modulus, or elastic modulus. The shear modulus is defined as the ratio of shear stress to shear strain. The shear modulus of a hydrogel can be measured using a viscometer. Briefly, a pre-formed hydrogel disc with a thickness of 1–1.4 mm is inflated in complete cell culture medium for at least 3 hours, and then placed between parallel plates of a viscometer. 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 measurement of the mechanical properties of the gel.

[0093] How to create a matrix array of hydrogels The array of the hydrogel matrix of the present invention can generally be prepared as described in International Publication No. 2014 / 180970.

[0094] In short, the preferred method involves the following steps: a) A step of providing one or more different hydrogel precursor molecules and, optionally, at least one crosslinking agent; b) A step of distributing, in an automated manner, a combination of the hydrogel precursor molecules from step a) and, optionally, at least one crosslinking agent, on the surface of a substrate or in separate spaces of the substrate, preferably in a multiwell plate; c) Adding one or more biologically active molecules to separate spaces, binding the molecules to at least one of the hydrogel precursor molecules present or the hydrogel formed in step e), or allowing them to diffuse freely; d) Adding cells to / into the surface of the separate spaces of the substrate; and e) A step of crosslinking the precursor molecules of the hydrogel by a cytocompatible crosslinking reaction, such as an enzyme-catalyzed reaction, or a Michael addition reaction, to form a hydrogel matrix. Includes.

[0095] The hydrogels used, obtained by crosslinking the precursor molecules of the hydrogel, can be essentially selected from any type of fully defined synthetic or semi-synthetic hydrogel known in the art. Examples include photocrosslinkable hydrogels, e.g., hydrogels prepared using a reaction mechanism via radical-mediated thiol-norbornene (thiol-ene) photopolymerization to form the hydrogel (Anseth et al., Adv Mater. 2009 December 28; 21(48): 5005-5010; Nature scientific reports 2015, 5:17814), or hydrogels produced by click chemistry (e.g., Michael addition reaction), physical crosslinking, or enzymatic crosslinking.

[0096] The hydrogels used are obtained by crosslinking hydrogel precursor molecules, and are preferably hydrophilic polymers, such as polymers based on poly(ethylene glycol) (PEG), most preferably polymers based on multi-arm PEG crosslinked by a cytocompatible crosslinking reaction. The specific hydrogel used depends on the results of prior selection regarding the specific tissue type and is considered in the following preferred embodiments.

[0097] Preferably, a PEG-based hydrogel is used, which is crosslinkable using thrombin-activated thrombin XIIIa under physiological conditions via a crosslinking mechanism detailed in Ehrbar et al. (Ehrbar, M., Rizzi, SC, Schoenmakers, RG, Miguel, BS, Hubbell, JA, Weber, FE, and Lutolf, MP, Biomolecular hydrogels formed and degraded via site-specific enzymatic reactions, Biomacromolecules 8 (2007), 3000-3007), or via a mild chemical reaction via a crosslinking mechanism detailed in, for example, Lutolf et al. (Lutolf, MP, and Hubbell, JA, Synthesis and physicochemical characterization of end-linked poly(ethylene glycol)-co-peptide hydrogels formed by Michael-type addition, Biomacromolecules 4, 713-722).

[0098] The preferred hydrogel of the present invention is based on a multi-arm PEG (poly(ethylene glycol)) containing an ethylene unsaturated group selected from the group consisting of vinyl sulfone and / or acrylate moieties as a precursor molecule.

[0099] According to a preferred embodiment of the present invention, the multi-arm PEG is selected from the group consisting of PEGs having 2 to 12 arms, preferably 4 arms or 8 arms, i.e., preferably a 4-arm or 8-arm PEG. PEG may have molecular weights of 1,000-1,000,000, 1,000-500,000, 1,000-250,000, 1,000-150,000, 1,000-100,000, 1,000-50,000, 5,000-100,000, 5,000-50,000, 10,000-100,000, 10,000-50,000, 20,000-100,000, 20,000-80,000, 20,000-60,000, 20,000-40,000, or 40,000-60,000. The above molecular weights are the average molecular weights in Da determined by methods such as GPC or MALDI.

[0100] Such PEGs are known and commercially available in the art. They consist of a core that may be pentaerythritol in the case of 4-arm PEGs, and a core that may be tripentaerythritol or hexaglycerol in the case of 8-arm PEGs.

[0101] [ka]

[0102] In 4-arm PEG-VS or 8-arm PEG-VS, the free OH groups at the ends of the above approximately 4-arm or 8-arm PEG are converted to vinyl sulfone groups under conditions known in the art, thereby in the above formula R is, for example,

[0103] [ka]

[0104] This is the result.

[0105] In 4-arm PEG-Acr or 8-arm PEG-Acr, the free OH groups at the ends of the approximately 4-arm or 8-arm PEG are converted to acrylate groups under conditions known in the art, thereby in the above formula R is, for example,

[0106] [ka]

[0107] This is the result.

[0108] Preferably, all of the free OH groups at the ends of the 4-armed PEG or 8-armed PEG are converted to vinyl sulfone or acrylate groups.

[0109] The vinyl sulfone or acrylate moiety is an ethylene unsaturated group suitable for crosslinking PEG precursor molecules via a Michael addition reaction. The Michael addition reaction is a well-known chemical reaction involving the reaction of a suitable nucleophile with a suitable electrophile. For example, it is well known that the acrylate or vinyl sulfone moiety is a suitable Michael acceptor (i.e., an electrophile) that reacts with, for example, a thiol moiety, as a suitable Michael donor (i.e., a nucleophile).

[0110] A hydrogel (gel) is a matrix containing a network structure of hydrophilic polymer chains. A biofunctional hydrogel is a hydrogel that contains bioadhesive (or bioactive) molecules and / or cell signaling factors that interact with living cells to promote cell viability and desired cell phenotypes.

[0111] To obtain a hydrogel according to a preferred embodiment of the present invention, the above-mentioned PEG precursor molecule is reacted with a crosslinking molecule containing at least two, preferably two, nucleophiles capable of reacting with the ethylene unsaturated group of the multi-armed PEG in a Michael addition reaction. The crosslinking molecule is a molecule that connects at least two of the above-mentioned PEG precursor molecules to each other. For its purpose, the crosslinking molecule must have at least two, preferably two, of the above-mentioned nucleophiles such that one nucleophile reacts with the first PEG precursor molecule and the other nucleophile reacts with the second PEG precursor molecule. According to a preferred embodiment of the present invention, the crosslinking molecule is a peptide comprising at least two RGD motifs and at least two cysteine ​​moieties. Cysteine ​​is an amino acid containing a thiol group, i.e., a Michael donor moiety.

[0112] Crosslinking of the hydrogel precursor molecules is performed in the presence of the tissue type being tested in separate spaces of the array, thereby encapsulating the cells by the hydrogel matrix, i.e., placing them in separate cell culture microspaces.

[0113] The mechanical properties of the matrix of the three-dimensional hydrogel according to the present invention can be varied by varying the polymer content of the cell culture microenvironment and the molecular weight and / or functionality (number of sites available for crosslinking) of the polymer gel precursor. Thus, for example, the stiffness of the matrix, expressed by the shear modulus (G'), can vary from 10 to 10,000 Pa, preferably 50 to 1,000 Pa, for soft gels, or 1,000 to 2,000 Pa for moderate gels, or 2,000 to 3,000 Pa for hard gels. The shear modulus of a hydrogel is equivalent to the stiffness coefficient, G, elastic modulus, or modulus of elasticity of the hydrogel. The shear modulus is defined as the ratio of shear stress to shear strain. The shear modulus of a hydrogel can be measured using a viscometer. Briefly, a pre-formed hydrogel disc with a thickness of 1 to 1.4 mm is expanded in an aqueous solution (e.g., buffer or complete cell culture medium) for at least 3 hours, and then placed between parallel plates of a viscometer. The mechanical response of the gel is recorded by performing frequency sweep measurements (0.1–10 Hz) in constant strain (0.05) mode at room temperature. The modulus of elasticity (G') is reported as a measurement of the gel's mechanical properties.

[0114] Furthermore, the physicochemical properties of the matrix can be altered over time by imparting degradation characteristics to the gel matrix through the incorporation of peptides with different sensitivities to cell-secreted proteases, such as matrix metalloproteinases (MMPs), plasmin, or cathepsin K. This makes the hydrogel matrix "enzyme-degradable." The changes in sensitivity to proteases when proteases are secreted by cells, and the resulting changes in the physicochemical properties of the matrix, enable efficient cell proliferation and migration in the three-dimensional matrix. To match the mechanical properties of hydrogel matrices with different sensitivities to proteolysis, the matrix precursor content can be fine-tuned by varying the matrix polymer precursor content, molecular weight of the polymer gel precursor, and / or functionality (number of sites available for crosslinking). The desired stiffness is achieved by fixing the total polymer (PEG) content and crosslinking agent content in the hydrogel to preferably 1.0-10 wt / vol%. In PEG-based hydrogel matrices, the sensitivity to proteases can be altered, for example, by incorporating different peptide sequences with different sensitivities to cellular secretory proteases into the matrix precursor molecule.

[0115] The biological characteristics of a cell culture microenvironment can be modulated by adding one or more biologically active molecules to the matrix. In this specification, these biologically active molecules are, for example, the following groups. i) Extracellular matrix-derived factors (ex vivo culture); ii) Cell-cell interaction factors; and / or iii) Cell signaling factors You may choose from the following.

[0116] The extracellular matrix-derived factors i) used may be, for example, ECM proteins such as laminin, collagen, elastin, fibronectin, or elastin; proteoglycans such as heparin sulfate or chondroitin sulfate; non-proteoglycan polysaccharides such as hyaluronic acid; or matrix cell proteins such as fibrin, osteopontin, periostin, SPARC family members, tenacin, or thrombospondin. These ECM factors can be used in full form, or as smaller functional building blocks such as peptides and oligosaccharides, or as glycosaminoglycans such as hyaluronic acid (also called hyaluronan).

[0117] The cell-cell interaction proteins used (ii) are often transmembrane proteins and may be proteins involved in cell-cell adhesion, such as cadherins, selectins, or cell adhesion molecules (CAMs) belonging to the Ig superfamily (ICAM and VCAM), or components of transmembrane signaling pathways, such as Notch ligands, Delta-like and Jagged.

[0118] The cell signaling factors used (iii) are growth factors or developmental morphogens, for example, growth factors or developmental morphogens of the following families: adrenomedullin (AM), angiopoietin (Ang), autologous motility stimulant, bone morphogenetic protein (BMP), brain-derived neurotrophic factor (BDNF), epidermal growth factor (EGF), erythropoietin (EPO), fibroblast growth factor (FGF), glial cell line-derived neurotrophic factor (GDNF), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), growth and differentiation factor-9 (GDF9), hepatocyte growth factor This may include hepatoma-derived growth factor (HGF), hepatoma-derived growth factor (HDGF), insulin-like growth factor (IGF), leukemia inhibitory factor (LIF), migration stimulant, myostatin (GDF-8), nerve growth factor (NGF) and other neurotrophins, platelet-derived growth factor (PDGF), thrombopoietin (TPO), transforming growth factor α (TGF-α), transforming growth factor β (TGF-β), tumor necrosis factor α (TNF-α), vascular endothelial growth factor (VEGF), Wnt signaling pathway, placental growth factor (PlGF), or large-class cytokines or chemokines.

[0119] Extracellular matrix-derived factors i) and cell-cell interaction factors ii) can site-specifically bind to the hydrogel matrix before or during crosslinking. Functionalization of the gel with biologically active molecules can be achieved by direct covalent bond formation between the peptide substrate of a free functional group (e.g., amine or thiol group) on a biomolecule or a crosslinking enzyme (e.g., transglutaminase) and the gel network structure, or by affinity binding between a domain on a chimeric / tagged protein and an accessory protein bound to the gel. Tagged proteins include, for example, protein A (or protein G, protein A / G), streptavidin (or NeutrAvidin), or proteins with Fc tags, biotin tags, or His tags that enable binding to NTA.

[0120] Alternatively, these factors may be part of the crosslinking agent and may be incorporated into the hydrogel polymer for the crosslinking reaction described in this specification.

[0121] Biomolecules may require different gel anchoring strategies for the hydrogel network structure. Larger ex vivo culture-derived or ex vivo culture-mimicking proteins and peptides are preferably attached to the hydrogel by nonspecific anchoring using a linear heterobifunctional linker. One functional group of this linker is reactive with a functional group, preferably a thiol, attached to the end of the polymer chain. The other functional group of the linker is capable of nonspecific anchoring to the biomolecule of interest via its amine group. The latter functional group is selected from the group consisting of succinimidyl active esters, e.g., N-hydroxysuccinimide (NHS), succinimidyl α-methylbutanoate, succinimidylpropionate; aldehydes; thiols; thiol-selective groups, e.g., acrylates, maleimide, or vinyl sulfones; pyridylthioesters and pyridyl disulfide. Preferably, an NHS-PEG-maleimide linker is attached to the biomolecule.

[0122] Cell signaling factors iii) can be added to a cross-linked hydrogel matrix that encloses cells in a soluble form in spatially separated regions, thus allowing them to diffuse freely within the matrix and reach the cells. Alternatively, they can be anchored to the matrix in the same manner as for extracellular matrix-derived factors i) and cell-cell interaction factors ii).

[0123] Step b) of the preferred method described above is performed using an automated method for gel preparation and miniaturization of samples in order to achieve the necessary diversity in the preparation of 3D cell-containing matrices with a number of different cell culture microenvironments, and also to achieve the necessary repeatability. For this purpose, preferably using a commercially available liquid handling robot, small volumes of 100-500 nL each of the unique mixtures of precursor molecules according to step a) are precisely synthesized, preferably in triplicate, in a fully automated manner on the surface of a substrate, such as a glass slide, or preferably in a multiwell plate, such as a standard 1536-well plate. The latter format is preferred because it presents an ideal surface-to-volume ratio for the selected hydrogel droplets and represents a standard format that can be adapted to various experimental settings. Once the 3D hydrogel matrix is ​​produced, the system can function as a multimodal assay platform from which multiple reads can be obtained simultaneously.

[0124] In another preferred embodiment, the components constituting the final hydrogel are freeze-dried and provided as a non-reactive powder, which is then redissolved manually or automatically using a handling robot to form the hydrogel. The desired cell suspension is added before gelation occurs, and the matrix of the 3D hydrogel is produced as described above.

[0125] In step e), crosslinking the hydrogel precursor molecules to form a three-dimensional hydrogel matrix can be achieved by using at least one crosslinking agent. When using PEG-based precursor molecules, for example, a chemically reactive bifunctional peptide can be selected as the crosslinking agent. An example of this is a mild chemical reaction via a crosslinking mechanism detailed in Lutolf et al. (Lutolf, MP, and Hubbell, JA, Synthesis and physicochemical characterization of end-linked poly(ethylene glycol)-co-peptide hydrogels formed by Michael-type addition, Biomacromolecules 4, 713-722 (2003)). However, crosslinking can also occur immediately when two different precursor molecules that readily react with each other are combined (e.g., by highly selective so-called click chemistry, e.g., Michael addition reaction or other chemical reactions).

[0126] Similarly, crosslinking may occur by two different precursor molecules that are reactive with each other, or by a combination of one type of precursor molecule having different types of moieties that are reactive with each other in the presence of a catalyst, such as an enzyme. An example of this is a PEG (polyethylene glycol) precursor molecule that can be crosslinked using thrombin-activated factor XIIIa under physiological conditions by a crosslinking mechanism detailed by Ehrbar et al. (Ehrbar, M., Rizzi, SC, Schoenmakers, RG, Miguel, BS, Hubbell, JA, Weber, FE, and Lutolf, MP, Biomolecular hydrogels formed and degraded via site-specific enzymatic reactions, Biomacromolecules 8 (2007), 3000-3007). Briefly, to create a suitable hydrogel precursor, 8-armed PEG-VS and / or 8-armed PEG-Acr macromers are terminally functionalized with lysine and glutamine-presenting peptides that act as substrates for activated transglutaminase XIII (FXIIIa). The crosslinking of the macromer and the resulting gel formation were achieved by FXIIIa-mediated crosslinking of the ε-(α-glutamyl)lysine isopeptide side chains between the two peptide substrates.

[0127] The distributed array of hydrogel precursors can be stored and used later (in contact with cells for screening experiments). Storage is preferably carried out in multi-well plates (e.g., 96-well, 384-well, or 1536-well plates) and can be done using precursors in solution (without crosslinking agents yet) or lyophilized precursors, i.e., powders. The powders are unreacted or remain unreacted. For example, with the addition of a buffer, the lyophilized precursors may be solubilized and then react with each other.

[0128] Prior selection According to the present invention, the ex vivo conditions used (e.g., extracellular matrix conditions) are pre-selected for the type of tissue being tested.

[0129] According to one aspect of the present invention, prior selection can be carried out in the manner described in International Publication No. 2014 / 180970.

[0130] More specifically, according to a preferred embodiment, cells from a particular type of tissue to be used are subjected to a method under randomly selected ex vivo conditions, and the method a) A step of providing one or more different hydrogel precursor molecules and, optionally, at least one crosslinking agent to construct a cell culture microenvironment; b) A step of combining and distributing different combinations of the hydrogel precursor molecules from step a), and optionally at least one crosslinking agent, on the surface of the substrate or in separate spaces of the substrate, preferably in a multiwell plate, preferably in an automated manner; c) Adding one or more biologically active molecules to the separate spaces of the substrate, thereby binding the molecules to at least one of the existing hydrogel precursor molecules or to the hydrogel formed in step e), or dispersing them freely; d) Adding cells of a specific tissue type to / into the surface of the separate spaces of the substrate: e) A step of crosslinking the hydrogel precursor molecules by a cytocompatible crosslinking reaction, such as an enzyme-catalyzed reaction, or a Michael addition reaction to form a hydrogel matrix; f) A step that allows cells of the specific tissue type to grow in the separate spaces of the hydrogel matrix; g) A step of monitoring cells of the specific tissue type over time during step f); h) A step to determine the behavior of different cell culture microenvironments; i) A step of identifying a specific cell culture microenvironment or range of cell culture microenvironments that provides suitable conditions for growing different cell populations from the specific tissue type. Includes.

[0131] The specific cell culture microenvironment or range of cell culture microenvironments identified in step i) is used as pre-selected extracellular matrix conditions for the method of the present invention.

[0132] In this specification, in the method of the present invention, an array of hydrogel matrices is provided together with pre-selected extracellular matrix conditions and alternative embodiments thereof. Compared to the method described in International Publication No. 2014 / 180970, the use of pre-selected extracellular matrix conditions and alternative embodiments thereof allows for more intensive and accurate assays, enabling the identification of specific therapeutics, for example, the different behaviors of multiple phenotypic and / or genotypic subpopulations of tissue types.

[0133] If suitable ex vivo conditions (e.g., ECM conditions) are known from prior art, it is not necessary to perform the method described in International Publication No. 2014 / 180970. Instead, known suitable ex vivo conditions (e.g., extracellular matrix conditions) can be directly used in the method and kit of the present invention.

[0134] Kit contents According to one aspect of the present invention, the contents of a kit can be provided that include pre-selected extracellular matrix conditions for a particular tissue type. Thus, the contents of such a kit can be easily used to perform operations on a particular tissue type under optimal conditions.

[0135] Thus, the present invention relates to the contents of a kit for operating on or using one type of tissue: a) A component for producing an array of perfectly clear hydrogel matrices in order to create conditions for a perfectly clear three-dimensional extracellular matrix in which the biological, biophysical and / or biochemical properties are distinct from each other, wherein the component is - One or more different hydrogel precursor molecules, - At least one crosslinking agent as needed, - One or more biologically active molecules as needed Ingredients, b) One or more different culture media, The kit contents include a specific combination of hydrogel characteristics, pre-selected for the type of tissue being tested.

[0136] Kits according to the present invention are readily usable for targeting specific tissue types and for working with or using specific tissue types, for example, for testing the effects of drugs on specific tissue types, or for using grown cells (e.g., 3D cell structures) in basic scientific research, or for isolating grown cells for implantation into humans for the purposes of personalized medicine or regenerative medicine. Kits according to the present invention are indicated correspondingly by the instructions for use provided with the kit, for example, relating to the specific tissue type in which they are used.

[0137] The contents of the kit are known in the art. Typically, they include one or more containers in which the components defined above are stored individually or together within the package.

[0138] In a preferred embodiment, the hydrogel precursor preparation in the form of an unreactive powder is provided in one container of the kit contents. The unreactive powder can be resuspended in a suitable buffer for use and distributed on the surface of a substrate or in separate spaces of a substrate, preferably in a multiwell plate. The hydrogel precursor preparation in the form of an unreactive powder comprises all the components necessary for the formation of the hydrogel of the present invention, namely, one or more different hydrogel precursor molecules, at least one optional crosslinking agent molecule, and one or more optional bioactive agents.

[0139] The provision of unreactive powders of hydrogel precursor formulations is known in the art, for example, from International Publication No. 2011 / 131642, in which freeze-drying is used as a means to provide the powder.

[0140] Typical examples The present invention will be described below with reference to non-limiting representative embodiments and drawings. [Brief explanation of the drawing]

[0141] [Figure 1a] Figure 1a shows the results of c-met expression and drug testing experiments in different ex vivo examples using non-small cell lung cancer cells overexpressing c-met grown in different gels. [Figure 1b] Figure 1b shows the effects of SoC treatment and c-met inhibitor therapy in an example according to the present invention. [Figure 1c] Figure 1c shows the effects of SoC treatment and c-met inhibitor therapy in a comparative example (Matrigel®). [Figure 1d] Figure 1d shows the results of c-met and EGFR expression in different ex vivo examples. [Figure 1e] Figure 1e shows the effects of SoC treatment and EGFR inhibitor therapy in an example according to the present invention. [Figure 2a] Figure 2a shows the growth of PDX pancreatic ductal adenocarcinoma (PDAC) cells in different gels. [Figure 2b] Figure 2b shows the drug sensitivity of PDX pancreatic ductal adenocarcinoma (PDAC) cells in different gels. [Figure 2c] Figure 2c shows the growth of PDX pancreatic ductal adenocarcinoma (PDAC) in soft and moderate gels. [Figure 3] Figure 3 shows bright-field images of 33% PDAC cells co-cultured with 67% fibroblasts in different gels. [Figure 4] Figure 4 shows bright-field images of human colon cancer organoids grown for 0 and 11 days. [Figure 5]Figure 5 shows bright-field images of the resulting growth of human primary or metastatic (Mets) breast cancer cells from four patients with HER2-positive breast cancer or triple-negative breast cancer (TNBC) (from patient-derived xenograft models). [Figure 6a] Figure 6a shows bright-field images of the results from healthy human prostate cells grown for 1 and 14 days. [Figure 6b] Figure 6b shows bright-field images of human prostate cancer cells grown for 1, 13, and 20 days.

[0142] Lung cancer treatment Well-characterized, patient-cell-derived preclinical models are essential components for conducting reliable translational cancer research, including identifying molecular pathways of tumorigenesis and evaluating potential therapies.

[0143] Tumor cell lines have long been a convenient platform for research, with countless cell lines being well-characterized and used to establish tumors in animal models (xenograft tumors). However, xenograft tumors derived from cell lines do not accurately reproduce the human tumor microenvironment, and there is no predictable relationship between the treatment response in preclinical models and the response in human clinical trials (Johnson et al., British Journal of Cancer (2001) 84(10), 1424-1431).

[0144] Patient-derived tumor xenograft models (PDXs) are frequently used in translational cancer studies and are expected to exhibit consistent behavior even after serial passage. Correlations of histopathological and genotypic characteristics between original patient samples and PDX models have been well reported (Rubio-Viqueira et al., Clin. Cancer Res. 2006, 12(15), 4652). In addition, PDX models grown over multiple passages maintain a correlation between the treatment response of the original human tumor and the response in these same patient-derived PDXs. However, the throughput of PDX-based screening models is low, and furthermore, such screening trials are expensive.

[0145] This invention provides an improved method for cancer research. It provides an improved alternative model to the PDX model that enables high-throughput screening in a highly cost-effective manner.

[0146] In a preferred embodiment, prior selection of extracellular matrix conditions suitable for cancer cell growth can be performed by using cells from PDX and assaying them as described in the “Prior Selection” section. Under such conditions, the histopathological and genotypic features of cells grown ex vivo can be correlated with one of the in vivo-established PDX models, and the therapeutic response of PDX tumors derived from those PDX models can be used as an in vivo criterion to evaluate which extracellular matrix conditions can reproduce the in vivo behavior of cancer cells.

[0147] As shown in the above literature, the microenvironment (i.e., extracellular matrix conditions) can influence how cancer cells respond to drug therapy in vivo and ex vivo. The method of the present invention makes it possible to establish ex vivo cell culture conditions for drug screening / testing that can capture different patient tumor characteristics (e.g., different cancer subtypes) in order to more accurately predict the outcomes of drug therapy for patients.

[0148] This involves growing cells and testing possible drug therapies on the ex vivo grown cells using the patient's own cells cultured within a pre-selected microenvironment. The method of the present invention makes it possible to capture patient heterogeneity in drug responses, both within and between tumors (including resistance to targeted therapy).

[0149] Currently, established methods in prior art still involve growing cells extracted from patient tissue using a single culture condition consisting of, for example, a reference Matrigel®. This single condition does not necessarily allow the patient's cells to grow in a way that captures all the characteristics and potential heterogeneity of the original patient's tumor. Similarly, some components of the matrix, which are sometimes unclear, may interfere with the drug response to the cells being tested.

[0150] The present invention makes it possible to culture a patient's cells and then expose them to different drug therapies to elucidate their sensitivity and potential resistance (and underlying mechanisms) to drug therapies that better reflect what is happening in the original patient's tumor (e.g., tumor heterogeneity, drug resistance). The present invention makes it possible to help select or exclude drug therapies for cancer patients and / or select second-line therapies to overcome resistance to one or more previous therapies.

[0151] Following this approach, it can be shown that a pre-selected condition suitable for testing the effects of c-Met inhibitors on non-small cell lung cancer (NSCLC) cells overexpressing c-Met is characterized by the absence of any RGD adhesion motifs in the hydrogel (see Example 1).

[0152] This is particularly surprising, given that prior art has predicted the opposite result (the necessity of the presence of RGD adhesion motifs under extracellular matrix conditions). Mitra et al. (Oncogene 2011, March 31; 30(13): 1566-1576) showed that c-Met can be activated via fibronectin-mediated activation of α5β1-integrin, independently of its ligand (HGF), leading to its interaction with the c-Met receptor. Inhibition of α5β1-integrin reduced c-Met phosphorylation in vitro and in vivo (ovarian cancer cells). Crosstalk between integrin β1 and c-Met was also explored in NSCLC by Ju et al. (Cancer Cell International 2013, 13:15). This literature has shown that the interaction between integrin β1 and c-Met induces c-Met activation (i.e., phosphorylation), allowing cancer cells sensitive to EGFR receptor inhibition to become resistant to EGFR-targeted drugs by bypassing the EGF pathway. Both studies demonstrate that c-Met can interact with integrin β1, a known RGD linker. They also demonstrate that this interaction leads to c-Met phosphorylation and activation of its downstream pathways (FAK, AKT), inducing prolonged cell proliferation and survival. In summary, all studies clearly highlight the relationship between the c-Met receptor and fibronectin.

[0153] However, according to the present invention, we were able to demonstrate that the presence of RGD motifs in the hydrogel matrix leads to downregulation of the c-Met receptor, and the absence of activated c-Met receptors (i.e., phosphorylated receptors) in NSCLC cells leads to their resistance to treatment with c-Met inhibitors. A key finding of the present invention was that the absence of any RGD adhesion motifs in the hydrogel provided precisely pre-selected conditions for identifying suitable drug candidates for NSCLC cancer cells exhibiting activated c-Met receptors. On the other hand, NSCLC cancer cells growing in the presence of RGD motifs do not possess or depend on activated c-Met receptors, indicating that these cancer cells must be treated with other drug candidates (perhaps in conjunction with c-Met inhibitors) rather than c-Met inhibitors. Without using “pre-selected growth conditions,” it would not have been possible to understand that these cells may depend on growth mechanisms other than c-Met. One of the main added values ​​of using “pre-selected growth conditions” compared to the single growth conditions used in prior art is that it allows for the identification of heterogeneity (e.g., genetic, phenotypic) specific to cancer tissue and cancer type, as well as a potentially better therapeutic range needed to cure the cancer. This is relevant in personalized medicine and drug discovery development applications.

[0154] Thus, according to this embodiment, the present invention is a method for testing the effect of a c-Met inhibitor on lung cancer cells, preferably non-small cell lung cancer cells, that overexpress c-Met. a) A step of providing an array having separate spaces of a completely distinct, non-self-degrading hydrogel matrix, by crosslinking one or more different combinations of hydrogel precursor molecules, optionally at least one crosslinking agent, on the surface of a substrate or in separate spaces of a substrate, preferably in a multiwell plate, optionally in the presence of one or more biologically active molecules, with the lung cancer cells, preferably non-small cell lung cancer cells; b) A step of enabling the lung cancer cells, preferably non-small cell lung cancer cells, to grow in the separate spaces of the hydrogel matrix in the presence of one or more different media, preferably comprising FBS (serum) or a Wnt agonist, such as R-spongin, and particularly preferably comprising FGF-7, FGF-10, and TGF-β inhibitors; c) Adding a drug that targets the c-Met receptor or the c-Met pathway to cells grown in the separate spaces of the hydrogel matrix; The present invention relates to a method comprising the crosslinking agent and the optional bioactive agent, wherein neither contains any RGD motif.

[0155] In a very preferred embodiment, the present invention relates to a method for testing the effects of c-Met inhibitors and other drugs on lung cancer cells, preferably non-small cell lung cancer cells, a) A step of providing pre-selected extracellular matrix conditions, comprising an array having separate spaces of a matrix of completely distinct, non-self-degrading hydrogels, produced by crosslinking one or more different combinations of hydrogel precursor molecules, at least one crosslinking agent, and the lung cancer cells, preferably non-small cell lung cancer cells, on the surface of the substrate or in separate spaces of the substrate, preferably in a multiwell plate, in the presence of one or more biologically active molecules as needed, wherein the crosslinking agent and the optional biologically active agent do not contain any RGD motifs, and A step of providing a pre-selected extracellular matrix condition in a second array of substrates, which differs from the pre-selected extracellular matrix condition in the first array due to the presence of the RGD motif in the crosslinking agent and / or the optional bioactive agent; b) A step of enabling the lung cancer cells, preferably non-small cell lung cancer cells, to grow in the separate spaces of the first and second arrays of the hydrogel matrix in the presence of one or more different media, preferably comprising FBS (serum) or a Wnt agonist, such as R-spongin, and particularly preferably comprising FGF-7, FGF-10, and TGF-β inhibitors; c) Adding a drug that targets the c-Met receptor or the c-Met pathway to cells grown in the separate spaces of the first and second arrays of the hydrogel matrix; d) Adding at least one other drug, preferably an EGFR receptor inhibitor, to cells grown in the separate spaces of the first and second arrays of the hydrogel matrix in wells that do not contain a drug targeting the c-Met receptor or c-Met pathway; Regarding methods including

[0156] Preferably, the array of the hydrogel matrix has a soft or moderate stiffness of 50 to 2000 Pa.

[0157] Preferably, the precursor molecule of the PEG hydrogel is PEG-VS (polyethylene glycol having a terminal vinyl sulfone moiety), and particularly preferably 4-arm or 8-arm PEG-VS.

[0158] More preferably, a completely distinct, non-self-degrading hydrogel matrix array is prepared by crosslinking PEG-VS, particularly preferably 4-arm or 8-arm PEG-VS, with a peptide containing at least two, preferably two cysteine ​​moieties, as a crosslinking agent, wherein the crosslinking agent does not contain any RGD motifs. Particularly preferably, no bioactive ligands bind to the hydrogel matrix.

[0159] As needed, a ligand containing a bioactive motif other than the RGD adhesion motif may be used as a bioactive ligand.

[0160] Preferably, the bioactive ligand, as needed, is selected from the group consisting of natural laminins, such as laminin-111, particularly mouse laminin-111, recombinant laminin isoforms, and their biofunctional fragments. Examples of suitable recombinant laminin isoforms are laminin-111, laminin-211, laminin-332, laminin-411, laminin-511, or laminin-521.

[0161] As needed, glycosaminoglycans, such as hyaluronic acid and hyaluronan, may be used as bioactive ligands. Examples of hyaluronic acid include hyaluronic acid 50k, hyaluronic acid 1000k, hyaluronatethiol 50k, or hyaluronatethiol 1000k.

[0162] Preferably, the culture medium is characterized by the presence of FBS (serum) or a Wnt agonist, such as R-spongin. In a preferred embodiment, a modified medium of the medium described by Sachs et al. (The EMBO Journal e 100300|2019) may be used. The preferred medium comprises AdDMEM / F12 medium supplemented with glutamine, noggin, EGF, fibroblast growth factor 7 and 10 [FGF7 and FGF10], HGF, R-spongin-conditioned medium, primocin, penicillin / streptomycin, N-acetyl-L-cysteine, nicotinamide, A83-01, SB202190 (p38 inhibitor), Y-27632 (rock inhibitor), B27 supplement and HEPES. Other culture media may be used, such as the media described by Lancaster et al. (Nat Biotechnol 2017 35(7): 659-666), or commercially available media such as PromoCell (Small Airway Epithelial Cell Growth Medium (C-39175)), or Invitrogen (StemPro® hESC SFM).

[0163] Particularly preferred are c-Met-overexpressing lung cancer cells, preferably non-small cell lung cancer cells, which are newly isolated or frozen human cells obtained from human biopsy or tissue resection, or from patient-derived xenograft (PDX) tissue.

[0164] By the method described above, lung cancer cells, preferably non-small cell lung cancer cells, can be grown and proliferated in a selected culture medium under extracellular matrix conditions that reproduce drug outcomes observed in vivo, and then tested.

[0165] In a particularly preferred embodiment, the extracellular matrix conditions are selected so as not to use any naturally derived matrix, such as Matrigel®.

[0166] This invention provides a method using pre-selected extracellular matrix conditions to maintain the growth and proliferation of lung cancer cells, preferably NSCLC cells, using a fully clear, or preferably totally synthetic, hydrogel matrix. The method enables the reproduction of target expression and drug responses observed in vivo in a PDX lung model but not achieved with Matrigel®. Prior selection is important because different ex vivo conditions can promote different drug responses, and using single culture conditions ensures that it does not reflect what is happening in the original patient's tumor.

[0167] According to this embodiment, the present invention also relates to the contents of a kit for testing the effects of c-Met inhibitors on lung cancer cells, preferably non-small cell lung cancer cells, that overexpress c-Met, the following: a) A component for producing an array of fully distinct, non-self-degrading hydrogel matrices in order to create conditions for a fully distinct three-dimensional extracellular matrix in which the biological, biophysical and / or biochemical properties are distinct from each other, wherein the component - One or more different PEG hydrogel precursor molecules, preferably PEG-VS, particularly preferably 4-arm or 8-arm PEG-VS, - A peptide comprising at least one crosslinking agent, preferably at least two, preferably two cysteine ​​moieties, - One or more biologically active molecules as needed, The ingredients include, and the crosslinking agent and the bioactive agent do not contain any RGD motifs. b) Preferably one or more different media containing FBS (serum) or a Wnt agonist, such as R-spongin. This includes the contents of the kit.

[0168] According to this embodiment, the present invention also relates to the contents of a kit for testing the effects of c-Met inhibitors on lung cancer cells, preferably non-small cell lung cancer cells, that overexpress c-Met, the following: a) A component for producing an array of fully distinct, non-self-degrading hydrogel matrices in order to create conditions for a fully distinct three-dimensional extracellular matrix in which the biological, biophysical and / or biochemical properties are distinct from each other, wherein the component - One or more different PEG hydrogel precursor molecules, preferably PEG-VS, particularly preferably 4-arm or 8-arm PEG-VS, - A peptide comprising at least one crosslinking agent, preferably at least two, preferably two cysteine ​​moieties, - One or more biologically active molecules as needed A component comprising the crosslinking agent and the bioactive agent, wherein neither contains any RGD motif; b) Preferably one or more different media containing FBS (serum) or a Wnt agonist, such as R-spongin. c) Cells from a cell repository / biobank created using the same extracellular matrix conditions as needed. This includes the contents of the kit.

[0169] In another preferred embodiment, the present invention also comprises the contents of a kit for testing the effects of c-Met inhibitors and other drugs on lung cancer cells, preferably non-small cell lung cancer cells, the following: a) A component for producing an array of fully distinct, non-self-degrading hydrogel matrices in order to create conditions for a fully distinct three-dimensional extracellular matrix in which the biological, biophysical and / or biochemical properties are distinct from each other, wherein the component - One or more different PEG hydrogel precursor molecules, preferably PEG-VS, particularly preferably 4-arm or 8-arm PEG-VS, - A peptide comprising at least one crosslinking agent, preferably at least two, preferably two cysteine ​​moieties, - One or more biologically active molecules as needed A component comprising the crosslinking agent and the bioactive agent, wherein neither contains any RGD motif; b) A component for producing an array of fully distinct, non-self-degrading hydrogel matrices in order to create conditions for a fully distinct three-dimensional extracellular matrix in which the biological, biophysical and / or biochemical properties are distinct from each other, wherein the component - One or more different PEG hydrogel precursor molecules, preferably PEG-VS, particularly preferably 4-arm or 8-arm PEG-VS, - A peptide comprising at least one crosslinking agent, preferably at least two, preferably two cysteine ​​moieties, - One or more biologically active molecules as needed, A component comprising the crosslinking agent and / or the bioactive agent containing the RGD motif; c) Preferably one or more different media containing FBS (serum) or a Wnt agonist, such as R-spongin. This includes the contents of the kit.

[0170] In another preferred embodiment, the present invention relates to the contents of a kit for testing the effects of c-Met inhibitors and other drugs on lung cancer cells, preferably non-small cell lung cancer cells, the following: a) A component for producing an array of fully distinct, non-self-degrading hydrogel matrices in order to create conditions for a fully distinct three-dimensional extracellular matrix in which the biological, biophysical and / or biochemical properties are distinct from each other, wherein the component - One or more different PEG hydrogel precursor molecules, preferably PEG-VS, particularly preferably 4-arm or 8-arm PEG-VS, - A peptide comprising at least one crosslinking agent, preferably at least two, preferably two cysteine ​​moieties, - One or more biologically active molecules as needed, A component comprising the crosslinking agent and the bioactive agent, wherein neither contains any RGD motif; b) A component for producing an array of fully distinct, non-self-degrading hydrogel matrices in order to create conditions for a fully distinct three-dimensional extracellular matrix in which the biological, biophysical and / or biochemical properties are distinct from each other, wherein the component - One or more different PEG hydrogel precursor molecules, preferably PEG-VS, particularly preferably 4-arm or 8-arm PEG-VS, - A peptide comprising at least one crosslinking agent, preferably at least two, preferably two cysteine ​​moieties, - One or more biologically active molecules as needed, A component comprising the crosslinking agent and / or the bioactive agent containing the RGD motif; c) Preferably one or more different culture media containing FBS (serum) or a Wnt agonist, such as R-spongin; d) Cells from a cell repository / biobank created using the same extracellular matrix conditions, if necessary. This includes the contents of the kit.

[0171] The methods and kits of the present invention also make it possible to culture additional lung cancer cells with different disease characteristics (e.g., different lung cancer subtypes, mutations, e.g., mutations in EGFR, KRAS, or PI3 kinase) and test drugs on these lung cancer cells. In the same manner, the extracellular matrix conditions can be pre-selected for these other cells.

[0172] In a particularly preferred embodiment, the hydrogel does not contain any RGD binding sites, and especially preferably does not contain any integrin binding sites at all.

[0173] As can be seen from Example 1 and Figure 1, when testing a drug for its activity against non-small cell lung cancer cells that overexpress c-Met, precise prior selection of conditions is crucial. It is impossible to identify drug candidates that target c-Met using Matrigel®, i.e., the reference matrix in the prior art. This is likely because, as found in Comparative Example 1, under conditions using Matrigel®, the drug target c-Met is not sufficiently expressed and activated. Therefore, under conditions using Matrigel®, it is impossible to identify suitable drug candidates that act on the most important target in those lung cancer cells, namely overexpressed c-Met.

[0174] Similarly, in Example 1 and Figure 1, it was shown that targeting of c-Met is indeed a key feature for inhibiting the growth of non-small cell lung cancer cells that overexpress c-Met.

[0175] In addition, the pre-selected conditions of this embodiment of the present invention also make it possible to better identify the optimal treatment regimen for a particular patient. A particular patient may have been found not to respond to drug therapy with a c-met inhibitor alone, perhaps because such a patient possessed a cellular phenotype that could complement c-met inhibition. The pre-selected conditions of this embodiment of the present invention allow for screening for combination drug therapy using a c-met inhibitor in combination with another type of drug. As discussed above, this is not possible under the prior art conditions using Matrigel®. The present invention provides better predictability regarding patient responses.

[0176] The pre-selected extracellular matrix conditions in this configuration are also, a) A step of providing newly isolated or frozen lung cancer cells, preferably non-small cell lung cancer cells, from a biopsy or tissue resection from a cancer patient; b) The step of immobilizing and growing organoids from the cells, and applying one or more drugs to the organoids by the method described above; c) A step of comparing the activity of one or more drugs applied in step b) with the result of treatment of the patient with one of the drugs applied in step b); d) A process to provide the results of drug activity on the patient's organoids, as well as the corresponding genetic and phenotypic data of the disease, to help a physician determine how to treat the patient. It can also be used in methods to test the effectiveness of treatments for cancer patients, including [mention specific examples of other methods].

[0177] A patient biopsy or resection specifically for the isolation of lung cancer cells, preferably non-small cell lung cancer (NSCLC) cells, can be collected during a standard diagnostic procedure for organoid implantation in step b) and then transported to the location where step b) is performed.

[0178] Step b) of this method provides pre-selected extracellular matrix conditions, comprising arrays having separate spaces of a completely distinct, non-self-degrading hydrogel matrix having a rigidity of 50 to 2000 Pa, prepared by crosslinking one or more different combinations of PEG hydrogel precursor molecules, at least one crosslinking agent, and lung cancer cells, preferably non-small cell lung cancer cells, on the surface of a substrate or in separate spaces of a substrate, preferably in a multi-well plate, in the presence of one or more biologically active molecules as needed, i.e., to create conditions for a completely distinct three-dimensional extracellular matrix having biological, biophysical and / or biochemical properties that are distinct from one another, as described above; The present medium preferably comprises FBS (serum) or a Wnt agonist, such as R-spongin, and more preferably also comprises FGF-7, FGF-10, and TGF-β inhibitors, enabling the lung cancer cells, preferably non-small cell lung cancer cells, to grow in the separate spaces of the hydrogel matrix; Furthermore, adding one or more drugs to cells grown in the separate spaces of the hydrogel matrix; The process is carried out by the crosslinking agent and the optional bioactive agent, and the crosslinking agent and the optional bioactive agent do not contain any RGD motifs.

[0179] One or more drugs added to cells grown in separate spaces within the hydrogel matrix include drugs used for the patient's standard of care (SoC) for cancer treatment.

[0180] In a preferred embodiment, the biopsy or excision can be further processed for histological analysis and / or for omics testing (e.g., NGS) to establish a reference baseline. The results of these additional analyses can also be used to compare and / or correlate the ex vivo and in vivo tests described above.

[0181] Based on this method, it is possible to reliably evaluate whether the applicable standard anti-cancer treatment (SoC) is suitable, or whether different drug treatment regimens tested ex vivo as described above are more promising. In this way, the present invention enables the individualization and optimization of cancer treatment. It can generate functional in vitro data that can increase the accuracy of treatment decisions made by healthcare professionals.

[0182] Patient-derived organoids (PDOs) in precision medicine Cancer is a multifactorial disease resulting from the genetic and epigenetic transformation of normal cells, leading to their abnormal proliferation. Conventional cancer treatments include surgical resection, radiotherapy, nonspecific or targeted chemotherapy, and immunotherapy to inhibit cell division or induce apoptosis in cancer cells.

[0183] Different cancers respond differently to treatment, and therefore some cancers can be treated better than others. Despite the development of powerful chemotherapy agents and oncogene-specific targeted therapies, permanent or sustained cures for this disease have not been achieved in many patients.

[0184] Recent improvements in DNA sequencing technology allow for the rapid identification of specific genomic mutations in tumors of patients with the potential to tailor cancer treatment based on the tumor's molecular profile. Significant improvements have been demonstrated in the treatment of leukemia, lung cancer, and melanoma cancer (Druker et al. (N Engl J Med, Vol. 344, No. 14 (2001), 1031), Lynch et al. (N Engl J Med 350; 21 (2004), 2129), Flaherty et al. (N Engl J Med 2010; 363: 809-19)). However, the clinical benefits of genome-guided precision medicine remain largely debatable (Le Tourneau et al. (www.thelancet.com / oncology, Published online September 3, 2015, http: / / dx.doi.org / 10.1016 / S1470-2045(15)00188-6), Prasad (Nature 537 (2016), S63), Letai (NATURE MEDICINE, VOLUME 23 | NUMBER 9 | SEPTEMBER 2017, 1028). Recent clinical trials evaluating the proportion of patients with solid tumors assigned to targeted therapy have shown that only a small fraction of patients (10-50%) have mutations consistent with clinically validated and approved available therapies (Letai 2017, Sicklick (Nature Medicine)). https: / / doi.org / 10.1038 / s41591-019-0407-5 (2019))). In addition to this, there are two fundamental biological aspects: - Resistance to specific treatments due to genetic and phenotypic heterogeneity (presence of different subclones) in cancer cells among patients (Tannock et al. (N Engl J Med 375;13 (2016), 1289), Flavahan et al., (Science 357, 266 (2017)); - Insufficient biological understanding of the effect of the tumor microenvironment on the modulation of drug response (Friedmann et al. (Nature Reviews Cancer AOP, published online 5 November 2015; doi:10.1038 / nrc4015 2015)) However, this undermines the effectiveness of genome-guided precision medicine.

[0185] Drug screening using patient-derived cells (functional precision medicine) addresses these limitations, complements genomics and pathology data to support the prediction of patient outcomes, and therefore assists in guiding decision-making regarding treatment methods.

[0186] Novel in vitro tumor biology models that replicate the tumor microenvironment in vivo, such as patient-derived organoids (PDOs), have the advantage of growing in a 3D environment and replicating the spatial structure of the original tissue. Organoids are small 3D in vitro structures grown from patient-derived cells that mimic the key features and functions of the original healthy or diseased tissue. A diverse range of PDOs have become established in many tumors, including, but not limited to, colorectal cancer (Sato et al. (Nature vol. 469 (2011), 415), van de Wetering et al. (Cell 161 (2015), 933-945), pancreatic ductal adenocarcinoma (Boj et al. (Cell 160, 324-338, January 15, 2015), Huang et al. (Nature medicine, published online 26 October 2015; doi:10.1038 / nm.3973)), breast cancer (Sachs et al. (Cell 172 2018, 1-14)), and lung cancer (Sachs et al. (The EMBO Journal e 100300|2019)). Overall, these studies have shown that PDOs can maintain the same genetic mutations identified in the primary tumor.

[0187] Recently, patient organoids originating from different locations within the same tumor were used to test the nature and degree of intertumor heterogeneity and evaluate their response to a panel of drugs (Roerink et al. Nature 556, 457-462, 2018). Significant differences in drug response were observed among closely related cells within the same tumor.

[0188] The use of organoids as a functional diagnostic tool in clinical practice has already been demonstrated for rectal cancer (Ganesh et al., Nature Medicine, 10, 1067-1614 (2019)), metastatic colorectal cancer (Vlachogiannis et al., Science 359, 920-926 (2018) and Ooft et al., Science Translational Medicine, 11, (2019), DOI: 10.1126 / scitranslmed.aay2574), pancreatic cancer (Tiriac, CANCER DISCOVERY, SEPTEMBER 2018, DOI: 10.1158 / 2159-8290.CD-18-0349), and appendiceal cancer (Votanopoulos et al., Ann Surg Oncol (2019) 26:139-147). These studies show that the drug response to PDOs correlates with the outcomes of the same treatment in patients from whom organoids originated.

[0189] Despite these promising results showing that PDO drug responses correlate with corresponding patient outcomes, these studies are limited to a small number of patients, and the methods used rely on basement membrane extracts (BMEs), e.g., Matrigel®, which have unclear compositions and batch-to-batch variability. This represents a significant limitation in the standardization of PDOs for bridging to relevant clinical applications. Similarly, only single culture conditions were used for each type of cancer, despite potential differences in genetic and / or phenotypic tumor features, including, non-limitingly, the expression of biomarkers that may require different extracellular matrix conditions. This may favor the growth of specific cell populations or the induction of only a limited phenotypic expression during cell proliferation in ex vivo (International Publication 2010 / 090513; International Publication 2016 / 015158; International Publication 2015 / 173425), and therefore cannot mimic in vivo tumor characteristics and drug responses. This is summarized above with respect to lung cancer cells, preferably non-small cell lung cancer cells, that overexpress c-Met.

[0190] To overcome the limitations of naturally derived matrices, such as Matrigel®, completely clear and similarly synthetic hydrogels have been developed for use with mouse and human intestinal and colon organoids (Gjorevski et al., Designer matrices for intestinal stem cell and organoid culture, Nature, Vol 539, 24 November 2016, 560-56, International Publication No. 2017 / 037295; or Cruz-Acuna et al., Synthetic hydro-gels 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, International Publication No. 2018 / 165565), cells from appendiceal cancer, pancreatic cancer, and mesothelioma cancer patients (Votanopoulos 2019 (above), Broguiere et al., Adv. Mater. It has already been studied for growing diverse organizations, including (2018, 1801621 (2018), Mazzocchi et al., SCIENTIFIC Reports (2018) 8:2886 DOI:10.1038 / s41598-018-21200-8).

[0191] While some of these studies use fully distinct or totally synthetic matrices, they still rely on the use of single-culture conditions, regardless of tumor characteristics.

[0192] Pancreatic cancer The present invention makes it possible to culture a patient's pancreatic cells, preferably pancreatic ductal adenocarcinoma (PDAC) cells, under conditions that maintain the growth and proliferation of these cells. Subsequently, the cells are exposed to different drug therapies to select an effective drug treatment for the cancer patient.

[0193] According to the present invention, it has been demonstrated that PDAC cells can be successfully cultured and tested using a combination of a completely distinct soft (50-1000 Pa stiffness), moderate (1000-2000 Pa), or stiff (2000-3000 Pa) non-autodegradable hydrogel matrix containing at least one RGD motif and a culture medium, preferably containing a Wnt agonist, such as R-spongin and Wnt3a.

[0194] Thus, according to this embodiment, the present invention is a method for testing the effects of a drug on pancreatic ductal adenocarcinoma (PDAC) cells, a) A step of providing pre-selected extracellular matrix conditions, comprising an array having separate spaces of a matrix of completely distinct, non-self-degrading hydrogels having a stiffness of 50-3000 Pa, preferably 50-2000 Pa, most preferably 50-1000 Pa, prepared by crosslinking one or more different combinations of hydrogel precursor molecules, at least one crosslinking agent, and pancreatic ductal adenocarcinoma cells, on the surface of a substrate or in separate spaces of a substrate, preferably in a multiwell plate, in the presence of one or more biologically active molecules as needed, to create conditions for a completely distinct three-dimensional extracellular matrix with biological, biophysical and / or biochemical properties distinct from one another; b) A step of enabling the pancreatic ductal adenocarcinoma cells to grow in the separate spaces of the hydrogel matrix, preferably in the presence of one or more different media containing Wnt agonists, such as R-spongin and Wnt3a; c) Adding one or more drugs to cells grown in the separate spaces of the hydrogel matrix; The present invention relates to a method comprising the crosslinking agent and at least one of the optional biological agents comprising the RGD motif.

[0195] Preferably, the precursor molecule of the PEG hydrogel is PEG-VS (polyethylene glycol having a terminal vinyl sulfone moiety), and particularly preferably 4-arm or 8-arm PEG-VS.

[0196] More preferably, a completely distinct non-self-degrading hydrogel matrix array is prepared by crosslinking PEG-VS, particularly preferably 4-arm or 8-arm PEG-VS, with a peptide containing at least two, preferably two cysteine ​​moieties, as a crosslinking agent, the crosslinking agent may contain an RGD motif.

[0197] As needed, a bioactive ligand containing a bioactive motif including an RGD adhesion motif may be used. Examples of suitable RGD motifs include RGD, RGDS, RGSSP, RGDSPG, RGDSPK, RGSTP, RGDSPASSKP, PHSRNSGSGSGSGSGRGDSPG, or any cyclic RGD motif, such as cyclo(RGDfC), but basically any RGD sequence that is known and successfully utilized in the fields of hydrogels and cell culture can be used.

[0198] Preferably, the bioactive ligand, as needed, is selected from the group consisting of natural laminins, such as laminin-111, particularly mouse laminin-111, recombinant laminin isoforms, and their biofunctional fragments. Preferred recombinant laminin isoforms are laminin-111, laminin-211, laminin-332, laminin-411, laminin-511, or laminin-521, with laminin-511 being preferred.

[0199] As needed, a bioactive ligand containing a collagen peptide motif may be used. A suitable example of a collagen peptide is DGEA.

[0200] As needed, glycosaminoglycans, such as hyaluronic acid and hyaluronan, may be used as bioactive ligands. Examples of hyaluronic acid include hyaluronic acid 50k, hyaluronic acid 1000k, hyaluronatethiol 50k, or hyaluronatethiol 1000k.

[0201] Preferably, the culture medium is characterized by the presence of a Wnt agonist, such as R-spongin and Wnt3a. In a preferred embodiment, the culture medium described in Boj et al. (Cell 160, 324-338, January 15, 2015), page 335, right column, second paragraph, or Huang et al. (Nature medicine, published online 26 October 2015; doi:10.1038 / nm.3973) may be used. Particularly preferred is a modified medium of Boj et al., which includes AdDMEM / F12 medium supplemented with HEPES, Glutamax, penicillin / streptomycin, B27, Primocin, N-acetyl-L-cysteine, Wnt3a-condition medium [50 v / v%] or recombinant protein [100 ng / ml], RSPO1-condition medium [10 v / v%] or recombinant protein [500 ng / ml], Noggin-condition medium [10 v / v%] or recombinant protein [0.1 μg / ml], epidermal growth factor [EGF, 50 ng / ml], gastrin [10 nM], fibroblast growth factor 10 [FGF10, 100 ng / ml], nicotinamide [10 mM], prostaglandin E2 [PGE2, 1 μM], and A83-01 [0.5 μM].

[0202] Particularly preferably, pancreatic ductal adenocarcinoma cells can be obtained from newly isolated or frozen cells obtained from human biopsy or tissue resection, or from patient-derived xenograft (PDX) tissue.

[0203] By the aforementioned method, pancreatic ductal adenocarcinoma cells can be grown and proliferated in a selected culture medium under extracellular matrix conditions that reproduce the drug effects observed in vivo, and then tested.

[0204] In a particularly preferred embodiment, the extracellular matrix conditions are selected so as not to use any naturally derived matrix, such as Matrigel®.

[0205] According to this embodiment, the present invention relates to the contents of a kit for testing the effects of a drug on pancreatic ductal adenocarcinoma cells, which are as follows: a) A component for producing an array of completely distinct, non-self-degrading hydrogel matrices having a stiffness of 50-3000 Pa, preferably 50-2000 Pa, most preferably 50-1000 Pa, in order to create conditions for a completely distinct three-dimensional extracellular matrix in which the biological, biophysical and / or biochemical properties are distinct from each other, wherein the component is - One or more different PEG hydrogel precursor molecules, preferably PEG-VS, particularly preferably 4-arm or 8-arm PEG-VS, - A peptide comprising at least one crosslinking agent, preferably at least two, preferably two cysteine ​​moieties, - One or more biologically active molecules as needed A component comprising the crosslinking agent and / or at least one of the optional bioactive agents comprising the RGD motif; b) Preferably one or more different media containing a Wnt agonist, such as R-spongin and Wnt3a. This includes the contents of the kit.

[0206] According to this embodiment, the present invention also relates to the contents of a kit for testing the effects of a drug on pancreatic ductal adenocarcinoma cells, the following: a) A component for producing an array of completely distinct, non-self-degrading hydrogel matrices having a stiffness of 50-3000 Pa, preferably 50-2000 Pa, most preferably 50-1000 Pa, in order to create conditions for a completely distinct three-dimensional extracellular matrix in which the biological, biophysical and / or biochemical properties are distinct from each other, wherein the component is - One or more different PEG hydrogel precursor molecules, preferably PEG-VS, particularly preferably 4-arm or 8-arm PEG-VS, - A peptide comprising at least one crosslinking agent, preferably at least two, preferably two cysteine ​​moieties, - One or more biologically active molecules as needed A component comprising the crosslinking agent and / or at least one of the optional bioactive agents comprising the RGD motif; b) Preferably one or more different media containing a Wnt agonist, such as R-spongin and Wnt3a, c) Cells from a cell repository / biobank created using the same extracellular matrix conditions as needed. This includes the contents of the kit.

[0207] The pre-selected extracellular matrix conditions in this configuration are also, a) A step of providing newly isolated or frozen pancreatic ductal adenocarcinoma cells from a biopsy or tissue resection from a cancer patient; b) The step of immobilizing and growing organoids from the cells, and applying one or more drugs to the organoids by the method described above; c) A step of comparing the activity of one or more drugs applied in step b) with the result of treatment of the patient with one of the drugs applied in step b); d) A process to provide results of drug activity on the patient's organoids and corresponding genetic and phenotypic data of the disease to help a physician determine how to treat the patient. It can be used in methods to test the effectiveness of treatments for cancer patients, including [mention specific examples of this method].

[0208] For the isolation of pancreatic ductal adenocarcinoma (PDAC) cells, a dedicated patient biopsy or resection can be collected during a standard diagnostic procedure for organoid implantation in step b) and then transported to the location where step b) is performed.

[0209] Step b) of this method provides pre-selected extracellular matrix conditions, including arrays having separate spaces of a matrix of completely distinct, non-self-degrading hydrogels having a stiffness of 50-3000 Pa, preferably 50-2000 Pa, most preferably 50-1000 Pa, prepared by crosslinking pancreatic ductal adenocarcinoma cells with one or more different combinations of PEG hydrogel precursor molecules, at least one crosslinking agent, on the surface of a substrate or in separate spaces of a substrate, preferably in a multi-well plate, in the presence of one or more biologically active molecules as needed; Preferably, the presence of one or more different media containing Wnt agonists, such as R-spongin and Wnt3a, allows the pancreatic ductal adenocarcinoma cells to grow in the separate spaces of the hydrogel matrix; Furthermore, adding one or more drugs to cells grown in the separate spaces of the hydrogel matrix; The method is implemented by including at least one of the crosslinking agent and the optional bioactive agent in the RGD motif.

[0210] One or more drugs added to separate spaces in the hydrogel matrix include drugs used for the patient's standard anti-cancer treatment (SoC).

[0211] In a preferred embodiment, the biopsy or excision can be further processed for histological analysis and / or for omics testing (e.g., NGS) to establish a reference baseline. The results of these additional analyses can also be used to compare and / or correlate the ex vivo and in vivo tests described above.

[0212] Based on this method, it is possible to reliably evaluate whether the applicable standard anti-cancer treatment (SoC) is suitable, or whether different drug treatment regimens tested ex vivo as described above are more promising. In this way, the present invention enables the individualization and optimization of cancer treatment. It can generate functional in vitro data that can increase the accuracy of treatment decisions made by healthcare professionals.

[0213] Co-culture of PDAC cells According to another preferred embodiment of the present invention, cancer cells and preferably pancreatic ductal adenocarcinoma (PDAC) cells can be co-cultured in combination with stromal cells, preferably fibroblasts. In this embodiment, the hydrogel matrix is ​​a non-self-degrading PEG hydrogel pre-selected having a rigidity of 50 to 3000 Pa, preferably 50 to 2000 Pa, most preferably 50 to 1000 Pa, at least one of the crosslinking agents comprises an enzymatically degradable motif, preferably an MMP-sensitive motif, and at least one of the crosslinking agents and / or the any bioactive agents comprises an RGD motif.

[0214] Preferably, the culture medium used in the above embodiment comprises a Wnt agonist, such as R-spongin and Wnt3a, and preferably further comprises FBS.

[0215] Thus, according to this embodiment, the present invention is a method for testing cancer cells, preferably pancreatic ductal adenocarcinoma (PDAC) cells, that are co-cultured with stromal cells, preferably fibroblasts. a) Providing pre-selected extracellular matrix conditions comprising arrays having separate spaces of a matrix of completely distinct, non-self-degrading hydrogels having a stiffness of 50-3000 Pa, preferably 50-2000 Pa, most preferably 50-1000 Pa, produced by crosslinking one or more different combinations of PEG hydrogel precursor molecules, at least one crosslinking agent, and the cancer cells, preferably pancreatic ductal adenocarcinoma (PDAC) cells, and the stromal cells, preferably fibroblasts, on the surface of a substrate or in separate spaces of a substrate, preferably in a multiwell plate, in the presence of one or more biologically active molecules as needed, to create conditions for a completely distinct three-dimensional extracellular matrix with distinct biological, biophysical and / or biochemical properties from one another; b) A step of enabling the pancreatic ductal adenocarcinoma cells and stromal cells, preferably fibroblasts, to grow in the separate spaces of the array of the hydrogel matrix in the presence of one or more different media, preferably comprising a Wnt agonist, such as R-spongin and Wnt3a, and preferably further comprising FBS; c) Adding one or more drugs to cells grown in the separate spaces of the hydrogel matrix; The present invention relates to a method comprising, wherein at least one crosslinking agent comprises an enzymatically degradable motif, preferably an MMP-sensitive motif, and at least one of the crosslinking agent and / or the any bioactive agent comprises an RGD motif.

[0216] In a preferred embodiment, the method is carried out such that the arrays are different in terms of the presence or absence of enzymatically degradable motifs, preferably MMP-sensitive motifs, to provide at least two different arrays. In the arrays where the enzymatically degradable motifs, preferably MMP-sensitive motifs, are present, PDAC cells can be co-cultured with stromal cells, preferably fibroblasts. In the arrays where the enzymatically degradable motifs, preferably MMP-sensitive motifs, are absent, PDAC cells grow in a single culture that impairs the growth of stromal cells, such as fibroblasts.

[0217] Preferably, the precursor molecule of the PEG hydrogel is PEG-VS (polyethylene glycol having a terminal vinyl sulfone moiety), particularly preferably 4-arm or 8-arm PEG-VS. In another preferred embodiment, the self-degrading PEG may be prepared from one or more PEG-Acr precursor molecules, which may be used alone or in combination with PEG-VS precursor molecules.

[0218] More preferably, the array of the completely clear, preferably non-self-degrading, hydrogel matrix is ​​produced by crosslinking PEG-VS, particularly preferably 4-arm or 8-arm PEG-VS, with a peptide comprising at least two, preferably two cysteine ​​moieties, as a crosslinking agent, the crosslinking agent comprising an enzymatically degradable motif, preferably an MMP-sensitive motif, and may further comprise an RGD motif.

[0219] As needed, a bioactive ligand containing a bioactive motif including an RGD adhesion motif may be used. Examples of suitable RGD motifs include RGD, RGDS, RGSSP, RGDSPG, RGDSPK, RGSTP, RGDSPASSKP, PHSRNSGSGSGSGSGRGDSPG, or any cyclic RGD motif, such as cyclo(RGDfC), but basically any RGD sequence that is known and successfully utilized in the fields of hydrogels and cell culture can be used.

[0220] Preferably, the bioactive ligand, as needed, is selected from the group consisting of natural laminins, such as laminin-111, particularly mouse laminin-111, recombinant laminin isoforms, and their biofunctional fragments. Preferred recombinant laminin isoforms are laminin-111, laminin-211, laminin-332, laminin-411, laminin-511, or laminin-521, with laminin-511 being preferred.

[0221] As needed, a bioactive ligand containing a collagen peptide motif may be used. A suitable example of a collagen peptide is DGEA.

[0222] As needed, glycosaminoglycans, such as hyaluronic acid and hyaluronan, may be used as bioactive ligands. Examples of hyaluronic acid include hyaluronic acid 50k, hyaluronic acid 1000k, hyaluronatethiol 50k, or hyaluronatethiol 1000k.

[0223] Preferably, the culture medium is characterized by the presence of a Wnt agonist, such as R-spongin and Wnt3a, and preferably further, the presence of FBS. In a preferred embodiment, the culture medium described in Boj et al. (Cell 160, 324-338, January 15, 2015), page 335, right column, second paragraph, or Huang et al. (Nature medicine, published online 26 October 2015; doi:10.1038 / nm.3973) may be used. Particularly preferred is a modified medium of Boj et al., which includes AdDMEM / F12 medium supplemented with HEPES, Glutamax, penicillin / streptomycin, B27, Primocin, N-acetyl-L-cysteine, Wnt3a-condition medium [50 v / v%] or recombinant protein [100 ng / ml], RSPO1-condition medium [10 v / v%] or recombinant protein [500 ng / ml], Noggin-condition medium [10 v / v%] or recombinant protein [0.1 μg / ml], epidermal growth factor [EGF, 50 ng / ml], gastrin [10 nM], fibroblast growth factor 10 [FGF10, 100 ng / ml], nicotinamide [10 mM], prostaglandin E2 [PGE2, 1 μM], and A83-01 [0.5 μM].

[0224] Particularly preferred are pancreatic ductal adenocarcinoma cells that are newly isolated or frozen cells obtained from human biopsy or tissue resection, patient-derived xenograft (PDX) tissue, or patient-derived organoid (PDO), which have been pre-immobilized in BME, for example, Matrigel®, as needed.

[0225] Preferably, the stromal cells are isolated from the patient. Particularly preferably, the stromal cells are fibroblasts.

[0226] By the aforementioned method, pancreatic ductal adenocarcinoma cells can be grown and proliferated in a selected culture medium under extracellular matrix conditions that reproduce the drug results observed in vivo, and then tested.

[0227] In a particularly preferred embodiment, the extracellular matrix conditions are selected so as not to use any naturally derived matrix, such as Matrigel®.

[0228] According to this embodiment, the present invention also relates to the contents of a kit for testing the effects of a drug on cancer cells, preferably pancreatic ductal adenocarcinoma (PDAC) cells, co-cultured with stromal cells, preferably fibroblasts, and further: a) Components for producing a matrix array of perfectly clear, preferably non-self-degrading hydrogels having a stiffness of 50-3000 Pa, preferably 50-2000 Pa, most preferably 50-1000 Pa, in order to create conditions for a perfectly clear three-dimensional extracellular matrix in which the biological, biophysical and / or biochemical properties are distinct from each other, wherein the components are: - One or more different PEG hydrogel precursor molecules, preferably PEG-VS, particularly preferably 4-arm or 8-arm PEG-VS, - A peptide comprising at least one crosslinking agent, preferably at least two, preferably two cysteine ​​moieties, - One or more biologically active molecules as needed A component comprising, preferably, at least one crosslinking agent comprising an enzymatically degradable motif, preferably an MMP-sensitive motif, and at least one of the crosslinking agents and / or any of the bioactive agents comprising an RGD motif; b) One or more different media preferably comprising a Wnt agonist, such as R-spongin and Wnt3a, and preferably further comprising FBS. This includes the contents of the kit.

[0229] According to this aspect, the present invention also comprises the contents of a kit for testing the effects of a drug on cancer cells, preferably pancreatic ductal adenocarcinoma (PDAC) cells, co-cultured with stromal cells, preferably fibroblasts. a) Components for producing a matrix array of perfectly clear, preferably non-self-degrading hydrogels having a stiffness of 50-3000 Pa, preferably 50-2000 Pa, most preferably 50-1000 Pa, in order to create conditions for a perfectly clear three-dimensional extracellular matrix in which the biological, biophysical and / or biochemical properties are distinct from each other, wherein the components are: - One or more different PEG hydrogel precursor molecules, preferably PEG-VS, particularly preferably 4-arm or 8-arm PEG-VS, - A peptide comprising at least one crosslinking agent, preferably at least two, preferably two cysteine ​​moieties, - One or more biologically active molecules as needed, A component comprising, preferably, at least one crosslinking agent comprising an enzymatically degradable motif, preferably an MMP-sensitive motif, and at least one of the crosslinking agents and / or any of the bioactive agents comprising an RGD motif; b) Preferably one or more different culture media comprising a Wnt agonist, such as R-spongin and Wnt3a, and preferably further comprising FBS. c) Cells from a cell repository / biobank created using the same extracellular matrix conditions as needed. This includes the contents of the kit.

[0230] The preselected extracellular matrix conditions of this aspect also a) providing newly isolated or frozen cancer cells, preferably pancreatic ductal adenocarcinoma cells, from a biopsy or tissue resection of a cancer patient and stromal cells isolated from the patient to establish a co-culture system; b) allowing the cells from the co-culture system to attach and proliferate and applying one or more drugs to the cells by the method described above; c) comparing the activity of one or more drugs applied in step b) with the outcome of treatment of the patient with one of the drugs applied in step b); d) and / or providing results of drug activity and corresponding genetic and phenotypic data of the disease regarding the patient's organoids to assist a physician in determining how to treat the patient can also be used in a method for testing the effectiveness of treating a cancer patient, which comprises the steps of:

[0231] A dedicated patient biopsy or resection for the isolation of pancreatic ductal adenocarcinoma (PDAC) cells can be collected during standard diagnostic procedures for establishing organoids in step b) and then transported to the location where step b) is performed.

[0232] Step b) of this method provides preselected extracellular matrix conditions as described above, i.e., on the surface of a substrate or in separate spaces of a substrate, preferably in a multiwell plate, to create distinct three-dimensional extracellular matrix conditions with different biological, biophysical, and / or biochemical properties, in the presence of one or more biologically active molecules as required, different combinations of precursor molecules of one or more different PEG hydrogels, at least one crosslinking agent and stromal cells, preferably fibroblasts, and cancer cells, preferably pancreatic ductal adenocarcinoma cells, co-cultured simultaneously, and crosslinking them to produce an array having separate spaces of a well-defined, preferably non-self-degrading hydrogel matrix having a stiffness of 50 - 3000 Pa, preferably 50 - 2000 Pa, most preferably 50 - 1000 Pa. In the presence of one or more different media, preferably containing a Wnt agonist, such as R-spondin and Wnt3a, and preferably further containing FBS, enabling the cancer cells, preferably pancreatic ductal adenocarcinoma cells, and the stromal cells, preferably fibroblasts, to grow in the separate spaces of the matrix of the hydrogel; And adding one or more drugs to the cells grown in the separate spaces of the matrix of the hydrogel; Performed by, the at least one cross-linking agent preferably includes a motif degradable by an enzyme, preferably an MMP-sensitive motif, and at least one of the cross-linking agent and / or the optional bioactive agent includes an RGD motif.

[0233] One or more drugs added to the cells grown in the separate spaces of the matrix of the hydrogel include one or more drugs used for the patient's standard of care (SoC) for cancer treatment.

[0234] According to a preferred embodiment, the biopsy or resection specimen can be further processed for histological analysis and / or for omics tests (e.g., NGS) to establish a reference baseline. The results of these additional analyses can also be used to compare and / or correlate the ex vivo and in vivo tests described above.

[0235] Based on this method, it is possible to reliably evaluate whether the applied standard of care (SoC) for cancer treatment is suitable or whether different drug treatment regimens tested ex vivo as described above are more promising. Thus, in the present invention, cancer treatment can be individualized and optimized. Functional in vitro data can be generated that can increase the accuracy of treatment decisions by healthcare providers.

[0236] Colorectal cancer Colorectal cancer (CRC) cells are known to exhibit heterogeneity (Roerink et al., (Nature, published online https: / / doi.org / 10.1038 / s41586-018-0024-3 (2018))). Significant differences in drug response were observed among closely related cells within the same tumor. Considerations for pancreatic cells are applicable to colorectal cancer.

[0237] The present invention makes it possible to culture colorectal cancer (CRC) cells from a patient under conditions that maintain their growth and proliferation. The cells are then exposed to different drug therapies to select an effective drug treatment for the cancer patient. Thus, the present invention provides a precision medicine platform that enables the growth and drug testing of CRC tissue in different microenvironments, and therefore captures the specificity of multiple clones within a single tumor.

[0238] According to the present invention, it has been demonstrated that colorectal cancer (CRC) cells can be successfully cultured and tested using a combination of a perfectly clear, soft or moderately stiff (50-2000 Pa) hydrogel matrix containing at least one RGD adhesion motif and, optionally, additional bioactive ligands such as laminin, preferably laminin-111, or laminin-511, particularly preferably natural mouse laminin-111 or recombinant human laminin-511, and preferably a Wnt agonist, such as R-spongin and Wnt3a.

[0239] Thus, according to this aspect, the present invention is a method for testing the effects of a drug on colorectal cancer (CRC) cells, a) A step of providing pre-selected extracellular matrix conditions, comprising an array having separate spaces of a matrix of completely clear, preferably non-self-degrading hydrogels having a rigidity of 50 to 2000 Pa, prepared by crosslinking one or more different combinations of PEG hydrogel precursor molecules, at least one crosslinking agent, and the colorectal cancer cells, on the surface of the substrate or in separate spaces of the substrate, preferably in a multiwell plate, in the presence of one or more biologically active molecules as needed, to create conditions for a completely clear three-dimensional extracellular matrix with biological, biophysical and / or biochemical properties that are distinct from one another; b) A step of enabling the colorectal cancer cells to grow in the separate spaces of the hydrogel matrix, preferably in the presence of one or more different media containing Wnt agonists, such as R-spongin and Wnt3a; c) Adding one or more drugs to cells grown in the separate spaces of the hydrogel matrix; The present invention relates to a method comprising, wherein at least one of the crosslinking agent and / or the bioactive agent comprises an RGD motif.

[0240] Preferably, the precursor molecules of the PEG hydrogel are PEG-VS (polyethylene glycol having a terminal vinyl sulfone moiety), particularly preferably 4-arm or 8-arm PEG-VS, and / or PEG-Acr (polyethylene glycol having a terminal acrylate moiety), particularly preferably 4-arm or 8-arm PEG-Acr.

[0241] More preferably, a matrix array of completely distinct self-degradable hydrogels is produced by crosslinking a 50:50 mixture of PEG-VS, particularly preferably 4-arm or 8-arm PEG-VS, and PEG-Acr, particularly preferably 4-arm or 8-arm PEG-Acr, with a peptide containing at least two, preferably two cysteine ​​moieties, as a crosslinking agent, the crosslinking agent may contain an RGD motif.

[0242] More preferably, a completely distinct non-self-degrading hydrogel matrix array is prepared by crosslinking PEG-VS, particularly preferably 4-arm or 8-arm PEG-VS, with a peptide containing at least two, preferably two cysteine ​​moieties, as a crosslinking agent, the crosslinking agent may contain an RGD motif.

[0243] As needed, a bioactive ligand containing a bioactive motif including an RGD adhesion motif may be used. Examples of suitable RGD motifs include RGD, RGDS, RGSSP, RGDSPG, RGDSPK, RGSTP, RGDSPASSKP, PHSRNSGSGSGSGSGRGDSPG, or any cyclic RGD motif, such as cyclo(RGDfC), but basically any RGD sequence that is known and successfully utilized in the fields of hydrogels and cell culture can be used.

[0244] Preferably, the bioactive ligand, as needed, is selected from the group consisting of natural laminins, such as laminin-111, particularly mouse laminin-111, recombinant laminin isoforms, such as recombinant human laminin-511, and their biofunctional fragments. Suitable recombinant laminin isoforms are laminin-111, laminin-211, laminin-332, laminin-411, laminin-511, or laminin-521.

[0245] Preferably, the culture medium is characterized by the presence of Wnt agonists, such as R-spongin and Wnt3a. In a preferred embodiment, the culture medium described in Vlachogiannis et al., Science 359, 920-926 (2018) may be used (see, for example, supplementary material, page 5, human PDO medium). Alternatively, the commercially available medium Intesticult® may be used. Particularly preferred is the medium by Vlachogiannis et al., which contains B27 additive, N2 additive, BSA, L-glutamine, penicillin-streptomycin, EGF, noggin, R-spongin 1, gastrin, FGF-10, basic FGF, Wnt-3A, prostaglandin E2, Y-27632, nicotinamide, A83-01, SB202190 and, if necessary, HGF supplemented into Advanced DMEM / F12.

[0246] Particularly preferred are colorectal cancer cells that are newly isolated or frozen cells obtained from human biopsy or tissue resection, or from patient-derived xenograft (PDX) tissue.

[0247] By the method described above, colorectal cancer cells can be grown and proliferated in a selected culture medium under extracellular matrix conditions that reproduce the drug effects observed in vivo, and then tested.

[0248] In a particularly preferred embodiment, the extracellular matrix conditions are selected so as not to use any naturally derived matrix, such as Matrigel®.

[0249] According to this aspect, the present invention relates to the contents of a kit for testing the effects of a drug on colorectal cancer cells, which are as follows: a) A component for producing an array of perfectly clear hydrogel matrices having a rigidity of 50-2000 Pa, in order to create conditions for perfectly clear three-dimensional extracellular matrices in which the biological, biophysical and / or biochemical characteristics are distinct from each other, wherein the component is: - One or more different PEG hydrogel precursor molecules, preferably PEG-VS, particularly preferably 4-arm or 8-arm PEG-VS, and / or PEG-Acr, particularly preferably 4-arm or 8-arm PEG-Acr - At least one crosslinking agent, preferably at least two, preferably a peptide containing two cysteine moieties - Optionally, one or more biologically active molecules including laminin, preferably laminin-111 or laminin-511, particularly preferably natural mouse laminin-111 or recombinant human laminin-511 Comprising, wherein at least one of the crosslinking agent and / or the bioactive agent contains an RGD motif; b) One or more different media preferably containing Wnt agonists such as R-spondin and Wnt3a Regarding the contents of the kit.

[0250] According to this aspect, the present invention also relates to the contents of a kit for testing the effect of a drug on colorectal cancer cells, comprising: a) Components for manufacturing an array of well-defined hydrogel matrices having a stiffness of 50 to 2000 Pa to create well-defined three-dimensional extracellular matrix conditions with different biological, biophysical and / or biochemical characteristics from each other, said components being; - One or more different PEG hydrogel precursor molecules, preferably PEG-VS, particularly preferably 4-arm or 8-arm PEG-VS, and / or PEG-Acr, particularly preferably 4-arm or 8-arm PEG-Acr, - At least one crosslinking agent, preferably at least two, preferably a peptide containing two cysteine moieties - Optionally, one or more biologically active molecules including laminin, preferably laminin-111 or laminin-511, particularly preferably natural mouse laminin-111 or recombinant human laminin-511 Comprising, wherein at least one of the crosslinking agent and / or the optional bioactive agent contains an RGD motif; b) Preferably one or more different media containing a Wnt agonist, such as R-spongin and Wnt3a; c) Cells from a cell repository / biobank created using the same extracellular matrix conditions as needed. This includes the contents of the kit.

[0251] The pre-selected extracellular matrix conditions in this configuration are also, a) A step of providing newly isolated or frozen colorectal cancer cells from a biopsy or tissue resection from a cancer patient; b) The step of immobilizing and growing organoids from the cells, and applying one or more drugs to the organoids by the method described above; c) A step of comparing the activity of one or more drugs applied in step b) with the result of treatment of the patient with one of the drugs applied in step b); d) A process to provide results of drug activity on the patient's organoids and corresponding genetic and phenotypic data of the disease to help a physician determine how to treat the patient. It can also be used in methods to test the effectiveness of treatments for cancer patients, including [mention specific examples of other methods].

[0252] For the isolation of colorectal cancer cells, a patient biopsy or resection can be collected during a standard diagnostic procedure for organoid implantation in step b) and then transported to the location where step b) is performed.

[0253] Step b) of this method provides pre-selected extracellular matrix conditions, including, as described above, arrays having separate spaces of a completely clear hydrogel matrix having a rigidity of 50 to 2000 Pa, prepared by crosslinking colorectal cancer cells with one or more different combinations of PEG hydrogel precursor molecules, at least one crosslinking agent, and the presence of one or more biologically active molecules as needed, including laminin, preferably laminin-111 or laminin-511, particularly preferably natural mouse laminin-111 or recombinant human laminin-511, on the surface of the substrate or in separate spaces of the substrate, preferably in a multiwell plate, to create conditions for a completely clear three-dimensional extracellular matrix having biological, biophysical and / or biochemical characteristics distinct from one another; Preferably, the presence of one or more different media containing Wnt agonists, such as R-spongin and Ant 3a, allows the colorectal cancer cells to grow in the separate spaces of the hydrogel matrix; Furthermore, adding one or more drugs to cells grown in the separate spaces of the hydrogel matrix; The method is implemented by including the crosslinking agent and at least one of the optional bioactive agents in the RGD motif.

[0254] One or more drugs added to cells grown in separate spaces within the hydrogel matrix include drugs used for the patient's standard of care (SoC) for cancer treatment.

[0255] In a preferred embodiment, the biopsy or excision can be further processed for histological analysis and / or for omics testing (e.g., NGS) to establish a reference baseline. The results of these additional analyses can also be used to compare and / or correlate the ex vivo and in vivo tests described above.

[0256] Based on this method, it is possible to reliably evaluate whether the applicable standard anti-cancer treatment (SoC) is suitable, or whether different drug treatment regimens tested ex vivo as described above are more promising. In this way, the present invention enables the individualization and optimization of cancer treatment. It can generate functional in vitro data that can increase the accuracy of treatment decisions made by healthcare professionals.

[0257] Breast cancer Breast cancer has distinct subtypes, each requiring different culture conditions. Considerations regarding pancreatic cells are applicable to breast cancer.

[0258] The present invention makes it possible to culture breast cancer cells, such as non-limiting triple-negative (TNBC) or HER2+ receptor breast cancer cells, under conditions that maintain the growth and proliferation of these cells. Subsequently, the cells are exposed to different drug therapies to select an effective drug treatment for the cancer patient. Thus, the present invention provides a precision medicine platform that enables the growth and drug testing of breast cancer tissue in different microenvironments, and thus captures the specificity of multiple clones within a single tumor.

[0259] According to the present invention, it was possible to demonstrate that breast cancer cells can be successfully cultured and tested, preferably under hypoxic (5% O2) conditions, using a medium preferably containing a hydrogel matrix that is completely degradable by a clear enzyme, and preferably FBS (serum) or a Wnt agonist, such as R-spongin. In some subtypes (particularly TNBC subtypes), laminin, preferably laminin-111, and especially preferably natural mouse laminin-111, was preferred as at least one RGD adhesion motif and optionally additional bioactive ligands.

[0260] Thus, according to this embodiment, the present invention is a method for testing the effects of a drug on breast cancer cells, a) A step of providing pre-selected extracellular matrix conditions, comprising an array having separate spaces of a completely clear, preferably enzymatically degradable, hydrogel matrix, prepared by crosslinking one or more different combinations of PEG hydrogel precursor molecules, at least one crosslinking agent, and the breast cancer cells, on the surface of the substrate or in separate spaces of the substrate, preferably in a multiwell plate, in the presence of one or more biologically active molecules as needed, to create conditions for a completely clear, preferably enzymatically degradable, three-dimensional extracellular matrix having distinct biological, biophysical and / or biochemical properties from one another; b) A step of enabling the breast cancer cells to grow in the separate spaces of the hydrogel matrix, preferably in the presence of one or more different media containing FBS (serum) or a Wnt agonist, such as R-spongin; c) Adding one or more drugs to cells grown in the separate spaces of the hydrogel matrix; The present invention relates to a method comprising, wherein at least one crosslinking agent preferably comprises an enzyme-degradable motif, preferably an MMP-sensitive motif.

[0261] Preferably, the array of the hydrogel matrix has a soft or moderate stiffness of 50 to 2000 Pa.

[0262] Preferably, the precursor molecules of the PEG hydrogel are PEG-VS (polyethylene glycol having a terminal vinyl sulfone moiety), particularly preferably 4-arm or 8-arm PEG-VS, and / or PEG-Acr (polyethylene glycol having a terminal acrylate moiety), particularly preferably 4-arm or 8-arm PEG-Acr.

[0263] More preferably, a perfectly clear hydrogel matrix array is produced by crosslinking PEG-VS, particularly preferably 4-arm or 8-arm PEG-VS, or a 50:50 mixture of PEG-VS, particularly preferably 4-arm or 8-arm PEG-VS, and PEG-Acr, particularly preferably 4-arm or 8-arm PEG-Acr, with a peptide containing at least two, preferably two cysteine ​​moieties, as a crosslinking agent, the crosslinking agent may contain an enzymatically degradable motif, preferably an MMP-sensitive motif.

[0264] As needed, a bioactive ligand containing a bioactive motif including an RGD adhesion motif may be used. Examples of suitable RGD motifs include RGD, RGDS, RGSSP, RGDSPG, RGDSPK, RGSTP, RGDSPASSKP, PHSRNSGSGSGSGSGRGDSPG, or any cyclic RGD motif, such as cyclo(RGDfC), but basically any RGD sequence that is known and successfully utilized in the fields of hydrogels and cell culture can be used.

[0265] A ligand containing a bioactive motif may be used as a bioactive ligand as needed. Preferably, the bioactive ligand as needed is selected from the group consisting of natural laminins, such as laminin-111, particularly mouse laminin-111, recombinant laminin isoforms, and their biofunctional fragments. Examples of suitable recombinant laminin isoforms are laminin-111, laminin-211, laminin-332, laminin-411, laminin-511, or laminin-521.

[0266] As needed, glycosaminoglycans, such as hyaluronic acid and hyaluronan, may be used as bioactive ligands. Examples of hyaluronic acid include hyaluronic acid 50k, hyaluronic acid 1000k, hyaluronatethiol 50k, or hyaluronatethiol 1000k.

[0267] In a preferred embodiment, the culture medium described by Sachs et al. (Cell 172 2018, 1-14 (see, for example, table S2 of supplementary material)) may be used. Alternatively, commercially available culture media such as Intesticult®, Mammocult®, WIT-P®, MEBM®, or StemPro® hESC SFM may be used. Particularly preferred is the culture medium described by Sachs et al., which comprises R-spongin 1 condition medium or R-spongin 3, Neureglin 1, FGF7, FGF10, EGF, Noggin, A83-01, Y-27632, SB202190, B27 supplement, N-acetylcysteine, nicotinamide, GlutaMax 100×, Hepes, penicillin / streptomycin, Primocin, and Advanced DMEM / F12. Other media may be used, such as IMDM + FBS (serum), or the media described in Liu et al. (Sci Rep 2019, (9):622) or Lancaster et al. (Nat Biotechnol 2017 35(7): 659-666).

[0268] In a preferred embodiment, low-oxygen (5% O2) conditions are preferred.

[0269] Particularly preferred are breast cancer cells that are newly isolated or frozen cells obtained from human biopsy or tissue excision, or from patient-derived xenograft (PDX) tissue.

[0270] By the method described above, breast cancer cells can be grown and proliferated in a selected culture medium under extracellular matrix conditions that reproduce the drug effects observed in vivo, and then tested.

[0271] In a particularly preferred embodiment, the extracellular matrix conditions are selected so as not to use any naturally derived matrix, such as Matrigel®.

[0272] According to this embodiment, the present invention relates to the contents of a kit for testing the effects of a drug on breast cancer cells, and furthermore: a) A component for producing a completely clear, preferably enzymatically degradable, hydrogel matrix array in order to create conditions for a completely clear three-dimensional extracellular matrix having distinct biological, biophysical and / or biochemical characteristics from one another, wherein the component is: - One or more different PEG hydrogel precursor molecules, preferably PEG-VS, particularly preferably 4-arm or 8-arm PEG-VS, and / or PEG-Acr, particularly preferably 4-arm or 8-arm PEG-Acr, - A peptide comprising at least one crosslinking agent, preferably at least two, preferably two cysteine ​​moieties, - One or more biologically active molecules as needed A component comprising, wherein at least one crosslinking agent preferably comprises an enzyme-degradable motif, preferably an MMP-sensitive motif; b) One or more different culture media, preferably FBS (serum) or a Wnt agonist, such as R-sponge. This includes the contents of the kit.

[0273] According to this embodiment, the present invention relates to the contents of a kit for testing the effects of a drug on breast cancer cells, and furthermore: a) A component for producing a completely clear, preferably enzymatically degradable, hydrogel matrix array in order to create conditions for a completely clear three-dimensional extracellular matrix having distinct biological, biophysical and / or biochemical characteristics from one another, wherein the component is: - One or more different PEG hydrogel precursor molecules, preferably PEG-VS, particularly preferably 4-arm or 8-arm PEG-VS, and / or PEG-Acr, particularly preferably 4-arm or 8-arm PEG-Acr, - A peptide comprising at least one crosslinking agent, preferably at least two, preferably two cysteine ​​moieties, - One or more biologically active molecules as needed, A component comprising, wherein at least one crosslinking agent preferably comprises an enzyme-degradable motif, preferably an MMP-sensitive motif; b) Preferably one or more different culture media containing FBS (serum) or a Wnt agonist, such as R-spongin; c) Cells from a cell repository / biobank created using the same extracellular matrix conditions as needed. This includes the contents of the kit.

[0274] The pre-selected extracellular matrix conditions in this configuration are also, a) A step of providing newly isolated or frozen breast cancer cells from a biopsy or tissue excision from a cancer patient; b) A step of immobilizing and growing organoids from the cells, and applying one or more drugs to the organoids by the method described above; c) A step of comparing the activity of one or more drugs applied in step b) with the result of treatment of the patient with one of the drugs applied in step b); d) A process to provide results of drug activity on the patient's organoids and corresponding genetic and phenotypic data of the disease to help a physician determine how to treat the patient. It can also be used in methods to test the effectiveness of treatments for cancer patients, including [mention specific examples of other methods].

[0275] For the isolation of breast cancer cells, a patient biopsy or excision can be collected during a standard diagnostic procedure for organoid implantation in step b), and then transported to the location where step b) is performed.

[0276] Step b) of this method provides pre-selected extracellular matrix conditions, including, as described above, that is, arrays having separate spaces of a completely clear, preferably enzymatically degradable, hydrogel matrix, prepared by crosslinking one or more different combinations of PEG hydrogel precursor molecules, at least one crosslinking agent, and the breast cancer cells, on the surface of the substrate or in separate spaces of the substrate, preferably in a multiwell plate, in the presence of one or more biologically active molecules as needed, to create conditions for a completely clear, preferably enzymatically degradable, three-dimensional extracellular matrix having different biological, biophysical and / or biochemical properties from one another; Preferably, the presence of one or more different media containing FBS (serum) or a Wnt agonist, such as R-spongin, allows the breast cancer cells to grow in the separate spaces of the hydrogel matrix; Furthermore, adding one or more drugs to cells grown in the separate spaces of the hydrogel matrix; This is carried out by the method, and at least one of the crosslinking agents preferably comprises an enzyme-degradable motif, preferably an MMP-sensitive motif.

[0277] The one or more drugs added to the separate spaces of the hydrogel matrix include drugs used for the patient's standard anti-cancer treatment (SoC).

[0278] In a preferred embodiment, the biopsy or excision can be further processed for histological analysis and / or for omics testing (e.g., NGS) to establish a baseline for reference. The results of these additional analyses can also be used to compare and / or correlate the ex vivo and in vivo tests described above.

[0279] Based on this method, it is possible to reliably evaluate whether the applicable standard anti-cancer treatment (SoC) is suitable, or whether different drug treatment regimens tested ex vivo as described above are more promising. In this way, the present invention enables the individualization and optimization of cancer treatment. It can generate functional in vitro data that can increase the accuracy of treatment decisions made by healthcare professionals.

[0280] prostate cancer This aspect demonstrates the benefits provided by the present invention with respect to the problem of selective growth of cancer cells compared to normal cells (e.g., wild-type healthy cells, stromal cells).

[0281] With respect to certain organs, patient-derived cancer cells that form tumor organoids tend to proliferate more slowly ex vivo than their healthy (wild-type) counterpart cells, and / or the ex vivo conditions currently used based on Matrigel® or equivalent matrices cannot select one tissue type from others and cannot be favorable to its growth.

[0282] Therefore, normal cells tend to overgrow more than cancer organoid cultures unless specific measures are taken. Despite the fact that in some cases modifications of the culture medium composition can solve this problem, the overgrowth of normal cells (healthy cells) compared to cancer cells remains a problem, for example, in the case of prostate cancer. This impairs the establishment of ex vivo growth of patient-derived cancer cells as a physiological preclinical model for determining which drugs or drug combinations may be effective in treating a particular patient.

[0283] Overgrowth of normal cells has been observed particularly in prostate cancer organoids (Drost et al., Development (2017) 144, 968-975). For example, there are currently fewer than 10 existing prostate cancer cell lines (compared to over 50 for colon cancer), and none of them adequately reflect the exact nature of the cancer (ATCC and ECACC cell banks). Therefore, if prostate cancer organoids could be grown while inhibiting the growth of normal cells, for example, this would significantly impact the development of new drugs by using a more physiologically accurate preclinical model and enable the use of patient-specific cell models for personalized medicine applications.

[0284] Drost et al., Development (2017) 144, 968-975 (see “Personalized cancer therapy” on pages 971-972) summarizes the selections made between (i) cancer cells and (ii) normal cells (wild-type) to grow a pure cell population of (i) ex vivo compared to (ii). Briefly, this is achieved, where possible, by adding or omitting chemicals / growth factors in the cell culture medium. However, in the case of prostate cancer, it is not as straightforward as, for example, certain colon cancers with specific gene mutations, where successful cultivation of its cells is possible independently of specific chemicals compared to wild-type counterpart cells. As described in Drost et al., Nature protocols, Vol.11 no.2 (2016) 347 (see “Limitation of the method” on pages 347-348), their culture protocol was not good enough to grow organoids derived from primary prostate cancer, most likely because tumor cells do not have a selective advantage over normal cells ex vivo. As a result, normal prostate cells, which are normally present in each cancer tissue sample, appear to overgrow compared to tumor cells (see also the last paragraph of “Discussion” on page 171, Karthaus et al., Cell 159, 163-175, September 25, 2014). Furthermore, similar issues have also been observed in biopsies of prostate metastases in bone and soft tissue (see “Results” on pages 177-178, Gao et al., Cell 159, 176-187, September 25, 2014), where normal host tissue cells (e.g., stromal cells and / or epithelial cells) outnumbered cancer cells.

[0285] Other examples of normal cell overgrowth that are not generally reported include ex vivo cultures of breast and lung cancer. In a study published by Sachs et al. (Cell 172 2018, 1-14) demonstrating the engraftment of over 100 primary and metastatic breast cancer organoids, there are a pair of cases where the organoid pathology was classified as normal, but the original histopathology was classified as tumorous (Sachs et al., Cell 172 2018, 1-14, Table S3). This can also be observed in lung cancer organoids derived from patients with p53 mutations, for example, where normal cells and cancer-like cells can be selected by adding chemicals to the cell culture medium (Sachs et al., The EMBO Journal e 100300|2019). However, in the absence of p53 mutations, there is no way to prevent normal cell overgrowth.

[0286] According to this aspect of the present invention, pre-selected extracellular matrix conditions that promote the growth of prostate cancer cells but simultaneously prevent the establishment of their normal counterpart cells have been identified. This makes it possible to establish a reliable screening method for cancer cells in which, otherwise, their normal counterpart cells would overgrow.

[0287] In detail, normal prostate cells grew only in gel formulations containing RGD adhesion motifs, and their growth was found to be better in softer gels compared to moderately stiff or rigid gels. On the other hand, prostate cancer cells isolated from patients or patient-derived xenograft (PDX) tumors showed similar growth in gels in and without the presence of RGD motifs. Some prostate cancer cells isolated from patient-derived xenograft (PDX) tumors grew even better in gels without RGD (soft or moderately stiff).

[0288] Thus, according to this embodiment, the present invention is a method for testing the effects of a drug on cancer cells, preferably prostate cancer cells, that proliferate more slowly than their normal corresponding cells or associated stromal cells in ex vivo. a) A step of providing pre-selected extracellular matrix conditions, comprising an array having separate spaces of a completely distinct hydrogel matrix, prepared by crosslinking one or more different combinations of PEG hydrogel precursor molecules, at least one crosslinking agent, and cancer cells, on the surface of a substrate or in separate spaces of a substrate, preferably in a multiwell plate, in the presence of one or more biologically active molecules as needed, in order to create conditions for a completely distinct three-dimensional extracellular matrix with biological, biophysical and / or biochemical properties distinct from one another; b) A step that allows the cancer cells to grow in the separate spaces of the hydrogel matrix in the presence of one or more different culture media; c) Adding one or more drugs to cells grown in the separate spaces of the hydrogel matrix; The present invention relates to a method comprising the crosslinking agent and the optional biological activator, wherein the crosslinking agent and the optional biological activator do not contain any RGD motifs.

[0289] Preferably, the precursor molecules of the PEG hydrogel are PEG-VS (polyethylene glycol having a terminal vinyl sulfone moiety), particularly preferably 4-arm or 8-arm PEG-VS, and / or PEG-Acr (polyethylene glycol having a terminal acrylate moiety), particularly preferably 4-arm or 8-arm PEG-Acr.

[0290] More preferably, arrays of completely distinct self-degradable hydrogel matrices are produced by crosslinking a 50:50 mixture of PEG-Acr, particularly preferably 4-arm or 8-arm PEG-Acr, or PEG-VS, particularly preferably 4-arm or 8-arm PEG-VS, with a peptide containing at least two, preferably two, cysteine ​​moieties as a crosslinking agent.

[0291] Preferably, the array of the hydrogel matrix has a soft or moderate stiffness of 50 to 2000 Pa.

[0292] In another preferred embodiment, a matrix array of completely distinct, non-self-degrading hydrogels is produced by crosslinking PEG-VS, particularly preferably 4-arm or 8-arm PEG-VS, with a peptide containing at least two, preferably two cysteine ​​moieties, as a crosslinking agent.

[0293] A ligand containing a bioactive motif may be used as a bioactive ligand as needed. Preferably, the bioactive ligand as needed is selected from the group consisting of tenacin C and glypican, natural laminins, such as laminin-111, particularly mouse laminin-111, recombinant laminin isoforms, and their biofunctional fragments. Examples of suitable recombinant laminin isoforms are laminin-111, laminin-211, laminin-332, laminin-411, laminin-511, or laminin-521.

[0294] Preferably, the culture medium was pre-selected for growing prostate cancer cells. Commercial culture media, such as Mammocult®, WIT-P®, StemPro® hESC SFM, Lonza’s PrEGM® BulletKit® (ref. CC-3166), and NutriStem® hPSC XF, may also be used. Furthermore, the culture media described in International Publication No. 2015 / 173425, Drost et al. (Nature Protocol 11, 347-358, January 2016), Beshiri et al. (Clinical Cancer Research 24, 4332-4345, May 2018), or Puca et al. (Nature Communications 9:2404, 1-10, June 2018), or Inc et al. (Cancer Cell 12, 160-170, August 2007) are suitable for cancer cell growth. The culture medium described in International Publication No. 2015 / 173425 has been found to be favorable for the growth of cancer cells. Particularly preferred are culture media containing glutamine, BSA, transferrin, noggin, FGF (2 or basic), FGF10, EGF, R-spongin-conditioned medium or recombinant, penicillin / streptomycin, glutathione, nicotinamide, DHT, prostaglandin E2, A83-01, Y-27632, N-acetylcysteine, SB202190, and Hepes.

[0295] Particularly preferred are cancer cells that are newly isolated or frozen human cells obtained from human biopsy or tissue excision, or from patient-derived xenograft (PDX) tissue.

[0296] By the method described above, cancer cells can be grown in a selected culture medium under conditions that reproduce drug outcomes observed in vivo, without overgrowth of their normal corresponding cells or associated stromal cells, and then tested.

[0297] In a particularly preferred embodiment, the extracellular matrix conditions are selected so as not to use any naturally derived matrix, such as Matrigel®.

[0298] According to this embodiment, the present invention also relates to the contents of a kit for testing the effects of a drug on cancer cells, preferably prostate cancer cells, that proliferate more slowly than their normal corresponding cells or associated stromal cells in ex vivo, the following: a) Components for producing a matrix array of perfectly clear hydrogels to create conditions for a perfectly clear three-dimensional extracellular matrix having distinct biological, biophysical and / or biochemical properties from one another, wherein the components are as follows: - One or more different PEG hydrogel precursor molecules, preferably PEG-VS, particularly preferably 4-arm or 8-arm PEG-VS, and / or PEG-Acr, particularly preferably 4-arm or 8-arm PEG-Acr, - A peptide comprising at least one crosslinking agent, preferably at least two, preferably two cysteine ​​moieties, - One or more biologically active molecules as needed, The components include, wherein the crosslinking agent and the optional biological agent do not contain any RGD motif; b) One or more different culture media This includes the contents of the kit.

[0299] According to this embodiment, the present invention also relates to the contents of a kit for testing the effects of a drug on cancer cells, preferably prostate cancer cells, that proliferate more slowly than their normal corresponding cells or associated stromal cells in ex vivo, the following: a) Components for producing a matrix array of perfectly clear hydrogels to create conditions for a perfectly clear three-dimensional extracellular matrix having different biological, biophysical and / or biochemical properties from one another, wherein the components are as follows: - One or more different PEG hydrogel precursor molecules, preferably PEG-VS, particularly preferably 4-arm or 8-arm PEG-VS, and / or PEG-Acr, particularly preferably 4-arm or 8-arm PEG-Acr, - A peptide comprising at least one crosslinking agent, preferably at least two, preferably two cysteine ​​moieties, - One or more biologically active molecules as needed, The components include, wherein the crosslinking agent and the optional biological agent do not contain any RGD motif; b) One or more different culture media; c) Cells from a cell repository / biobank created using the same extracellular matrix conditions as needed. This includes the contents of the kit.

[0300] The pre-selected extracellular matrix conditions in this configuration are also, a) A process of providing newly isolated or frozen cancer cells from a biopsy or tissue excision from a cancer patient; b) A step of immobilizing and growing organoids from the cells, and applying one or more drugs to the organoids by the method described above; c) A step of comparing the activity of one or more drugs applied in step b) with the result of treatment of the patient with one of the drugs applied in step b); d) A process to provide drug activity results and / or corresponding genetic and phenotypic data on the patient's organoids to help a physician determine how to treat the patient. It can also be used in methods to test the effectiveness of treatments for cancer patients, including [mention specific examples of other methods].

[0301] For the isolation of prostate cancer cells, a patient biopsy or resection can be collected during a standard diagnostic procedure for organoid implantation in step b) and then transported to the location where step b) is performed.

[0302] Step b) of this method provides pre-selected extracellular matrix conditions, including, as described above, arrays having separate spaces of a matrix of completely distinct, non-self-degrading hydrogels, prepared by crosslinking one or more different combinations of PEG hydrogel precursor molecules, at least one crosslinking agent, and the cancer cells, on the surface of the substrate or in separate spaces of the substrate, preferably in a multi-well plate, in the presence of one or more biologically active molecules as needed, to create conditions for a completely distinct three-dimensional extracellular matrix with biological, biophysical and / or biochemical properties that are distinct from one another; To enable the cancer cells to grow in the separate spaces of the hydrogel matrix in the presence of one or more different culture media; Furthermore, adding one or more drugs to cells grown in the separate spaces of the hydrogel matrix; The process is carried out by the crosslinking agent and the optional bioactive agent, and the crosslinking agent and the optional bioactive agent do not contain any RGD motifs.

[0303] One or more drugs added to separate spaces in the hydrogel matrix include drugs used for the patient's standard anti-cancer treatment (SoC).

[0304] In a preferred embodiment, the biopsy or excision can be further processed for histological analysis and / or for omics testing (e.g., NGS) to establish a baseline for reference. The results of these additional analyses can also be used to compare and / or correlate the ex vivo and in vivo tests described above.

[0305] Based on this method, it is possible to reliably evaluate whether the applicable standard anti-cancer treatment (SoC) is suitable, or whether different drug treatment regimens tested ex vivo as described above are more promising. In this way, the present invention enables the individualization and optimization of cancer treatment. It can generate functional in vitro data that can increase the accuracy of treatment decisions made by healthcare professionals.

[0306] Dynamic organoid growth image analysis method It is necessary to accurately and easily identify different parameters of organoid growth (e.g., growth rate, number of organoids, organoid size). In conventional 2D cell culture, the quantification of organoid growth can be achieved indirectly using fluorescence, colorimetric, or luminescence methods to measure the amount of metabolites in the culture well (e.g., Alamar Blue, MTT, Cell Titer Glow 3D). All of these indirect assays can affect organoid suitability, even if they are not lethal to cells, and completely hinder the possibility of further use of the grown organoids (e.g., for drug testing, regenerative medicine).

[0307] Therefore, using non-invasive (and label-free) methods such as optical microscopy to quantify organoid growth is a favorable alternative in long-term culture and / or when organoids need to be maintained as "natural" and "unused" as possible (e.g., for regenerative medicine, biobanking). The need for high-throughput imaging quantification of 3D organoid cultures has led to modifications of 2D methods (Carpenter et al., Genome Biology 2006, 7:R100) or the development of novel automated detection and image segmentation algorithms that enable rapid, reproducible, and unbiased counting and measurement of organoids. These methods are easily and accurately performed using fluorescent markers (Robinson et al., PloS ONE 10(12): e0143798. doi:10.1371 / journal.pone.01437982015, Boutin et al., Nature scientific reports (2018) 8:11135, DOI:10.1038 / s41598-018-29169-0 2018), and even in these cases, they require immunofluorescence staining or fluorescent transgene expression, making them incompatible with the use of "unused" and "unlabeled" patient-derived cells or re-implantation protocols.

[0308] Recently, in Borten et al., Nature scientific reports (2018) 8:5319, DOI:10.1038 / s41598-017-18815-8 2018, an algorithm based on Matlab® (Mathworks Inc.) called OrganoSeg was developed to specifically analyze organoids from 3D bright-field images, thereby enabling the detection, segmentation (i.e., distribution of digital images into specific pixel sets), and quantification of numerous parameters from living, naturally occurring organoids that have grown in 3D (Borten 2018). This open-source software enables the identification and multiparametric morphological classification of organoids based on the size, sphericity, and shape of features detected at a given time point.

[0309] However, while accurate and powerful, this tool cannot account for the time dimension and requires multiple analyses at different points in time, thus necessitating complex subsequent work to adequately assess the dynamics of organoid growth.

[0310] This invention provides a novel analytical method. Based on MATLAB® code, a novel method has been developed that can align bright-field images acquired at different time points and automatically identify and segment organoids based on their intensity. This program segments objects from bright-field images using the same method as OrganoSeg. The main difference lies in the use of these segmented objects: OrganoSeg classifies different types of organoids at given separate time points using size and morphology, whereas this novel program enables dynamic tracking of organoid growth in a single analysis, thus enabling the calculation of OFE / AIF and drug response. Therefore, the program provides the following dynamic information regarding organoid growth: • Organoid formation efficiency (OFE) and • Area Increasing Factor (AIF).

[0311] The method of the present invention also makes it possible to create aligned time-lapse videos for each acquired well, as well as "time projections" representing the overall growth of the organoids over the culture period in single images. These time projections can be easily included in presentations and publications.

[0312] Single cells or small aggregates of cells are encapsulated within a 3D extracellular matrix, and only a subset of these cells can grow to form organoids; this is designed as the "organoid formation efficiency" (OFE) of the culture. The method of the present invention quantifies the number of encapsulated cells on day 0. Furthermore, this method allows the user to define a threshold size for what is considered an organoid. This then provides the OFE for any particular point in time in the assay.

[0313] OFE provides an indicator of the proportion of original cultured cells (e.g., stem cells) that are capable of developing in the organoid, but by the method of the present invention, the growth rate of the entire organoid culture is also quantified by calculating the increase in area along time after segmentation of time-lapse photography. This is the “Area Increase Factor” (AIF), which corresponds to the ratio of the total area occupied by the organoid at any given day to the total area occupied by a single cell at day 0. The method of the present invention allows for the selection of the first and last days to calculate the AIF.

[0314] Combining OFE and AIF scores provides useful information regarding the suitability and performance of a given extracellular matrix condition.

[0315] This semi-automated image analysis method according to the present invention enables time-based study and analysis of organoid growth in high-throughput settings. It provides unbiased and reproducible scoring that reflects the suitability and performance of extracellular matrix conditions for organoid culture without the need for markers and / or harmful assays. It also enables semi-automated quantification of drug test results for patient-derived organoids (e.g., IC). 50 Determining the value).

[0316] Example 1: Testing of lung cancer cells Lung cancer cells overexpressing 1a c-Met receptor Lung cancer cells overexpressing the c-Met receptor were obtained from patients using PDX cells. Upon activation by ligand binding, the c-Met receptor autophosphorylates, activating several intracellular signaling cascades.

[0317] When xenograft (PDX) cells derived from patients with the non-small cell lung cancer (NSCLC) model LXFA-1647 are treated with a c-Met-targeting drug (c-Met inhibitor: PF-04217903, Selleck Chemicals), their autophosphorylation is inhibited, inducing tumor growth regression in vivo.

[0318] PEG was used as a precursor molecule for hydrogels to create non-self-degrading hydrogels. As crosslinking agents, peptides containing at least two, preferably two cysteine ​​moieties, differing in their amino acid sequences, particularly with respect to the presence or absence of the RGD adhesion motif, and with respect to the presence or absence of the MMP degradation sequence, were used. Another aspect performed on some hydrogels was the binding of bioactive ligands containing the RGD adhesion motif and / or ligands selected from the group consisting of natural laminin, recombinant laminin isoforms, and their biofunctional fragments. Examples of preferred recombinant laminin isoforms are laminin-111, laminin-211, laminin-332, laminin-411, laminin-511, or laminin-521. Arrays of pre-selected, characterized hydrogels were established by the methods described above.

[0319] The mechanical properties of the hydrogels also differed (soft (50-1000 Pa), moderate (1000-2000 Pa), or hard (2000-3000 Pa) gels).

[0320] For comparison, the same procedure was also performed with Matrigel®, a naturally derived matrix whose origin is not clearly defined.

[0321] The culture medium was pre-selected to contain the c-Met inhibitor PF-04217903 (Selleck Chemicals), i.e., a drug that targets c-met and inhibits its autophosphorylation, or a drug used in the standard of care (SoC) for this cancer (docetaxel). Each drug was added to the medium after 1–8 days of culture (1–8 days after cell encapsulation). The drug response was measured 5–10 days after drug addition. Preferred media were pre-selected to be those characterized by the presence of FBS (serum) or a Wnt agonist, such as R-spongin. Following this example, a modified medium from the one described in Sachs et al. (The EMBO Journal e 100300|2019) was used. The preferred medium contained glutamine, noggin, EGF, fibroblast growth factor 7 and 10 [FGF7 and FGF10], HGF, R-spongin-conditioned medium, primocin, penicillin / streptomycin, N-acetyl-L-cysteine, nicotinamide, A83-01, SB202190 (p38 inhibitor), Y-27632 (rock inhibitor), B27 supplement, and AdDMEM / F12 supplemented with HEPES. Target expression (c-Met and phospho-c-Met) was detected by Western blotting under the corresponding growth conditions.

[0322] The results are shown in Figures 1a, 1b, and 1c.

[0323] These pre-selected conditions identified ex vivo growth conditions (ex vivo culture, extracellular matrix) that reproduced the drug outcomes observed in vivo (i.e., activity of the c-Met inhibitor PF-04217903 (Selleck Chemicals)). These extracellular matrix conditions were considered "responder conditions."

[0324] In this embodiment, the most preferred responder condition was the use of a non-self-degrading hydrogel made from a peptide containing two cysteine ​​moieties but no RGD motif (either in the crosslinker or bound to the hydrogel) as a precursor molecule of each PEG hydrogel and as a crosslinking agent. The hydrogel has a soft rigidity of 50 to 1000 Pa (Example 1a), more preferably 250 to 500 Pa.

[0325] In the same assay, other conditions that induce drug resistance were identified. Drug resistance was found to be dependent on the microenvironment, i.e., the extracellular matrix or soluble factors. In particular, we were able to demonstrate that when a 1 mM bioactive ligand containing an RGD motif bound to a hydrogel, tumor cells became resistant to the c-Met inhibitor PF-04217903 (Selleck Chemicals). These conditions are considered "non-responder conditions" (Example 1b).

[0326] Finally, in the same assay, the use of Matrigel® as the matrix (Comparative Example 1) also provided a “non-responsive condition” (Comparative Example 1) in which tumor cells did not respond to the c-Met inhibitor PF-04217903 (Selleck Chemicals).

[0327] Figure 1b shows the effects of standard treatment (SoC) with docetaxel and treatment with the c-met inhibitor PF-04217903 (Selleck Chemicals) under the conditions of Example 1a. Both drugs were clearly effective.

[0328] In contrast, Figure 1c shows that under the conditions of Comparative Example 1 (Matrigel®), only the effect of standard treatment (SoC) with docetaxel was observed. No therapeutic effect was observed with the c-met inhibitor PPF-04217903 (Selleck Chemicals). Therefore, Figures 1a-1c show that c-met inhibitors have an effect on the cells examined only under the prior selection conditions of the present invention. When tested under conventional conditions (i.e., Matrigel®), no potential therapeutic effect of c-met inhibitors was recognized.

[0329] 1b Lung cancer cells overexpressing the EGFR receptor Example 1a was repeated with lung cancer cells overexpressing the EGFR receptor. These cells were obtained from PDX cells.

[0330] In Example 1a, using the “responder condition” (i.e., without any RGD motifs (bound to the crosslinking agent or hydrogel)), no therapeutic effect with c-met inhibitors was observed in Example 1b, as expected due to the lack of autophosphorylation of the c-met receptor in the cells tested in Example 1b (see Figure 1d). On the other hand, EGFR receptors and their phosphorylated forms were overexpressed under these conditions (Figure 1d), but SoC treatment with drugs acting on the EGFR receptor (erlotinib and cetuximab) and paclitaxel showed clear effects (similar to the conditions in Comparative Example 1 using Matrigel® (data not shown)) (Figure 1e).

[0331] Example 2: Testing of pancreatic cancer cells We first propagated pancreatic ductal adenocarcinoma (PDAC) cancer cells from patients in mice as a PDX model. PDX-derived cells were then grown within a range of extracellular matrix conditions.

[0332] Treating patient-derived xenograft (PDX) cells from the pancreatic ductal adenocarcinoma (PDAC) cancer model PAXF736 with an EGFR-targeting drug (EGFR inhibitor: cetuximab) reduces tumor growth in vivo.

[0333] PEG was used as a precursor molecule for hydrogels to create non-self-degrading hydrogels. As a crosslinking agent, peptides containing at least two, preferably two cysteine ​​moieties, differing in their amino acid sequences, particularly with respect to the presence or absence of the RGD adhesion motif and the presence or absence of the MMP degradation sequence, were used. Another aspect performed on some hydrogels was the binding of a bioactive ligand containing an RGD or cyclic RGD adhesion motif, or a bioactive ligand containing a DGEA motif. Arrays of pre-selected hydrogels with differing characteristics were established by the methods described above.

[0334] The mechanical properties of the hydrogels were also diverse (hard (2000-3000 Pa), moderate (1000-2000 Pa), or soft (50-1000 Pa) gels).

[0335] A variety of different known, commonly used, and / or commercially available culture media were used.

[0336] Figures 2a and 2b show the results for a soft, non-self-degrading PEG hydrogel (Example 2a) having a crosslinked portion without an RGD motif and a bioactive ligand with an RGD adhesion motif, and a soft, non-self-degrading PEG hydrogel (Example 2b) having a crosslinked portion with an RGD motif and a bioactive ligand containing a DGEA adhesion motif. For comparison, tests were also conducted on an unidentified naturally derived Matrix Matrigel® (Comparative Example 2).

[0337] Figure 2a shows that all the hydrogels tested resulted in comparable growth of patient-derived xenograft (PDX) cells from the pancreatic ductal adenocarcinoma (PDAC) cancer model PAXF736.

[0338] Figure 2b shows that the hydrogel of Example 2a exhibited drug sensitivity (to cetuximab) equivalent to that of Matrigel® (Comparative Example 2). On the other hand, the hydrogel of Example 2b showed considerably higher drug sensitivity.

[0339] Figure 2c shows that when a soft gel (50-1000 Pa, Examples 2c and 2d) or a moderate gel (1000-2000 Pa, Examples 2e and 2f) was used in the presence of the RGD motif and in the presence (Examples 2c and 2e) or absence (Examples 2d and 2f) of the MMP-sensitive motif, very good growth of PDAC cells was achieved, which was comparable to the growth of PDAC cells using Matrigel® (Comparative Example 2).

[0340] The presence of Wnt agonists in the culture medium, such as R-spongin and Wnt3a, was found to be important for cell growth. Similarly, it was found that the hydrogel matrix must contain at least one RGD motif.

[0341] This example demonstrates the advantages of prior selection according to the present invention. When tested under conventional extracellular matrix conditions using Matrigel® (Comparative Example 2), no effect of EGFR inhibitors on the tested cells was observed. Therefore, a potentially effective treatment for this cancer has not yet been identified.

[0342] A comparison of Examples 2a and 2b demonstrates another advantage of the prior selection according to the present invention. By using different prior selection criteria that are essentially favorable for a particular cell type (in this example, the presence of the RGD motif), it is possible to identify potential resistance in the cells being tested. In Example 2a, the drug sensitivity observed to the EGFR inhibitor cetuximab was considerably lower compared to Example 2b, indicating that treating this particular type of cancer cell with an EGFR inhibitor alone is insufficient.

[0343] Example 3: Testing of pancreatic cancer cells co-cultured with fibroblasts PDAC cells (from PDOs pre-implanted in Matrigel®) and a different ratio cancer-associated fibroblasts Co-culture with fibroblasts (isolated from patients and pre-grown in 2D culture) was investigated within a range of extracellular matrix conditions. Fibroblasts in co-culture were identified using a specific marker (CD90).

[0344] Figure 3 shows the results of co-culturing 33% PDAC cells with 67% fibroblasts for hydrogels containing both the RGD motif and the enzymatically (MMP)-degradable portion (Example 3a), a hydrogel containing only the RGD motif and no enzymatically degradable portion (Example 3b), a hydrogel containing only the enzymatically degradable portion and no RGD motif (Example 3c), and a hydrogel containing neither the RGD motif nor the enzymatically degradable portion (Example 3d). For comparison, the results for Matrigel® (Registered Trademark), a conventional matrix of unclear natural origin (Comparative Example 3), are also shown.

[0345] It can be seen that the best co-culture results were obtained in Example 3a, preferably in a soft PEG hydrogel containing both the RGD motif and the enzymatically degradable portion.

[0346] This embodiment demonstrates that conditions can be pre-selected depending on whether or not the simultaneous growth of other cells, such as fibroblasts, should be permitted.

[0347] Example 4: Test for colorectal cancer Colorectal cancer (CRC) cells from patients and from pre-implanted Matrigel® were grown under specific extracellular matrix conditions.

[0348] PEG was used as a precursor molecule for hydrogels to provide non-self-degrading PEG hydrogels, or a 50:50 mixture of non-self-degrading PEG hydrogels and self-degrading PEG hydrogels. As crosslinking agents, peptides containing at least two, preferably two cysteine ​​moieties, with different amino acid sequences, particularly with respect to the presence or absence of RGD adhesion motifs and the presence or absence of MMP degradation sequences, were used. Another aspect performed on some hydrogels was the binding of a bioactive ligand containing an RGD adhesion motif, or a ligand selected from the group consisting of natural laminins, e.g., laminin-111, recombinant laminin isoforms, e.g., recombinant human laminin-511, and their biofunctional fragments. Examples of suitable recombinant laminin isoforms are laminin-111, laminin-211, laminin-332, laminin-411, laminin-511, or laminin-521. Arrays of hydrogels with pre-selected characteristics were established by the method described above.

[0349] The mechanical properties of the hydrogels also differed (hard (2000-3000 Pa), moderate (1000-2000 Pa), or soft (50-1000 Pa) gels).

[0350] A variety of different known, commonly used, and / or commercially available culture media were used.

[0351] The results are shown in Figure 4. Figure 4 shows bright-field images of human colon cancer organoids grown for 0 and 11 days.

[0352] Examples 4a-4c used non-autodegradable and non-enzymatically degradable hydrogels. In Example 4b, the crosslinking agent contained an RGD motif, and in Examples 4a and 4c, a bioactive ligand containing the RGD motif was bound in a hanging manner. In Example 4b, the bioactive ligand laminin-111 was bound in a hanging manner. The hydrogels of Examples 4a and 4b were soft (less than 500 Pa), while the hydrogel of Example 4c was moderately soft (greater than 1000 Pa). In Example 4d, an autodegradable but non-enzymatically degradable hydrogel with an initial stiffness of 400-600 Pa was used. This hydrogel contained a crosslinking agent containing an RGD motif and laminin-111 as the bioactive ligand. For comparison, tests were also conducted with Matrigel® (Comparative Example 4), a matrix of unclear natural origin.

[0353] Examples 4a-4d showed cell growth comparable to that of standard Matrigel® in the hydrogels, but this was not observed under specific conditions (different from those of Matrigel®).

[0354] On the other hand, in Example 4e, a hydrogel was used that was non-autodegradable but enzymatically degradable and contained no RGD motifs. Under these conditions, the tested CRC cells did not grow.

[0355] In Example 4f, a self-degrading hydrogel with an initial stiffness of approximately 400-600 Pa, containing an RGD motif (integrated as a crosslinking agent) and recombinant human laminin-511 as a bioactive agent, was used. Very good growth of the tested CRC cells was observed.

[0356] The presence of Wnt agonists, such as R-spongin and Wnt3a, in the culture medium was found to be advantageous for cell growth. Similarly, it was found that the hydrogel matrix must contain at least one RGD motif, as well as at least one bioactive ligand selected optionally from the group consisting of native laminins, such as laminin-111, recombinant laminin isoforms, such as recombinant human laminin-511, and their biofunctional fragments.

[0357] Example 5: Testing of breast cancer cells Breast cancer cells derived from patients with distinct cancer subtypes (triple-negative (TNBC) or HER2+ receptor status) were first grown in mice as a PDX model. Next, PDX-derived cells were grown under conditions of a specific range of extracellular matrix.

[0358] A non-self-degrading hydrogel was provided using PEG as a precursor molecule for the hydrogel. As a crosslinking agent, a peptide containing at least two, preferably two cysteine ​​moieties, which differ in their amino acid sequences, particularly with respect to the presence or absence of the RGD adhesion motif and the presence or absence of the MMP degradation sequence, was used.

[0359] Another approach performed on some hydrogels involved the binding of bioactive ligands containing RGD adhesion motifs, and / or ligands selected from the group consisting of natural laminins, e.g., laminin-111, recombinant laminin isoforms, and their biofunctional fragments. Examples of suitable recombinant laminin isoforms include laminin-111, laminin-211, laminin-332, laminin-411, laminin-511, or laminin-521. Arrays of pre-selected hydrogels with differing characteristics were established by the method described above.

[0360] The mechanical properties of the hydrogels also differed (hard (2000-3000 Pa), moderate (1000-2000 Pa), or soft (50-1000 Pa) gels).

[0361] A variety of different known, commonly used, and / or commercially available culture media were used.

[0362] The tests were conducted under hypoxic (5% O2) or normal oxygen (18% O2) conditions.

[0363] The presence of FBS (serum) or a Wnt agonist, such as R-spongin, in the culture medium was found to be advantageous for cell growth. Similarly, it was found that the hydrogel matrix should preferably be enzymatically soluble.

[0364] Figure 5 shows the results of different breast cancer cell growth. Bright-field images (4× objective) of human primary or metastatic (Mets) breast cancer cells from four patients with HER2+ breast cancer or triple-negative breast cancer (TNBC) (from patient-derived xenograft models).

[0365] As can be seen from the bottom row of the figure, the hydrogel of Example 5a (a non-autodegradable, enzymatically degradable soft (<500 Pa) PEG hydrogel containing the RGD motif and laminin-111 as a bioactive ligand) after the same time interval provided similar growth conditions for TNBC lung metastases and TNBC primary cells as Comparative Example 5 (Matrigel®) in the top row of the figure.

[0366] With respect to TNBC brain metastasis cells, the hydrogel of Example 5b (a non-self-degrading, enzymatically degradable, soft (<500 Pa) PEG hydrogel containing an RGD motif but without a laminin bioactive ligand) provided similar growth conditions to Comparative Example 5 (Matrigel®).

[0367] Regarding HER2+ cutaneous metastatic cells, the hydrogel of Example 5c (a non-autodegradable, enzymatically degradable moderate (>1000 Pa) PEG hydrogel that does not contain RGD motifs or laminin bioactive ligands) provided similar growth conditions to Comparative Example 5 (Matrigel®).

[0368] Generally, TNBC subtypes are more difficult to grow. Hypoxic conditions improved the growth of breast cancer organoids compared to normoxic conditions. In addition, the morphology of HER2+ and TNBC cells grown under pre-selected extracellular matrix conditions was consistent with the morphology of previously established breast cancer organoids in Matrigel® (Sachs et al., 2018, Cell 172, 1-14).

[0369] Example 6: Testing of prostate cancer cells Prostate cancer cells from commercially available primary cultured healthy prostate cells and PDX cells were encapsulated under different extracellular matrix conditions.

[0370] A non-self-degrading hydrogel was provided using PEG as a precursor molecule for the hydrogel. As a crosslinking agent, a peptide containing at least two, preferably two cysteine ​​moieties, which differed in their amino acid sequences, particularly in the presence or absence of the RGD adhesion motif and in the presence or absence of the MMP degradation sequence, was used.

[0371] Another approach performed on some hydrogels involved the binding of bioactive ligands containing RGD adhesion motifs and / or ligands selected from the group consisting of natural laminins, e.g., laminin-111, recombinant laminin isoforms, and their biofunctional fragments. Examples of suitable recombinant laminin isoforms include laminin-111, laminin-211, laminin-332, laminin-411, laminin-511, or laminin-521. Arrays of hydrogels with pre-selected characteristics were established by the method described above.

[0372] The mechanical properties of the hydrogels also differed (hard (2000-3000 Pa), moderate (1000-2000 Pa), or soft (50-1000 Pa) gels).

[0373] A variety of different known, commonly used, and / or commercially available culture media were used. Preferably, the media were characterized by the presence of a Wnt agonist, such as R-spongin. In a preferred embodiment, modified media described in Drost et al. (Nature Protocol 11, 347-358, January 2016) or Beshiri et al. (Clinical Cancer Research 24, 4332-4345, May 2018) may be used. The preferred medium includes AdDMEM / F12 medium supplemented with glutamine, BSA, transferrin, noggin, fibroblast growth factor 2 or basic and FGF10 [FGF2 or FGF-basic and FGF10], EGF, R-spongin-conditioned medium, penicillin / streptomycin, glutathione, and optionally N-acetyl-L-cysteine, nicotinamide, DHT (dihydrotestosterone), insulin, prostaglandin E2, A83-01, SB202190 (p38 inhibitor), Y-27632 (rock inhibitor), and HEPES.

[0374] The results are shown in Figures 6a and 6b. The hydrogels of Examples 6a and 6b were soft, enzymatically degradable hydrogels. Example 6a is a hydrogel that does not contain the RGD motif. Example 6b is a hydrogel that contains the RGD motif.

[0375] Normal (healthy) cells were found to grow only in extracellular matrices containing the bioactive peptide RGD (Figure 6a, Example 6b). Similarly, the growth of healthy cells was not significantly observed in moderate gels containing RGD compared to soft gels containing RGD. On the other hand, prostate cancer cells grew in Examples 6a (without RGD) and 6b (with RGD) (Figure 6b).

[0376] In contrast, in comparative examples using Matrigel®, a naturally derived matrix, no difference in the growth of healthy prostate cells and prostate cancer cells was achieved in either medium (Figures 6a and 6b).

Claims

1. A method performed in a single tissue type, which is arbitrarily combined with other cells such as stromal cells or immune cells, wherein the method is a) A step of providing an array having separate spaces of a completely distinct hydrogel matrix by crosslinking cells of the type of tissue to be tested with one or more different combinations of one or more different hydrogel precursor molecules, optionally at least one crosslinking agent, on the surface of a substrate or in separate spaces of a substrate, preferably in a multiwell plate, in the presence of optionally one or more biologically active molecules; b) A step of enabling the cells to grow and proliferate in the separate spaces of the array of the hydrogel matrix in the presence of one or more different media; c) A step of manipulating cells grown in the separate spaces of the array of the hydrogel matrix; A method comprising a specific combination of hydrogel characteristics, wherein a particular combination of hydrogel characteristics is pre-selected for the type of tissue being tested.

2. The method according to claim 1, wherein the prior selection of the precursor molecule of the hydrogel or at least one of the features of the hydrogel and the culture medium is based on selecting suitable extracellular matrix conditions from a method using random extracellular matrix conditions.

3. The method according to claim 1 or 2, wherein the type of tissue is selected from the group consisting of cancer cells and normal / healthy cells.

4. The method according to any one of claims 1 to 3, wherein newly isolated or frozen cells are used from a human biopsy or tissue excision, or from patient-derived xenograft (PDX) tissue.

5. The method according to any one of claims 1 to 4, wherein one or more drugs are added to the separate spaces of the hydrogel matrix in step c).

6. The method according to any one of claims 1 to 5, wherein the tissue type is c-Met-overexpressing lung cancer cells, preferably non-small cell lung cancer cells, the matrix of the hydrogel is pre-selected to be a non-autodegradable PEG hydrogel, and the crosslinking agent and the optional bioactive agent do not contain an RGD motif.

7. The method according to claim 6, wherein the culture medium comprises a Wnt agonist such as R-spongin or FBS (serum).

8. The method according to any one of claims 1 to 5, wherein the tissue type is pancreatic ductal adenocarcinoma (PDAC) cells, the matrix of the hydrogel is a non-self-degrading PEG hydrogel pre-selected having a stiffness of 50 to 3000 Pa, preferably 50 to 2000 Pa, most preferably 50 to 1000 Pa, and at least one of the crosslinking agent and / or the optional bioactive agent comprises an RGD motif.

9. The method according to claim 8, wherein the culture medium comprises R-spongin and a Wnt agonist such as Wnt3a.

10. The method according to any one of claims 1 to 5, wherein the tissue type is colorectal cancer (CRC) cells, the matrix of the hydrogel is selected in advance and further comprises one or more bioactive molecules, optionally including laminin, preferably laminin-111 or laminin-511, particularly preferably natural mouse laminin-111 or recombinant human laminin-511, and at least one of the crosslinking agent and / or the optional bioactive agent comprises an RGD motif.

11. The method according to claim 10, wherein the culture medium comprises R-spongin and a Wnt agonist such as Wnt3a.

12. The method according to any one of claims 1 to 5, wherein the tissue type is breast cancer cells, the matrix of the hydrogel is preferably pre-selected as an enzymatically degradable PEG hydrogel, preferably at least one of the crosslinking agents comprises an enzymatically degradable motif, preferably an MMP-sensitive motif, and the hydrogel optionally further comprises one or more biologically active molecules, including laminin, preferably laminin-111, and particularly preferably natural mouse laminin-111.

13. The method according to claim 12, wherein the culture medium comprises a Wnt agonist such as R-spongin or FBS (serum).

14. The method according to any one of claims 1 to 5, wherein the tissue type is cancer cells that proliferate more slowly ex vivo than normal corresponding cells, preferably prostate cancer cells, the matrix of the hydrogel is preferably a PEG hydrogel with a stiffness of 50 to 2000 Pa selected in advance, and the crosslinking agent and the optional bioactive agent do not contain an RGD motif.

15. The contents of a kit for operating on one or more tissue types, or using one or more tissue types, a) A component for producing an array of perfectly clear hydrogel matrices in order to create conditions for a perfectly clear three-dimensional extracellular matrix in which the biological, biophysical and / or biochemical properties are distinct from each other, wherein the component is - One or more different hydrogel precursor molecules, - Optionally, at least one crosslinking agent, - Optionally, one or more biologically active molecules, Ingredients b) One or more different culture media The kit contents include a specific combination of hydrogel characteristics, pre-selected for the type of tissue being tested.

16. The kit according to claim 15 for testing the effects of a drug on c-Met-overexpressing lung cancer cells, preferably non-small cell lung cancer cells, wherein the matrix of the hydrogel is pre-selected to be a non-self-degrading PEG hydrogel, the crosslinking agent and the optional bioactive agent do not contain an RGD motif, and preferably the culture medium contains a Wnt agonist such as R-spongin or FBS (serum).

17. The hydrogel matrix is ​​a non-self-degrading PEG hydrogel pre-selected having a stiffness of 50 to 3000 Pa, preferably 50 to 2000 Pa, most preferably 50 to 1000 Pa, and at least one of the crosslinking agent and the optional bioactive agent comprises an RGD motif, and preferably the culture medium comprises R-spongin and a Wnt agonist such as Wnt3a, the kit according to claim 15 for testing the effects of a drug on pancreatic ductal adenocarcinoma (PDAC) cells.

18. A PEG hydrogel is pre-selected for testing the effects of a drug on colorectal cancer (CRC) cells, wherein the matrix of the hydrogel has an initial stiffness of at least 50 to 2000 Pa and further comprises one or more bioactive molecules, optionally including laminin, preferably laminin-111 or laminin-511, particularly preferably natural mouse laminin-111 or recombinant human laminin-511, and at least one of the crosslinking agent and the optional bioactive agent comprises an RGD motif, and preferably the culture medium comprises R-spongin and a Wnt agonist such as Wnt3a.

19. The hydrogel matrix is ​​preferably pre-selected to be an enzymatically degradable PEG hydrogel, preferably at least one of the crosslinking agents comprises an enzymatically degradable motif, preferably an MMP-sensitive motif, the hydrogel optionally further comprises one or more biologically active molecules, including laminin, preferably laminin-111, particularly preferably natural mouse laminin-111, and preferably the culture medium comprises a Wnt agonist such as R-spongin or FBS (serum), the kit according to claim 15 for testing the effects of a drug on breast cancer cells.

20. The kit according to claim 15, wherein the matrix of the hydrogel is preferably a PEG hydrogel with a stiffness of 50 to 2000 Pa, which is pre-selected, and the crosslinking agent and the optional bioactive agent are free of RGD motifs, for testing the effects of a drug on cancer cells, preferably prostate cancer cells, that proliferate more slowly in ex vivo than normal corresponding cells.

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