WO2019226120A1

WO2019226120A1 - A tumour cell culture system and a method of preparing a tumour cell culture system

Info

Publication number: WO2019226120A1
Application number: PCT/SG2019/050272
Authority: WO
Inventors: Motoichi Kurisawa; Shengyong Ng; Min Han TAN; Wai Jin TAN; Swarnalatha Lucky SASIDHARAN
Original assignee: Agency For Science, Technology And Research
Priority date: 2018-05-23
Filing date: 2019-05-23
Publication date: 2019-11-28

Abstract

There is provided a tumour cell culture system comprising a hydrogel matrix comprising a phenol-conjugated polymer, such as a hyaluronic acid-tyramine (HA-Tyr) hydrogel or a gelatin-hydroxypropionic acid (Gelatin-HPA) hydrogel, which is configured to support in vitro growth of tumour cells thereon, in particular a patient-derived tumour organoid. The hydrogel matrix has a storage modulus of at least 1000 Pa. There is also provided a method of preparing the tumour cell culture system, wherein the storage modulus of the hydrogel can be defined by selecting the concentration of horseradish peroxidase (HRP) or hydrogen peroxide (H₂0₂).

Description

A TUMOUR CELL CULTURE SYSTEM AND A METHOD OF PREPARING A TUMOUR CELL CULTURE SYSTEM

TECHNICAL FIELD

The present disclosure relates broadly to a tumour cell culture system and a method of preparing a tumour cell culture system.

BACKGROUND

Patient-derived cell or tissue models of cancer have emerged as important tools in cancer biology and drug discovery due to limitations associated with conventional cancer cell lines, chiefly their poor predictive accuracy of clinical efficacy of potential cancer therapies. In contrast to conventional cell lines, patient-derived xenograft (PDX) lines, which involve the xenotransplantation of human tumour fragments into immunocompromised mice, can be established from various solid tumours with higher efficiency and better retain the morphological features, genomic (transcriptomic and mutational) profile and intratumoural heterogeneity of the parental tumour.

In some well-characterized cases, PDX drug responses correlate with donor patient responses and may even be predictive. While PDX models provide greater predictive value in cancer drug discovery, the cost and complexity of such in vivo models limit their statistical power, and therefore feasibility, in early-stage therapeutic screens involving larger chemical libraries. In vitro patient-derived tumour organoid models offers the potential to vastly expand the efficiency of primary tumour-based cancer drug screens by providing physiologically relevant in vitro models that are more scalable and complement existing in vivo PDX models.

A major requirement for the maintenance of patient-derived tumour organoid (PTO) cultures ex vivo is a sufficient recapitulation/reproduction of the insoluble tumour-specific microenvironment.

However, existing PTO cultures are inadequate in recapitulating mechanical properties of tumour tissue in three dimensional (3D) organoid culture matrix and defining biochemical components of 3D organoid culture matrix. Existing PTO culture methods rely almost exclusively on poorly defined animal-derived matrices such as Matrigel or type I collagen gels regardless of tumour type. In addition to posing limitations on recapitulating tumour- associated mechanical properties, both matrices are relatively costly and practically challenging to work with due to their lack of customisation options, lack of rigidity and intrinsic biological variability. Matrigel (even in its growth- factor reduced formulation) may contain various mouse-derived growth factors which may lead to spurious and physiologically irrelevant pleiotropic effects on PTOs.

Thus, there is a need for a tumour cell culture system and a method of preparing a tumour cell culture system which seek to address or at least ameliorate one or more of the above problems.

SUMMARY

In one aspect, there is provided a tumour cell culture system comprising, a hydrogel matrix comprising a phenol-conjugated polymer that is configured to support in vitro growth of tumour cells thereon, wherein the hydrogel matrix has a storage modulus (G’) of at least 1000 Pa. In one embodiment of the tumour cell culture system disclosed herein, the polymer comprises gelatin.

In one embodiment of the tumour cell culture system disclosed herein, the phenol-conjugated polymer comprises one or more phenols selected from the group consisting of hydroxyphenylpropionic acid (HPA), tyramine, and hydroxyphenylacetic acid.

In one embodiment of the tumour cell culture system disclosed herein, the phenol-conjugated polymer is configured to support in vitro growth of tumour organoids thereon.

In one embodiment of the tumour cell culture system disclosed herein, the tumour cell culture system further comprises tumour cells encapsulated within the matrix.

In one embodiment of the tumour cell culture system disclosed herein, the tumour cells are derived from one or more organoids isolated from colorectal cancer tumours, and/or nasopharyngeal carcinoma tumours.

In one embodiment of the tumour cell culture system disclosed herein, the tumour cells are in the form of one or more tumour organoids on the hydrogel matrix. In one embodiment of the tumour cell culture system disclosed herein, the tumour cell culture system is maintained at an oxygen level that is no more than 10%.

In one embodiment of the tumour cell culture system disclosed herein, the tumour cell culture system comprises one or more therapeutic compounds. In one embodiment of the tumour cell culture system disclosed herein, the hydrogel matrix has an elastic modulus (E) from 2000 Pa to 35,000 Pa.

In one embodiment of the tumour cell culture system disclosed herein, the hydrogel matrix has a storage module (G’) in the range of from 2500 Pa to 7500 Pa.

In one embodiment of the tumour cell culture system disclosed herein, the hydrogel matrix comprises at least 2% (w/v) of hydroxyphenylpropionic acid (HPA)-conjugated gelatin polymer.

In one embodiment of the tumour cell culture system disclosed herein, the backbone structure of the hydrogel matrix consists essentially of hydroxyphenylpropionic acid (HPA)-conjugated gelatin polymer.

In one aspect, there is provided a method of preparing a tumour cell culture system, the method comprising, crosslinking a precursor solution comprising phenol-conjugated polymer in the presence of an enzyme and an oxidising agent to form a phenol-conjugated hydrogel matrix that is configured to support in vitro growth of tumour cells, wherein the hydrogel matrix has a storage modulus (G’) of at least 1000 Pa.

In one embodiment of the method disclosed herein, the polymer comprises gelatin.

In one embodiment of the method disclosed herein, the method further comprises, prior to crosslinking the precursor solution, adding gelatin to a reaction mixture comprising one or more phenols selected from the group consisting of hydroxyphenylpropionic acid (HPA), tyramine, and hydroxyphenylacetic acid to form phenol conjugated gelatin polymer. In one embodiment of the method disclosed herein, the step of adding gelatin to a reaction mixture comprising one or more phenols comprises (i) adding gelatin to a reaction mixture comprising HPA, N-hydroxysuccinimide (NHS) and 1 -ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC- HCI) dissolved in a solvent, or (ii) adding gelatin to a reaction mixture comprising HPA-NHS, to form HPA-conjugated gelatin polymer.

In one embodiment of the method disclosed herein, the enzyme comprises horseradish peroxidase (HRP) and the oxidizing agent comprises hydrogen peroxide.

In one embodiment of the method disclosed herein, the method further comprises defining the storage modulus of the hydrogel matrix by selecting a predetermined concentration of HRP and hydrogen peroxide, wherein the HRP concentration is in a range from 0.030 U/mL to 0.20 U/mL and the hydrogen peroxide concentration is in a range from 0.14 mM to 4.0 mM.

In one embodiment of the method disclosed herein, the method further comprises seeding tumour cells into the hydrogel matrix.

In one embodiment of the method disclosed herein, the tumour cells have been isolated from colorectal cancer tumours, and/or nasopharyngeal carcinoma tumours. In one embodiment of the method disclosed herein, the method further comprises culturing the seeded tumour cells to obtain one or more tumour organoids disposed on the hydrogel matrix, wherein the seeded tumour cells are in (i) an organoid form or (ii) in a non-organoid form that is suitable to be subsequently cultured into an organoid form.

In one embodiment of the method disclosed herein, the step of seeding the tumour cells on the hydrogel matrix comprises encapsulating tumour cells in the hydrogel matrix by adding horseradish peroxidase (FIRP) and tumour cells to the precursor solution, followed by adding hydrogen peroxide to the precursor solution.

In one embodiment of the method disclosed herein, the method further comprises culturing the tumour cells at no more than 10% oxygen levels.

In one embodiment of the method disclosed herein, the method further comprises introducing one or more therapeutic compounds to the hydrogel matrix.

In one embodiment of the method disclosed herein, the method further comprises engrafting the hydrogel matrix comprising the tumour cells into an animal model for in-vivo culture.

DEFINITIONS

The term“hydrogel” as used herein is to be interpreted broadly to include a polymeric matrix which is capable of absorbing liquid e.g. water. A hydrogel may refer to the polymeric matrix with any absorbed liquid, or the polymeric matrix in its dry state without any absorbed liquid.

The term“organoid” as used herein is to be interpreted broadly to include a 3D multicellular in vitro tissue construct that substantially mimics its corresponding in vivo organ. It may be used to study aspects of that organ in the tissue culture dish.

The term“effective amount” as used herein is to be interpreted broadly as an amount that is sufficient to carry out its intended effect. For example, when an“effective amount” is used to refer to the administration of a compound, it can refer to the situation where the compound is administered at a dosage and/or for a period of time necessary to achieve the desired result.

The term“treating” as used herein is to be interpreted broadly to mean attempting to inhibit the progression of a disease (e.g. cancer) temporarily or attempting to stop the progression of the disease permanently. The disease may not need to be effectively treated eventually.

The term“storage modulus” as used herein is to be interpreted broadly as a measure of elastic response of a material. It measures the stored energy.

The term“loss modulus” as used herein is to be interpreted broadly as a a measure of viscous response of a material. It measures the energy dissipated as heat.

The term“gelation rate” as used herein is to be interpreted broadly as the degree of gel formation as a function of time and may refer to the rate of crosslinking in the gelation solution. The term“gel point” as used herein is to be interpreted broadly as an abrupt change in the viscosity of a solution containing polymerisable components. Typically, at the gel point, a solution undergoes gelation as reflected in a loss in fluidity. The gel point, which may also be related to the crossover of the storage modulus and the loss modulus, is typically employed to evaluate the gelation rate of hydrogel.

The term“substrate” as used herein is to be interpreted broadly to refer to a supporting structure. The term“micro” as used herein is to be interpreted broadly to include dimensions from about 1 micron to about 1000 microns. The term“nano” as used herein is to be interpreted broadly to include dimensions less than about 1000 nm.

The terms "coupled" or "connected" as used in this description are intended to cover both directly connected or connected through one or more intermediate means, unless otherwise stated.

The term "associated with", used herein when referring to two elements refers to a broad relationship between the two elements. The relationship includes, but is not limited to a physical, a chemical or a biological relationship. For example, when element A is associated with element B, elements A and B may be directly or indirectly attached to each other or element A may contain element B or vice versa. The term "adjacent" used herein when referring to two elements refers to one element being in close proximity to another element and may be but is not limited to the elements contacting each other or may further include the elements being separated by one or more further elements disposed therebetween.

The term "and/or", e.g., "X and/or Y" is understood to mean either "X and Y" or "X or Y" and should be taken to provide explicit support for both meanings or for either meaning. Further, in the description herein, the word“substantially” whenever used is understood to include, but not restricted to, "entirely" or“completely” and the like. In addition, terms such as "comprising", "comprise", and the like whenever used, are intended to be non-restricting descriptive language in that they broadly include elements/components recited after such terms, in addition to other components not explicitly recited. For example, when “comprising” is used, reference to a“one” feature is also intended to be a reference to“at least one” of that feature. Terms such as“consisting”,“consist”, and the like, may in the appropriate context, be considered as a subset of terms such as "comprising", "comprise", and the like. Therefore, in embodiments disclosed herein using the terms such as "comprising", "comprise", and the like, it will be appreciated that these embodiments provide teaching for corresponding embodiments using terms such as“consisting”,“consist”, and the like. Further, terms such as "about", "approximately" and the like whenever used, typically means a reasonable variation, for example a variation of +/- 5% of the disclosed value, or a variance of 4% of the disclosed value, or a variance of 3% of the disclosed value, a variance of 2% of the disclosed value or a variance of 1 % of the disclosed value.

Furthermore, in the description herein, certain values may be disclosed in a range. The values showing the end points of a range are intended to illustrate a preferred range. Whenever a range has been described, it is intended that the range covers and teaches all possible sub-ranges as well as individual numerical values within that range. That is, the end points of a range should not be interpreted as inflexible limitations. For example, a description of a range of 1% to 5% is intended to have specifically disclosed sub-ranges 1 % to 2%, 1 % to 3%, 1 % to 4%, 2% to 3% etc., as well as individually, values within that range such as 1 %, 2%, 3%, 4% and 5%. The intention of the above specific disclosure is applicable to any depth/breadth of a range.

Additionally, when describing some embodiments, the disclosure may have disclosed a method and/or process as a particular sequence of steps. Flowever, unless otherwise required, it will be appreciated that the method or process should not be limited to the particular sequence of steps disclosed. Other sequences of steps may be possible. The particular order of the steps disclosed herein should not be construed as undue limitations. Unless otherwise required, a method and/or process disclosed herein should not be limited to the steps being carried out in the order written. The sequence of steps may be varied and still remain within the scope of the disclosure. DESCRIPTION OF EMBODIMENTS

Non-limiting embodiments of a tumour cell culture system and a method of preparing a tumour cell culture system are disclosed hereinafter.

In various embodiments, the tumour cell culture system comprises a hydrogel matrix, said hydrogel matrix comprising a phenol-conjugated polymer that is configured to support in vitro growth of tumour cells thereon/therewithin. The hydrogel matrix may serve as a scaffold/platform in tissue engineering applications for culturing tumour cells/tissues/organoids.

In various embodiments, the hydrogel matrix is configured to support in vitro growth of patient-derived tumour organoids (PTOs). The tumour cells may be present as or arranged to form one or more organoids in the matrix. Tumour organoids may be isolated from tumour biopsies taken from a patient, i.e. after biopsy collection, cells can be isolated and cultured as organoids. Tumour organoids may also be derived/isolated from patient-derived xenograft (PDX) tumours. Isolated tumour cells may be in a non-organoid form that is suitable to be subsequently cultured into an organoid form. In various embodiments, the tumour cell culture system serves as PDX models. For example, tumour tissue from patients may be subcutaneously or orthotopically transplanted into immunodeficient mice using embodiments of the disclosed tumour cell culture system as a support platform.

Advantageously, tumour organoids or cells derived from tumour organoids may be more representative of the in vivo situation for expanded populations of cells and organoids grown from diseased tissue. Patient-derived tumour organoids may be superior to cancer cell lines in terms of mimicking the biological characteristics of the primary tumours. Thus, in various embodiments, tumour organoids supported on the tumour cell culture system recapitulate basic features of primary tumours such as histological complexity and genetic heterogeneity of cancer. In various embodiments, tumour organoids supported on the tumour cell culture system preserve the genomic and transcriptomic characteristics of their primary tumours, such as having similar mutation status, copy number alteration and identification of fusion genes and transcriptional landscape to the primary tumour. Therefore, embodiments of the presently disclosed tumour cell culture system of tumour organoids may be used as ex vivo disease models in applications such as drug screening, drug target discovery and validation, toxicology and toxicology screens, personalized medicine, regenerative medicine and/or as ex vivo cell/organ models, such as disease models.

Examples of tumour cells which may be cultured using the tumour cell culture system include but are not limited to colorectal cancer, liver cancer, squamous cell cancer, nasopharyngeal cancer, lung cancer, cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, bladder cancer, hepatoma, breast cancer, colon cancer, rectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, as well as head and neck cancer and combinations thereof. Various embodiments of the tumour cell culture system are suitable for culturing tumour cells from the above cancers due to its customisability. Cells from different tumour types could vary significantly in their matrix requirements for culture in vitro and therefore, the customisability of the presently disclosed tumour cell culture system may be advantageous in providing a viable platform for in vitro culture of a variety of tumour cells. In one embodiment, the tumour cells are tumour organoids derived/isolated from colorectal cancer patient-derived xenograft (CRC-PDX) tumours. For example, the tumour cell culture system may be used for culturing colorectal cancer (CRC), in particular, CRC-PTOs. In another embodiment, the tumour cells are tumour organoids derived/isolated from nasopharyngeal carcinoma tumours, for e.g. nasopharyngeal carcinoma-PDX tumours. The inventors have recognised that cells from different parts of the body, or even different regions within the same organ, may have different microenvironments of the extracellular matrix in vivo. Thus, in various embodiments, the phenol-conjugated polymer of the hydrogel matrix is configured to have its mechanical and biochemical properties easily/effectively configured/tuned to substantially mimic the microenvironment of the extracellular matrix in vivo. In various embodiments, the phenol-conjugated polymer e.g. phenol-conjugated gelatin polymer of the hydrogel matrix is configured to have its biochemical composition varied by adding other polymers e.g. non-gelatin polymers such as HA, laminin, synthetic polymers etc. on top of the phenol-conjugated gelatin hydrogel which is crosslinkable via a gelation mechanism comprising an enzyme e.g. HRP and an oxidant e.g. H2O2. This may advantageously provide a tumour cell culture system with customisable biochemical composition for various tumour types. In addition to composition, other biochemical properties that are varied may include but are not limited to pH, polymer concentration, crosslinkability, degradability etc. The mechanical properties may include but are not limited to mechanical strength/ stiffness (e.g. shear modulus, compression modulus), shear relaxation and swelling ratio etc. In various embodiments, pH, polymer concentration, crosslinkability and degradability of the phenol-conjugated gelatin polymer can be controlled solely by a gelation mechanism comprising the use of an enzyme e.g. HRP and an oxidant e.g. H2O2. In various embodiments, mechanical strength/stiffness (as measured by shear modulus or compression modulus) and swelling ratio can also be controlled solely by the gelation mechanism comprising the use of an enzyme e.g. HRP and an oxidant e.g. H2O2. Thus, the hydrogel matrix of the tumour cell culture system may be capable of maintaining patient-derived tumour organoid cultures ex vivo by achieving sufficient recapitulation/reproduction of the insoluble tumour-specific microenvironment.

In various embodiments, the hydrogel is tuned/selected to have a storage modulus in the range of about 1000 Pa to about 8000 Pa. In various embodiments, the storage modulus of the hydrogel matrix is at least about 1000 Pa, at least about 2000 Pa, at least about 3000 Pa, at least about 4000 Pa, at least about 5000 Pa, at least about 6000 Pa, or at least about 7000 Pa. In various embodiments, the storage modulus of the hydrogel matrix is from about 1000 Pa to about 8000 Pa, from about 1500 Pa to about 7500 Pa, from about

2000 Pa to about 7000 Pa, or from about 2500 Pa to about 6500 Pa. The inventors have recognised that a relatively stiffer hydrogel matrix of at least 1000 Pa may advantageously support growth and viability of tumour cells encapsulated within the hydrogel matrix. Without being bound by theory, it is believed that this may be due to the finding that increased tissue stiffness is a common hallmark of tumours. For example, in the tumour stroma, both increased expression of collagen and lysyl oxidase (LOX), which crosslink collagen, contribute to increased tumour stiffness. Thus, in various embodiments, the matrix rigidity may advantageously drive tumour progression in various types of cancer.

In various embodiments, the hydrogel is tuned/selected to have an elastic modulus in the range of about 2000 Pa to about 35000 Pa. In various embodiments, the elastic modulus of the hydrogel matrix is from about 2000 Pa to about 35000 Pa, from about 5000 Pa to about 30000 Pa, from about 10000 Pa to about 25000 Pa, or from about 15000 Pa to about 20000 Pa. The range of elastic modulus of the hydrogel matrix may advantageously enable mimicking of both healthy and diseased tissue e.g. tumour tissue. In various embodiments, the phenol-conjugated gelatin polymer comprises a gelatin polymer conjugated to 3,4-hydroxyphenylpropionic acid (HPA), i.e. gelatin-HPA. Other phenol components which may be conjugated to the gelatin polymer include but are not limited to tyramine (Tyr), hydroxyphenylacetic acid, derivatives thereof, or a combination thereof. Without being bound by theory, it is believed that conjugation of HPA, tyramine, and hydroxyphenylacetic acid may result in similar gelation behaviour. In various embodiments, the tumour cell culture system is tuned/selected/maintained at a pH from about 4 to about 8, from about 4.5 to about 7.5, from about 5 to about 7, from about 5.5 to about 6.5, or from about 6 to about 6.5. Recapitulation of different pH conditions may advantageously allow the hydrogel to mimic the in vivo chemical microenvironment of different tissues e.g. diseased tissue such as tumours. In one embodiment, the pH of the tumour cell culture system is maintained at normal physiological levels, i.e. pH of about 7.4. In various embodiments, the temperature of the tumour cell culture system is maintained at a range of from about 35°C to about 40°C, from about 36°C to about 39°C, or from about 37°C to about 38°C. In one embodiment, the temperature of the hydrogel is maintained at normal physiological levels, i.e. temperature of about 37°C.

In various embodiments, the hydrogel matrix comprises a phenol- conjugated polymer which includes but is not limited to gelatin, hyaluronic acid (hyaluronan), chitosan, heparin, cellulose, dextran, dextran sulfate, chondroitin sulfate, keratan sulfate, dermatan sulfate, alginate, collagen, albumin, fibronectin, laminin, elastin, vitronectin, fibrinogen, derivatives thereof and combinations thereof. The phenol conjugate may be selected from the group consisting of tyramine (Tyr), hydroxyphenylacetic acid, hydroxypropionic acid e.g. 3,4-hydroxyphenylpropionic acid, derivatives thereof, and combinations thereof. Advantageously, the biochemical composition of the hydrogel matrix may be customised by adding/combining different polymers to achieve desired characteristics, while maintaining control over mechanical properties with the gelation mechanism comprising the use of an enzyme e.g. HRP and an oxidant e.g. H2O2. Such customisability of the hydrogel matrix may provide significant flexibility for optimisation for tumour organoids derived from different tumour types, or different tumour organoid lines from the same tumour type. Such customisability of the hydrogel matrix may also facilitate systematic interrogation of the potential effects of tumour matrix components in combination with matrix rigidity and soluble factors on tumour organoids behaviour, thereby providing a suitable system for drug target discovery.

In various embodiments, the backbone structure of the hydrogel matrix consists essentially of or is mainly composed of hydroxyphenylpropionic acid (HPA)-conjugated gelatin polymer. For example, the majority e.g. more than about 50% of the polymer is a hydroxyphenylpropionic acid (HPA)-conjugated gelatin polymer. In some embodiments, the hydrogel matrix is substantially devoid of other polymers apart from phenol-conjugated gelatin polymer. In one embodiment, the hydrogel matrix comprises phenol-conjugated gelatin polymer only, i.e. not a composite hydrogel which may contain more than one type of polymer. In one embodiment, the hydrogel matrix comprises phenol-conjugated gelatin polymer and is substantially devoid of polymer/macromolecule selected from the group consisting of hyaluronic acid (hyaluronan), chitosan, heparin, cellulose, dextran, dextran sulfate, chondroitin sulfate, keratan sulfate, dermatan sulfate, alginate, collagen, albumin, fibronectin, laminin, elastin, vitronectin, fibrinogen, derivatives thereof and combinations thereof.

In various embodiments, the tumour cell culture system is maintained at an oxygen level that is below atmospheric oxygen level. In one embodiment, tumour cells are subject to hypoxia by maintaining the tumour cell culture system under hypoxic conditions. Hypoxic conditions may be achieved e.g. using modular gas chambers inside a standard CO2 incubator, or using a specialised hypoxia incubator or the like. Oxygen sensors may be provided to measure and monitor the oxgen level of the incubation chamber where the tumour cell culture system is placed. The tumour cell culture system may be maintained under hypoxic conditions wherein the concentration of oxygen is no more than about 10%, no more than about 5%, no more than about 4%, no more than about 3%, no more than about 2%, no more than about 1%, no more than about 0.5%, no more than about 0.2%, or no more than about 0.1 %. The use of hypoxic conditions may mimic the in vivo hypoxic tumour microenvironment where tissue oxygen levels in vivo are from about 3% to about 7.4% and may even be lower in a tumour. This may advantageously promote survival and growth of the cells/organoids cultured within the tumour cell culture system. In various embodiments, the hydrogel matrix contains additives such as a drug or protein. Drugs or proteins which may be suitable or potentially suitable for treatment may be added. For example, if the tumour cell culture system is used for studying colorectal cancer, therapeutic compounds for treating colorectal cancer such as 5-fluorouracil (5FU), oxaliplatin or cetuximab may be added. The additives may be added before gelation, i.e. before the hydrogel is formed, or after gelation, i.e. after the hydrogel is formed. It would be appreciated that the addition of other additive(s) may affect the mechanical strength or other properties of the formed hydrogel or on the formation process, such as the gelation rate. As such, depending on which and how much other additive(s) are included, the concentration of oxidizing agent (e.g. FI2O2) or enzyme (e.g. FIRP), or both, may be adjusted to off-set such impact.

In one embodiment, the tumour cell culture system is used to test libraries of chemicals, antibodies, etc for suitability for use as drugs in the treatment or prevention of cancer. For example, the tumour cell culture system using a patient’s own tumour cells may be treated with a chemical compound or a chemical library to determine which compounds effectively modify, kill and/or treat the patient’s cells. This advantageously facilitates the study of a patient’s response to a test drug and allows treatment to be customised to the patient.

In various embodiments, the hydrogel matrix is a synthetic hydrogel matrix. Synthetic hydrogel matrices may advantageously provide practical alternatives to natural matrices by affording control over both biochemical and biophysical features that may vary with tissue origin, thus providing a synthetic 3D microenvironment that is better at recapitulating the biochemical and mechanical properties of tumour extracellular matrix (ECM). In another embodiment, the synthetic hydrogel matrix is substantially devoid of unknown/uncharacterised substances/factors. The presence of factors which are not typically found in the native tumour microenvironment may lead to spurious or irrelevant pleiotropic effects that are difficult to ascertain. Embodiments of the synthetic hydrogel matrix disclosed herein make it possible to minimise or prevent the inadvertent introduction of unknown/ uncharacterised substances into the matrix.

In various embodiments, a method of preparing a tumour cell culture system is provided. The method may comprise crosslinking a precursor solution comprising phenol-conjugated polymer e.g. phenol-conjugated gelatin polymer in the presence of an enzyme and an oxidising agent to form a phenol- conjugated hydrogel matrix configured to support in vitro growth of tumour cells. The phenol-conjugated polymer may have one or more properties described above. The precursor solution may be a macromer solution.

In various embodiments, the method of preparing a tumour cell culture system comprises, prior to crosslinking the precursor solution, adding a polymer e.g. gelatin to a reaction mixture comprising a phenol moiety to form phenol- conjugated polymer e.g. phenol-conjugated gelatin polymer. The phenol moiety may be selected from the group consisting of hydroxyphenylpropionic acid (HPA), tyramine, and hydroxyphenylacetic acid.

In one embodiment, the phenol-conjugated gelatin polymer e.g. HPA- conjugated gelatin polymer is synthesised using a general carbodiimide/active ester-mediated coupling reaction. For example, a reaction mixture comprising a phenol moiety e.g. hydroxyphenylpropionic acid, N-hydroxysuccinimide (NHS) and 1 -ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC-HCI) may be formed by dissolving in a solvent mixture. In another embodiment, the phenol-conjugated gelatin polymer e.g. HPA-conjugated gelatin polymer is synthesised by adding the HPA-NHS (N-hydroxysuccinimide) to gelatin solution without the carbodiimide coupling agent. In various embodiments, prior to crosslinking the precursor solution, the method of preparing a tumour cell culture system comprises preparing a precursor solution of phenol-conjugated polymer e.g. phenol-conjugated gelatin polymer. The precursor solution may be formed by reconstituting a lyophilised form of phenol-conjugated polymer in a suitable solvent having desired properties. The reconstituting step may be carried out prior to the step of crosslinking the precursor solution. For example, the phenol-conjugated polymer may be dissolved in water, buffered or saline solution e.g. phosphate buffered solution (PBS), Hank’s balanced salt solution (HBSS), cell culture medium etc. Dissolution of the lyophilised phenol-conjugated polymer in a suitable solvent may supply substances e.g. growth factors or chemical compounds which are present in the in vivo environment of the tumour cells. A higher concentration of phenol-conjugated polymer may produce a hydrogel with an increasing level of stiffness as quantified by its storage modulus.

In various embodiments, the precursor solution comprises at least 1% by weight, at least 2% by weight, at least 3% by weight, at least 4% by weight, at least 5% by weight, at least 6% by weight, at least 7% by weight, at least 8% by weight, at least 9% by weight, at least 10% by weight, at least 15% by weight, or at least 20% by weight of phenol-conjugated polymer e.g. phenol-conjugated gelatin polymer dissolved in a volume of a suitable solvent. In one embodiment, the precursor solution may comprise from about 1.5% (w/v) to about 15% (w/v) of phenol-conjugated gelatin polymer e.g. gelatin-HPA. The amount of phenol- conjugated polymer may affect the stiffness and degradability of the hydrogel matrix. In various embodiments, the higher the content of phenol-conjugated polymer, the higher the stiffness and the poorer the degradability of the hydrogel matrix. In various embodiments, the amount of phenol-conjugated polymer is about 5% (w/v). Advantageously, 5% (w/v) has been found to be a concentration that can relatively easily form hydrogels with G’ greater than 2000 Pa. In various embodiments, the method of preparing a tumour cell culture system comprises crosslinking a precursor solution comprising phenol- conjugated polymer e.g. phenol-conjugated gelatin polymer in the presence of an enzyme and an oxidising agent to form a phenol-conjugated gelatin hydrogel matrix. In one embodiment, the hydrogel matrix comprising phenol-conjugated gelatin polymer is formed by adding selected concentrations of an enzyme and an oxidising agent to a precursor solution comprising a selected concentration of phenol-conjugated gelatin polymer. The enzyme may be horseradish peroxidase (HRP). The oxidising agent may include but is not limited to hydrogen peroxide and benzoyl peroxide. The hydrogel matrix comprising phenol-conjugated gelatin polymer may be formed via enzymatic crosslinking of the phenol groups in the presence of the oxidising agent. The amount of enzyme to be added may be selected so as to crosslink the phenol groups at a desired gelation rate. The amount of enzyme e.g. HRP may be of at least a threshold amount which is effective for crosslinking the polymer to form the hydrogel. The amount of enzyme is generally measured in units (U). For example, one unit of enzyme is the amount of enzyme that catalyses the reaction of 1 pmol of the substrate in 1 minute under the standard conditions. In various embodiments, depending on the gelation rate to be achieved, the concentration of enzyme may be from about 0.030 U/mL (unit/mL) to about 0.20 U/mL, from about 0.040 U/mL to about 0.18 U/mL, from about 0.060 U/mL to about 0.16 U/mL, from about 0.080 U/mL to about 0.14 U/mL, or from about 0.10 U/mL to about 0.12 U/mL. The inventors have also found that when the enzyme concentration is above a certain amount, variation in its concentration may not have a substantial impact on the crosslinking density at a given H2O2 concentration. In various embodiments therefore, the concentration of the enzyme may be at least about 0.020 U/mL, at least about 0.030 U/mL, at least about 0.040 U/mL, at least about 0.050 U/mL, at least about 0.060 U/mL, at least about 0.070 U/mL, at least about 0.080 U/mL, at least about 0.090 U/mL, or at least about 0.10 U/mL. The concentration of enzyme may be chosen in order to reach a pre-specified, desired gel point. The amount of oxidizing agent to be added may be chosen in order to adjust or control the crosslinking density in the resulting hydrogel. The crosslinking density in the resulting hydrogel may affect the stiffness which may be measured in terms of its storage modulus (G’). Therefore, the storage modulus may be adjusted/tuned/configured based on the added amount of oxidizing agent. Independent tuning of gelation rate and stiffness of hydrogel may be based on a catalytic system where an enzyme (e.g. HRP) catalyses the crosslinking reaction in the presence of an oxidant/oxidising agent (e.g. H2O2). Without being bound by theory, it is believed that after successive oxidations of two phenol molecules, the enzyme (e.g. HRP) returns to its original state and re-enters the crosslinking cycle. Thus, the independent tuning achieved in phenol-conjugated gelatin hydrogel may be due to the catalytic reaction of an enzyme (e.g. HRP) and an oxidising agent (e.g. H2O2). Advantageously, the mechanical properties of the hydrogel can be adjusted or controlled during the formation process. Independent tuning of the phenol-conjugated gelatin hydrogel may allow hydrogels to be formed at an efficient gelation rate with a wide range of stiffness. Without being bound by theory, it is believed that appropriate mechanical properties, e.g. stiffness, and biochemical properties play a role in maintaining growth and viability of cells culture in the hydrogel. This may be achieved by adjusting the gelation rate and crosslinking density of the hydrogel.

The inventors have found that in various embodiments, below a certain H2O2 concentration, an increase in H2O2 concentration does not affect the gelation rate and only increases the crosslinking density. Above such H2O2 concentration, further increases in H2O2 concentration may decrease the crosslinking density and decrease the gelation rate due to denaturation of HRP. In this respect, the concentration of oxidizing agent may be varied singly such that the phenol-conjugated gelatin hydrogel formed from the precursor solution possesses the desired storage modulus. In various embodiments, depending on the degree of crosslinking to be achieved, the concentration/molarity of oxidising agent may be from about 0.14 mM to about 4.0 mM, from about 0.16 mM to about 3.8 mM, from about 0.18 mM to about 3.6 mM, from about 0.20 mM to about 3.4 mM, from about 0.22 mM to about 3.2 mM, from about 0.24 mM to about 3.0 mM, or from about 0.26 mM to about 2.8 mM. The storage modulus may be selected depending on the particular application e.g. type of cell to be cultured. In various embodiments, the storage modulus is selected to be at least about 1000 Pa for supporting in vitro culture of tumour cells.

In various embodiments, the method of preparing a tumour cell culture system comprises tuning mechanical or biochemical properties apart from gelation rate and/or stiffness. Additional/further tuning of the hydrogel matrix may be performed to impart stability to the hydrogel matrix in various biological environments, and also to impart improved properties in terms of biocompatibility and biodegradability.

In various embodiments, the method of preparing a tumour cell culture system comprises measuring the stiffness of the crosslinked hydrogel matrix. Stiffness of the hydrogel matrix may be studied using techniques such as oscillatory rheometry which measures the storage modulus (G’) and loss modulus (G”) against the shear strain. Rheological method may be employed to study the viscoelastic behavior of materials and G’ may be used as an indication of stiffness of a given viscoelastic material. In addition, the gel point may be employed to evaluate the gelation rate of hydrogel. For example, the value of G’ when it reaches a plateau may indicate that crosslinking is completed.

In various embodiments, the method of preparing a tumour cell culture system comprises isolating tumour cells/organoids/tissues for seeding into the hydrogel matrix. Sources of tumour cells for seeding may include but are not limited to cancer cell lines, resected tumours from patients, tumour biopsies from patients, and patient-derived xenografts. In one embodiment, isolation of tumour cells comprises harvesting tumours e.g. patient-derived xenograft tumours from an animal model e.g. mouse model. In one embodiment, isolation of tumour cells comprises physically cutting/mincing a tumour sample into fragments. In one embodiment, isolation of tumour cells further comprises chemically digesting fragments of a tumour sample using one or more enzymes.

In one embodiment, isolation of tumour cells further comprises passing/straining tumour fragments through one or more cell strainers for one or more times to obtain tumour organoids within a defined range of sizes. The tumour organoids may be within a range from about 20 pm to about 150 pm, from about 30 pm to about 140 pm, from about 40 pm to about 130 pm, from about 50 pm to about 120 pm, from about 60 pm to about 1 10 pm, from about 70 pm to about 100 pm, or from about 80 pm to about 90 pm. In various embodiments, initial organoid sizes for subsequent seeding/encapsulation may be kept at less than 100 pm, because the diffusion limits for exchange/transportation of materials e.g. nutrients and waste are typically around 150-200 pm. Without being bound by theory, it is believed that organoids which are significantly bigger than 200 pm tend to lose viability due to inefficient nutrient and waste exchange by diffusion. However, if the organoids are intended for use within a relatively short period or immediately after encapsulation, e.g. in a short-term drug study where organoid growth is substantially negligible, and the untreated organoids are only expected to maintain their current viability level, then an upper limit of 150 pm may be acceptable.

In one embodiment, isolation of tumour cells comprises mashing/cutting of larger tumour fragments, redigesting tumour fragments with different enzymes and/or re-straining tumour fragments to increase tumour cell/organoid yield. In one embodiment, isolation of tumour cells comprises resuspending tumour cells/organoids in solution e.g. in buffer solution prior to use. In one embodiment, isolation of tumour cells comprises quantifying organoid yield by counting the number of organoids per volume e.g. 10 mI aliquot under a microscope. In one embodiment, isolation of tumour cells comprises adjusting the organoid suspension to an appropriate density for seeding/ encapsulation.

In various embodiment, the method of preparing a tumour cell culture system comprises seeding/encapsulating tumour cells into the hydrogel matrix. Prior to seeding, the tumours cells may be in an organoid form or non-organoid form that is suitable to be subsequently cultured into an organoid form.

In one embodiment, seeding of tumour cells into the hydrogel matrix comprises reconstituting the lyophilised phenol-conjugated polymer e.g. phenol- conjugated gelatin polymer with the tumour cells to form a precursor solution comprising said tumour cells.

In one embodiment, seeding of tumour cells into the phenol-conjugated hydrogel comprises adjusting the pH of the precursor solution by adding a suitable base/acid. The pH level of the precursor solution may be from about 4 to about 8, from about 4.5 to about 7.5, from about 5 to about 7, from about 5.5 to about 6.5, or from about 6 to about 6.5. In one embodiment, the pH level of the precursor solution is adjusted to a physiological pH of about 7.4.

In one embodiment, seeding of tumour cells into the phenol-conjugated hydrogel comprises filtering the precursor solution. The precursor solution may be filtered using e.g. a 0.2 pm filter to remove unwanted particles e.g. bacteria and larger particles prior to organoid encapsulation.

In one embodiment, seeding of tumour cells into the phenol-conjugated hydrogel comprises separately dissolving an enzyme and an oxidising agent in a buffer solution e.g. PBS. In one embodiment, seeding of tumour cells into the phenol-conjugated hydrogel comprises adding a volume of the enzyme to the precursor macromer solution and mixing/vortexing for a period of time e.g. 5 seconds, before adding and mixing organoids e.g. by pipetting.

In one embodiment, seeding of tumour cells into the phenol-conjugated hydrogel further comprises adding and mixing the oxidising agent to the gelation mixture. For example, the gelation mixture may be pipetted for a number of times e.g. 10-12 times before dispensing a volume e.g. 50 pL into a culture dish e.g. well of an ultra-low attachment cell culture plates e.g. 96-well plate. In one embodiment, seeding of tumour cells into the phenol-conjugated hydrogel comprises selecting a gel point of a time duration to ensure an even organoid distribution throughout the hydrogel. The time duration may be from about 10 seconds to about 120 seconds, from about 20 seconds to about 1 10 seconds, from about 30 seconds to about 100 seconds, from about 40 seconds to about 90 seconds, from about 50 seconds to about 80 seconds, or from about 60 seconds to about 70 seconds.

In one embodiment, seeding of tumour cells into the phenol-conjugated hydrogel comprises incubating the tumour cells encapsulated within the hydrogel within a time period. After the enzyme and oxidising agent are added and mixed with the phenol-conjugated polymer, gelation is initiated in the precursor solution to form a hydrogel within a time period, e.g. within about one second to about 20 minutes, depending on the concentration of enzyme in the solution. Gelation rate may also depend on the temperature. In various embodiments, the gelation process may be increased by incubating at a relatively higher temperature, such as from about 25°C to about 40°C, from about 26°C to about 39°C, from about 27°C to about 38°C, from about 28°C to about 37°C, from about 29°C to about 36°C, from about 30°C to about 35°C, from about 31 °C to about 34°C, or from about 32°C to about 33°C.

In one embodiment, seeding of tumour cells into the phenol-conjugated hydrogel comprises seeding a selected number/density of cells/organoids. For example, the organoid seeding density may range from about 100 organoids/ml to about 4 x 10³ organoids/ml, about 200 organoids/ml to about 3 x 10³ organoids/ml, about 400 organoids/ml to about 2 x 10³ organoids/ml, about 600 organoids/ml to about 1 x 10³ organoids/ml, about 800 organoids/ml to about 900 organoids/ml, about 5 x 10³ organoids/ml to about 10 x 10⁴ organoids/ml, from about 6 x 10³ organoids/ml to about 9 x 10⁴ organoids/ml, from about 7 x 10³ organoids/ml to about 8 x 10⁴ organoids/ml, from about 8 x 10³ organoids/ml to about 7 x 10⁴ organoids/ml, from about 9 x 10³ organoids/ml to about 6 x 10⁴ organoids/ml, from about 1 x 10⁴ organoids/ml to about 5 x 10⁴ organoids/ml, from about 2 x 10⁴ organoids/ml to about 4 x 10⁴ organoids/ml, from about 2.5 x 10⁴ organoids/ml to about 3 x 10⁴ organoids/ml.

In various embodiments, the method of preparing a tumour cell culture system comprises maintaining the hydrogel matrix with the tumour cells at any oxygen level that is below atmospheric oxygen level. Maintaining under hypoxic condition may advantageously improve the organoid microenvironment in the hydrogel matrix by mimicking the in vivo hypoxic tumour microenvironment. This may promote organoid survival and growth in vitro, since hypoxia could induce the expression of growth factors that promote tumour cell survival and proliferation. In one embodiment, the hydrogel matrix with the tumour cells is maintained at no more than about 10% or no more than about 5% oxygen levels in a hypoxia chamber.

In various embodiments, the method of preparing a tumour cell culture system comprises introducing one or more compounds to the hydrogel matrix. The one or more compounds may include but are not limited to therapeutic agents, hormones, hormone releasing agents, hormone analogs, and anti proliferative agents. The therapeutic agent may be selected from the group consisting of a chemotherapeutic agent, a toxin, a radiotherapeutic agent, a radiosensitizing agent, a genetic construct, and combinations thereof. The chemotherapeutic agent may be selected from the group consisting of a small molecule or macromolecular anti-tumour drug, a cytokine, an anti metabolite, an alkylating agent, a hormone, methotrexate, doxorubicin, daunorubicin, cytosine arabinoside, etoposide, 5-4 fluorouracil, melphalan, chlorambucil, a nitrogen mustard, cyclophosphamide, cis-platinum, vindesine, vinca alkaloids, mitomycin, bleomycin, purothionin, macromomycin, 1 ,4- benzoquinone derivatives, trenimon, steroids, aminopterin, anthracyclines, demecolcine, etoposide, mithramycin, doxorubicin, daunomycin, vinblastine, neocarzinostatin, macromycin, -amanitin, and combinations thereof. The toxin may be selected from the group consisting of Russell's Viper Venom, activated Factor IX, activated Factor X, thrombin, phospholipase C, cobra venom factor, ricin, ricin A chain, Pseudomonas exotoxin, diphtheria toxin, bovine pancreatic ribonuclease, pokeweed antiviral protein, abrin, abrin A chain, gelonin, saporin, modeccin, viscumin, volkensin and combinations thereof.

In various embodiments, the tumour cell culture system is advantageously compatible with, and is configured to test proven/ FDA- approved anti-cancer drugs and novel/ investigational/ repurposed drug candidates, of both small and large molecular weights. The term“molecular weight” refers to the sum of the atomic weights of all atoms constituting a molecule and can be numerically expressed in Dalton (Da). Low molecular weight compounds may have a molecular weight of less than 900 Da and large molecular weight compounds may have a molecular weight greater than 900 Da. Advantageously, tumour organoids cultured in the tumour cell culture system may retain chemosensitivity, organoid histomorphology, cellular polarity and mutational profile, thus providing a suitable platform for drug testing/ screening. In one embodiment, chemotherapeutic compounds for treating colorectal cancer e.g. 5-fluorouracil, oxaliplatin, irinotecan and cetuximab are introduced to the hydrogel matrix.

In various embodiments, the method of preparing a tumour cell culture system comprises performing tests to monitor the development of the tumour cell culture system. For example, assays such as cytotoxicity assays and cell viability assays may be performed at selected time points during culture of the tumour cell culture system. Histological techniques such as immunofluorescence staining may be performed on hydrogel matrix comprising 5 tumour cells.

In various embodiments, the crosslinking of precursor solution comprising phenol-conjugated gelatin polymer is substantially non-cytotoxic and may be safely carried out in a conventional biosafety hood. The phenol ic) conjugated gelatin polymer may be crosslinked through the phenol groups to form the hydrogel matrix in an enzyme-mediated oxidation crosslinking process, in the presence of an enzyme and oxidising agent. This may advantageously obviate use of physical crosslinking or chemical crosslinkers that may otherwise introduce cytotoxicity and reduce bioactivity of the surrounding biological is agents.

In various embodiment, the method of preparing a tumour cell culture system comprises engrafting/transplanting the hydrogel matrix comprising the tumour cells into an animal model for in vivo culture. The tumour cells may be 20 coated with the hydrogel matrix prior to transplantation into the animal e.g. mouse model. A suitable animal model may be an immunocompromised mouse e.g. patient-derived xenograft mouse models. Other animal species with appropriate immunosuppression may have the potential to support patient- derived xenograft tumour engraftment as well. Advantageously, the hydrogel 25 matrix comprising the tumour cells may exhibit accelerated PDX tumour take- rate and tumour progression in vivo.

BRIEF DESCRIPTION OF FIGURES

30 FIG. 1 is a schematic diagram of a general carbodiimide/active ester- mediated coupling reaction of 3,4-hydroxyphenylpropionic acid (FIPA) and gelatin for synthesis of gelatin-FIPA conjugates in an embodiment. FIG. 2A is a schematic diagram of processes for forming different hydrogels in an embodiment.

FIG. 2B is a schematic diagram of a process for forming a gelatin-FIPA hydrogel in an embodiment.

FIG. 3A is a bar chart showing organoid areas measured from FIA-Tyr hydrogel samples, with Geltrex acting as a positive control.

FIG. 3B are microscope images of organoids from FIA-Tyr hydrogel and Geltrex samples that are stained with calcein AM and propidium iodide (PI) and are depicted in grayscale for clarity. Scale bars: 100 pm.

FIG. 4A are microscope images of CRC-PDX organoids encapsulated in gelatin-FIPA hydrogels and Geltrex samples that are stained with calcein AM and propidium iodide (PI), and are depicted in grayscale for clarity. Scale bars: 50 pm.

FIG. 4B is a bar chart showing organoid areas measured from gelatin- HPA hydrogel samples, with Geltrex acting as a positive control.

FIG. 4C are microscope images of CRC-PDX organoids were encapsulated in gelatin-FIPA hydrogels and FIA-Tyr hydrogel samples that are stained with calcein AM and propidium iodide (PI), and are depicted in grayscale for clarity. Scale bars: 100 pm.

FIG. 4D are microscope images of CRC-PDX organoids encapsulated in gelatin-FIPA, gelatin-H PA/FI A-Tyr, FIA/Tyr hydrogels and Geltrex samples that are stained with calcein AM and propidium iodide (PI), and are depicted in grayscale for clarity. Scale bars: 100 pm.

FIG. 4E is a bar chart showing organoid areas measured from gelatin- HPA and gelatin-HPA/HA-Tyr hydrogel samples, with Geltrex acting as a positive control.

FIG. 4F is a bar chart showing organoid areas measured from FIA-Tyr hydrogel samples, with Geltrex acting as a positive control.

FIG. 5A is a chart showing measurements of storage modulus and Young’s modulus of gelatin-FIPA hydrogels prepared at various concentrations of H2O2. FIG. 5B is a chart showing mean organoid areas measured from CRC-

PDX organoids encapsulated in different gelatin-FIPA hydrogels, with Geltrex acting as a positive control.

FIG. 5C is a chart showing organoid size distributions measured from CRC-PDX organoids encapsulated in different gelatin-FIPA hydrogels, with Geltrex acting as a positive control.

FIG. 5D is a chart showing ATP contents of CRC-PDX organoids encapsulated in different gelatin-FIPA hydrogels, with Geltrex acting as a positive control.

FIG. 5E is a chart showing DNA contents of CRC-PDX organoids encapsulated in different gelatin-FIPA hydrogels, with Geltrex acting as a positive control.

FIG. 5F are microscope images of CRC-PDX organoids encapsulated in gelatin-FIPA hydrogels and Geltrex samples that are stained with calcein AM and propidium iodide (PI), and are depicted in grayscale for clarity. Scale bars: 50 pm.

FIG. 5G are microscope images of CRC-PDX organoids encapsulated in gelatin-HPA hydrogels and Geltrex samples that are immunohistochemically stained for ITGA6 and F-actin, which are depicted in grayscale for clarity. Scale bars: 50 pm.

FIG. 6A are microscope images of CRC-PDX organoids isolated from various CRC-PDX tumours and encapsulated in G1 k-5% and G3k gelatin-FIPA hydrogels that are stained with calcein AM and propidium iodide. Only calcein images are shown because the propidium iodide staining is substantially undetectable. Scale bars: 100 pm. FIG. 6B are microscope images of CRC-PDX organoids isolated from various CRC-PDX tumours and encapsulated in GO.5k and G3k gelatin-FIPA hydrogels that are stained with calcein AM and propidium iodide. Only calcein images are shown because the propidium iodide staining is substantially undetectable. Scale bars: 100 pm.

FIG. 6C is a chart showing organoid areas measured from gelatin-FIPA hydrogel and Geltrex samples containing CRC-PDX organoids (line 106-p12, subcutaneous implantation site). FIG. 6D is a chart showing ATP contents measured from gelatin-FIPA hydrogel and Geltrex samples containing CRC-PDX organoids (line 106-p12, subcutaneous implantation site).

FIG. 6E is a chart showing organoid areas measured from gelatin-FIPA hydrogel and Geltrex samples containing CRC-PDX organoids (line 106-p1 1 , caecal implantation site). FIG. 6F is a chart showing ATP contents measured from gelatin-FIPA hydrogel and Geltrex samples containing CRC-PDX organoids (line 106-p1 1 , caecal implantation site). FIG. 7A are microscope images showing CRC-PDX organoids encapsulated in hydrogels that are mixed/not mixed with laminin and fibronectin, and that are stained with calcein AM and propidium iodide. Only calcein images are shown because the propidium iodide staining is substantially undetectable. Scale bars: 100 pm.

FIG. 7B is a chart showing organoid areas measured from gelatin-FIPA hydrogels with and without incorporation of LF.

FIG. 7C are microscope images showing CRC-PDX organoids encapsulated in hydrogels that are mixed/not mixed with Geltrex, and that are stained with calcein AM and propidium iodide. Only calcein images are shown because the propidium iodide staining is substantially undetectable. Scale bars: 100 pm. FIG. 7D is a chart showing organoid areas measured from G1 k gelatin-

FIPA hydrogels, with and without incorporation of Geltrex.

FIG. 7E is a chart showing organoid areas measured from G3k gelatin- FIPA hydrogels, with and without incorporation of Geltrex.

FIG. 8A are microscope images showing CRC-PDX organoids from multiple lines and tumour implantation sites encapsulated in hydrogels that are maintained under normoxia or hypoxia conditions, and that are stained with calcein AM and propidium iodide. Only calcein images are shown because the propidium iodide staining is substantially undetectable. Scale bars: 100 pm. FIG. 8B is a chart showing organoid areas measured from gelatin-FIPA hydrogel and Geltrex cultured under normoxia and hypoxia conditions. Dotted line indicates day 1 (D1 ) level. FIG. 8C is a chart showing ATP contents measured from gelatin-FIPA hydrogel and Geltrex cultured under normoxia and hypoxia conditions. Dotted line indicates day 1 (D1 ) level.

FIG. 8D is a chart showing organoid areas measured from gelatin-FIPA hydrogel and Geltrex cultured under normoxia and hypoxia conditions. Dotted line indicates day 1 (D1 ) level.

FIG. 8E is a chart showing ATP contents measured from gelatin-FIPA hydrogel and Geltrex cultured under normoxia and hypoxia conditions. Dotted line indicates day 1 (D1 ) level.

FIG. 9A are microscope images showing H&E staining of organoids (line 106-p14-s.c.) cultured in G3k or Geltrex in either normoxic or hypoxic conditions, or the parental PDX tumour. Scale bars: 50 pm.

FIG. 9B are microscope images showing H&E staining of organoids (line 1 1 1 -p12-cae) cultured in G3k or Geltrex in either normoxic or hypoxic conditions, or the parental PDX tumour. Scale bars: 50 pm. FIG. 9C are microscope images showing immunohistochemical staining of cellular polarity markers, ITGA6 or CK20, on organoids cultured in G3k or Geltrex. The colour images are depicted in grayscale for clarity. Scale bars: 50 pm. FIG. 9D is a chart showing mutation profiles of primary tumour, CRC-

PDX tumour and organoids. FIG. 10A is a dose response curve showing ATP content in a first group of G3k- or Geltrex- cultured CRC-PDX organoids with various concentrations of 5-fluorouracil (5-FU) for 9 days (n = 4). FIG. 10B is a dose response curve showing ATP content in a second group of G3k- or Geltrex- cultured CRC-PDX organoids with various concentrations of 5-fluorouracil (5-FU) for 9 days (n = 4).

FIG. 10C is a dose response curve showing ATP content in a first group of G3k- or Geltrex- cultured CRC-PDX organoids with various concentrations of oxaliplatin for 9 days (n = 4).

FIG. 10D is a dose response curve showing ATP content in a second group of G3k- or Geltrex- cultured CRC-PDX organoids with various concentrations of oxaliplatin for 9 days (n = 4).

FIG. 10E are microscope images of CRC-PDX organoids encapsulated in gelatin-FIPA hydrogels and Geltrex treated with chemotherapeutic drugs. Representative calcein AM and bright-field images of organoids (line 89) after various treatments for 9 days were taken (n = 4). Scale bars: 50 pm.

FIG. 11 A is a dose response curve showing ATP content in G3k- or Geltrex- cultured CRC-PDX organoids from KRAS mutant (line 1 11 -p16) with various concentrations of cetuximab for 9 days (n = 4).

FIG. 11 B is a dose response curve showing ATP content in G3k- or Geltrex- cultured CRC-PDX organoids from KRAS mutant (line 97-p7) with various concentrations of cetuximab for 9 days (n = 4). FIG. 1 1 C is a dose response curve showing ATP content in G3k- or

Geltrex- cultured CRC-PDX organoids from KRAS/NRAS/BRAF wild-type lines (line 89-p12) with various concentrations of cetuximab for 9 days (n = 4). FIG. 1 1 D is a dose response curve showing ATP content in G3k- or Geltrex- cultured CRC-PDX organoids from KRAS/NRAS/BRAF wild-type lines (line 1 13-p4) with various concentrations of cetuximab for 9 days (n = 4).

FIG. 11 E is a dose response curve showing ATP content in G3k- or Geltrex- cultured CRC-PDX organoids from KRAS/NRAS/BRAF wild-type lines (line 1 17-p3) with various concentrations of cetuximab for 9 days (n = 4). FIG. 12A is a chart showing take rate of uncoated CRC-PDX tumour,

CRC-PDX tumour coated with G3k gelatin-FIPA hydrogel and Geltrex.

FIG. 12B is a chart showing tumour volume of uncoated CRC-PDX tumour, CRC-PDX tumour coated with G3k gelatin-FIPA hydrogel and Geltrex.

FIG. 12C is a chart showing survival rate of uncoated CRC-PDX tumour, CRC-PDX tumour coated with G3k gelatin-FIPA hydrogel and Geltrex.

FIG. 13A are microscope images of nasopharyngeal carcinoma (NPC)- PDX organoids encapsulated in various gelatin-FIPA hydrogels and Geltrex that are stained with calcein AM and propidium iodide (PI), and are depicted in grayscale for clarity. Scale bars: 200 pm.

FIG. 13B is a chart showing ATP content of NPC-PDX organoids encapsulated in various gelatin-FIPA hydrogels, with Geltrex acting as a positive control.

EXAMPLES Example embodiments of the disclosure will be better understood and readily apparent to one of ordinary skill in the art from the following examples, tables and if applicable, in conjunction with the figures. The materials used in the examples were obtained from the following sources. Gelatin (MW = 80 - 140 kDa, pi = 5) and horseradish peroxidase (HRP) (150 units/mg) were obtained from Wako Pure Chemical Industries (Japan). 3,4-Hydroxyphenylpropionic acid (HPA), tyramine hydrochloride (Tyr-HCI), /V-hydroxysuccinimide (NHS), 1 -ethyl-3-(3-dimethylaminopropyl)- carbodiimide hydrochloride (EDC-HCI), type IV collagenase and fibronectin (from bovine plasma) were purchased from Sigma-Aldrich (Singapore). Hyaluronic acid (HA) (90 KDa) was supplied by Chisso Corporation (Tokyo, Japan). Geltrex (LDEV-free reduced growth factor), mouse laminin-11 1 , dispase, LIVE/DEAD™ Viability/Cytotoxicity Kit, Quant-iT™ PicoGreen™ dsDNA Assay Kit, were purchased from Life Technologies. Hydrogen peroxide (H2O2) was from MGC Pure Chemicals Singapore.

For statistical analysis of data, Kruskal-Wallis test with Dunn’s multiple comparison test was used for comparisons of organoid size distributions. Tukey’s method was used to plot the boxplot whiskers for the organoid size boxplots. The one-way ANOVA was used with Tukey’s multiple comparison test for comparisons of ATP or DNA content. Error bars show standard error unless otherwise stated. Unless otherwise defined in the captions, p-values are defined as follows: ^* p < 0.05, ^** p < 0.01 , ^*** p < 0.001 , ^**** p < 0.0001.

Example 1 - Synthesis of gelatin-HPA, HA-Tyr and hydrogel characterization

Gelatin-HPA conjugates were synthesised from a general carbodiimide/active ester-mediated coupling reaction of 3,4- hydroxyphenylpropionic acid (HPA) and gelatin. HA-Tyr conjugates were synthesised in a similar manner from the carbodiimide/active ester-mediated coupling reaction. To synthesise gelatin-HPA conjugates, a solvent mixture of distilled water and N,N-dimethylformamide (DMF) was first prepared by mixing the distilled water and DMF in a 3:2 weight ratio. FIPA (3.32 g, 20 mmol), N- hydroxysuccinimide (NFIS) (3.20 g, 27.8 mmol) and 1 -ethyl-3-(3- dimethylaminopropyl)-carbodiimide hydrochloride (EDC-FICI) (3.82 g, 20 mmol) were dissolved in 250 ml of the solvent mixture to form a reaction mixture. The reaction mixture was stirred at room temperature for 5 hours, and the pH of the reaction mixture was maintained at 4.7. Next, 150 ml of gelatin aqueous solution (6.25 wt.%) was added to the reaction mixture and stirred overnight at room temperature at pH 4.7. As shown in FIG. 1 , the gelatin may contain other amino acid residues (represented by X and Y). The percentage of FIPA introduced to the amine groups of gelatin was determined by the conventional 2,4,6-trinitrobenzene sulfonic acid (TNBS) method. The solution of the reaction mixture and gelatin was transferred to dialysis tubes with molecular cut-off of 1000 Da. The dialysis tubes were dialyzed against 100 mM sodium chloride solution for 2 days, a mixture of distilled water and ethanol (in a 3:1 volume ratio) for 1 day and distilled water for 1 day, successively, to obtain a purified solution. The purified solution was lyophilised to obtain the gelatin-FIPA conjugate.

To synthesise FIA-Tyr conjugates, FIA (1 g, 2.5 mmol) was first dissolved in 100 ml of distilled water. To this solution tyramine hydrochloride (202 mg, 1.2 mmol) was added. EDC-FICI (479 mg, 2.5 mmol) and NFIS (290 mg, 2.5 mmol) were then added to initiate the conjugation reaction. As the reaction proceeded, the pH of the mixture was maintained at 4.7 with 0.1 M NaOFI. The reaction mixture was stirred overnight at room temperature and then the pH was brought to 7.0. The solution was transferred to dialysis tubes with a molecular cut-off of 1000 Da. The tubes were dialyzed against 100 mM sodium chloride solution for 2 days, a mixture of distilled water and ethanol (in a 3:1 volume ratio) for 1 day and distilled water for 1 day, successively, to obtain a purified solution. The purified solution was lyophilized to obtain the FIA-Tyr. As shown in FIG. 2A, gelatin-HPA conjugate 202 and HA-Tyr conjugate 204 were mixed with cells 206, an oxidising agent (H2O2) and an enzyme (HRP) which crosslinked the conjugates to form hydrogels e.g. gelatin-HPA hydrogel 208, gelatin-HPA/HA-Tyr hydrogel 210, and HA-Tyr hydrogel 212.

As shown in FIG. 2B, the gelatin-HPA hydrogel 208 was formed by mixing gelatin-HPA conjugates 202 with cells 206 via oxidative coupling of HPA moieties 214 catalysed by H2O2 and HRP. As shown in the enlarged view 216 of the hydrogel 208, the gelatin-HPA conjugates were crosslinked to one another via the phenol groups.

Rheological measurements of the hydrogel formation were performed with a HAAKE Rheoscope 1 rheometer (Karlsruhe, Germany) using a cone and plate geometry of 35 mm diameter and 0.945° cone angle. The measurements were taken at 37°C in the dynamic oscillatory mode with a constant deformation of 1 % and frequency of 1 Hz. A roughened glass bottom plate was used to avoid slippage of samples during the measurement. HRP and H2O2 solutions with different concentrations were added sequentially to an aqueous solution of gelatin-HPA (2% or 5% w/v, 250 ml in PBS). The solution was vortexed and then immediately applied to the bottom plate. The upper cone was then lowered to a measurement gap of 0.025 mm and a layer of silicon oil was carefully applied around the cone to prevent solvent evaporation during the experiment. The measurement parameters were determined to be within the linear viscoelastic region in preliminary experiments. Rheological measurement was allowed to proceed until the storage modulus (G’) reached a plateau, indicating that crosslinking was completed. Compression tests of the hydrogels were performed with Instron microtester (Model 5848P8600). Cylindrical hydrogels were prepared with a plastic mold and allowed to swell in HBSS for 2 hours. Gel dimensions were measured with calipers before loading between the parallel plates of the microtester. Compression tests were carried out using a 50 N load cell, with a compressive strain rate of 1 mm/min and no preload. Young’s modulus (E) was determined using the slope of the linear regime of the stress versus strain curve at low strains (5 - 15%).

Example 2 - Isolation of CRC-PDX organoids

Organoid isolation from PDX tumours that were stored in an isolation buffer comprising RPMI (Roswell Park Memorial Institute) containing 20mM HEPES (4-(2-hydroxyethyl)-1 -piperazineethanesulfonic acid) and 5% penicillin/streptomycin (P/S) on ice started within 1 hour after tumour harvest from mice, and was performed as previously described by Kondo et al, Proc. Natl. Acad. Sci. U.S.A. 2011 Apr 12; 108 (15): 6235-40.

Briefly, grossly necrotic tissue was excised and the remaining tumour tissue was minced into 1 -2 mm fragments prior to digestion in 0.28 U/mL Liberase DH (Roche) in isolation buffer containing 1 U/mL DNase I (New England Biolabs) and 5 mM Y-27632 ROCK I inhibitor (Stem Cell Technologies) at 37°C with manual agitation every 10 - 15 minutes.

After 1.5 - 2 hours, tumour fragments were washed twice via centrifugation, resuspended in fresh isolation buffer, and strained through a 100 pm cell strainer with gentle mashing of larger fragments to increase the yield. The fraction smaller than 100 pm was strained through a reversible 37 pm cell strainer (Stem Cell Technologies) to obtain a 37 - 100 pm fraction which was defined as CRC-PDX organoids.

In some experiments, the fraction greater than 100 pm was re-digested with trypsin for 10 - 15 minutes at 37°C, and re-strained through a 100 pm strainer to increase the organoid yield. Organoids were resuspended in HBSS + 20mM HEPES (bHBSS) before use. Organoid yield was quantified by counting the number of organoids per 10 pi aliquot under a microscope, and the final organoid suspension was adjusted to an appropriate density for encapsulation.

Example 3 - Organoid encapsulation and 3D culture in gelatin-HPA, HA-Tyr and Geltrex

Gelatin-HPA or HA-Tyr macromers were reconstituted in bHBSS to the desired working concentration at 5% (w/v) or 1 % (w/v) respectively. Both macromer solutions were pH-adjusted to about 7.4 by adding 1 M NaOH and 0.2 pm-filtered prior to organoid encapsulation. HRP and H2O2 were prepared in PBS. An appropriate volume of HRP was added to the diluted macromer solution and vortexed for 5 seconds, before organoids were added and mixed by pipetting. Lastly, H2O2 was added and the gelation mixture was pipetted I Q- 12 times before 50 pL was dispensed per well in ultra-low attachment 96-well plates (Corning). A gel point of about 1 minute was chosen to ensure an even organoid distribution throughout the hydrogel. Gelatin-HPA/HA-Tyr composite hydrogels encapsulating organoids were similarly fabricated.

In parallel, organoids were resuspended in Geltrex at 1.2% (w/v) and dispensed as above. CRC organoid medium was added after complete gelation (about 45 minutes at 37°C) and prepared as previously described for human intestinal organoids except Wnt3A [Sato, T. et al. Gastroenterology 141 , 1762- 1772 (2011 )]. Advanced DMEM/F12 (Life Technologies) culture medium was supplemented with penicillin/streptomycin, 10 mM HEPES (Life Technologies), 2 mM Glutamax (Life Technologies), 1 x B27 (Life Technologies), 1 mM N- acetylcysteine (Sigma), 10nM [Leu15]-gastrin I (Sigma), 50 ng/mL recombinant human noggin (Sigma), 50pg/mL recombinant human R-spondin-1 (Sigma), 500 nM A83-01 (Tocris), and 10 mM SB202190 (Sigma). Plates were incubated at 5% O2 in a hypoxia chamber (Billups-Rothenberg Inc.) and media was changed every 2-3 days.

Example 3A - Organoid characterization Cvtoxicitv and viability assays

Live/Dead cytotoxicity assay was performed according to the manufacturer’s instructions. At appropriate time points, gels were degraded (gelatin-HPA: 150 U/mL collagenase IV; Geltrex: 1 U/mL dispase) for 1 hour at 37°C. Organoids were pelleted and washed twice in bHBSS by centrifugation before proceeding to the CellTiter-Glo® Luminescent Cell Viability Assay (Promega), which was performed according to the manufacturer’s instructions In some experiments, the cell lysate from the CellTiter-Glo assay was used as starting material in the Picogreen assay. Preliminary experiments with DNA standards confirmed that the presence of the CellTiter-Glo reagent in the DNA samples did not interfere with the Picogreen assay in a non-linear fashion, and that the background fluorescence signal from a blank CellTiter-Glo reagent sample can simply be subtracted from the measured fluorescence in organoid samples.

Immunofluorescence staining of organoids Organoids from degraded hydrogels were immunostained with a whole- mount-type protocol. Briefly, organoids were fixed in 10% neutral-buffered formalin for 15 - 20 minutes at room temperature and washed thrice in 1 x PBS. Fixed organoids were incubated with 50 mM NH₄CI for 15 minutes at room temperature to quench autofluorescence, permeabilised with 0.1 % Triton X-100 for 10-15 minutes at room temperature, blocked with 5% BSA/PBS for 1 hour at room temperature or overnight at 4°C and incubated with primary antibodies (rat anti-human ITGA6: clone NKI-GoH3, 20 gg/mL; mouse anti-human CK20: clone Ks 20.8, 1.6 gg/mL) overnight at 4°C. Organoids were washed thrice in PBS/0.1 % Tween20 and incubated in secondary antibodies (goat anti-rat IgG- AlexaFluor488: 1 :400; goat anti-mouse lgG2a-AlexaFluor488: 1 :400) overnight at 4°C. All antibodies were diluted in 5% BSA/PBS/0.1 % Tween20/0.05% sodium azide. Organoids were counterstained with 10 pg/mL Hoechst 33258 and stored in PBS/0.05% sodium azide at 4°C until imaging.

Microscopy

Live/Dead images were acquired on an Olympus IX-70 epifluorescence microscope. Immunofluorescence images were acquired on an Olympus FV- 1000 inverted laser scanning confocal microscope (IMB Microscope Unit, A^*STAR, Singapore).

Histology

Organoids were fixed in neutral buffered formalin for about 15 - 20 minutes and washed thrice in 1 x PBS before dehydration through graded ethanol into xylene and paraffin infiltration. Organoids were then embedded into paraffin blocks and sectioned with rotary microtome into 5pm thick sections. Slides with sections were dried and placed into incubator at 60°C for 15 minutes before haematoxylin and eosin staining with Leica Autostainer XL. Sections were deparaffinised and rehydrated through graded ethanol into water. Rehydrated sections were then subjected to Hematoxylin solution, Bluing, Clarifier and Eosin-Phloxine B Solution and were further dehydrated through graded ethanol into xylene.

Mutational profiling

Custom library panel for 40 genes (including HRAS (exon 1 -3), KRAS (exon 1 -3), NRAS (exon 1 -3) and BRAF (exon 1 1 , 12, 15)) were designed using Ion AmpliSeq™ Designer (Life Technologies) and library preparation was performed with Ion AmpliSeq™ Custom Panels and Library Kits 2.0 (Life Technologies) following the manufacturer’s instructions. Panel includes two primer pools with 1 132 primer pairs and starting DNA template of up to a total of 20 ng (10 ng per pool) was used for each sample. Ion Xpress™ Barcode Adapter 1-96 kit (Life Technologies) was used for sample barcoding. Libraries were quantified with 2100 Agilent Bioanalyzer System (Agilent Technologies, Santa Clara, CA, USA). Template preparation, enrichment of Ion Sphere™ Particles and chip loading were performed using the Ion Chef™ System with Ion PI™ Hi-Q™ Chef kit and PI™ Chip v3 and sequencing data was generated on the Ion Proton™ sequencer. Filtering, trimming and alignment were processed with the Torrent Suite and BAM files were analyzed on the Ion Reporter™ software for final somatic variant calling using the paired tumour-normal workflow. Mean sequencing depth of 2000x and 540x were obtained for tumour (primary/PDX/organoids) and matched adjacent normal respectively.

Example 3B - Effects of HA-Tyr hydrogels on CRC-PDX organoid growth and organoid viability CRC-PDX organoids were encapsulated in 1 % (w/v) HA-tyramine (Tyr) hydrogels, which were enzymatically crosslinked with horseradish peroxidase (HRP) in the presence of hydrogen peroxide (H2O2), at various crosslinking densities by controlling the H2O2 concentration, which resulted in hydrogels of various storage moduli (G’ = 91 ± 0.3, 281 ± 10 and 1231 ± 154 Pa, which are abbreviated respectively, as H0.1 k, HO.3k and H1 2k).

HO.1 k, HO.3k and H1.2k represent HA-Tyr hydrogel. GO.1 k, GO.5k, G1 k, G3k, G5.5k G7k represent gelatin-HPA hydrogel. GH1 k represent a composite of gelatin-HPA and HA-Tyr. Table 1 summarises the preparation and rheological properties of the various hydrogels.

Table 1. Preparation and rheological properties of hydrogels.³

Sample Gelatin- HA- Geltrex HRP H2O2 G’ Gel point^b Time needed

HPA Tyr (wt.%) (U/mL) (mM) (Pa) (min) for G’ to reach

(wt.%) (wt.%) plateau

(min)

91 ± 0.98 ± 2.94 ±

H0.1 k 0 0 0.188 0.164

0.3 0.01 0.06 281 ± 1.57 ± 5.40 ±

H0.3k

0 0.188 0.221

10 0.01 0.60

1231 ± 1.57 ± 16.38 ±

H1.2k

0 0.188 0.374

154 0.01 0.04

113 ± 1.19 ± 2.03 ±

GO.1k

0 0.090 0.547

22 0.13 0.21

510 ± 1.40 ± 5.21 ±

GO.5k

0 0.090 0.910

42 0.14 0.04

G1k- 1000 ± 0.97 ± 4.70 ±

0 0.090 0.999

2% 2 0.05 0.07

998 ± 0.96 ± 9.66 ±

GH1k 0 0.090 0.956

27 0.28 0.53

G1k- 1030 ± 1.06 ± 3.12 ±

0 0.090 1.406

5% 108 0.04 0.06

3115 ± 1.04 ± 4.52 ±

G3k

0 0.090 2.060

204 0.09 0.27

5545 ± 1.07 ± 7.04 ±

G5.5k

0 0.090 3.000

330 0.09 0.30

7152 ± 1.15 ± 11.37 ±

G7k

0 0.090 4.000

93 0.06 0.69

G1k-

1000 ± 1.36 ± 8.94 ± 2% +

0.1 0.090 1.352

18 0.10 0.33 Geltrex

G3k + 3076 ± 1.01 ± 5.14 ±

0.1 0.090 2.222

Geltrex 308 0.07 0.61 ^a Measurement was taken with constant deformation of 1% at 1 Hz and 37°C ( n = 3). Results are shown as the average values ± standard deviation.

b Gel point is defined as the time at which the crossover of storage modulus (G’) and loss modulus (G”) occurred. It is used herein as an indicator of the rate of gelation.

Hyaluronan (HA) is a major component of the extracellular matrix in CRC and regulates CRC survival and invasion. CD44, the major cell surface receptor for HA, is expressed in CRC and is also a key marker of cancer stem cells that may sustain tumour growth and progression. Therefore, it was initially hypothesised that an HA-based biomaterial may support the culture of CRC- PDX organoids in vitro.

However, the results show that HA-Tyr hydrogels support CRC-PDX organoid growth over time but do not maintain organoid viability. As shown in FIG. 3A, CRC-PDX organoids that were encapsulated in mechanically defined 1 % (w/v) HA-Tyr hydrogels exhibited growth over time, as shown by an increase in the measured organoid areas on microscopy images. However, organoid viability was suboptimal, as shown with the positive viability marker, calcein AM, and negative viability marker, propidium iodide in FIG. 3B. Geltrex served as a positive biological control matrix for PDX organoid viability. Kruskal- Wallis test (p < 0.0001 ) with Dunn’s multiple comparison test (n = 100, 294, 387 and 412 organoids measured for HO.1 k, HO.3k, H1.2k and Geltrex respectively). Therefore, the presence of other ECM molecules may be necessary to support better organoid viability.

Example 3C - Effects of gelatin-HPA hydrogels on CRC-PDX organoid growth and viability As shown in FIG. 4A, CRC-PDX organoids (line 106) encapsulated in mechanically defined 2% (w/v) gelatin-HPA hydrogels exhibited more intense calcein AM and less intense propidium iodide staining in stiffer G1 k-2% compared to GO.5k hydrogels. Geltrex served as a positive biological control matrix for organoid viability. As shown in FIG. 4B, CRC-PDX organoids encapsulated in stiffer G1 k-2% exhibited faster growth over time than those in GO.5k, as quantified via microscopy. Kruskal-Wallis test (p < 0.0001 ) with Dunn’s multiple comparison test were performed for statistical analysis (n = 168, 221 and 160 organoids measured for GO.5k, G1 k and Geltrex respectively). As shown in FIG. 4C, CRC-PDX organoids (line 92) exhibited more intense calcein AM and less intense propidium iodide staining in gelatin-HPA compared to HA- Tyr hydrogels at 6 days post-encapsulation. Next, the effects of gelatin-HPA hydrogels on CRC-PDX organoids were compared against gelatin-HPA/HA-Tyr and HA/Tyr hydrogels, with Geltrex serving as a positive control. As shown in FIG. 4D, CRC-PDX organoids encapsulated in gelatin-HPA hydrogels exhibited more intense calcein AM and less intense propidium iodide staining as compared to gelatin-HPA/HA-Tyr, HA/Tyr hydrogels and Geltrex which served as a positive biological control matrix for organoid viability. FIG. 4D shows that gelatin-HPA improves organoid viability as compared to gelatin- HPA/HA-Tyr and HA-Tyr. As shown in FIG. 4E and 4F, CRC-PDX organoids encapsulated in gelatin-HPA hydrogel exhibited comparable growth over time as those in gelatin-HPA/HA-Tyr and HA-Tyr hydrogels, as quantified via microscopy. FIG. 4E shows that G1 k increases organoid sizes as compared to GH1 k, but still supports smaller organoid sizes than Geltrex.

The above results demonstrate that gelatin-HPA hydrogels support CRC- PDX organoid growth and viability. The CRC-PDX organoids were encapsulated in gelatin-HPA hydrogels and enzymatically crosslinked with HRP in the presence of H2O2. Collagen is a major component of CRC tumour ECM and purified type I collagen gels may support the 3D culture of primary CRC organoids. Initial experiments revealed that very soft 2% w/v gelatin-HPA hydrogels (G’ = 113 ± 22 Pa; GO.1 k ) rapidly degraded within 2 days after organoid encapsulation, possibly due to organoid-secreted gelatinases, and that stiffer 1000 Pa hydrogels (G’ = 1000 ± 2 Pa; G1 k-2%) supported better organoid viability than 500 Pa hydrogels (G’ = 510 ± 42 Pa; GO.5k) See FIG. 4A and Table 2-1. In addition, as shown in FIG. 4C, G1 k-2% maintained better organoid viability than HA-Tyr gels of matched stiffness (H1 k) and supported faster organoid growth than GO.5k gels (see also FIG. 4B and Table 2-1 ). Together, these data suggest that increased gelatin-HPA hydrogel stiffness not only promoted organoid growth, but also better maintained organoid survival. Table 2-1 . Effect of Gelatin-HPA hydrogel stiffness on PDX organoids.³

PDX line^b Tumour Gl k v.s. GO.5k G1 k v.s. Geltrex

site⁰

Viability^® Size ATP Viability^® Size ATP

106-p4 s.c. > > n.d.^f = < n.d.

Table 2-2. Effect of Gelatin-HPA hydrogel stiffness on PDX organoids. ^£

PDX Iine^b Tumour G3k v.s. G1 k G3k v.s. Geltrex

site⁰

Viability^® Size ATP Viability^® Size ATP

106-p7 s.c. > = n.d. = < n.d. 106-p7 cae. > > n.d. n.d. 103-p6 s.c. > = n.d. n.d. 103-p6 cae. > > n.d. n.d.

106-p12 s.c. > (N, H) > (N, H) > (N, H) = (N, H) = (N, H) - (N, H) 106-p1 1 cae. = (N, H) = (N, H) = (N, H) <(N), >(H) < (N, H) - (N, H)

106-p14 s.c. n.d. n.d. > (N, H) n.d. n.d. - (N, H)

1 17-p3 s.c. - (H) > (H) > (H) - (H) > (H) - (H)

89-p12 s.c. n.d. n.d. - (H) n.d. n.d. < (H)

97-p7 s.c. > (H) n.d. - (H) - (H) n.d. < (H) ^a 3D organoid culture was conducted in either normoxia (N) or hypoxia (H) condition. If not specified, then a particular experiment was conducted only under N.

b p: passage number for a particular PDX line.

⁰ s.c.: subcutaneous, cae. : caecal.

d Or maximum G’ tested in a particular experiment if < 3 kPa.

^® Increased viability is defined as a decrease in % of organoids with Pl⁺ cells and/or an increase in relative calcein staining intensities. ^f n.d.: not determined

Example 3D - Effects of mechanically optimized gelatin-HPA hydrogels on CRC-PDX organoid viability and growth

As shown in FIG. 5A, dynamic rheological measurements of 5% (w/v) gelatin-HPA mixed with 0.09 U/mL HRP and various H2O2 concentrations revealed increased maximal storage modulus (G’) with increasing H2O2 concentration (left axis). Young’s moduli (E) of equilibrium-swollen gelatin-HPA hydrogels increased with increasing H2O2 concentration used during gel formation (right axis).

As shown in FIGs. 5B and 5C, CRC-PDX organoids (line 1 17) encapsulated in mechanically defined 5% (w/v) gelatin-HPA hydrogels exhibited optimal growth rates in moderately stiff G3k hydrogels, as quantified via microscopy in terms of mean organoid areas per well (FIG. 5B) and organoid size distributions (FIG. 5C). For FIG. 5B, one-way ANOVA (p < 0.0001 ) with Tukey’s multiple comparison test (n = 4) was performed for statistical analysis. For FIG. 5C, Kruskal-Wallis test (p < 0.0001 ) with Dunn’s multiple comparison test was performed for statistical analysis (n = 405, 483, 506, 471 and 510 organoids measured for G1 k, G3k, G5.5k, G7k and Geltrex respectively).

As shown in FIG. 5D, organoid ATP content (line 1 17) was optimally maintained in G3k hydrogels, as quantified via the CellTiter-Glo assay performed on organoids retrieved from degraded hydrogels. ^** p = 0.0036, ^***p = 0.0002, one-way ANOVA (p < 0.0001 ) with Tukey’s multiple comparison test (n = 4).

As shown in FIG. 5E, organoid DNA content (line 1 17) was decreased in stiffer G7k hydrogels but similar to Geltrex in other gelatin-HPA hydrogels tested, as quantified via the Picogreen assay. ^* p = 0.03, one-way ANOVA (p > 0.05) with Tukey’s multiple comparison test (n = 4). As shown in FIG. 5F, organoids (line 1 17) exhibited more intense calcein AM and less intense propidium iodide staining in G3k, G5.5k and Geltrex compared to G1 k-5% and G7k hydrogels at 7 days post-encapsulation.

As shown in FIG. 5G, organoids (line 1 17) exhibited basolateral ITGA6 and apical F-actin in G3k, G5.5k, G7k and Geltrex but not in G1 k-5% hydrogels at 7 days post-encapsulation. Table 3 summarises the frequencies in which organoid sizes and ATP contents of G3k samples are larger than/ equal to/ smaller than those of G1 k samples. In general, G3k samples have larger organoids sizes and ATP contents than G1 k samples. Notably, none of the G3k samples had organoid sizes and ATP contents smaller than G1 k samples.

Table 3. Comparison of organoid sizes and ATP contents of G3k samples versus G1 k samples.

Organoid size ATP

G3k > G1 k 5/8 3/7

G3k = G1 k 3/8 4/7^"

G3k < G1 k 0/8 0/ T

Generally, human CRC tumour ECM is mechanically stiffer than its paired perilesional or normal ECM counterparts. In this study, the inventors sought to address the question of whether gelatin-FIPA hydrogels stiffer than 1 kPa could provide a better microenvironment for CRC-PDX organoids, and whether there might be an optimal stiffness for the maintenance of CRC-PDX organoids.

In this respect, stiffer hydrogels comprising 5% (w/v) gelatin-FIPA were fabricated. Flowever, the inventors found that a progressive increase in FI2O2 concentration, which should lead to an increase in crosslinking density and hence an increase in equilibrium G’, did not lead to a monotonic increase in G’, but instead led to apparently mechanically unstable gels that exhibited transient peaks in G’ and with no measurable equilibrium G’ above H2O2 concentrations of 2.3 mM. These transient peaks were found to be due to rapid gel shrinkage during rheology rather than inherent gel instability, and the measurement of the Young’s moduli of equilibrium-swollen gelatin-HPA gels fabricated with various H2O2 concentrations confirmed that stably crosslinked gelatin-HPA hydrogels as stiff as E = 35 kPa could be fabricated (see FIG. 5A). This expanded range of gelatin-HPA hydrogel stiffness enabled the mimicking of both human CRC tumour tissue (E = 15 - 40 kPa) and healthy colon tissue (E = 2 - 5 kPa) in terms of their stiffness.

As shown in FIG. 5B and 5C, encapsulation of CRC-PDX organoids in 5% (w/v) gelatin-HPA hydrogels ranging from G’ = 1 - 7 kPa (G’ = 1030 ± 108, 31 15 ± 204, 5545 ± 330, 7152 ± 93 Pa corresponding to E = 2.6, 14.6, 22.3 and

34.0 kPa respectively, and abbreviated as G1 k-5%, G3k, G5.5k and G7k respectively) revealed that organoids grew larger in moderately stiff G3k hydrogels than in softer G1 k-5% or much stiffer G7k hydrogels. Consistent with organoid sizes, the overall ATP content of gelatin-HPA-cultured organoids exhibited a bimodal trend with the highest ATP levels in G3k organoids, as shown in FIG. 5D. In contrast, organoids exhibited a significant decrease in DNA content in the stiffest G7k hydrogel (see FIG. 5E).

Organoid viability was well-maintained at day 7 post-encapsulation in all gelatin-HPA hydrogels tested, but were qualitatively better in G3k or G5.5k hydrogels (see FIG. 5F). Organoids cultured in all gelatin-HPA hydrogels generally exhibited a cystic/luminal morphology that is reminiscent of those cultured in Geltrex (see FIG. 5F) and of other patient-derived CRC tumour organoids cultured in Matrigel. However, while organoids in stiffer hydrogels (G3k, G5.5k, G7k) universally exhibit appropriate epithelial polarity, most organoids in G1 k-5% hydrogels did not appropriately express integrin a6 (ITGA6) on the basolateral surface (see FIG. 5G). Organoids cultured in Geltrex were observed to adopt a smooth/rounded morphology that is typical of CRC organoids faster than those in stiff gelatin-HPA hydrogels, which may be related to a relative lack of rigidity and hence increased mechanical pliancy of Geltrex. Gelatin-HPA-cultured organoids also had a tendency to exhibit multiple, in contrast to single, lumen compared to Geltrex-cultured organoids, although such morphologies are observed in both matrices. These findings suggested that G3k hydrogels provided an optimal mechanical environment for culturing CRC-PDX organoids. The above results demonstrate that mechanically stiffer gelatin-HPA hydrogels support optimal CRC-PDX organoid growth and viability. In particular, gelatin-HPA hydrogels of moderately increased stiffness (Young’s modulus E « 15 - 20 kPa) relative to normal colon tissue (E = 2 - 5 kPa) supported increased viability, sizes and ATP levels in organoids derived from multiple CRC-PDX lines. However, further increases in gel stiffness resulted in suboptimal organoid viability, growth and metabolism, which suggest that an optimum exists. The variability in the effect of increased stiffness on organoid growth and metabolism could reflect an intrinsic variability in the stiffness of patient-derived CRC tumours (E = 13 - 50 kPa), and suggest that the optimal stiffness for organoid culture may vary from donor to donor. This putative donor- dependent variation could partially account for the finding that G3k did not always support organoid growth rates and metabolism that were comparable to Geltrex, which served as a biological positive control. Example 3D - Generalisabilitv of increased gelatin-HPA hydrogel stiffness on CRC-PDX organoid viability and growth

To determine whether the effect of increased gelatin-HPA hydrogel stiffness on CRC-PDX organoid viability and growth rates is generalizable, organoids were isolated from various CRC-PDX tumours and cultured in either G1 k-5%, G3k or Geltrex for up to two weeks. As shown in FIGs. 6A and 6B, CRC-PDX organoids encapsulated in G3k exhibited higher viability, as shown by more intense calcein AM compared to G1 k-5% hydrogels in 3 out of 4 tumours studied from line 106, obtained from various passages and either subcutaneous (s.c.) or caecal implantation sites (see FIG. 6A), and both tumours studied from line 103, obtained from passage 6 at either subcutaneous (s.c.) or caecal implantation sites (see FIG. 6B).

As shown in FIGs. 6C and 6D, CRC-PDX organoids studied exhibited increased growth in G3k in 5 out of 8 tumours, and increased ATP content in G3k in 3 out of 6 tumours studied. An example of CRC-PDX organoids that exhibited increased organoid sizes (FIG. 6C) and ATP content (FIG. 6D) in G3k compared to G1 k-5%. For FIG. 6C, Kruskal-Wallis test (p = 0.0007) with Dunn’s multiple comparison test was performed for statistical analysis (n = 59, 102, 39, organoids measured for G1 k, G3k, Geltrex respectively). For FIG. 6D, ^* p = 0.03, one-way ANOVA (p > 0.05) with Tukey’s multiple comparison test was performed for statistical analysis (n = 3).

As shown in FIGs. 6E and 6F, CRC-PDX organoids exhibited similar organoid sizes (FIG. 6E) and ATP content (FIG. 6F) in G3k and G1 k-5%. For FIG. 6E, Kruskal-Wallis test (p < 0.0001 ) with Dunn’s multiple comparison test was performed for statistical analysis (n = 221 , 142, 71 , organoids measured for G1 k, G3k, Geltrex respectively).

The above results demonstrate that mechanically optimized gelatin-FIPA hydrogels support CRC-PDX organoid viability and growth from multiple PDX tumour lines. Increased organoid viability in G3k compared to G1 k-5% hydrogels was observed in a majority of CRC-PDX lines characterized with the calcein-AM/propidium iodide assay (7/9 PDX tumours) (See FIG. 6A, 6C and Table 2-2). For a particular PDX line (106), increased organoid viability in G3k was independent of tumour implantation site and tumour passage number (which potentially included an independent effect of murine host variability) (see FIG. 6A), and this phenotype was also observed in other parental PDX lines (FIG. 6B, Table 2-2). In contrast to its more consistent effects on organoid viability, increased gelatin-HPA stiffness either increased (see FIG. 6C) or had no effect (see FIG. 6D) on organoid growth (increased in 5/8 PDX tumours) and organoid ATP content (increased in 3/6 PDX tumours) (see Table 2-2). This phenotype varied with the tumour passage number or murine host (for example, 106-p4 versus 106-p7), but was not restricted to a particular tumour implantation site or parental PDX line (see Table 2). Nevertheless, increasing gelatin-HPA stiffness from 1 kPa to 3 kPa was not detrimental to organoid growth in any PDX line. Importantly, G3k supported comparable organoid size distributions and ATP content to Geltrex in 5/8 and 4/6 experiments respectively (see Table 2-2). Taken together, these data suggest that G3k hydrogels could provide a suitable alternative matrix for the 3D culture of CRC-PDX organoids.

Example 3E - Effects of Gelatin-HPA-based composite hydrogels on CRC-PDX Organoid Survival or Growth

CRC-PDX organoids were encapsulated in 2% (w/v) gelatin-HPA hydrogels (G1 k-2%), composite hydrogels comprising co-crosslinked gelatin- HPA and HA-Tyr mixed in a 1 :1 ratio (GH1 k), or composite hydrogels comprising semi-interpenetrating networks of chemically crosslinked G1 k-2% or GH1 k that are physically mixed with 50 pg/ml laminin and 50 pg/ml fibronectin (LF).

As shown in FIG. 7A, organoid viability was decreased in the GH1 k based composite hydrogels regardless of LF incorporation (compared with G1 k- 2%), and remained unchanged in the G1 k-2% + LF hydrogels, as shown by the positive viability marker, calcein AM.

As shown in FIG. 7B, organoid growth (line 106) in G1 k-2% hydrogels remained unchanged in the presence of LF, but decreased in GH1 k composite hydrogels regardless of LF incorporation, as quantified by microscopy. ^* p = 0.01 , ^*** p = 0.0003, one-way ANOVA (p < 0.0001 ) with Tukey’s multiple comparison test was performed for statistical analysis (n = 221 , 356, 415 and 310 organoids measured for G1 k-2%, G1 k-2% + LF, GH1 k and GH1 k + LF respectively). As shown in FIG. 7C, composite hydrogels comprising semi- interpenetrating networks of gelatin-FIPA (G1 k-2% or G3k) and 0.1 % (w/v) Geltrex generally exhibited similar levels of organoid viability compared with gelatin-FIPA alone (3 out of 4 tumours tested) and increased viability in 1 out of 4 tumours tested.

As shown in FIGs. 7D and 7E, organoids exhibited similar growth rates in gelatin-FIPA + Geltrex composites and gelatin-FIPA alone in 3 out of 4 PDX tumours studied, as quantified via microscopy. Two representative organoid size quantification plots showing gelatin-FIPA + Geltrex composites based either on G1 k-2% or G3k hydrogels are shown. For FIG. 7D, p > 0.05, two-tailed Mann-Whitney U-test was performed for statistical analysis (n = 98 and 102 organoids measured for G1 k-2% and G1 k-2% + Geltrex respectively). For FIG. 7E, p > 0.05, two-tailed unpaired t-test was performed for statistical analysis (n = 149 and 1 18 organoids measured for G3k and G3k + Geltrex respectively).

The above results demonstrate that composite gelatin-FIPA hydrogels do not improve CRC-PDX organoid survival or growth. In the above stiffness optimization studies with gelatin-FIPA, the inventors concurrently attempted to tune its biochemical properties in parallel by incorporating ECM molecules that are commonly overexpressed in various cancers, in particular HA, laminin and fibronectin. The optimization studies exploited the ability of HA-Tyr and gelatin- HPA to be chemically crosslinked by the same enzymatic mechanism to fabricate a co-crosslinked network of gelatin-HPA/HA-Tyr (GH1 k) with matched mechanical properties (G’ = 998 ± 27 Pa) to unmodified G1 k-2% (G’ = 1000 ± 2 Pa). Laminin and fibronectin were physically mixed into either G1 k-2% or GH1 k composite hydrogels during gelation. However, the results show that GH1 k composite gels negatively affected organoid viability and significantly decreased CRC-PDX organoid growth compared to unmodified G1 k-2% (see FIG. 7 A and 7B). The addition of laminin and fibronectin to either G1 k-2% or GH1 k hydrogels neither improved organoid viability nor organoid growth. Next, Geltrex was physically mixed into gelatin-HPA gels during gelation since the former contains a complex mix of mouse-derived growth factors that may provide beneficial pleiotropic effects on CRC-PDX organoids. Gelatin- HPA/Geltrex composite hydrogels, which contained 10% of the concentration used for Geltrex-only gels and were tuned to match the mechanical properties of the unmodified gelatin-HPA gels, led to modest improvements on organoid viability in earlier studies with softer G1 k-2% gels but had minimal effects in later studies with stiffer G3k gels compared with unmodified gels (see FIG. 7C). However, the composite gels did not increase organoid growth rates at either stiffness tested (see FIG. 7D). Therefore, these data suggest that unmodified gelatin-HPA hydrogels are adequate for the maintenance of CRC-PDX organoid survival and growth.

The ability of gelatin-HPA alone to support CRC-PDX organoid survival, but not HA-Tyr, suggests that integrin-dependent, rather than CD44 - dependent, cell adhesion is necessary and sufficient for maintaining organoid viability, given that gelatin contains exposed Arg-Gly-Asp (RGD) motifs that mediate integrin-dependent cell adhesion. The observation that addition of laminin-11 1 or fibronectin to soft gelatin-HPA gels (G1 k-2%) did not noticeably improve PDX organoid viability or growth could be due to the relatively low concentrations tested (50 pg/ml), or some degree of integrin-binding redundancy in fibronectin, where RGD motifs are the major integrin-binding sites. Laminin-1 1 1 may also affect more differentiated cellular phenotypes than viability or growth, as revealed by the requirement for both RGD and laminin- 1 1 1 to support the formation of differentiated intestinal organoids from normal murine ISC colonies, but only RGD for ISC colony survival and expansion in PEG hydrogels. Deeper molecular characterization of gelatin-HPA-cultured CRC-PDX organoids alongside the original donor tumour tissue should be performed to ascertain whether further optimization of scaffold biochemistry is necessary in order to maximize the maintenance of patient characteristics in these organoids. Example 3F - Effects of hypoxia on CRC-PDX organoid growth and viability

As shown in FIG. 8A, hypoxia (ambient 5% O2) increased CRC-PDX organoid viability in multiple lines and tumour implantation sites (subcutaneous (s.c.) and caecal). Organoids were stained with the positive viability marker, calcein AM.

As shown in FIGs. 8B and 8C, hypoxia increased organoid growth rates and ATP content in both gelatin-FIPA and Geltrex in 3 out of 5 PDX tumours studied, as quantified by microscopy or CellTiter-Glo assay respectively. Representative plots of line 106 organoids seeded at 1 x 10⁴ organoids/mL at 13 days post-encapsulation is shown. For FIG. 8B, two-tailed unpaired t-test was performed for statistical analysis (n = 59, 102, 49 and 39 organoids measured for G3k with or without hypoxia and Geltrex with or without hypoxia respectively). For FIG. 8C, ^** p = 0.0018, ^*** p = 0.0002, two-tailed unpaired t- test was performed for statistical analysis (n = 3).

As shown in FIGs. 8D and 8E, hypoxia decreased organoid growth rates and ATP content in line 122 organoids seeded at 5 x 10⁴ organoids/mL at 13 days post-encapsulation, as quantified by microscopy or CellTiter-Glo assay respectively. For FIG. 8D, two-tailed unpaired t-test was performed for statistical analysis (n = 54, 164, 126 and 218 organoids measured for G3k with or without hypoxia and Geltrex with or without hypoxia respectively). For FIG. 8E, ^** p = 0.0059, ^*** p = 0.0004, two-tailed unpaired t-test was performed for statistical analysis (n = 3).

The above results demonstrate that hypoxia supports optimal CRC-PDX organoid viability and growth. To further improve the organoid microenvironment in G3k hydrogels, the inventors have found that mimicking the in vivo hypoxic tumour microenvironment may promote organoid survival and growth in vitro, since hypoxia could induce the expression of growth factors that promote tumour cell survival and proliferation. Hypoxia generally increased the long-term viability of CRC-PDX organoids derived from tumours of various parental lines, and from both subcutaneous and caecal origins, in both G3k and Geltrex gels (see FIG. 8A and Table 4), except in certain cases where the viability of the normoxia controls was already high (e.g. Geltrex-cultured organoids from 106-p1 1 -cae and G3k-cultured organoids from 106-p14-sc). Hypoxia increased organoid size distributions (see FIG. 8B) in 3 out of 5 tumours studied for which organoid sizes could be measured, and increased ATP content (see FIG. 8C) in all 5 tumours studied when organoid seeding densities were between 7.5-13 x 10³ organoids/mL (see Table 4). The need to optimize the organoid density emerged in an early experiment where a high organoid density of 5.0 x 10⁴ organoids/mL resulted in an inhibitory effect of hypoxia on both organoid growth and ATP content (see FIG. 8D and 8E), presumably due to anoxia, although a parental line dependence cannot be ruled out since the experiment could not be subsequently repeated with the same line. Nevertheless, hypoxia did not inhibit organoid growth in subsequent studies where lower organoid densities were used. The broadly conserved trends across organoid sizes and ATP content (see Table 4) confirmed that ATP levels could provide a convenient readout for organoid growth in future studies. For any given PDX tumour, hypoxia often induced similar effects on organoid viability, growth and ATP levels in G3k and Geltrex (see Table 4), which suggests that similar mechanisms of hypoxia-induced organoid growth may have occurred in both matrices. Moreover, hypoxic G3k-cultured organoids largely exhibited comparable organoid sizes and ATP levels to hypoxic Geltrex- cultured organoids (see Table 4). These observations suggest that hypoxia can be generally applied to both G3k and Geltrex as it may improve, but not adversely affect, CRC-PDX organoid growth and metabolism. Table 4. Effect of hypoxia on PDX organoids.

G3k v.s. hypoxia v.s. normoxia Geltrex

(hypoxia)

CO co co

CD CD CD

X X X

103-p6 cae 7500

122-p4 s.c. 50000 13 > >

106-p12 s.c. 10000 13 > >

106-p1 1 cae 10000 13 >

89-p8 s.c. 9200 21 > > n.d.^d n.d.^d > > n.d.

106-pM s.c. 12000 21

1 1 1 -p12 cae 13000 21 > ^a p: passage number for a particular PDX line.

b s.c.: subcutaneous, cae.: caecal.

c Increased viability is defined as a decrease in % of organoids with Pl⁺ cells and/or an increase in relative calcein staining intensities.

d Normoxic organoids in this experiment exhibited extensive cell death leading to a loss of organoid integrity and poorly defined organoid boundaries.

The finding that hypoxia promoted CRC-PDX organoid viability, growth and metabolism in multiple lines highlights the importance of mimicking physiological oxygen levels in vitro. Under physiological normoxia, normal peripheral tissues are exposed to 3 - 7.4% O2, and intratumoural oxygen levels are significantly lower, ranging from 0.3 -4.2% O2. In contrast, in vitro normoxia is conventionally defined as 20% O2 (atmospheric), but the actual oxygen level at the cell surface depends on various culture parameters such as cell density, cellular oxygen consumption rate and oxygen diffusion distance (depth of culture medium).

Further complications arise in 3D hydrogel-based cultures due to decreased oxygen diffusion rates and generation of oxygen gradients within the hydrogels. Hence, the atmospheric oxygen levels employed in the hypoxia studies was set at a relatively high 5% O2 to avoid organoid anoxia, in conjunction with a control of organoid encapsulation densities. However, the variable organoid growth response to hypoxia suggests that certain PDX lines do not exhibit hypoxia-dependent growth in vitro and could reflect intrinsic parental line-dependent biological variability.

Example 3G - Effects of Gelatin-HPA on CRC-PDX Organoid Histological and Mutational Profile

As shown in FIGs. 9A and 9B, the images show that organoids generally resemble the parental tumour, with compact regions that are frequently interspersed with one or more lumens. As shown in FIG. 9C, organoids (line 1 17) maintained basolateral ITGA6

(top) and basolateral CK20 (bottom) and predominantly apical F-actin in G3k and Geltrex at 15 days post-encapsulation.

As shown in FIG. 9D, mutational profiles of primary tumour, CRC-PDX tumour and organoids were obtained by next generation sequencing (NGS) using a panel of 40 mutations commonly observed in colorectal cancer. This representative NGS data set shows that 5 out of 40 mutations present in the CRC-PDX tumour (line 106) was also present in organoids cultured in G3k and Geltrex at similar levels. Normoxia and hypoxia growth conditions had no effect on the mutation profile of the organoids. The above results demonstrate that gelatin-HPA maintains CRC-PDX organoid histological and mutational profile. To further establish the suitability for G3k for culturing CRC-PDX organoids as an alternative for Geltrex, G3k- cultured organoids were processed to examine the histomorphological and molecular characteristics of Geltrex-cultured organoids. H&E staining revealed that organoids cultured in G3k resemble those cultured in Geltrex, and organoids in both matrices generally recapitulate their original tumour histology. For example, organoids derived from poorly differentiated 106-p14 exhibited compact regions that were frequently interspersed with small lumens (see FIG. 9A), whereas those from moderately differentiated 1 11 -p12 retained cells with elongated nuclei and columnar morphology (see FIG. 9B). Immunofluorescence assays also showed that G3k organoids maintained appropriate epithelial polarity like Geltrex organoids, with predominantly basolateral expression of ITGA6 and apical localization of F-actin, and a comparable expression pattern of the differentiation marker CK20 at 2 weeks after encapsulation (see FIG. 9C). The mutational profiles of the parental tumour and cultured organoids were compared via next generation sequencing using a panel of hotspot mutations commonly observed in colorectal cancer, and the overall pattern of mutations in G3k- or Geltrex- cultured organoids was highly similar to the parental tumour (see FIG. 9D). Together, these data suggest that G3k maintained the histological and mutational characteristics of the parental tumour in a comparable fashion to Geltrex.

Example 4 - Effects of Gelatin-FIPA on CRC-PDX Organoid treated with chemotherapeutic drugs

Encapsulated organoids were treated with 5-fluorouracil (5FU, Selleck), oxaliplatin (Selleck) or cetuximab (Absolute Antibody) from day 1 to 9 or 10 after encapsulation. On day 9 or 10, organoids were retrieved from degraded gels and CellTiter-Glo assay was performed as above. ATP content for 5FU-treated organoids was normalized to the DMSO-treated control, whereas ATP content for oxaliplatin- or cetuximab- treated organoids was normalized to the untreated control. ATP levels of DMSO-treated or untreated controls are plotted at a drug concentration of 0.001 mM in the log x-axis.

The results shown in FIGs. 10A to 1 1 E demonstrate that CRC-PDX organoids cultured in gelatin-HPA retain chemosensitivity, organoid histomorphology, cellular polarity and mutational profile. Utilising CRC-PDX organoids cultured in G3k under hypoxia, an in vitro drug study was performed using conventional chemotherapies for CRC (5-FU, oxaliplatin and irinotecan), and using the CellTiter-Glo ATP assay to quantify drug efficacy.

Initial studies revealed both similarities and differences in chemosensitivity between G3k- and Geltrex- cultured organoids. For example, Geltrex-cultured organoids from line 89 responded to 5-fluorouracil but not to oxaliplatin whereas G3k organoids responded to both compounds (see FIG. 10A and 10B), which was validated by a viability assay (see FIG. 10E). Follow up studies performed with subsequent PDX tumour passages from the same parental line revealed that G3k supported reproducible trends in organoid chemosensitivity and consistent differential chemosensitivity patterns between G3k and Geltrex organoids (see FIG. 10C and 10D).

Overall, gelatin-FIPA-cultured CRC-PDX organoids demonstrated reproducible chemosensitivity to conventional CRC drugs such as 5-fluorouracil and oxaliplatin, but exhibited drug-dependent differences in sensitivity compared to Geltrex- cultured organoids. This differential sensitivity may reflect the vast differences in mechanical and biochemical properties between G3k and Geltrex, and their distinct effects on organoid phenotypes.

Example 5 - Effects of Gelatin-FIPA on CRC-PDX Organoid treated with anti- EGFR therapies

The results in FIGs. 1 1 A to 11 E demonstrate that CRC-PDX organoids cultured in gelatin-FIPA retain cetuximab sensitivity. In addition to chemotherapies, anti-EGFR therapies like cetuximab are important therapeutic options for CRC. Biomarkers that predict cetuximab efficacy, such as KRAS, NRAS and BRAF, are clinically used to stratify CRC patients into different treatment regimes, since a mutation in any of these oncogenes potentially renders the tumour unresponsive to cetuximab due to constitutive EGFR pathway activation.

Next-generation sequencing of the parental tumours from five CRC-PDX lines used in the cetuximab studies was performed in KRAS exons 1 -3, NRAS exons 1 -3 and BRAF exons 1 1 , 12, 15, which harbour hotspot mutations in CRC as well as functionally important domains. Mutational analyses revealed that three lines were wild-type for these genes, whereas the other two exhibited KRAS mutations (see Table 5). G3k-cultured organoids from both KRAS mutant lines predictably did not respond to cetuximab when cultured in G3k, whereas Geltrex-cultured organoids from line 1 1 1 showed a significant response (see FIG. 11 A and 1 1 B). Among the three KRAS/NRAS/BRAF wild-type lines studied, organoids from two lines (89, 1 13) exhibited matrix-independent decreases in ATP content, albeit only at relatively high cetuximab concentrations (20 - 50 pg/ml) (see FIG. 1 1 C and 1 1 D). Flowever, organoids from line 117 were cetuximab-sensitive only when cultured in G3k (see FIG. 1 1 E).

Table 5. KRAS/NRAS/BRAF mutational profiles of various CRC-PDX lines as determined by next-generation sequencing.

PDX Line KRAS NRAS BRAF

89 WT WT WT

1 13 WT WT WT

1 17 WT WT WT

1 1 1 G12V WT WT 97 Q22K WT WT

Gelatin-HPA-cultured organoids from all three wild-type KRAS/NRAS/BRAF lines studied exhibited cetuximab sensitivity, albeit at relatively high concentrations, whereas one of these three lines (117) was unresponsive when cultured in Geltrex. However, the discrepancy in cetuximab sensitivity observed between G3k- and Geltrex-cultured organoids in line 1 17 suggests the possibility of matrix-dependent effects on cetuximab sensitivity.

Nevertheless, the relatively high cetuximab doses required to elicit a response in this study reflect (i) the generally low sensitivities reported in

Matrigel-cultured PDX organoids, (ii) the lower efficacy of cetuximab monotherapy compared with combination therapy in KRAS wild-type CRC patients, and (iii) the existence of indirect, immune-mediated inhibitory mechanisms of cetuximab that may not be captured in vitro, for example, antibody-dependent cell-mediated cytotoxicity. As with chemotherapies, the relative predictivity of cetuximab sensitivity in G3k- and Geltrex-cultured organoids cannot be resolved unless the treatment history of the donor patient is available for comparison. As most CRC patients are treated with combination therapies, future studies may also test relevant drug combinations (e.g. FOLFIRI, FOLFOX) in order to enable validation of in vitro drug responses.

Example 6 - In vivo PDX expansion

Since G3k hydrogels optimally supported organoid growth in vitro, the G3k hydrogels were next evaluated for its ability to support PDX tumour engraftment in vivo.

CRC-PDX tumours (line 89) were harvested from existing PDX tumour- engrafted mice and cut into fragments of approximately 0.5 cm dimensions after removal of necrotic tissue. Tumour fragments were kept on ice in RPMI/metronidazole/BAytril (no gel control) or in a Matrigel mixture (50% v/v phenol red-free Matrigel, 50% v/v antibiotics mixture of metronidazole + BAytril), or kept in a gelatin-HPA/HRP/H202/metronidazole/BAytril mixture that was undergoing gelation at room temperature. The amount of H2O2 added was adjusted to maintain a final hydrogel G’ of 3 kPa to account for the presence of metronidazole and BAytril in the gelation mixture, and the gelation time was adjusted to be ~10 min to allow gelatin-HPA-coated tumour fragments to be incubated for a comparable length of time as those incubated in the Matrigel mixture. Tumour fragments were subcutaneously implanted in 5 - 10 weeks old NOD/SCID mice (InVivos). Tumour lengths (L) and widths (W) were measured two to three times per week with calipers, and tumour volumes were estimated as follows: (L ^* W²)/2. Mice were sacrificed when tumour volumes reached 1500 mm³ or when the tumour exhibited visible necrosis.

The results in FIGs. 12A to 12C show that gelatin-HPA supports accelerated CRC-PDX tumour engraftment and growth in vivo. CRC-PDX tumour fragments (line 89) were subcutaneously implanted in NSG mice either uncoated (No gel, black) or coated with Geltrex or G3k. G3k and Geltrex supported faster tumour take, faster tumour growth and shorter median time to sacrifice than the No gel control (125, 96 and 84 days for No gel, Geltrex and G3k respectively). Mice were sacrificed when tumour volumes reached 1500 mm³. For FIG. 12B, ^**** p < 0.0001 , two-way ANOVA was performed for statistical analysis (p < 0.0001 for matrix type) with Tukey’s multiple comparison test (n = 8). For FIG. 12C, ^*p < 0.05, versus no gel control by log-rank (Mantel- Cox) test was performed for statistical analysis (n = 8).

The conventional model of PDX tumour expansion involves dividing a suitable PDX tumour into several fragments, and coating each fragment with Matrigel is a common strategy to improve tumour take rates upon transplantation. CRC-PDX tumour fragments coated with either G3k or Geltrex reached a 100% take-rate between days 50-60 post-transplantation, whereas the no-gel control fragments took 80 days to reach stable 100% engraftment (see FIG. 12A). Consistent with this observation, both G3k and Geltrex-coated tumour fragments exhibited measurable tumour growth by day 30, in contrast to day 45 for no-gel fragments (see FIG. 12B). Gel-coated fragments also exhibited a shorter a median time to sacrifice of 80-90 days as compared to 120 days for no-gel fragments (see FIG. 12C). These data suggest that G3k gelatin- FI PA hydrogels could accelerate PDX tumour take-rate and promote tumour progression in vivo in a comparable fashion to Geltrex.

Example 7 - Effects of Gelatin-FIPA-based hydrogels on NPC-PDX Organoid Survival or Growth

To investigate whether gelatin-FIPA-based hydrogels are suitable for culturing other types of organoids, NPC-PDX organoids were encapsulated in mechanically defined 5% (w/v) gelatin-FIPA hydrogels of various storage moduli.

As shown in FIG. 13A, NPC-PDX organoids encapsulated in G1 k, G3k, G5k and G7k hydrogels exhibited good viability, as shown by calcein AM staining. Geltrex served as a positive biological control matrix for organoid viability. Flowever, NPC-PDX organoids were smaller in G7k hydrogels compared with G1 k, G3k, G5k and Geltrex hydrogels.

As shown in FIG. 13B, NPC-PDX organoids encapsulated in G1 k, G3k and G5k hydrogels exhibited higher ATP content compared to G7k hydrogels. NPC-PDX organoids encapsulated in G1 k hydrogels exhibited the highest ATP content and is comparable to the ATP content of NPC-PDX organoids encapsulated in Geltrex. NPC-PDX organoids cultured in G3k and G5k hydrogels exhibited similar ATP content but are lower than the ATP content of NPC-PDX organoids encapsulated in G1 k hydrogels. The above results demonstrate that gelatin-FIPA hydrogels support NPC-

PDX organoid growth and viability. These data suggest that gelatin-FIPA hydrogels are suitable for culturing tumour cells from different tumour types. The customisability of the gelatin-HPA hydrogels may be advantageous in providing a viable platform for in vitro culture of a variety of tumour cells from different tumour types. Summary of findings

In this study, the inventors reported chemically and mechanically defined synthetic hydrogels for the 3D culture of tumour organoids (for e.g. CRC-PDX organoids, NPC-PDX organoids etc.) in vitro. Gelatin-HPA hydrogels were found to support better organoid survival than HA-Tyr hydrogels of comparable stiffness. Subsequently, gelatin-HPA hydrogels of moderate stiffness (G3k) were found to maintain better organoid viability and increase organoid growth compared with softer gelatin-HPA hydrogels (G1 k-5%) in a majority of PDX tumours studied. Inclusion of common ECM molecules, like HA, laminin-11 1 and fibronectin, to gelatin-HPA hydrogels neither increased organoid growth rate nor altered organoid viability under the conditions tested. However, hypoxia significantly increased organoid viability, growth and metabolism in a majority of PDX lines tested. Further studies revealed that G3k gels under hypoxia maintained parental tumour-specific histological and mutational characteristics in cultured organoids, supported organoid sensitivity to chemotherapies and cetuximab, and improved PDX tumour take and tumour growth in a comparable fashion to Geltrex in vivo.

Robust ex vivo culture methods for PDX tumour-derived organoids are urgently needed to enable the transition from the conventional use of cancer cell lines in cancer drug discovery. However, existing methods invariably utilise animal-derived matrices that offer limited control over their biochemical and mechanical properties and hence pose significant challenges in recapitulating the tumour-specific microenvironment. In contrast to natural matrices, chemically defined synthetic hydrogels that exhibit a larger range of mechanical stiffness can be fabricated without necessitating concomitant changes in matrix concentration, and may potentially better mimic naturally occurring mechanical changes that underlie tumourigenesis, such as increased matrix crosslinking. In the specific case of CRC, human CRC tumour ECM is mechanically stiffer than its paired perilesional or normal ECM counterparts, and increased lysyl oxidase (LOX) expression in CRC tumours and the increased collagen crosslinking that LOX mediates plays a causal role in this association. To incorporate the relevant mechanical rigidity associated with human CRC tumours while representing major tumour ECM components, the inventors employed enzymatically crosslinkable synthetic macromers of gelatin and HA that are conjugated to phenolic groups (HPA or tyramine respectively) to fabricate mechanically diverse hydrogels with tunable stiffness.

The ability of gelatin-HPA alone to support CRC-PDX organoid survival, but not HA-Tyr, suggests that integrin-dependent, rather than CD44 - dependent, cell adhesion is necessary and sufficient for maintaining organoid viability (see FIG. 3 and FIG. 7), given that gelatin contains exposed Arg-Gly- Asp (RGD) motifs that mediate integrin-dependent cell adhesion. The observation that addition of laminin-11 1 or fibronectin to soft gelatin-HPA gels (G1 k-2%) did not noticeably improve PDX organoid viability or growth could be due to the relatively low concentrations tested (50 pg/ml), or some degree of integrin-binding redundancy in fibronectin, where RGD motifs are the major integrin-binding sites.

Gelatin-HPA hydrogels of moderately increased stiffness (E « 15 - 20 kPa) relative to normal colon tissue (E = 2 - 5 kPa) supported increased viability, sizes and ATP levels in organoids derived from multiple CRC-PDX lines (see Table 1 , FIG. 5 and FIG. 6). However, further increases in gel stiffness resulted in suboptimal organoid viability, growth and metabolism (see FIG. 5), which suggest that an optimum exists. The variability in the effect of increased stiffness on organoid growth and metabolism (see Table 2) could reflect an intrinsic variability in the stiffness of patient-derived CRC tumours (E = 13 - 50 kPa), and suggest that the optimal stiffness for organoid culture may vary from donor to donor. This putative donor-dependent variation could partially account for the finding that G3k did not always support organoid growth rates and metabolism that were comparable to Geltrex (see Table 2, 4), which served as a biological positive control. The finding that hypoxia promoted CRC-PDX organoid viability, growth and metabolism in multiple lines (see FIG. 8) highlights the importance of mimicking physiological oxygen levels in vitro. Under physiological normoxia, normal peripheral tissues are exposed to 3 - 7.4% O2, and intratumoural oxygen levels are significantly lower, ranging from 0.3 - 4.2% O2. In contrast, in vitro normoxia is conventionally defined as 20% O2 (atmospheric), but the actual oxygen level at the cell surface depends on various culture parameters such as cell density, cellular oxygen consumption rate and oxygen diffusion distance (depth of culture medium). Further complications arise in 3D hydrogel-based cultures due to decreased oxygen diffusion rates and generation of oxygen gradients within the hydrogels. Flence, the atmospheric oxygen levels employed in the hypoxia studies was set at a relatively high 5% O2 to avoid organoid anoxia, in conjunction with a control of organoid encapsulation densities. Flowever, the variable organoid growth response to hypoxia suggests that certain PDX lines do not exhibit hypoxia-dependent growth in vitro and could reflect intrinsic parental line-dependent biological variability.

Gelatin-FIPA-cultured CRC-PDX organoids demonstrated reproducible chemosensitivity to conventional CRC drugs such as 5-fluorouracil and oxaliplatin, but exhibited drug-dependent differences in sensitivity compared to Geltrex- cultured organoids (see FIG. 10). This differential sensitivity may reflect the vast differences in mechanical and biochemical properties between G3k and Geltrex, and their distinct effects on organoid phenotypes.

Gelatin-FIPA-cultured organoids from all three wild-type KRAS/NRAS/BRAF lines studied exhibited cetuximab sensitivity, albeit at relatively high concentrations, whereas one of these three lines (117) was unresponsive when cultured in Geltrex (see FIG. 11 ). Flowever, the discrepancy in cetuximab sensitivity observed between G3k- and Geltrex-cultured organoids in line 1 17 suggests the possibility of matrix-dependent effects on cetuximab sensitivity. Nevertheless, the relatively high cetuximab doses required to elicit a response in this study reflect (i) the generally low sensitivities reported in Matrigel-cultured PDX organoids, (ii) the lower efficacy of cetuximab monotherapy compared with combination therapy in KRAS wild-type CRC patients, and (iii) the existence of indirect, immune-mediated inhibitory mechanisms of cetuximab that may not be captured in vitro, for example, antibody-dependent cell-mediated cytotoxicity. As with chemotherapies, the relative predictivity of cetuximab sensitivity in G3k- and Geltrex-cultured organoids cannot be resolved unless the treatment history of the donor patient is available for comparison. As most CRC patients are treated with combination therapies, future studies may also test relevant drug combinations (e.g. FOLFIRI, FOLFOX) in order to enable validation of in vitro drug responses.

In conclusion, by exploiting enzymatically crosslinkable phenol- conjugated gelatin derivatives, the inventors have developed a chemically and mechanically defined matrix for the successful culture of patient-derived tumour organoids (for e.g. CRC-PDX organoids, NPC-PDX organoids etc.). It should be appreciated that the specific examples provided herein are not meant to be exhaustive and the matrix can extend to other types of phenol-conjugated ECM- derived macromers that may also provide well-defined matrices with tailorable mechanical and chemical properties for the culture of various types of patient- derived tumour organoids.

APPLICATIONS

Various embodiments of the disclosure provided herein provide a tumour cell culture system and a method of preparing a tumour cell culture system. The tumour cell culture system may advantageously be chemically and mechanically defined synthetic 3D hydrogels which are developed based on phenol- conjugated polymer e.g. phenol-conjugated gelatin polymer for 3D culture of tumour cells/organoids/tissues. Advantageously, the method may allow independent control of hydrogel mechanical strength and gelation rate at a pre- defined hydrogel concentration.

Various embodiments of the phenol-conjugated polymer provide a chemically and mechanically defined matrix for the successful culture of patient- derived tumour organoids. By mimicking the mechanical properties of tumour ECM with mechanically defined phenol-conjugated hydrogels of moderate stiffness, tumour cells can be optimally maintained in vitro, including their survival, growth, mutational profile and drug response, and also support PDX tumour engraftment in vivo. Various embodiments of the tumour cell culture system serve as a 3D organoid culture platform, where the hydrogels comprising phenol-conjugated polymers may be applied to encapsulate a variety of patient-derived tumour organoids in a chemically and mechanically tailorable microenvironment for various applications. Embodiments of the tumour cell culture system are therefore well-defined matrices with tailorable mechanical and chemical properties for the culture of other types of tumour cells e.g. patient-derived tumour organoids for future studies involving other types of PTOs.

Various embodiments of the tumour cell culture system serve as a practical alternative matrix/platform for in vitro PTO culture. The tumour cell culture system may facilitate robust ex vivo culture methods for PDX tumour- derived organoids, which are urgently needed to enable the transition from the conventional use of cancer cell lines in cancer drug discovery. Existing methods invariably utilise animal-derived matrices that offer limited control over their biochemical and mechanical properties and hence pose significant challenges in recapitulating the tumour-specific microenvironment. In contrast to natural matrices, various embodiments of the tumour cell culture system comprise chemically defined synthetic hydrogels which may exhibit a larger range of mechanical stiffness and may be fabricated without necessitating concomitant changes in matrix concentration, and may potentially better mimic naturally occurring mechanical changes that underlie tumourigenesis, such as increased matrix crosslinking.

Advantageously, embodiments of the tumour cell culture system may be capable of supporting organoid viability, growth, metabolism that are comparable to Geltrex/Matrigel in a majority of PDX lines. Various embodiments of the tumour cell culture system are configured to support in vitro culture of tumour organoids. Using organoids isolated from PDX tumours as a model of PTOs, and by fabricating hydrogels of diverse mechanical stiffness within the physiological range of tumours, phenol- conjugated hydrogels of increased stiffness (relative to normal colon tissue), together with hypoxia, may be capable of supporting PDX organoid viability, growth, metabolism that are comparable to Geltrex (i.e. Matrigel) in a majority of PDX lines. Mechanically-defined phenol-conjugated hydrogels may also support organoid sensitivity to various conventional therapeutic drugs, as well as PDX tumour engraftment and growth in vivo, and may provide a suitable culture matrix with tailorable mechanical properties.

It will be appreciated by a person skilled in the art that other variations and/or modifications may be made to the embodiments disclosed herein without departing from the spirit or scope of the disclosure as broadly described. For example, in the description herein, features of different example embodiments may be mixed, combined, interchanged, incorporated, adopted, modified, included etc. or the like across different example embodiments. The present embodiments are, therefore, to be considered in all respects to be illustrative and not restrictive.

Claims

1. A tumour cell culture system comprising,

a hydrogel matrix comprising a phenol-conjugated polymer that is configured to support in vitro growth of tumour cells thereon,

wherein the hydrogel matrix has a storage modulus (G’) of at least 1000 Pa.

2. The tumour cell culture system of claim 1 , wherein the polymer comprises gelatin.

3. The tumour cell culture system of claim 1 or 2, wherein the phenol- conjugated polymer comprises one or more phenols selected from the group consisting of hydroxyphenylpropionic acid (HPA), tyramine, and hydroxyphenylacetic acid.

4. The tumour cell culture system of any one of claims 1 to 3, wherein the phenol-conjugated polymer is configured to support in vitro growth of tumour organoids thereon.

5. The tumour cell culture system of any one of claims 1 to 4, further comprising tumour cells encapsulated within the matrix.

6. The tumour cell culture system of any one of claims 1 to 5, wherein the tumour cells are derived from one or more organoids isolated from colorectal cancer tumours, and/or nasopharyngeal carcinoma tumours.

7. The tumour cell culture system of any one of claims 1 to 6, wherein the tumour cells are in the form of one or more tumour organoids on the hydrogel matrix.

8. The tumour cell culture system of any one of claims 1 to 7, wherein the tumour cell culture system is maintained at an oxygen level that is no more than

10%.

9. The tumour cell culture system of any one of claims 1 to 8, wherein the tumour cell culture system comprises one or more therapeutic compounds.

10. The tumour cell culture system of any one of claims 1 to 9, wherein the hydrogel matrix has an elastic modulus (E) from 2000 Pa to 35,000 Pa.

1 1. The tumour cell culture system of any one of claims 1 to 10, wherein the hydrogel matrix has a storage module (G’) in the range of from 2500 Pa to 7500 Pa.

12. The tumour cell culture system of any one of claims 1 to 11 , wherein the hydrogel matrix comprises at least 2% (w/v) of hydroxyphenylpropionic acid (HPA)-conjugated gelatin polymer.

13. The tumour cell culture system of any one of claims 1 to 12, wherein the backbone structure of the hydrogel matrix consists essentially of hydroxyphenylpropionic acid (HPA)-conjugated gelatin polymer.

14. A method of preparing a tumour cell culture system, the method comprising,

crosslinking a precursor solution comprising phenol-conjugated polymer in the presence of an enzyme and an oxidising agent to form a phenol- conjugated hydrogel matrix that is configured to support in vitro growth of tumour cells,

wherein the hydrogel matrix has a storage modulus (G’) of at least 1000 Pa.

15. The method of claim 14, wherein the polymer comprises gelatin.

16. The method of claim 14 or 15, further comprising, prior to crosslinking the precursor solution, adding gelatin to a reaction mixture comprising one or more phenols selected from the group consisting of hydroxyphenylpropionic acid (HPA), tyramine, and hydroxyphenylacetic acid to form phenol conjugated gelatin polymer.

17. The method of claim 16, wherein the step of adding gelatin to a reaction mixture comprising one or more phenols comprises (i) adding gelatin to a reaction mixture comprising HPA, N-hydroxysuccinimide (NHS) and 1 -ethyl-3- (3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC-HCI) dissolved in a solvent, or (ii) adding gelatin to a reaction mixture comprising HPA-NHS, to form HPA-conjugated gelatin polymer.

18. The method of claim 14, wherein the enzyme comprises horseradish peroxidase (HRP) and the oxidizing agent comprises hydrogen peroxide.

19. The method of claim 18, further comprising defining the storage modulus of the hydrogel matrix by selecting a predetermined concentration of HRP and hydrogen peroxide, wherein the HRP concentration is in a range from 0.030 U/mL to 0.20 U/mL and the hydrogen peroxide concentration is in a range from 0.14 mM to 4.0 mM.

20. The method of any one of claims 14 to 19, further comprising seeding tumour cells into the hydrogel matrix.

21. The method of claim 20, wherein the tumour cells have been isolated from colorectal cancer tumours, and/or nasopharyngeal carcinoma tumours.

22. The method of claim 20 or 21 , further comprising culturing the seeded tumour cells to obtain one or more tumour organoids disposed on the hydrogel matrix, wherein the seeded tumour cells are in (i) an organoid form or (ii) in a non-organoid form that is suitable to be subsequently cultured into an organoid form.

23. The method of any one of claims 20 to 22, wherein the step of seeding the tumour cells on the hydrogel matrix comprises encapsulating tumour cells in the hydrogel matrix by adding horseradish peroxidase (HRP) and tumour cells to the precursor solution, followed by adding hydrogen peroxide to the precursor solution.

24. The method of claim 22, further comprising culturing the tumour cells at no more than 10% oxygen levels.

25. The method of any one of claims 14 to 24, further comprising introducing one or more therapeutic compounds to the hydrogel matrix.

26. The method of any one of claims 14 to 25, further comprising engrafting the hydrogel matrix comprising the tumour cells into an animal model for in-vivo culture.