Hydrogel composition and method of use thereof

Abstract

A two-component hydrogel matrix system is provided, which is useful in a variety of cell growth applications, including, but not limited to, three-dimensional culture systems. The components include modified hyaluronic acid (HA) and modified elastin-like protein (ELP). Variables of HA, including matrix stiffness, matrix stress relaxation rate, and cell adhesion ligand concentration, can be independently and quantitatively specified.

Description

[Background technology]

[0001] Human tissues, including human organoids derived from primary tissue or pluripotent cells from patient biopsies, have the potential to revolutionize personalized medicine and preclinical models of disease. However, human patient-derived organoids in synthetic matrices often require a spheroidization process, such as in a decellularized Engelbreth-Holm-Swarm (EHS) matrix or co-culture with feeder cells.

[0002] Recent studies developing manipulative matrices for patient-derived organoids rely on polyethylene glycol (PEG) as the synthetic matrix backbone, but PEG is known to interact with the immune system and induce antibody formation.

[0003] Reproducible, biodegradable, minimal matrix free from animal-derived products or synthetic PEGs is of great interest for clinical translation and is addressed herein. [Overview of the Initiative]

[0004] A two-component hydrogel matrix system is described that offers various advantages for cell and tissue culture, including but not limited to providing a three-dimensional culture environment. The components comprise (1) chemically modified hyaluronic acid (HA) and (2) chemically modified elastin-like protein (ELP). By mixing the two modified biopolymers together, the formation of hydrazone bonds is induced, resulting in a hydrogel network referred to herein as "HELP". The selection of the ratio of HA to ELP, and the ratios of variants of these components, allows for the adjustment of key variables of HELP, including, for example, matrix stiffness, matrix stress relaxation rate, and cell adhesion ligand concentration and identity. These variables can be defined independently and quantitatively.

[0005] Hyaluronic acid components are chemically modified to include pendant benzaldehyde or aldehyde side groups. The ratio of these two chemical groups in the final hydrogel formulation controls the stress relaxation parameter. A key feature of natural extracellular matrix and EHS matrix is ​​their ability to undergo stress relaxation through physical crosslinking, which can be easily remodeled. Compositions containing a larger proportion of pendant aldehyde-modified hyaluronic acid increase the mean kinetic exchange rate of the gel, resulting in a faster stress relaxation rate. A higher proportion of pendant benzaldehyde-modified hyaluronic acid reduces the mean kinetic exchange rate of the gel, resulting in a slower stress relaxation rate. Modification of the stress relaxation rate can be achieved independently of the matrix ligand composition and stiffness. The ratio of HA-benzaldehyde to HA-aldehyde may be pre-selected for the desired hydrogel, typically in the range of approximately 100:0 to 0:100, for example, ratios such as approximately 95:5, 90:10, 75:25, 50:50, or 25:75.

[0006] The ELP component optionally includes a recombinant elastin-like sequence interspersed with cell adhesion sequences. To crosslink with chemically modified HA, the ELP is chemically modified to include pendant hydrazine groups. Any cell adhesion sequences within the ELP may be selected from integrin bonds, fibronectin systems, elongated RGD sequences, scrambled RGD sequences, cell adhesion sequences derived from collagen type I, e.g., (SEQ ID NO: 3) DGEA, cell adhesion sequences derived from tenacin, e.g., (SEQ ID NO: 4) PLAEIDGIELTY, (SEQ ID NO: 5) VFDNFVLK, etc., cell adhesion sequences derived from laminin, e.g., (SEQ ID NO: 6) IKVAV, (SEQ ID NO: 7) YIGSR, etc., and cell adhesion sequences derived from cadherin, e.g., (SEQ ID NO: 8) HAVDI, (SEQ ID NO: 9) HAVDIHAVDI, etc. For example, SEQ ID NO: 1 and SEQ ID NO: 2 are ELPs having an RGD sequence and a scrambled RGD sequence, respectively.

[0007] The cell adhesion sequence concentration of the hydrogel can be varied by adjusting the ratio of ELP containing RGD motifs to ELP lacking RGD motifs, or containing scrambled or non-RGD cell adhesion motifs, as disclosed above. This ratio is typically in the range of about 100:0 to 0:100, for example, about 75:25, 50:50, 25:75, or 10:90, and may be pre-selected for the hydrogel of interest. In some applications, the HELP hydrogel contains about 0.25 mM to about 1.5 mM RGD, for example, about 0.25 mM, 0.5 mM, 0.75 mM, 1 mM, 1.25 mM, or 1.5 mM. Concentrations above 0.75 mM are preferred for culturing intestinal organoids.

[0008] Matrix stiffness is determined by the concentration and ratio of hydrazine to aldehyde and benzaldehyde reactive groups, where a ratio of approximately 1:1 provides maximum crosslinking. The ratio can vary, for example, to approximately 1:3, 1:2, 1.5:1, 1.25:1, 1:1, 1:1.25, 1:1.5, 1:2, 1:3, etc. For cell culture purposes, a preferred gel may have a G' of approximately 1 kPa, for example, approximately 750-1250 Pa. The ratio of hydrazine to aldehyde or benzaldehyde groups can be modified by three variables: (1) the number of hydrazine groups per ELP molecule, (2) the number of aldehyde or benzaldehyde groups per HA molecule, and (3) the blend of ELP and HA.

[0009] In some embodiments, the hydrogel matrix disclosed herein is used in in vitro cell culture. In some embodiments, a 3D culture environment is provided that includes a matrix of adjustable HELP hydrogels. In some embodiments, a method is provided for culturing mammalian cells in vitro, comprising suspending a cell population of interest in the hydrogel described herein and culturing the embedded cells in a suitable medium, which may be primary cells, cell lines, in vitro reprogrammed cells, genetically modified cells, primary tissue grafts, etc. In some embodiments, the cells are primary cells from a human patient. In some embodiments, the culture system does not contain polyethylene glycol.

[0010] In some embodiments, as disclosed herein, cells cultured in a hydrogel matrix differentiate into cells of the tissue of interest. For example, intestinal stem cells or intestinal grafts can be differentiated into intestinal organoids, etc. The cells can further be passaged by a process of matrix dissociation. In matrix dissociation, the HELP matrix can be enzymatically degraded using elastase and hyaluronidase. The formed organoids can be further separated into single cells or small cell aggregates, e.g., trypsin, or passaged intact. Encapsulation of single cells or small cell aggregates into a fresh HELP matrix provides continuous organoid formation for at least 12 passages without any visible change in morphology. Alternatively, cells can be pre-formed into spheroids and then encapsulated within the HELP matrix.

[0011] Examples of tissues include, but are not limited to, intestinal tissue, lung tissue, stomach tissue, pancreatic tissue, bladder tissue, liver tissue, bone marrow stroma, muscle tissue, kidney tissue, and brain tissue. The cultured grafts of the present invention can be continuously grown in culture for a certain period, for example, one week, two weeks, three weeks, four weeks, or longer. Mammalian tissue grafts cultured by the method of the present invention can summarize the characteristics of in vivo tissue growth. These characteristics include, but are not limited to, long-term tissue expansion with proliferation, multi-system differentiation, and reproduction of the ultrastructure of cells and tissues, including epithelial tissue, submucosa, and stromal environment. The culture system provides the growth of various cells found in normal or diseased mammalian tissues, while the cultures are also useful in generating cells for selection and provide a single-lineage purified or concentrated population for any given tissue, including tissue-specific stem cells. Organoids cultured by these methods have found use in many applications, such as tissue engineering, disease modeling, and drug discovery.

[0012] Cultured cells can be experimentally modified before or during the culture period. In some embodiments, cells are modified by exposure to a viral or bacterial pathogen. In other embodiments, cells are modified by altering the pattern of gene expression, for example by providing reprogramming factors to induce pluripotency or otherwise alter differentiation potential, or by introducing cancer drivers that provide oncogenic conversion of cells to carcinoma. Experimentally modified cells are useful for investigating the effects of therapeutic agents, tumor therapy, and differentiation effects.

[0013] A method is provided for screening cells for the presence of stem cell potential in a population, such as a complex population of multiple cell types, or a population of purified cells isolated from a complex population by sorting, culturing, etc. This method involves co-culturing detectably labeled candidate cells with a tissue graft of the present invention. Candidate cells with stem cell potential are detected by increased proliferation of the cultured graft above the basal level and by co-localization of multiphylogenetic differentiation markers indicating the presence of tissue-specific stem cells with the labeled candidate cells. The stem cell properties of the candidate cells co-culturned with the graft are further assayed by determining long-term reconstitution activity via in vivo transplantation, etc.

[0014] In another aspect of the present invention, a method for in vitro screening of drugs for cytotoxicity to different tissues is provided by screening the toxicity of the transplanted cultures of the present invention. In yet another embodiment, a method for evaluating drug absorption by different tissues is provided by evaluating drug absorption by the transplanted cultures of the present invention.

[0015] In another aspect of the present invention, the matrix is ​​extruded through a syringe needle or catheter. After extrusion, the HELP matrix modifies the gel phase material. Cells encapsulated within the HELP matrix can be extruded in this manner for use as bio-ink for 3D bioprinting, or as bio-ink for injectable regenerative medicine. Shear-thinned materials having a fracture stress of less than 2000 Pa are injectable by hand force. The fracture stress can be adjusted by changing three variables: (1) the molecular weight of HA at MW, usually less than 100 kDa; (2) the kinetics of hydrazone bonding (for this purpose, a fast exchange kinetics of hydrazine-aldehyde is preferred over a slow exchange kinetics of hydrazine-benzaldehyde reaction); and (3) the overall polymer concentration, which is usually about 0.5–2 wt% of ELP and about 0.5–2 wt% of HA. [Brief explanation of the drawing]

[0016] [Figure 1] HELP matrix for patient-derived intestinal organoid formation, growth, and progression. a) Enteroids were generated from intestinal tissue biopsies from human patients. b) Schematic diagram of the HELP matrix, composed of benzaldehyde-modified hyaluronic acid (HA) and hydrazine-modified elastin-like protein (ELP). Hyaluronic acid can engage with the CD44 receptor on cells, while recombinant ELP contains an RGD peptide ligand that engages with cellular integrin receptors. c) Schematic diagram of the enteroid passage technique. Enteroids can be dissociated into single cells or directly re-embedded in new material as fully formed enteroids during passage. d) Bright-field and confocal fluorescence microscopy images of enteroids in different materials, during dissociation (left) and re-embedding (right) in these materials. e) Representative bright-field images of enteroids grown in passages 1 and 5 of HELP, dissociated into single cells at each passage. f) Growth curves of dissociated enteroids grown in passage 12 of EHS matrix or passage 12 of HELP. n=3, ns=not significant. g) Enteroid formation efficiency for enteroids grown from single cells in the material formulations shown in d). Data are one-way ANOVA with mean + / - standard deviation, Tukey's multiple comparison test. **=p<0.01, n=3, nd=not detected. [Figure 2]Differentiation of organoids grown in HELP and EHS matrices. a) Schematic diagram of the differentiation experiment timeline. Organoids were cultured from single cells in growth medium for 10 days, followed by culture in differentiation medium for 5 days (see Methods). b) Confocal micrographs showing the progression from early enteroids to polarized enteroids and differentiated organoids. c) Confocal micrographs showing observation of mature intestinal cell subtypes: Paneth cells (Lyz+, left), goblet cells (Muc2+, middle), and enteroendocrine cells (ChgA+, right). d) RT-qPCR quantification of changes in RNA expression of differentiated cell type markers compared to cells maintained in maintenance medium for 15 days and to the control gene BACT. Two-sided Student's t-tests were performed on the CT values ​​between differentiated vs. maintenance cultures, assuming a normal distribution of CT values. **=p<0.01, *=p<0.05, N=3 independent experiments, n=4 technical replicates. [Figure 3] The role of hyaluronic acid in the HELP matrix. a) Comparative confocal micrographs with CD44 staining at the same intensity and gain settings in EHS and HELP matrices. b) Flow cytometry analysis of enteroids grown in EHS and HELP matrices 11 days post-encapsulation compared to a negative control. c) Bright-field and confocal micrographs of enteroids grown in HELP and ELP-PEG matrices 6 days post-seeding. HA is absent in the ELP-PEG gel. d) Formation efficiency of enteroids grown in HELP and ELP-PEG. Data are mean + / - standard deviation, Student's t-test, **=p<0.01, n=3, nd=not detected. [Figure 4]Custom tailoring of HELP matrix properties. a) HELP schematic diagram showing specification of RGD ligand concentration by blending different ELP variants in the material. b) HELP schematic diagram showing that matrix stiffness is modulated by changing the number of crosslinks (top) (top), while matrix viscoelasticity is adjusted by replacing benzaldehyde with aldehyde portions. c) Shear storage coefficients (G') of rigid elastic (EL) and flexible EL HELP matrices. Student's t-test, **=p<0.01, N=3-5. d) Step strain stress relaxation curves comparing EL HELP formulations. e) Cross-sectional area measurement of enteroids in EL HELP material 12 days after encapsulation. Two-way ANOVA with Tukey multiple comparison test, *=p<0.05, **=p<0.01, n=3. f) Shear storage coefficients of rigid EL and rigid viscoelastic (VE) formulations. Student's t-test, N=3-5, ns=not significant. g) Step strain-stress relaxation curves comparing rigid EL and flexible VE formulations. h) Measurement of cross-sectional area of ​​enteroids in rigid material 12 days after encapsulation. Two-way ANOVA with Tukey multiple comparison test, *=p<0.05, **=p<0.01, n=3. [Figure 5-1] HELP protein backbone and chemical modifications. a) ELP amino acid sequence (SEQ ID NO: 30) that can be modified to contain a fibronectin-mimicking RGD or scrambled RDG motif. b) Nuclear magnetic resonance (NMR) of ELP modified with the hydrazine moiety. c) Hyaluronic acid structure modified with the benzaldehyde moiety. d) NMR of 30% modified hyaluronic acid and the corresponding peak from c). [Figure 5-2] HELP protein backbone and chemical modifications. e) NMR of 12% modified hyaluronic acid and corresponding peaks from c). [Figure 6] Intestinal organoids can be passaged within the EHS or HELP matrix. Intestinal cells derived from a single patient can be seeded into either the EHS or HELP matrix and repeatedly passaged from a single cell. [Figure 7]Human intestinal organoids are robustly formed in two or more patient-derived cell lines of HELP. a) Bright-field and confocal fluorescence microscopy images of spheroids in EHS, HELP, and ELP at dissociation (left) and re-embedding (right). b) Spheroid formation efficiency of spheroids grown from single cells in the material formulation shown in a). One-way ANOVA by Tukey's multiple comparison test, ****=p<0.0001, nd=not detected. c) Representative bright-field images of spheroids grown in passages 1 and 4 of HELP, with dissociation to single cells at each passage. [Figure 8] HELP supports organoid formation and growth from mouse intestinal cells and human iPSC-derived liver organoids. a) Bright-field micrographs of mouse small intestinal spheroids grown in HELP and EHS matrix at dissociation (left) and re-embedding (right). b) Micrographs of human liver organoids in HELP by bright-field (upper left), calcein AM fluorescence (upper right), and immunostaining for cytokeratin 19 (CK19) and hepatocyte nuclear factor 4α (HNF4A). Quantification of changes in human liver organoid size in a functional assay to demonstrate response to forskolin / IBMX (3-isobutyl-1-methylxanthine) treatment (lower right). ****=p<0.0001. [Figure 9] Flow cytometry analysis of CD44+ cells in enteroids grown in HELP and EHS matrices. a) Percentage of CD44+ gated cells without any antibody added. b) Percentage of CD44+ gated cells stained with isotype antibody control. c) Percentage of CD44+ gated cells formed in EHS matrix. d) Percentage of CD44+ gated cells grown in HELP. [Figure 10] Material Rheology. a-g) Vibrational shear rheology was performed to characterize the viscoelastic properties of all hydrogel formulations reported in this work. These frequency sweeps were performed between 0.1 and 10 Hz at 1% strain, and the nominal stiffness of each matrix was determined based on the storage coefficient value at 1 Hz. [Figure 11]Cross-sectional area of spheroids grown in separate HELP formulations. a) Cross-sectional area of enteroids grown in rigid EL compared to soft EL HELP with varying concentrations of RGD ligand on day 12 after encapsulation. b) Cross-sectional area of enteroids grown in rigid EL compared to rigid VE HELP with varying concentrations of RGD ligand on day 12 after encapsulation. Kruskal-Wallis test using Dunn's multiple comparison test, *= p < 0.05. [Figure 12] Determination of the lower critical solution temperature properties of engineered proteins designed for use in HELP gels. Six different variants of engineered elastin-like proteins (see Table 1) were synthesized, purified, and characterized for their lower critical solution temperature behavior. [Figure 13] To demonstrate the modularity of these engineered proteins within the HELP system, six different variants of engineered elastin-like proteins (see Table 1) were modified with hydrazine groups and crosslinked with benzaldehyde-modified hyaluronic acid to form HELP gels that exhibit six clearly different bioactive peptide sequences. The six HELP gels were characterized for their viscoelastic properties by oscillatory rheology and showed no statistically significant differences in their gel mechanics. [Figure 14]By adjusting the thermodynamic average molecular weight between crosslinks, the overall polymer molecular weight, and the dynamics of the hydrazone crosslink, the HELP gel is shear thinning and can thus be formulated to be injectable through a syringe needle or catheter when a force is applied. When the force is removed, the gel structure self-heals, restoring the mechanical properties of the gel phase. This extrusion and self-healing cycle can be repeated multiple times. The gel can be extruded when a hand force is applied through various medical devices including a 30-gauge syringe needle and a 150-cm catheter (0.75 mm in diameter). The qualitative injectability of five HELP gels was tested by injecting each formulation through a hooked 30G insulin needle. (A) The criteria for injectability include the following. (1) The ability to be injectable after complete gelation (30 minutes), (2) being injectable with one hand, and (3) no evidence of a sudden burst injection. (B) Qualitative still images taken from the recording of each injection. Each gel is stained with food coloring to improve visibility. (C) A table summarizing the qualitative evaluation. Check marks, crosses (×) indicate passing and failing the test, respectively. Since formulation HA-B was not injectable at all, criteria (2) and (3) could not be evaluated and are designated as (-).

BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Before further describing embodiments of the present disclosure, it is understood that the present disclosure is not limited to the specific embodiments described and can, of course, vary. Also, the terms used herein are for the purpose of describing only specific embodiments and are not intended to be limiting, as the scope of the present disclosure is limited only by the appended claims.

[0018] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of embodiments of the present disclosure.

[0019] It should be noted that, as used herein and in the appended claims, the singular forms "a," "and," and "the" refer to multiple objects unless the context explicitly indicates otherwise. For example, a reference to "compound" includes not only a single compound but also a combination of two or more compounds, and a reference to "substituent" includes a single substituent as well as two or more substituents, and so on.

[0020] In the description and claims of this invention, certain terms are used in accordance with the definitions set forth below. It is understood that the definitions provided herein are not intended to be mutually exclusive. Accordingly, some chemical terms may be included within the definitions of two or more terms.

[0021] Where used herein, the words “for example,” “for instance,” “etc.,” or “including” are intended to introduce examples that further clarify a more general subject matter. These examples are provided solely to aid in understanding this disclosure and do not constitute a limitation in any way.

[0022] The term "hydrogel" is used in its conventional sense to refer to a material that absorbs a solvent (e.g., water), undergoes expansion without measurable dissolution, and maintains a three-dimensional network capable of reversible deformation. "Expansion" as used herein refers to the isotropic expansion of the hydrogel structure as water molecules diffuse throughout the internal volume of the hydrogel. The properties of the copolymer hydrogels disclosed herein can be tuned as desired by varying the amounts of each component, the ratios of each component, or the density of particular components, as described in more detail below. The term "hydrogel" may include both dry and hydrated (e.g., solvent-swollen) hydrogels.

[0023] In some embodiments of the present invention, the hydrogel provides a scaffold for cell growth, including the growth of metabolically active cells, such as differentiated cells. The cells may be grown in vitro, for example, in a culture of one or more cell types. The cells may also be grown in vivo, for example, when the hydrogel provides a substrate for regenerative cell growth. The hydrogel of the present invention provides adequate mechanical strength for long-term structural stability.

[0024] Elastin-like proteins (ELPs) contain recombinant sequences of elastin-like sequences interspersed with optionally selected cell adhesion sequences. To crosslink with chemically modified HA, ELPs are chemically modified to include pendant hydrazine groups. Any cell adhesion sequences within an ELP may contain motifs involved in cell adhesion and may be selected from integrin bonds, fibronectin systems, elongated RGD sequences, scrambled RGD sequences, cell adhesion sequences derived from collagen type I, e.g., (SEQ ID NO: 3) DGEA, cell adhesion sequences derived from tenacin, e.g., (SEQ ID NO: 4) PLAEIDGIELTY, (SEQ ID NO: 5) VFDNFVLK, etc., cell adhesion sequences derived from laminin, e.g., (SEQ ID NO: 6) IKVAV, (SEQ ID NO: 7) YIGSR, etc., and cell adhesion sequences derived from cadherin, e.g., (SEQ ID NO: 8) HAVDI, (SEQ ID NO: 9) HAVDIHAVDI, etc.

[0025] The cell adhesion domain of engineered elastin-like proteins can be designed to include alternative peptide sequences known to interact with cell surface receptors. These sequences may include peptides derived from native extracellular matrix proteins (e.g., fibronectin, laminin, collagen, tenacin-C) or peptides derived from cell adhesion receptors (e.g., N-cadherin) (Table 1). Selecting cell adhesion peptide sequences along with elastin-like region sequences defines the overall hydrophobicity of the engineered protein and thus controls its lower critical solution temperature (LCST) behavior.

[0026] In some embodiments, the ELP includes the following structure:

[0027] [ka]

[0028] The cell adhesion domain is approximately 15 to 45 amino acids long and contains one or more cell adhesion sequence motifs, which may be selected from RGD, scrambled RGD, no RGD, or one of SEQ ID NOs: 3 to 9. SEQ ID NOs: 10-19 and 22 are illustrative.

[0029] The linker sequence is optionally adjacent to a cell adhesion sequence motif, and the peptide linker may be approximately 5–20, 5–15, 5–10, or 5–9 amino acid lengths. Exemplary linkers include linear peptides having at least two amino acid residues, such as Gly-Gly, Gly-Ala-Gly, Gly-Pro-Ala, and Gly-Gly-Gly-Ser (SEQ ID NO: 34). Preferred linear peptides include polyglycine, polyserine, polyproline, polyalanine, and oligopeptides consisting of alanyl and / or selinyl and / or prolinyl and / or glycyl amino acid residues. In one embodiment, the linker comprises the amino acid sequence GTSTGSGKSSEGKG (SEQ ID NO: 35) or (GGGGS)n (SEQ ID NO: 36), where n is 1, 2, 3, 4, 5, etc., but many such linkers are known and used in the art and may serve this purpose.

[0030] The elastin-like domain consists of elastin-like motifs, including but not limited to (SEQ ID NO: 23)VPGIG, (SEQ ID NO: 24)VPGKG, and (SEQ ID NO: 25)VPGYG. One or more of SEQ ID NOs: 23, 24, and 25 may be present in the protein. In some embodiments, the number of motifs may be 1-7, 1-6, 2-5, 3-5, and approximately 5 motifs. Exemplary domain sequences are provided, for example, in SEQ ID NOs: 20 and 21. Examples include, but are not limited to, SEQ ID NOs: 1, LQ(LDASTVYAVGRGDSPASSA[(VPGIG)2VPGKG(VPGIG)2]3)4 and SEQ ID NOs: 2, LQ(LDASTVYAVGRDGSPASSA[(VPGIG)2VPGKG(VPGIG)2]3)4.

[0031] ELP proteins are chemically modified to contain pendant hydrazine groups, and may contain approximately 3 to 20, 5 to 18, or 10 to 14 hydrazine groups. Standard bioconjugation chemistry can be used to attach pendant hydrazines to any of the lysine, cysteine, or tyrosine amino acid sites.

[0032] [Table 1]

[0033] Hyaluronic acid is an anionic, non-sulfated glycosaminoglycan widely distributed throughout connective tissue, epithelial tissue, and nerve tissue. It is a disaccharide polymer composed of D-glucuronic acid and N-acetyl-D-glucosamine, alternately linked via β-(1→4) and β-(1→3) glycosidic bonds. Hyaluronic acid can have up to 25,000 disaccharide repeats. Hyaluronic acid polymers can range in size from approximately 20 kDa to approximately 1.5 MDa, and from approximately 20 kDa.

[0034] Hyaluronic acid is chemically modified to contain pendant benzaldehyde or aldehyde side groups. HA is typically modified with about 5% to 30% of the available reactive groups, which can be about 7% to 20%, 10% to 15%, and can be about 12%.

[0035] In the case of an aldehyde functional group, the carboxylic acid group on HA is amidated with propargamine to produce an HA-alkyne intermediate. Then, using copper click chemistry, this alkyne is reacted on HA with the azide moiety of a heterobifunctional small molecule containing an aldehyde functional group to produce aldehyde-functionalized HA.

[0036] Benzaldehyde modification can be achieved, for example, by first modifying HA to contain an alkyne group at a desired concentration of about 3% to about 30%. Then, the HA-alkyne is modified with N-(2-azidoethyl)-4-formylbenzamide to produce HA-benzaldehyde.

[0037] The terms “activator,” “antagonist,” “inhibitor,” “drug,” and “pharmacologically active agent” are used interchangeably herein to refer to a chemical substance or compound that, when administered to a living organism (human or animal), induces a desired pharmacological and / or physiological effect by local and / or systemic action.

[0038] As used herein, terms such as “treatment” and “to treat” refer to obtaining a desired pharmacological and / or physiological effect, such as a reduction in viral titer. The effect may be prophylactic in that it completely or partially prevents the disease or its symptoms, and / or therapeutic in that it partially or completely cures the disease and / or adverse effects resulting from the disease. As used herein, “treatment” encompasses any treatment of a disease in mammals, in particular humans, and includes (a) preventing the development of a disease or its symptoms in a subject that is predisposed to the disease but has not yet been diagnosed with the disease (including, for example, a disease that may be associated with or caused by a primary disease), (b) inhibiting the disease, i.e., preventing its development, and (c) mitigating the disease, i.e., causing disease regression (e.g., a reduction in viral titer).

[0039] The terms “individual,” “host,” “subject,” and “patient” are used interchangeably herein and are not limited to refer to animals including humans and non-human primates, including monkeys and humans; rodents, including rats and mice; and animals including cattle, horses, sheep, cats, dogs, and birds. “Mammal” means one or more members of any mammalian species, including, for example, dogs, cats, horses, cattle, sheep, rodents, and primates, such as non-human primates and humans. Non-human animal models, such as mammals, such as non-human primates, murids, and lagomorphs, may be used in experimental studies.

[0040] As used herein, the terms “determine,” “measure,” “evaluate,” and “assay” are used interchangeably and include both quantitative and qualitative determinations.

[0041] The terms “polypeptide” and “protein” are used interchangeably herein and refer to polymeric forms of amino acids of any length, and may include coded and uncoded amino acids, chemically or biochemically modified or induced amino acids, and polypeptides having a modified peptide backbone. The term includes, but is not limited to, fusion proteins having heterologous amino acid sequences, fusions having heterologous and native leader sequences with or without an N-terminal methionine residue, immunotagged proteins, and fusion proteins having a fusion partner, such as a detectable fusion partner, including fluorescent proteins, β-galactosidase, luciferase, etc.

[0042] The terms “nucleic acid molecule” and “polynucleotide” are used interchangeably and refer to polymeric forms of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides can have any three-dimensional structure and can perform any known or unknown function. Non-limiting examples of polynucleotides include genes, gene fragments, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, regulatory regions, isolated RNA of any sequence, nucleic acid probes, and primers. Nucleic acid molecules may be linear or cyclic.

[0043] The "therapeutic dose" or "effective dose" means the amount of a compound that, when administered to a mammal or other subject for the treatment of a disease, condition, or disorder, is sufficient to produce an effect of such treatment for the disease, condition, or disorder. The "therapeutic dose" varies depending on the compound, the disease and its severity, and the age, weight, etc., of the subject being treated.

[0044] As used herein, the term “unit dosage form” refers to a physically distinct unit suitable as a unit dose for human and animal subjects, each unit containing a predetermined amount of compound calculated to be sufficient to produce the desired effect in conjunction with a pharmaceutically acceptable diluent, carrier, or vehicle. The specifications of a unit dosage form depend on the specific compound used, the effect to be achieved, and the pharmacodynamics associated with each compound in the host.

[0045] "Pharmacologically acceptable excipients," "pharmaceutically acceptable diluents," "pharmaceutically acceptable carriers," and "pharmaceutically acceptable adjuvants" mean excipients, diluents, carriers, and adjuvants that are useful in the preparation of pharmaceutical compositions, are generally safe, non-toxic, and are biologically and otherwise desirable, and include excipients, diluents, carriers, and adjuvants that are acceptable for veterinary and human pharmaceutical use. As used herein and in the claims, "pharmaceutically acceptable excipients, diluents, carriers, and adjuvants" includes both one and two or more such excipients, diluents, carriers, and adjuvants.

[0046] As used herein, “pharmaceutical composition” means a composition suitable for administration to subjects such as mammals, particularly humans. Generally, “pharmaceutical composition” is sterile and preferably free from contaminants that could induce an undesirable response in the subject (e.g., the compounds in the pharmaceutical composition are pharmaceutical grade). Pharmaceutical compositions may be designed for administration to subjects or patients in need of treatment via several different routes of administration, including oral, rectal, rectal, parenteral, intraperitoneal, intradermal, intratracheal, intramuscular, and subcutaneous.

[0047] The term "somatic cell" encompasses any cell in an organism that is not pluripotent, meaning it cannot produce all types of cells in the organism. In other words, a somatic cell is a cell that is sufficiently differentiated to not spontaneously produce cells of all three germ layers of the body: the ectoderm, mesoderm, and endoderm.

[0048] The term "pluripotent" refers to a cell that, under appropriate conditions, has the ability to produce offspring that can differentiate into a cell type that collectively exhibits characteristics related to the cell lineages of the three germ layers (endoderm, mesoderm, and ectoderm). "Stem cells" are cells characterized by their ability to regenerate through mitosis and their potential to differentiate into tissues or organs. In mammalian stem cells, embryonic stem cells and somatic stem cells can be distinguished. Pluripotent stem cells, including embryonic stem cells, embryonic germ cells, and induced pluripotent cells, can contribute to the tissues of prenatal, postnatal, or adult organisms.

[0049] The terms “primary cell,” “primary cell line,” and “primary culture” are used interchangeably herein and refer to cells and cell cultures derived from a subject and grown in vitro for a limited number of passages, i.e., cells grown in vitro for division. For example, a primary culture is a culture that has been passaged 0, 1, 2, 4, 5, 10, or 15 times, but may not be enough passages to reach a critical stage. Typically, the primary cell lines of the present invention are maintained in vitro for fewer than 10 passages.

[0050] The cells of the subject may be derived from humans, primates, livestock and farm animals, and any mammal, including zoo animals, laboratory animals, or pet animals such as dogs, cats, cattle, horses, sheep, pigs, goats, rabbits, rats, and mice. They may be established cell lines or they may be primary cells. "Primary cells," "primary cell lines," and "primary cultures" are used interchangeably herein and refer to cells and cell cultures derived from the subject and grown in vitro for a limited number of passages.

[0051] Cells of the subject can be isolated from fresh or frozen cells, which may be from neonates, infants, or adults, and from tissues, including skin, muscle, bone marrow, peripheral blood, umbilical cord blood, spleen, liver, pancreas, lungs, intestines, stomach, fat, and other differentiated tissues. Tissues can be obtained by biopsy or non-electrophoresis from live donors, or from dead or dying donors within about 48 hours postmortem, or from tissues frozen within about 12 hours postmortem, usually indefinitely at about liquid nitrogen temperature (-190°C), or freshly frozen tissues maintained below about -20°C. For the isolation of cells from tissues, suitable solutions can be used for dispersion or suspension. Such solutions are generally sterile equilibrium salt solutions, such as physiological saline, PBS, or Hanks equilibrium salt solution, conveniently supplemented with fetal bovine serum or other naturally occurring factors, in combination with an acceptable buffer at a low concentration, generally 5–25 mM. Convenient buffers include HEPES, phosphate buffer, and lactate buffer.

[0052] The terms "cell culture" or "culture" refer to the maintenance of cells in an artificial in vitro environment. However, it should be understood that "cell culture" is a general term and can be used to encompass the culture of tissues or organs, not just individual cells.

[0053] The target culture conditions provide a differentiation-permitting environment in which stem cells or progenitor cells proliferate, differentiate, or mature in vitro. Such conditions may also be called differential conditions. The characteristics of the environment include the culture medium in which the cells are cultured, any growth factors or differentiation-inducing factors that may be present, and the hydrogel support structures disclosed herein. Differentiation may be initiated by the formation of organoids or similar structures.

[0054] As used herein, “long-term culture” means a culture in which cells have grown, differentiated, and remain viable for at least about 10 days, or more than 30 days, or more than 60 days, or more than 100 days, or more than 150 days.

[0055] Stem cells. The term stem cells is used herein to refer to mammalian cells that possess both the ability to regenerate and the ability to produce differentiated offspring (see Morrison et al. (1997) Cell 88:287-298). Generally, stem cells also possess one or more of the following characteristics: the ability to undergo asynchronous or asymmetric replication, which can result in two daughter cells having different phenotypes after division; broad regenerative capacity; the ability to exist in mitotic quiescent form; and the ability to clonalize all tissues in which they reside, for example, the ability of hematopoietic stem cells to reconstruct all hematopoietic lineages. "Progenitor cells" differ from stem cells in that they typically do not possess broad regenerative capacity and are often only able to regenerate a subset of lineages within the tissue from which they originate, for example, the lymphoid lineage or erythroid lineage in a hematopoietic setting.

[0056] Stem cells can be characterized by both the presence of markers associated with specific epitopes identified by antibodies and the absence of specific markers identified by the lack of binding of specific antibodies. Stem cells can also be identified by functional assays, both in vitro and in vivo, particularly assays related to the ability of stem cells to produce multiple differentiated offspring.

[0057] In this specification, “differentiated lineage stem cells” is used to refer to cells of a specific lineage, such as pluripotent stem cells that give rise to germ layer stem cells (see, for example, Reyes et al. (2001) Blood 98:2615-2625, Eisenberg & Bader (1996) Circ Res. 78(2):205-16, etc.).

[0058] "Tissue-specific stem cells" is used herein to refer to pluripotent stem cells that reside within a specific tissue and are capable of clonal regeneration of cells in that tissue, such as hematopoietic stem cells having the ability to reconstruct all hematopoietic lineages, or neural stem cells having the ability to reconstruct all nervous / glial lineages. "Progenitor cells" differ from tissue-specific stem cells in that they typically do not possess broad regenerative capacity and are often only capable of regenerating a subset of lineages within the tissue from which they originate, such as only the lymphoid or erythroid lineage in a hematopoietic setting, or only neurons or glia in a nervous system.

[0059] Stem cells and their cultures: Pluripotent stem cells are cells derived from any type of tissue (usually embryonic tissue such as fetal or pre-fetal tissue), and are characterized by their ability, under suitable conditions, to produce offspring of different cell types that are derivatives of all three germ layers (endoderm, mesoderm, and ectoderm). These cell types can be provided in the form of established cell lines, or they can be obtained directly from primary embryonic tissue and used immediately for differentiation. This includes cells listed in the NIH Human Embryonic Stem Cell Registry, such as hESBGN-01, hESBGN-02, hESBGN-03, hESBGN-04 (BresaGen, Inc.), HES-1, HES-2, HES-3, HES-4, HES-5, HES-6 (ES Cell International), Miz-hES1 (MizMedi Hospital-Seoul National University), HSF-1, HSF-6 (University of California at San Francisco), and H1, H7, H9, H13, H14 (Wisconsin Alumni Research Foundation (WiCell Research Institute)).

[0060] The target stem cells include various types of embryonic cells, exemplified by human embryonic stem cells (hES) described by Thomson et al. (1998) Science 282:1145, embryonic stem cells derived from other primates such as rhesus monkey stem cells (Thomson et al. (1995) Proc. Natl. Acad. Sci USA 92:7844), marmoset stem cells (Thomson et al. (1996) Biol. Reprod. 5:254), and human embryonic germ cells (hEG) (Shamblott et al., Proc. Natl. Acad. Sci. USA 95:13726, 1998). Furthermore, differentiated stem cells such as mesodermal stem cells and other early cardiogenic cells are also included (see Reyes et al. (2001) Blood 98:2615-2625, Eisenberg & Bader (1996) Circ Res. 78(2):205-16, etc.). Stem cells can be obtained from any mammalian species, such as humans, horses, cattle, pigs, dogs, cats, rodents such as mice, rats, hamsters, and primates.

[0061] ES cells are considered undifferentiated if they have not been determined to a specific differentiation lineage. Such cells exhibit morphological features that distinguish them from differentiated cells derived from embryos or adults. Undifferentiated ES cells are readily recognizable to those skilled in the art and typically appear in two dimensions of microscopic images within colonies of cells with a high nucleus / cytoplasmic ratio and prominent nucleoli. Undifferentiated ES cells express genes that can be used as markers for detecting the presence of undifferentiated cells, and their polypeptide products can be used as markers for negative selection. See, for example, US2003 / 0224411A1, Bhattacharya (2004) Blood 103(8):2956-64, and Thomson (1998) (see above), respectively, incorporated herein by reference. Human ES cell lines express cell surface markers that characterize undifferentiated non-human primate ES and human EC cells, including stage-specific embryonic antigens (SSEA)-3, SSEA-4, TRA-1-60, TRA-1-81, and alkaline phosphatase. The globoseseries glycolipid GL7, which carries the SSEA-4 epitope, is formed by the addition of sialic acid to the globoseseries glycolipid Gb5, which carries the SSEA-3 epitope. Therefore, GL7 reacts with antibodies against both SSEA-3 and SSEA-4. Undifferentiated human ES cell lines did not stain for SSEA-1, but differentiated cells stained strongly for SSEA-1. Methods for proliferating hES cells in their undifferentiated form are described in WO99 / 20741, WO01 / 51616, and WO03 / 020920.

[0062] As used herein, “reprogramming factor” refers to one or more biologically active factors, i.e., a cocktail of biologically active factors, that act on cells to alter transcription, thereby reprogramming the cells to be multipotent or pluripotent. Reprogramming factors may be provided individually or as a single composition, i.e., as a pre-mixed composition of reprogramming factors, to cells, e.g., cells derived from an individual having a family history or genetic structure of cardiovascular disease of interest, such as fibroblasts or adipocytes. The factors may be provided in the same or different molar ratios. The factors may be provided once or multiple times in the process of culturing the cells of the subject invention. In some embodiments, the reprogramming factors are transcription factors, including, but not limited to, Oct3 / 4, Sox2, Klf4, c-Myc, Nanog, and Lin-28.

[0063] Somatic cells are brought into contact with reprogramming factors as defined above, in combinations and quantities sufficient to reprogram the cells to be pluripotent. The reprogramming factors may be provided to somatic cells individually or as a single composition, i.e., a pre-mixed composition of reprogramming factors. In some embodiments, the reprogramming factors are provided as multiple coding sequences on a vector.

[0064] Genes can be introduced into somatic cells or iPS cells derived therefrom for various purposes, such as replacing a gene with a loss of functional mutation, or providing a marker gene. Alternatively, vectors expressing antisense mRNA or ribozymes can be introduced to block the expression of undesirable genes. Another method of gene therapy is the introduction of drug resistance genes to enable normal progenitor cells to be advantageously exposed to selective pressures, such as multiple drug resistance genes (MDRs) or anti-apoptotic genes such as bcl-2. As discussed above, nucleic acids may be introduced into target cells using various techniques known in the art, such as electroporation, calcium-precipitated DNA, fusion, transfection, lipofection, and infection. The specific method by which the DNA is introduced is not important for the implementation of the present invention.

[0065] As used herein, the term “intestinal cell” refers to the cells that make up the mammalian intestinal epithelium. The mammalian intestinal epithelium of the gastrointestinal tract has a clearly defined tissue structure. The epithelium can be divided into two regions: a functional region that houses differentiated cells (villi) and a proliferative region (Lieberkühn’s crypts) that represents the epithelial stem cell niche. Pluripotent epithelial stem cells reside in the crypts and give rise to four major epithelial lineages: absorptive intestinal cells, mucin-secreting goblet cells, peptide hormone-secreting enteroendocrine cells, and Paneth cells.

[0066] The phrase "mammalian intestinal cells" refers to cells derived from the intestines of mammals. Typically, in the method of the present invention, intestinal fragments are obtained surgically and are approximately 1 mm in size. 3 Cut into pieces smaller than approximately 0.5 mm. 3 Less than, or approximately 0.1 mm 3The cells may be less than or alternatively dissociated into single cells. Mammals used herein include humans, horses, cattle, pigs, dogs, cats, rodents such as mice, rats, hamsters, and primates. Intestinal tissue can be obtained from humans by biopsy during endoscopy. "Mammalian intestinal cells," "intestinal cells," and "intestinal epithelial cells" are used interchangeably. Sources of intestinal tissue may be fetuses, neonates, juveniles, or adults.

[0067] "Intestine" refers to the small intestine and large intestine of mammals. For the methods described herein, intestinal tissue is obtained from either the small intestine or the large intestine.

[0068] The term "graft" means, for example, cells derived from mammalian intestinal tissue and grown in vitro according to the method of the present invention.

[0069] "Intestinal stem cells" are used interchangeably with "epithelial stem cells" and refer to stem cells that have the potential to proliferate and differentiate into intestinal epithelial cells. Pluripotent epithelial stem cells can give rise to various epithelial lineages, including all intestinal epithelial lineages, such as absorptive intestinal cells, mucin-secreting goblet cells, peptide hormone-secreting enteroendocrine cells, and Paneth cells.

[0070] The term "multiphylogenetic differentiation markers" refers to differentiation markers characteristic of different cell types. These differentiation markers can be detected by using marker-specific affinity reagents, such as marker-specific antibodies, or by using chemicals that specifically stain cell types, as is known in the art. Non-limiting examples of terminal differentiation markers include chromogranin A, NeuroD-enterotocrine cells, mucin-goblet cells, bilin, CD10-enterocytes, lysozyme, and Ang4-Paneth cells. Common progenitor cells of enterotocrine, goblet, and Paneth cells are detected by using antibodies against Math1. P-PTEN, SFRP5, and Musashi1 are specifically expressed in intestinal stem cells and intestinal progenitor cells. Intestinal alkaline phosphatase (IAP) marks intestinal cells.

[0071] The term "candidate drug" means oligonucleotides, polynucleotides, siRNAs, shRNA genes, gene products, small molecules, and pharmacological compounds introduced into intestinal cell cultures as described herein to assay their effects on grafts.

[0072] The term "contact" refers to placing either candidate cells or candidate drugs into a mammalian intestinal cell graft culture. Contact also includes co-culturing candidate cells with an intestinal graft in culture medium for at least one hour, or more than two or four hours, before placing them in a semipermeable substrate. Alternatively, contact refers to placing candidate cells into the lumen of a graft that will develop as a cyst via transluminal injection.

[0073] "Screening" refers to the process of either co-culturing candidate cells with the culture described herein or adding a candidate drug to the culture described herein. The effect of candidate cells or candidate drugs on the culture is evaluated by increased graft growth above the basal level, for example, by the presence of multi-system differentiation markers indicating intestinal stem cells. The effect of candidate cells or candidate drugs on intestinal grafts can be further evaluated by assaying the intestinal grafts for long-term reconstitution activity by sequential in vitro passage and by in vivo transplantation using subcutaneous implant assays and renal capsule assays.

[0074] "Container" refers to a glass, plastic, or metal container that can provide a sterile environment for culturing cells.

[0075] The term “graft” is used herein to mean, for example, tissue and cells derived from mammalian tissue cultured in vitro according to the method of the present invention. The mammalian tissue from which the graft is derived may be obtained from an individual, i.e., a primary graft, or it may be obtained in vitro, for example, by differentiation of induced pluripotent stem cells.

[0076] The term “organoid” is used herein to mean the three-dimensional growth of mammalian cells in a culture that preserves tissue characteristics in vivo, such as long-term tissue expansion with proliferation, multiphyletic differentiation, and reproduction of the ultrastructure of cells and tissues. Primary organoids are organoids cultured from grafts, i.e., cultured grafts. Secondary organoids are organoids cultured from a subset of primary organoid cells, i.e., primary organoids are fragmented, for example, by mechanical or chemical means, and the fragments are reproduced and cultured. Tertiary organoids are organoids cultured from secondary organoids, etc.

[0077] "Ultrological structure" refers to the three-dimensional structure of cells or tissues observed in vivo. For example, the ultrastructure of a cell may be its polarity or morphology in vivo, while the ultrastructure of a tissue may be the arrangement of different cell types relative to each other within the tissue.

[0078] "Screening" refers to the process of co-culturing candidate cells with the culture described herein, or adding a candidate drug to the culture, and evaluating the effect of the candidate cells or candidate drug on the culture. The effect can be evaluated by assessing any convenient parameters, such as graft growth rate, the presence of multi-lineage differentiation markers indicating stem cells, etc. The effect of candidate cells or candidate drug on the graft can be further evaluated by assaying the graft for long-term reconstitution activity by serial in vitro passaging and by in vivo transplantation.

[0079] Culture method Culture systems and methods for culturing various mammalian tissues are provided. In some embodiments, the tissue, i.e., primary tissue, is obtained from mammalian organs. The tissue may be derived from any mammalian species, e.g., humans, horses, cattle, pigs, dogs, cats, rodents, e.g., mice, rats, hamsters, primates, etc. The mammal may be of any age, e.g., fetus, neonatal, juvenile, or adult. Some non-limiting examples of tissues that can be obtained for the purpose of preparing organoids are given below. Cells or tissues may be obtained by any convenient method, e.g., biopsy, e.g., during endoscopy, surgery, by needle, etc., or from cell lines, in vitro differentiation, etc. In the case of tissue, it is immersed in ice-cold buffer solution, e.g., PBS, Ham's F12, MEM, culture medium, etc. Tissue fragments are about 1 mm 3 The tissue can be cut into pieces smaller than a certain size and dissociated into single cells. The cut tissue is mixed with the hydrogel of this disclosure. Subsequently, the cell-containing hydrogel is placed in a suitable culture medium.

[0080] In some embodiments, the tissue is grown in vitro from pluripotent stem cells, such as embryonic stem cells (ESCs), embryonic germ cells (EGCs), or induced pluripotent stem cells (iPSCs). Any convenient method may be followed to induce the desired tissue from the pluripotent stem cells. The manipulated tissue can then be transferred to a hydrogel substrate.

[0081] The continued growth of the graft can be confirmed by any convenient method, such as phase-contrast microscopy, stereomicroscopy, histology, immunohistochemistry, and electron microscopy. In some cases, the ultrastructure and multiphylogenetic differentiation of cells can be evaluated. The ultrastructure of intestinal grafts in culture can be determined by performing hematoxylin-eosin staining, PCNA staining, electron microscopy, etc., using methods known in the art. Multiphylogenetic differentiation can be determined by performing labeling with antibodies against terminal differentiation markers, for example, as described in more detail below. Antibodies for detecting differentiation markers are commercially available from many sources.

[0082] In some embodiments, cells in cultured grafts may be experimentally modified. For example, graft cells may be modified by exposure to a viral or bacterial pathogen to develop experimental reagents for evaluating the antiviral or antibacterial effects of a therapeutic agent. Explant cells may be modified by modifying the pattern of gene expression to provide reprogramming factors to induce pluripotency or otherwise alter differentiation potential, or to determine the effect of increased or lost gene activity on the ability of cells to form graft cultures or cells to undergo tumor transformation. Explant cells may be modified to transform into oncogenetic or oncogenic cells, for example, by providing nucleic acids that suppress the expression of cancer driver-oncogenic factors or tumor suppressor gene inhibitors, such as APC, p53, or Smad4, for overexpression of Kras.sup.G12D, in order to evaluate the effect of a therapeutic agent on tumors.

[0083] Experimental modification can be carried out by any method known in the art, for example, by providing candidate drugs, such as nucleic acids, polypeptides, small molecules, or viruses, to grafts and their cells for screening purposes, as described below.

[0084] Organoids prepared by the subject method have found use in many applications. For example, cancer, ischemia, congenital syndromes, trauma, and inflammation can cause significant loss of function or forced physical resection of a large portion of patient tissue large enough to impair organ physiology. The ability to grow mammalian tissue grafts in vitro and return them to such patients, or to use them as a source of tissue-specific stem cells for transplantation into such patients, is a valuable therapeutic option. Such cells can enhance the ex vivo expansion of tissue and provide an autogenous source of engineered tissue and / or tissue stem cells. As another example, organoids prepared by the subject method may be used to predict the responsiveness of individuals, e.g., individuals with cancer, individuals with infection, etc., to therapy. As yet another example, organoids prepared by the subject method may be used in basic research, e.g., to better understand the basis of disease, and in drug discovery, e.g., in reagents in screening, such as those further described below. Organoids are also useful for evaluating the pharmacokinetics and pharmacodynamics of drugs, such as the ability of mammalian tissues to absorb active drugs, and the cytotoxicity of drugs on primary mammalian tissues or carcinogenic mammalian tissues.

[0085] experiment The manipulated matrix enables the formation, growth, progression, and differentiation of human patient-derived intestinal organoids. Human intestinal organoids derived from primary tissue biopsies of patients have the potential to revolutionize personalized medicine and preclinical models of human gastrointestinal diseases. To date, most intestinal organoids have been grown in decellularized matrices derived from Engelbresholmeswam (EHS) mouse sarcoma, a material with limited tunability and reproducibility. To overcome this limitation, we report a designer matrix called hyaluronic acid elastin-like protein (HELP), which enables the formation, differentiation, and passage of epithelial-only intestinal organoids derived from adult primary tissue. These materials allow for the encapsulation of isolated patient-derived cells, which then undergo proliferation and new formation of enteroids, spherical structures with polarized internal lumens. After 12 passages, dissociation, and regeneration of human enteroids, the growth of HELP material was found to be statistically similar to that of EHS matrix. HELP material further supported the differentiation of human enteroids into more specialized cell types: Paneth cells, goblet cells, and enteroendocrine cells. The three key variables of HELP—matrix stiffness, matrix stress relaxation rate, and matrix integrin ligand concentration—can each be quantitatively specified independently, enabling fundamental research into organoid-matrix interactions and potential patient-specific optimization. Organoid formation in HELP materials was found to be most robust in gels with a stiffer coefficient (G' ~ 1 kPa), a slower stress relaxation rate (t1 / 2 ~ 18 hours), and a higher integrin ligand concentration (0.5 ~ 1 mM RGD peptide). Our material provides a 3D in vitro model for further understanding organoid development and disease in humans, and offers reproducible, biodegradable, minimal matrix free of synthetic polyethylene glycol for animal-derived products or potential clinical translation.

[0086] The synthetic matrix is ​​designed to support the formation of mouse and human induced pluripotent stem cell (iPSC)-derived organoids without requiring an EHS matrix or other cell types. In contrast, human patient-derived intestinal organoids in synthetic matrices often require either a spheroid formation process in an EHS matrix or co-culture with mesenchymal cells. Recent studies developing manipulative matrices for patient-derived intestinal organoids rely on polyethylene glycol (PEG) as the synthetic matrix backbone, but PEG is known to interact with the immune system and induce antibody formation. To create a PEG-free system, we report a designer matrix, hyaluronic acid elastin-like protein (HELP), which enables the formation, differentiation, and passage of intestinal organoids derived solely from adult primary tissue epithelium. Three key variables of HELP (matrix stiffness, matrix stress relaxation rate, and matrix integrin ligand concentration) can each be independently and quantitatively specified, enabling fundamental research into organoid-matrix interactions and potential patient-specific optimization. Our materials provide 3D in vitro models for a deeper understanding of human intestinal development and intestinal diseases, and reproducible, biodegradable, minimal matrix materials free of synthetic PEG for animal-derived products or potential clinical translation.

[0087] We hypothesized that a minimal matrix inspired by biopolymers found in the natural gut would support the formation of organoids derived from primary human tissue (Figure 1a). Our group previously reported a protein-modified matrix that supports the formation and growth of primary mouse intestinal organoids using amino acid sequences derived from human elastin and fibronectin. Elastin is one of the major components of the extracellular matrix (ECM), and fibronectin is expressed in the crypts of intestinal stem cells (ISCs). Recombinant elastin-like protein (ELP, MW 37.7 kDa) is interspersed with elastin-like sequences that have integrin-binding elongation RGD sequences borrowed from fibronectin (Figure 1b, Figure S1a). In vivo, ISC maintenance and proliferation are partially mediated by the CD44 receptor, and CD44 activation is associated with intestinal growth. The CD44 receptor can interact with hyaluronic acid (HA, MW 100kDa), a glycosaminoglycan important for normal intestinal growth, leading to the hypothesis that an engineered matrix containing HA could support patient-derived enteroid culture. To create a reproducible hydrogel material from these two biopolymer components, we developed a scheme in which ELP was chemically modified with a hydrazine group and HA was chemically modified with benzaldehyde, as previously reported (Figure 5b-e). Simply mixing the two modified biopolymers together induced the formation of hydrazone bonds, resulting in a hydrogel network called HELP.

[0088] Through this research, cells are classified as follows: Undifferentiated spheroids of intestinal epithelial cells are referred to as patient-derived intestinal enteroids (or more simply, enteroids), and when differentiated, these intestinal cellular structures are called patient-derived intestinal organoids. In some studies, what is referred to as the enteroid state is specifically Lgr5 +In mouse strains utilizing fluorescent reporter systems for ISC populations, these are referred to as ISC colonies or spheroids. Here, human enteroids were dissociated into single cells and embedded within HELP during covalent crosslinking of a hydrogel. Novel spheroid formation was observed within 3 days (Figure 1c, d). Several previous reports of human enteroids in synthetic materials require an initial step of enteroid formation in an EHS matrix before encapsulation in synthetic biomaterials, a process referred to as "re-embedding" (Figure 1c). Therefore, we compared the ability of different materials to support these two different culture methods: 1) encapsulation of dissociated single cells, and 2) re-embedding of pre-formed enteroids (Figure 1d). As expected and consistent with previous reports, the re-embedded enteroids grew and survived for at least 6 days in an ELP-only matrix containing fibronectin-derived integrin-binding RGD ligand, but it should be noted that enteroid polarization, as observed by ZO-1 and β-catenin staining, was not maintained (Figure 1d, top). In stark contrast, dissociated human intestinal cells in an ELP-only matrix (i.e., without HA) were unable to form enteroids, suggesting that RGD ligand alone is insufficient to support novel organoid formation in this material. Interestingly, the addition of HA to the manipulated matrix allowed a single intestinal cell to robustly form an enteroid with HELP and survive for at least 6 days when cultured as a re-embedded enteroid (Figure 6). Importantly, enteroids formed in HELP exhibited appropriate intestinal epithelial polarity similar to that of the EHS matrix, as indicated by the localization of the narrow junction protein tight junction-1 (ZO-1) to the apical lumen and the basolateral localization of the adhesion junction protein β-catenin (Figure 1d). Similar results were observed for a second distinct patient line in the HELP matrix (Figure 7). To demonstrate the potential broad applicability of the HELP matrix for supporting organoid growth, primary mouse intestinal enteroids and liver organoids derived from human induced pluripotent stem cells (iPSCs) were also viable in the HELP matrix (Figure 8).

[0089] In addition to robust organoid formation from single cells, human enteroids in HELP can be repeatedly passaged after each passage, continuing to form new enteroids (Figure 1e, 6, 7b, c). The HELP matrix is ​​enzymatically degraded using elastase and hyaluronidase, followed by enteroid lysis into single cells using trypsin (see Culture Method). By encapsulating these single cells in fresh HELP matrix, new enteroid formation for up to 12 passages was successfully achieved without any visible morphological changes (Figure 1e, f). The enteroids repeatedly passaged in the HELP matrix matched the enteroid growth rate in the EHS matrix, as observed by bright-field microscopy over 12 days of culture (Figure 1f). Furthermore, these repeatedly passaged enteroids had statistically similar formation rates in the HELP and EHS matrices (Figure 1g).

[0090] To evaluate whether HELP can support differentiation into patient-derived intestinal organoids, single cells embedded in HELP were initially allowed to form enteroids for 10 days in growth medium containing Wnt3A, epidermal growth factor, Noggin, and R-spondin1 (Figure 2a). In the growth medium, cells undergo initial enteroid formation (Figure 2b, left). As the enteroids develop, the cells become morphologically columnar and adopt intrinsic apical basal polarity, as demonstrated by a thick apical actin boundary within the organoid (Figure 2b, center) and localization of known polarizing markers (Figure 1d). On day 10, Wnt and R-spondin1 were removed to promote enteroid differentiation into organoids over a 5-day period.

[0091] This process resulted in the formation of undulating lumens containing differentiated enteric cell types (Figure 2b). Immunocytochemistry identified lysozyme-positive Paneth cells in organoids grown in both HELP and EHS matrices (Figure 2c). Higher expression of mucin-2 (Muc2)-positive goblet cells was observed in organoids grown in HELP compared to those grown in EHS matrices. Chromogranin-A (ChgA)-positive cells, a marker of differentiated enteroendocrine cells, were identified in the HELP matrix but not in the EHS matrix. To evaluate the transcriptional expression of these differentiation markers, reverse transcription quantitative polymerase chain reaction (RT-qPCR) was performed on organoids differentiated in either the HELP or EHS matrix compared to enteroids maintained for 15 days in the growth medium of each matrix (Figure 2d). Expression levels of LYZ1 (lysozyme) and the enteric cell marker VIL1 (virin-1) were relatively unchanged in both the HELP and EHS matrices compared to undifferentiated controls. High upregulation of MUC2 (mucin-2) and CHGA (chromogranin-A) gene expression was observed in both the HELP matrix and the EHS matrix.

[0092] Since enteroids formed normally in the HELP matrix, not just the ELP matrix (Figure 1), we then sought to further explore the permissive role of HA in these matrices. To confirm that patient-derived intestinal cells are rich in proliferative intestinal crypts and express CD44, which is also an HA receptor, we performed immunocytochemistry on enteroids formed from single cells in the HELP and EHS matrices. Interestingly, higher intensity of CD44 staining on the periphery of enteroids grown in the HELP matrix was observed compared to those grown in the EHS matrix (Figure 3a). Flow cytometry further confirmed this finding, as higher surface expression of CD44 was observed on single dissociated cells from enteroids grown in the HELP matrix compared to those grown in the EHS matrix (Figure 3b, Figure 9). Flow cytometry analysis revealed that approximately 90% of enteroid-derived cells grown in the HELP matrix were CD44-positive, compared to 70% of CD44-positive cells from enteroids grown in the EHS matrix. These results suggest that HA signaling in HELP may play a crucial role in promoting enteroid formation. To further investigate whether HA signaling is a key feature of the material that supported enteroid formation, a synthetic PEG polymer having the same benzaldehyde moiety as that used to modify the HA component was modified. Using this material, an ELP-PEG hydrogel matrix was generated that was rigidly matched to the HELP matrix (Figure 10) with an equivalent concentration of RGD peptide (1 mM). Bright-field and confocal fluorescence microscopy revealed that enteroid formation was not possible in the ELP-PEG gel with mechanical properties equivalent to HELP, suggesting that HA biochemical signaling is an essential component of the HELP matrix (Figure 3c, d).

[0093] HELP materials allow for the independent selection of multiple biomaterial properties, enabling the study of organoid growth in response to different biochemical and biophysical matrix cues. By tuning these parameters, it is possible to carefully study how cells in organoids respond to tissue mechanics. In fact, the interactions of matrix stiffness, matrix stress relaxation, and matrix RGD content have been important in other manipulated biomaterial systems. To explore these interactions within the HELP system, we first investigated non-integrin binding, sequence scrambled peptides (R), which are known to be non-cell adhesion. DG A variant of the ELP protein containing ) was prepared. Next, RGD- and R in the HELP matrix. DG By blending with ELP protein, the precise concentration of RGD ligand in the hydrogel could be adjusted to 0-1 mM without affecting matrix dynamics (Figures 4a, 10). To create matrices with various mechanical properties, we first varied the degree of hydrazine-benzaldehyde crosslinking (Figure 4b, top) to create a rigid elastic matrix (storage coefficient, G'~1 kPa, referred to as "rigid EL") and a more conformable elastic matrix (G'~400 Pa, "soft EL") (Figures 4c, d). These matrices had comparable quasi-elastic stress relaxation profiles, with negligible stress relaxation over 60 minutes and a stress decay half-life of approximately 18 hours (t 1 / 2 ) possessed.

[0094] The distribution of patient-derived enteroid sizes was measured as a function of various material properties in a set of HELP matrices (data shown as mean in Figure 4, violin plot distribution shown in Figure 11). In general, the cross-sectional area of ​​enteroids grown in stiffer gels (G' ~ 1 kPa) was larger than that of enteroids grown in softer gels (G' ~ 400 Pa, Figure 4e). This result is consistent with the stiffness range previously found to be optimal for primary human intestinal organoids grown in mouse and synthetic PEG-based matrices. In addition to this trend, we observed that enteroids did not grow as robustly in matrices lacking RGD ligand, regardless of matrix stiffness. This is consistent with previous reports on intestinal organoids in synthetic matrices, where a minimum threshold level of RGD was required for optimal organoid growth. However, in those previous reports, organoids cultured in RGD-free synthetic matrices were unable to form or survive. The fact that viable enteroids were formed in the HELP matrix without the RGD ligand suggests that while co-presentation of RGD and HA significantly improved organoid growth, signaling from HA may be sufficient to induce some organoid formation (Figure 4e).

[0095] Next, we explored the role of RGD concentration in the matrix, which is stress-relaxing and viscoelastic. The matrix, being "stress-relaxing," undergoes molecular-level remodeling to dissipate stress and relax after being deformed by cellular forces. Studies using other cell types have demonstrated that matrix stress relaxation may have a stronger impact on the cellular phenotype than matrix stiffness, and these effects differ depending on the integrin ligand concentration, as this is the primary mechanism cells use to exert forces on the matrix. Therefore, we sought to design a family of biomimetic HELP matrices that allow for independent tuning of matrix stiffness, RGD ligand concentration, and matrix stress relaxation. A key feature of native extracellular matrices and EHS matrices is their ability to undergo stress relaxation through physical crosslinking, which can be readily remodeled. The remodeling dynamics of dynamic covalent crosslinks, such as those used in HELP matrices, can be tuned by the selection of adjacent chemical moieties. By replacing the benzaldehyde group fraction on HA with aldehyde groups (Figure 4b, bottom), a semi-elastic "rigid EL" HELP matrix (Figure 4f, g) with the same stiffness but faster stress relaxation (t 1 / 2 A viscoelastic HELP matrix (referred to as "rigid VE") was formulated (~30 minutes). Interestingly, a greater dependence of enteroid growth on RGD ligand concentration was observed in the viscoelastic gel compared to the elastic gel (Figure 4e, h). The viscoelastic gel had a threshold of 0.75 mM RGD required for robust enteroid growth (Figure 4h). These data suggest that enteroids may be more sensitive to the presence of integrin ligands in matrices that can undergo greater remodeling, while enteroids can form more robustly across a wider range of matrix properties in matrices with more elastic mechanisms.

[0096] In summary, we present a HELP matrix that enables robust formation, growth, passage (Figure 1), and differentiation (Figure 2) of primary human intestinal organoids from dissociated single cells. Interestingly, the presence of hyaluronic acid in HELP is sufficient to enable novel enteroid formation from single cells, as ELP alone and ELP-PEG matrices with similar mechanisms and RGD-ligand concentrations did not support enteroid formation (Figures 1, 3). This observation correlates with increased expression of CD44, a well-known receptor for HA, in enteroids cultured in the HELP matrix (Figure 3). This receptor is known to be important in ISC regeneration, and therefore, the findings contribute to a collective understanding of matrix factors influencing intestinal cell proliferation and support further mechanistic studies of HA-containing designer matrices.

[0097] The HELP matrix is ​​not only suitable for this particular cell source, but also supports not only mouse intestinal organoids but also human iPSC-derived liver organoids (Figure 8). Therefore, it is useful to add the HELP matrix to a library of minimal matrices available for reproducible organoid culture. These results, combined with bespoke tailoring of several material properties including biochemical ligand density, matrix stiffness, and matrix stress relaxation rate (Figure 4), position the HELP matrix as a platform that can be optimized for culturing a wide variety of patient-derived organoids. A clearly defined, minimally manipulated matrix overcomes the main limitations of EHS matrices, particularly batch-to-batch variability, poor tunability, biological complexity, and clinical translationability, while avoiding the use of PEG. In the future, the HELP matrix could be customized to mimic patient-specific matrix properties, resulting in reproducible and personalized organoid culture. By enabling the culture of human intestinal and other organoids, the HELP material has many future applications, including research in intestinal disease pathology, developmental biology, and regenerative medicine.

[0098] Materials and methods Subculture and maintenance culture of human enteroids in EHS matrix. Human primary intestinal tissue was used for all experiments, between subcultures 4 and 24. Cells in the maintenance cultures were maintained by encapsulating them in 40 μL of EHS matrix in 24-well plates, particularly in Culturex basement membrane extracted reduced growth factor (BME-RGF) type 2 (Trevigen, Gaithersburg, MD). Enteroids were subcultured every 1-2 weeks depending on the growth rate. To subculture the enteroids, Culturex droplets were immersed in phosphate-buffered saline (PBS) with 5 mM ethylenediaminetetraacetic acid (EDTA) on ice to dissolve the gel, centrifuged at 500 × g for 5 minutes, and treated with TrypLE (Thermo Fisher Scientific, Waltham, MA) at 37°C for 10 minutes, vigorously mixed by pipette aspiration every 5 minutes to support single cell generation. Next, TrypLE was quenched in enteroid growth medium (described below) and centrifuged at 500×g for 5 minutes. The pellet was washed in growth medium for cell counting and then centrifuged at 500×g for 5 minutes. The cell pellet was resuspended in ice-cold Culturex at a concentration of 750,000 cells / mL and transferred to a cell culture incubator. After gelation at 37°C for 10 minutes, 500 μL of pre-warmed growth medium was added to each well. Small molecule inhibitors 10 μM Y-27632 and 2.5 μM CHIR-99021 (both obtained from Bio-Techne, Minneapolis, MN) were added to the medium for the first medium change of maintenance culture. The medium was completely replaced every 3-4 days.

[0099] Mouse enteroid isolation and culture. Mouse intestinal enteroids were produced as described above. Briefly, the isolated mouse small intestine was longitudinally cut and washed with cold PBS. The tissue was then crushed into approximately 5 mm square fragments and washed again with cold PBS. The tissue was then incubated in 2 mM EDTA in PBS on ice. After incubation, the EDTA solution was aspirated, and the tissue fragments were thoroughly mixed with a 10 mL serological pipette using cold PBS to allow the tissue to settle. The supernatant was discarded, and the precipitate containing intestinal crypts was resuspended in PBS. The sample was vigorously mixed and then centrifuged at 500 × g for 5 minutes, and the crypt-rich supernatant was passed through a 70 μm cell strainer (BD Biosciences, San Jose, CA). The crypts were centrifuged again at 200 × g for 3 minutes to isolate single cells. A portion of the mainly pure crypts was used for culture.

[0100] Differentiation and culture of human liver organoids. Liver organoids were generated as described above. Briefly, secondary liver organoids (HO2) derived from normal iPSCs were digested in 0.25% trypsin-EDTA for 5-10 minutes. Cells were collected by centrifugation at 200×g for 3 minutes, resuspended in 25 μl of 1% HA, and directly mixed with 25 μl of pre-loaded 1% ELP in a 24-well plate at a rate of 1,000 cells per well. After HELP solidification, 1 ml of growth medium was added, and the cells were cultured for 6 days. The growth medium consisted of RPMI+B27 (Thermo Fisher Scientific, Waltham, MA) medium containing 250 nM LDN-193189, 3 μM CHIR99021, 10 μM A83-01, 100 ng / ml EGF, 10 ng / ml FGF10, and 20 ng / ml HGF. Next, cells were cultured for a further 6 days in differentiation medium consisting of HCM (Lonza, Basel, SUI) medium supplemented with 10 μM DAPT, 10 ng / ml oncostatin M, 20 ng / ml HGF, 10 μM dexamethasone, and 10 ng / ml BMP4. To perform a forskolin-induced swelling assay on HO, both forskolin (FSK) and 3-isobutyl-1-methylxanthine (IBMX) (10 μM and 100 μM, respectively) were added to activate the cAMP pathway and increase CFTR (cystic fibrosis transmembrane conductance regulator) function in the HO cultures for 24 hours. After staining with a 10 μM solution of the cell-permeable fluorescent dye calcein green, swelling was visualized. Then, the difference in the total area of ​​each liver organoid after 24 hours of treatment was calculated and plotted.

[0101] Preparation of intestinal organoid growth medium. The organoid growth base medium consisted of a 1:1 mixture of ADMEM-F12 medium (Thermo Fisher Scientific, Waltham, MA) and L-WRN (ATCC CRL3276) condition medium. To prepare the L-WRN condition medium, L-WRN cells were plated onto T150 cell culture flasks in L-WRN growth medium (Dulbeccoo's Modified Essential Medium (DMEM) supplemented with 10% FBS and 1% penicillin-streptomycin-glutamine (PSQ)) and grown for 1-2 days. The growth medium was changed and supplemented with L-WRN selective medium (L-WRN growth medium supplemented with 500 μg / mL each of G418, and hygromycin antibiotic for selecting cells containing transgenic DNA encoding the secretion of Wnt-3A, R-spondin 3, and Noggin). The cells were grown to confluence, divided twice in a 1:4 ratio, and then divided into multiple T150 flasks. The cells were cultured to confluence in L-WRN growth medium, washed with L-WRN recovery medium (ADMEM-F12 containing 10% FBS and 1% PSQ), and cultured for 24 hours in fresh L-WRN recovery medium. After 24 hours, the conditional media were collected from each flask and combined. The fresh L-WRN recovery medium was replaced, and the process of preparing and collecting conditional media was repeated up to four times. ADMEM-F12 was mixed with L-WRN conditional medium in a 1:1 ratio, and the following reagents were added.1 mM HEPES (Thermo Fisher Scientific, Waltham, MA), 1x Glutamax (Thermo Fisher Scientific, Waltham, MA), 10 mM Nicotinamide (Sigma-Aldrich, St. Louis, MO), 1 mM N-Acetylcysteine ​​(Sigma-Aldrich, St. Louis, MO), 1x B-27 Supplement (Thermo Fisher Scientific, Waltham, MA), 0.5 μM A83-01 (Sigma-Aldrich, St. Louis, MO), 1x PSQ (Thermo Fisher Scientific, Waltham, MA), 10 nM Gastrin-I (Sigma-Aldrich, St. Louis, MO), 10 μM SB-202190 (Bio-Techne, Minneapolis, MN), 50 ng / mL Recombinant EGF (Thermo Fisher Scientific, Waltham, MA), and 1xNormocin (InvivoGen, San Diego, CA).

[0102] Intestinal Organoid Differentiation: To differentiate intestinal enteroids into organoids, cells were encapsulated as single cells in HELP or EHS matrix, maintained in growth medium for 10 days, briefly washed with PBS, and cultured in differentiation medium for 5 days. The differentiation medium was Advanced DMEM / F12 medium supplemented with 1x Glutamax, 1x Penicillin / Streptomycin, 1x Normocin, 100 ng / mL recombinant noggin (Peprotech, Rocky Hill, NJ), 1x B27, 1 mM N-acetylcysteine, 50 ng / mL recombinant EGF, 10 nM Gastrin-I, 10 μM Y-27632 ROCK inhibitor, 5 μM DAPT, and 500 nM A83-01.

[0103] ELP-hydrazine synthesis. Elastin-like protein (ELP) was prepared as described above. Briefly, the ELP sequence was cloned into the pET15b plasmid and protein expression was controlled using the T7 promoter. BL21(DE3)pLysS Escherichia coli (Life Technologies) containing the plasmid encoding ELP was cultured in terrific broth to an OD of 0.8 600 and expression was induced using 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG). The protein was expressed in the bacteria for 7 hours, then harvested by centrifugation, suspended in 10 buffer (10 mM Tris, 1 mM EDTA, and 100 mM NaCl, pH 8.0), and lysed by 3 cycles of freeze-thaw. The cell lysate was treated with deoxyribonuclease (DNase) and 1 mM phenylmethanesulfonyl fluoride (PMSF) to inhibit proteolysis. ELP was purified by an alternating sequence of centrifugation steps at 4 °C and 37 °C, followed by dialysis against deionized water for 4 shifts (48 hours, 4 L volume per shift), then frozen at -80 °C and lyophilized. To modify the ELP amine with hydrazine functional groups, the lyophilized ELP (210 mg) was completely dissolved at 7 wt% in 3 mL of anhydrous dimethyl sulfoxide (DMSO), then diluted to 3.5 wt% with 3 mL of anhydrous N,N-dimethylformamide (DMF). In a round-bottom flask, 3 mL of anhydrous DMF was used to separately dissolve triboc-hydrazinoacetic acid (2 equivalents: ELP amine), hexafluorophosphate azabenzotriazolium tetramethyluronium (HATU, 2 equivalents: ELP amine), and 4-methylmorpholine (4.5 equivalents: ELP amine), and the vessel was stirred for 5 minutes to allow HATU to activate the free acid on triboc-hydrazinoacetic acid. Next, the ELP solution was added dropwise to the round-bottom flask with stirring. The reaction was allowed to proceed overnight at room temperature (RT). The product was precipitated in ice-cold ether, centrifuged, and dried to obtain the Boc-protected ELP-hydrazine intermediate. This intermediate was 1Modification efficiency was quantified by 1H NMR analysis (500 Hz, DMSO-d6) δ7.00 (d,2H), 6.62 (d,2H), and 1.46 (m,27H). Modification efficiency was determined by comparing the integrated signal of Boc protons (δ1.5~1.35) with that of aromatic protons of tyrosine residues on ELP (δ7.00 and 6.62). To remove the Boc protecting group, the ELP-hydrazine intermediate was dissolved at 2 wt% in 1:1 DCM:TFA with 2.5% v / v triisopropylsilane and stirred in a vented round-bottom flask at room temperature for 4 hours. The product was precipitated in ether, centrifuged, dried, then dissolved in deionized water, dialyzed in 3 shifts (24 hours, 4 L per shift) against deionized water, and lyophilized.

[0104] Hyaluronic acid modification. 100 kDa sodium hyaluronate (HA, Lifecore Biomedical, Chaska, MN, USA) was modified to have an aldehyde functional group by the following overall procedure. First, the carboxylic acid group on HA was amidated with propargamine to produce an HA-alkyne intermediate. Then, using copper click chemistry, this alkyne was reacted with the azide moiety of a heterobifunctional small molecule containing an aldehyde functional group to produce aldehyde-functionalized HA.

[0105] A 12% modified HA alkyne was prepared. HA was dissolved in 2-(N-morpholino)ethanesulfonic acid (MES) buffer (0.2 M, pH 4.5) to a concentration of 10 mg / mL. N-hydroxysuccinimide (NHS, 0.8 equivalents relative to the HA dimer unit), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC, 0.8 equivalents), and propargylamine (0.8 equivalents) were successively added to this solution. After adjusting the pH to 6, the mixture was stirred at room temperature for 4 hours. The solution was then dialyzed against deionized water for 6 shifts (3 days, 4 L per shift) and lyophilized to obtain a white powder.

[0106] 30% modified HA alkyne. Sodium hyaluronate was dissolved in MES buffer (0.2 M, pH 4.5) to a concentration of 10 mg / mL. NHS (1.5 equivalents per HA dimer unit), EDC (1.5 equivalents), and propargylamine (1.0 equivalent) were successively added to this solution. After adjusting the pH to 6, the mixture was stirred at room temperature for 4 hours. The solution was then dialyzed against deionized water for 6 shifts (3 days, 4 L per shift) and freeze-dried to obtain a white powder.

[0107] Next, HA-alkyne was modified with the following small molecule, 1, to produce HA-benzaldehyde. The small molecule was produced as follows.

[0108] [ka]

[0109] N-(2-azidoethyl)-4-formylbenzamide (1) was synthesized according to the method published in Biomaterials, 2018, 154, 213-222. With minor modifications, HA was modified with molecule 1 according to a previously reported procedure. 300 mg of HA-alkyne was dissolved in PBS at 2% by weight, followed by the addition of 1 (1 equivalent to HA dimer units). 1 was dissolved using a minimal amount of DMSO before being added to the HA solution. The solution was then frothed with N2 for 30 minutes. Copper(II) sulfate pentahydrate (0.004 equivalents) and sodium ascorbate (0.06 equivalents) were dissolved in deionized water, frothed with N2, and added to the HA solution. After stirring at room temperature for 1 day, the mixture was dialyzed against deionized water for 3 days and lyophilized. Since the proton signals of the aromatic ring on the benzaldehyde moiety overlap with those of the triazole group, the degree of modification on HA-benzaldehyde was quantified by integrating the proton signals (δ=7.5-8,5H) for the modification of the methyl group on the N-acetylglucosamine of the HA skeleton (δ=1.8,3H).

[0110] Synthesis of HA-aldehyde. HA-aldehyde was synthesized according to the method published in Biomaterials, 2009, 30, 2499-506. HA was first dissolved in Milli-Q water at a concentration of 0.4 w / v% and stirred at room temperature. A 0.1 M aqueous solution of sodium periodate was added dropwise, and the reaction was stirred overnight in the dark at room temperature. The following day, ethylene glycol was added for 1 hour to inactivate any unreacted periodates. The solution was then purified for 3 days by dialysis using a 10,000 MWCO membrane in Milli-Q water, with fresh water being replaced in 12-hour shifts. After dialysis, the dried product was obtained by lyophilization.

[0111] Synthesis of polyethylene glycol-benzaldehyde (PEG-BZA). PEG-BZA was synthesized as described above. Briefly, 4-formylbenzoic acid (0.528 g, 3.52 mmol, 2.1 equivalents per amine, Sigma) was dissolved in 5 mL of anhydrous dimethylformamide (DMF, Sigma) and activated with HATU (1.216 g, 3.2 mmol, 2 equivalents, Sigma) and 4-methylmorpholine (0.792 mL, 7.2 mmol, 4.5 equivalents, Sigma). After stirring the reaction mixture for 5 minutes, 4 g, 0.2 mmol; Creative PEGworks, a 4-arm 10 kDa PEG-amine dissolved in 5 mL of DMF was added to bring the total reaction volume to 10 mL. The reaction mixture was stirred overnight at room temperature. The final polymer was precipitated in ethyl ether (Thermo Fisher), pelletized by centrifugation at 22,000 rcf for 20 minutes, and redissolved in Milli-Q water. PEG-BZA was dialyzed against Milli-Q water at 4°C for 3 days (MWCO: 3,500 Da, Spectrum), with the dialyzed water replaced 2-3 times per day. PEG-BZA was freeze-dried and stored at -20°C. The modifications of PEG-BZA were as follows: 1 The results were estimated using 1H-NMR (500 MHz). PEG-BZA was dissolved in heavy water (D2O; Sigma) at a concentration of 10 mg / mL. The concentrations were δ=9.9 ppm (1H, s, aldehyde), δ=7.93 and 7.82 ppm (2H, d, benzene ring, respectively), and δ=3.56 (217H, s, PEG per arm).

[0112] Formation and rheological characterization of manipulated hydrogels. Mechanical testing was performed using a stress-controlled ARG2 vibrating rheometer (TA) with a 20 mm diameter 1° cone plate shape having a 28 μm gap between the geometry and the rheometer stage. Two hydrogel components were dissolved separately in 2 wt% PBS and kept on ice. First, 25 μL of the HA gel component was pipetteed into the center of the rheometer stage, and then 25 μL of the ELP component was pipetteed directly into the HA droplet, and the components were mixed using the pipette tip. The rheometer head was rapidly lowered, and the hydrogel components were reacted at 1 Hz, under 1% strain vibrational shear, for 10 minutes at room temperature and 5 minutes at 37°C. Immediately after this protocol, frequency sweeps from 0.1–10 Hz were performed at 1% strain. Storage and loss factors were taken from these measurements at 1 Hz. For stress relaxation measurements, the samples were gelled in situ under 1% vibrational shear at 1 Hz at 37°C for 10 minutes at room temperature, and then a 5% step strain was applied. The stress relaxation response was measured for at least 45 minutes. 1 / 2 This was calculated as the time it took for the stress to decay to 50% of its initial, stable value. Measurements were taken at least three times.

[0113] To form a HELP hydrogel containing cell-encapsulated cells within a manipulated matrix, the ELP and HA gel components were dissolved separately to form a 2 w / v% stock solution in PBS. To generate a dissociated culture, the cells were passaged as described above, the pellet was suspended in the ELP-hydrazine component, and kept on ice. 3 μL of selected modified HA component was added to the bottom of a 6 μL silicone mold (plasma bound to a 4 mm diameter, 0.5 mm height, 12 mm circular #1 coverslip). Then, 3 μL of ELP cell solution was pipetted directly into the droplet of modified HA. The two hydrogel components were then mixed using the pipette tip to uniformly disperse the cells within the hydrogel. The hydrogel was crosslinked at room temperature for 10 minutes, then at 37°C for 10 minutes, after which 750 μL of growth medium was added. To form an ELP-only gel, unmodified ELP protein was dissolved in PBS at a concentration of 3.25 w / v% at 4°C. A 5-fold solution of the crosslinking agent tetrakis(hydroxymethyl)phosphonium chloride (THPC) was prepared by diluting it 1:750 in PBS. Cells were subculturized as described above, and the pellet was resuspended in the unmodified ELP component and kept on ice. Next, the ELP solution was mixed with THPC in a 4:1 ELP:THPC volume ratio, thoroughly mixed by pipetting, and then pipetted into a silicone glass mold. The ELP-only culture was crosslinked at room temperature for 15 minutes, followed by 15 minutes at 37°C. For the ELP-PEG gel, enteroids were subculturized as described above, and single cells were suspended in 4 w / v% ELP-hydrazine on ice. Next, 3 μL of 8 w / v% PEG-BZA component was pipetted to the bottom of a 6 μL silicone glass mold while the plate was kept on ice. Then, 3 μL of the ELP component containing cells was added to the PEG component and mixed by swirling with the pipette tip. Next, these gels were crosslinked at 4°C for 1 hour, followed by 15 minutes at room temperature, and then 15 minutes at 37°C, after which 750 μL of preheated growth medium was added. For re-embedding culture, the enteroids in the EHS matrix were incubated with 5 mM EDTA on ice for 45–60 minutes to completely dissociate the matrix, and then centrifuged at 500 × g for 5 minutes.Next, the cells were washed in growth medium and centrifuged again at 500 × g for 5 minutes. To maintain a nearly constant cell seeding density, the number of cells from equivalent maintenance culture wells that had dissociated into single cells was always measured, and it was assumed that equivalent volumes of maintenance cultures had approximately equivalent cell numbers so that the re-embedded enteroid seeding density could be controlled. The growth medium was changed every 3-4 days. In all intestinal enteroid experiments, the small molecule inhibitors Y-27632 and CHIR-99021 were not included in the culture medium, as in the EHS maintenance cultures.

[0114] Enteroid passage in the HELP matrix. Enteroids in HELP were passaged every 10–14 days. To passage the enteroids in HELP, the matrix was first degraded with 100 U / mL elastase derived from porcine pancreas (Thermo Fisher Scientific, Waltham, MA) and 2500 U / mL hyaluronidase derived from bovine testicle (Sigma-Aldrich, St. Louis, MO), dissolved in PBS. The culture medium was completely aspirated from the medium wells, including the top surface of the silicone mold. After drying this top surface, droplets of elastase-hyaluronidase mixture equal to the gel volume were added onto the gel. The gel was incubated at 37°C for 1 hour to allow for complete matrix degradation. Then, to dilute the enzymes, the enteroids were pipetteed into a 15 mL cone centrifuge tube in excess growth medium. The enteroids were spun down at 500×g for 5 minutes, then the pellet was washed with growth medium and centrifuged again at 500×g for 5 minutes. The enteroids were then passaged as described above, and single cells were encapsulated in HELP as described above.

[0115] Enteroid formation and growth analysis. To analyze enteroid formation efficiency, up to 100 enteroids were analyzed gel by gel for three separate gels under different conditions. Within 4–6 hours after encapsulation in EHS matrix or HELP material, initial cell cultures were observed under a bright-field microscope to ensure the presence of only single cells. Bright-field images of each well were taken at 10x magnification every 3 days. For each well at each time point, three fields of view were randomly selected, and three z-slices were taken in each field of view. To analyze enteroid growth, enteroid contours were traced using a Wacom Intuos tablet, and enteroid size was quantified using the particle analysis function of FIJI (ImageJ, NIH). Based on previously reported enteroid morphology, 2000 μm was used. 2 An enteroid formation threshold was selected. Morphological criteria were also applied to separate viable enteroids from severely deformed ones. From this analysis, enteroid size and number were collected for each well. Using the size of each image and the z-volume of approximately 250 μm for the three z-slices, the organoid number per z-stack volume in each well was extrapolated and the organoid formation efficiency for each well was calculated by comparing it to the initial cell seeding density, assuming a uniform cell distribution. The formation efficiency for each condition was then calculated as the average of the three wells. Distribution statistics were generated for the three pooled wells to calculate the average enteroid size. Outliers were excluded from the dataset as follows: The outlier is > 1.5 * Q3 - Q1 + Q3, where Q3 is the third quartile of the data and Q1 is the first quartile of the data. Next, the average enteroid cross-sectional area was calculated as the average of the three wells for each condition.

[0116] Immunocytochemistry. To prepare the samples for fixation, each well was briefly washed with pre-warmed PBS. Cells were fixed by adding 750 μL of pre-warmed 4% paraformaldehyde (PFA) containing 0.1% glutaraldehyde in PBS and incubating at 37°C for 30–45 minutes. The fixation solution was then aspirated and washed three times with PBS for 5 minutes each. Cells were permeabilized with 0.25% v / v TritonX-100 in PBS (PBST) for 30 minutes, and then blocked in PBS for 3 hours with 5% by wt bovine serum albumin (BSA), 5% v / v goat serum, and 0.5% v / v TritonX-100. A primary antibody diluent was prepared in PBS using 2.5 wt% BSA, 2.5% v / v goat serum, and 0.5% v / v TritonX-100 (antibody diluent), and a primary incubation was performed overnight at 4°C. The antibody solution was removed, and the sample was washed three times for 5 minutes each in PBST. The secondary antibody was diluted 1:500 in the antibody diluent and incubated overnight at 4°C. The secondary antibody solution was then removed, and each was washed twice for 30 minutes in PBST. A 1:2000 dilution of DAPI and a 1:250 dilution of Farodine were prepared in PBST, incubated for 45 minutes, and then washed three times for 5 minutes each in PBST. The sample was then dried from excess liquid and inverted onto a rectangular coverslip on a droplet of ProLong Gold Antifade mounting medium. The sample was cured at room temperature in the dark for 48 hours before imaging on a DMI4000 B confocal microscope (Leica, Wetzlar, Germany).

[0117] [Table 2]

[0118] Quantitative real-time RT-PCR analysis. The hydrogel was removed from the silicone mold using a pipette tip, and the gel was scraped onto a 1.5 mL Eppendorf tube containing 500 μL of Trizol reagent (Invitrogen, Carlsbad, CA) on ice to extract RNA. The solution was then sonicated to allow complete degradation of the hydrogel for optimal RNA extraction. RNA was isolated on a phase-locked gel (Quantabio, Beverly, MA) using phenol-chloroform extraction. A fixed amount of RNA (0.1–1 μg) was reverse transcribed using a high-volume cDNA reverse transcription kit (Applied Biosystems, Foster City, CA). Then, 1 μg of cDNA in 5 μL of nuclease-free water was mixed with 10 μL of Fast SYBR Green Master Mix (Applied Biosystems, Foster City, CA) and run on an Applied Biosystems StepOnePlus real-time PCR system. The primers used in this study are listed in Table S2.

[0119] [Table 3]

[0120] Flow cytometry analysis. Enteroids were dissociated into single cells according to the method described above (see human organoid passaging and maintenance culture in EHS matrix and enteroid passaging in HELP matrix). Cells were pelleted by centrifugation at 500 × g for 5 minutes. The medium was then removed from each pellet, and the cells were resuspended in FACS buffer (PBS + 1 mM EDTA [Invitrogen] + 2% v / v FBS [Atlanta Bio] + 1% penicillin / streptomycin [Gibco]) supplemented with fluorophore-conjugated primary antibody [BioLegend anti-human CD44 antibody, BioLegend IgG2B isotype control]. Antibody staining was performed in the dark at 4°C for 30 minutes. After staining, the cells were washed twice with FACS buffer and resuspended in 200 μL of FACS buffer with DAPI (1:10,000, BioLegend) to select viable cells. Flow cytometry was performed using a Beckman-Coulter CytoFlex analyzer (Stanford Stem Cell Institute FACS Core). To analyze the data, gates were determined using forward and lateral scattering with heights and widths used to identify cell doublets. Subsequently, live DAPI-negative cells were gated for all marker analyses and population frequency calculations.

[0121] Statistical analysis. All significance tests in this publication use the following statistical significance expression: *, #=p<0.05, **=p<0.01, ***=p<0.001, ***=p<0.0001. To compare individual means, data from Figure 1g and S3 were analyzed by one-way ANOVA with Tukey's post-hoc test. Data from Figure 2d were analyzed via a non-paired two-tailed Student's t-test to compare gene expression changes between undifferentiated and differentiated conditions for each material. Data from Figures 3d, 4c, 4f, and S4 were analyzed using a two-tailed Student's t-test. To compare individual means, data from Figures 4e and 4h were analyzed by two-way ANOVA with Tukey's post-hoc test. Data from Figure S7 were analyzed using the Kruskal-Wallis test with Dunn's multiple comparison test. All statistical analyses were performed using GraphPad Prism 8.0 software (GraphPad Software, La Jolla, CA, USA).

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[0123] Cross-reference of related applications This application claims the benefit and priority of U.S. Provisional Patent Application No. 63 / 110,667, filed on November 6, 2020, the entire disclosure of which is described herein.

[0124] Inclusion by referencing sequence listings provided as text files The sequence listing was created on November 3, 2021, and is provided with this specification in a text file (STAN-1763WO_SEQ_LIST_ST25.txt) with a size of 21,000 bytes. The contents of the text file are included in their entirety in this specification by reference. It will be incorporated.

Claims

1. A two-component hydrogel matrix system, Hyaluronic acid (HA) modified to contain the pendant reactive group benzaldehyde, or a first component comprising a defined ratio of HA modified to contain the pendant reactive group aldehyde and HA modified to contain the pendant reactive group benzaldehyde, A second component comprising an elastin-like protein (ELP) modified to contain the pendant reactive group hydrazine, and Includes, A crosslink bond is formed between the first component and the second component, and a hydrogel is generated when they are mixed. A two-component hydrogel matrix system.

2. The system according to claim 1, wherein the matrix stiffness is adjusted by changing the ratio of reactive groups present on the first component to reactive groups present on the second component.

3. The system according to claim 1 or 2, wherein the ratio of reactive groups is varied by specifying the number of reactive groups on HA, specifying the number of reactive groups on ELP, and specifying the ratio of HA:ELP.

4. The system according to any one of claims 1 to 3, wherein the matrix stress relaxation rate is adjusted by changing the ratio of hyaluronic acid modified to contain pendantaldehyde to hyaluronic acid modified to contain pendantbenzaldehyde in the first component, and the ratio of hyaluronic acid modified to contain pendantaldehyde to hyaluronic acid modified to contain pendantbenzaldehyde is 100:0 to 0:

100.

5. The system according to claim 1, wherein the ELP comprises 1 to 7 elastin-like motifs, and the elastin-like motifs are selected from sequence numbers 23, 24, and 25.

6. The system according to any one of claims 1 to 5, wherein the ELP comprises a cell adhesion domain of 15 to 45 amino acids in length, comprising one or more cell adhesion sequence motifs, or scrambled RGD or RGD deletion, and the cell adhesion sequence motif is selected from RGD or any of SEQ ID NOs: 3 to 9.

7. The system according to any one of claims 1 to 6, wherein the ELP includes one or both of sequence number 1 and sequence number 2.

8. The system according to claim 6 or 7, wherein the cell adhesion peptide concentration of the hydrogel is adjusted by changing the ratio of ELP containing cell adhesion motifs to ELP lacking cell adhesion motifs, and the ratio of ELP containing cell adhesion motifs to ELP lacking cell adhesion motifs is 100:0 to 0:

100.

9. The system according to any one of claims 1 to 8, wherein the ELP comprises 3 to 20 hydrazine groups.

10. The system according to any one of claims 1 to 9, wherein the hydrogel contains up to 1.5 mM RGD.

11. A hydrogel formed from the system described in any one of claims 1 to 10.

12. A method for culturing mammalian cells, Encapsulating a starting population of mammalian cells in the hydrogel described in claim 11, Maintaining the encapsulated mammalian cells in a suitable culture medium and Methods that include...

13. The method according to claim 12, wherein the initial population of mammalian cells comprises a single-cell suspension or a tissue graft, and the initial population of mammalian cells differentiates into organoids in a culture.

14. The method according to claim 12, wherein the initial population of mammalian cells includes stem cells and includes intestinal cells.

15. The method according to any one of claims 12 to 14, wherein the encapsulated cells are passaged by enzymatically degrading the hydrogel.

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