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WO2020239990A1 - Modèles tridimensionnels de fibrose tissulaire - Google Patents

Modèles tridimensionnels de fibrose tissulaire Download PDF

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WO2020239990A1
WO2020239990A1 PCT/EP2020/065006 EP2020065006W WO2020239990A1 WO 2020239990 A1 WO2020239990 A1 WO 2020239990A1 EP 2020065006 W EP2020065006 W EP 2020065006W WO 2020239990 A1 WO2020239990 A1 WO 2020239990A1
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liver
cells
hydrogel
kpa
tissue
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Inventor
Saba REZAKHANI
Ece YILDIZ
Kristina SCHOONJANS
Matthias Lutolf
Giovanni SORRENTINO
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Ecole Polytechnique Federale de Lausanne EPFL
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Ecole Polytechnique Federale de Lausanne EPFL
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/067—Hepatocytes
    • C12N5/0671—Three-dimensional culture, tissue culture or organ culture; Encapsulated cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2513/00—3D culture
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2537/00—Supports and/or coatings for cell culture characterised by physical or chemical treatment
    • C12N2537/10—Cross-linking

Definitions

  • the invention belongs to the field of regenerative medicine. Aspects of the invention provide for methods for generating an organotypic 3D model of liver fibrosis, as well as 3D model of liver fibrosis and uses thereof.
  • liver stroma is mechanically dynamic and encompasses a wide range of physical changes during the transition from normal to pathological conditions such as fibrosis [10, 11] Indeed, the increased liver stiffness in patients with chronic liver disease has been exploited as a reliable non-invasive diagnostic tool for fibrosis [12] Although liver fibrosis has been suggested as a key contributing factor in the impairment of hepatic regenerative capacity observed in cirrhotic patients [13], experimental models to identify the biological effects induced by increased liver stiffness, and therefore to identify compounds for the treatment of liver fibrotic disorders, are lacking.
  • hydrogels have been used extensively for culturing, expanding, proliferating and differentiation of various cell types and organoids, recreating a 3D model for a hepatic pathological state which recapitulates the changes in regenerative capacity during liver fibrotic progression has not been accomplished yet. Indeed, it is believed that although synthetic hydrogels provide sufficient support for cell proliferation and differentiation of cell lines, the microenvironment provided by the currently available hydrogels lacks the mechanical requirements and biochemical complexity required to achieve the above goal.
  • the invention is based upon the discovery that a mechanically tunable hydrogel can be used to generate an organotypic three-dimensional (3D) model of liver fibrosis.
  • the inventors found that chemically defined and mechanically tunable hydrogels are suitable for culturing mouse and human liver organoids derived from biliary epithelial cells (BEC).
  • BEC biliary epithelial cells
  • derivation of liver organoids within these 3D environments is highly sensitive to matrix stiffness and hydrogels mimicking the liver fibrosis stiffness (about 4 kPa and beyond), leading to a significant impairment of organoid formation and activation of an inflammatory and pro-fibrotic response.
  • the present invention relates to a method for obtaining a three-dimensional (3D) cellular model of tissue fibrosis, such as liver fibrosis, comprising culturing epithelial cells in a 3D hydrogel in conditions suitable for three-dimensional cellular cluster formation, wherein the hydrogel comprises a crosslinked hydrophilic polymer having a shear modulus of at least 4 kPa.
  • the shear modulus is between about 4 and about 10 kPa, more preferably between about 4 and about 6 kPa, and most preferably from about 4 kPa to about 5 kPa.
  • the epithelial cells may be comprised in organoids, preferably liver organoids; and/or may be progenitor cells, for example liver progenitor cells, which may be obtained from tissues or derived from organoids.
  • organoids preferably liver organoids
  • Other epithelial cell types which may be used include intestinal, colonic, gastric, pancreatic, rectal, mammary, renal, brain, skin or lung cells; stem cells are preferred.
  • condition suitable for three-dimensional cellular cluster formation is meant that growth of the epithelial cells results in formation of a three-dimensional cellular cluster.
  • the key factor in such conditions is the shear modulus of the gel (subject of course to the other elements of the gel and the environment being suitable for cell growth in general - for example, at 37 degrees C, in a suitable cell culture medium).
  • reference herein to“conditions suitable for organoid formation” may be interpreted in a similar way.
  • Measurement of shear modulus may be carried out by any suitable means, which will be known to those of skill in the art.
  • One suitable method is described herein.
  • a three-dimensional cellular model of tissue fibrosis comprising a three- dimensional cellular cluster of cultured epithelial cells (preferably liver cells) in a 3D hydrogel, wherein the hydrogel comprises a crosslinked hydrophilic polymer having a shear modulus of at least 4 kPa.
  • a three-dimensional cellular model of tissue fibrosis produced in accordance with a method described herein.
  • the epithelial cells may comprise a marker transgene; for example, a luciferase transgene, or a GFP transgene.
  • the marker transgene is preferably constitutively expressed, although in certain embodiments, it may be conditionally expressed in response to certain conditions. The use of a marker transgene permits rapid screening of cells in an assay, as described herein.
  • the method comprises the step of culturing epithelial cells, such as liver cells, in a 3D hydrogel in conditions suitable for organoid formation, prior to the step of culturing epithelial cells in a 3D hydrogel in conditions suitable for three-dimensional cellular cluster formation wherein the cells comprise one or more of cells, tissue fragments and organoids or fragments thereof.
  • cells comprise one or more of 1) isolated liver tissue, 2) liver organoid fragments, 3) differentiated hepatocytes, 4) liver stem cells, 5) clusters of liver-derived cells, 6) bile ducts cells or fragments as well as any combinations of the foregoing.
  • the step results in organoid formation.
  • said 3D hydrogel used to culture epithelial cells in conditions suitable for organoid formation has a shear modulus of between 0.05 and 3.1 kPa, such as between 0.5 and 2.5 kPa, preferably between 1 and 2 kPa, and most preferably between 1.3 kPa and 2kPa.
  • the 3D hydrogel used to culture epithelial cells in conditions suitable for organoid formation is preferably the same 3D hydrogel as used for culture in conditions suitable for three-dimensional cellular cluster formation; other than the different shear modulus.
  • the 3D hydrogel used to culture epithelial cells in conditions suitable for three- dimensional cellular cluster formation is a different 3D hydrogel, such as a Matrigel R or any other matrix commonly used for epithelial organoid derivation.
  • the method may further comprise a step of modifying the shear modulus of the 3D hydrogel - e.g. by modulation of the hydrogel’s crosslinking density, solid content or molecular weight and functionality of the pre-polymers - from that used in conditions suitable for organoid formation (for example, between 0.05 and 3.1 kPa) up to at least 4 kPa, such as between about 4 and about 10 kPa, thereby providing conditions suitable for three-dimensional cellular cluster formation, and obtaining a three-dimensional cellular model of tissue, such as liver, fibrosis.
  • the modifying step may take place after organoid formation has occurred.
  • the nature of the modifying step will depend on the nature of the gel; for example, where the gel is initially crosslinked by means of enzymatic activity, further enzymes may be added to the gel in order to promote additional crosslinking to thereby increase the shear modulus. Alternatively, additional chemical crosslinking may be promoted; or photopolymerization of cross-linkable sites that may initially be photoprotected (i.e. ‘caged’).
  • different gels may be used for the two steps carried out in a gel; in such embodiments, the formed organoids may be removed from the first gel (for example, by enzymatic digestion of the gel), followed by seeding into a second gel of the desired shear modulus.
  • kits comprising first and second 3D hydrogels, each hydrogel comprising a crosslinked hydrophilic polymer; the first hydrogel having a shear modulus of between 0.05 and 3.1 kPa, and the second having a shear modulus of at least 4 kPa.
  • a three-dimensional hydrogel for culturing adult epithelial, such as biliary or hepatic, stem or progenitor cells to form an organotypic three-dimensional model of liver fibrosis comprising a cross-linked hydrophilic polymer functionalized with an RGD containing peptide, wherein the concentration of the RGD-containing peptide is of at least 0.05% w/v (0.5 mM), and wherein the hydrogel has a shear modulus (stiffness) of at least 4 kPa, such as between 4 and 10 kPa.
  • adult epithelial such as biliary or hepatic
  • stem or progenitor cells in a 3D hydrogel comprising a cross-linked hydrophilic polymer functionalized with an RGD containing peptide, wherein the concentration of the RGD-containing peptide is of at least 0.05% w/v (0.5 mM), and
  • the hydrophilic polymer of the 3D hydrogel comprises multiarm polyethylene glycol (PEG) molecules. Additionally or alternatively, the hydrophilic polymer of the 3D hydrogel comprises polyethylene oxide, polyoxazoline, polyaliphatic polyurethanes, polyether polyurethanes, polyester polyurethanes, polyethylene copolymers, polyamides, polyvinyl alcohols, polypropylene oxide, polypropylene glycol, polytetramethylene oxide, polyvinyl pyrrolidone, polyacrylamide, polyhydroxy ethyl acrylate, polyhydroxyethyl methacrylate, or mixtures or co-polymers thereof.
  • PEG polyethylene glycol
  • the 3D hydrogel comprises a bioactive molecule such as an oligopeptide, a small molecule, a protein, an oligo- or polysaccharide, or an oligo- or poly nucleotide.
  • a bioactive molecule such as an oligopeptide, a small molecule, a protein, an oligo- or polysaccharide, or an oligo- or poly nucleotide.
  • the 3D hydrogel is a bioactive or biofunctional hydrogel.
  • the 3D hydrogel comprises an RGD-containing ligand, such as for instance at least one of fibronectin or a functional variant thereof (for instance llh-C fragment, FNIII9-10 fragment, and FNIII12-14 fragment), RGD, RGDS, RGDSP, RGDSPK, RGDTP, RGDSPASSKP, Ac-GRCGRGDSPG-NH2 and H-NQEQVSPLRGDSPG-NH2.
  • RGD-containing ligand such as for instance at least one of fibronectin or a functional variant thereof (for instance llh-C fragment, FNIII9-10 fragment, and FNIII12-14 fragment), RGD, RGDS, RGDSP, RGDSPK, RGDTP, RGDSPASSKP, Ac-GRCGRGDSPG-NH2 and H-NQEQVSPLRGDSPG-NH2.
  • the bioactive molecule comprised within the 3D hydrogel is selected from a group comprising a transglutaminase (TG) substrate peptide bearing a protease- sensitive moiety Ac-FKGG-GPQG/H/GQ-ERCG-NH2, Ac-GCRE-GPQG/H/GQ-ERCG-NH2, AC-FKGG-GDQGIAGF-ERCG-NH2 and/or H-NQEQ- ⁇ /SPLEPCGNH2, collagen IV, analogues and/or fragments thereof, laminin 111 , analogues and/or fragments thereof, and any combinations of the foregoing.
  • the invention provides a method for screening of pharmacologic compounds, biomolecules or evaluating cell-based therapies for efficacy in reducing or arresting tissue, such as liver, stiffness and/or fibrosis, reducing or arresting tissue, such as liver, inflammation and/or promoting tissue, such as liver, progenitor proliferation, the method comprising i) providing a three-dimensional cellular model of tissue fibrosis according to the present disclosure with pharmacologic compounds, biomolecules or cells to be tested, and ii) monitoring at least one of 1) reduction or arrest of one of tissue stiffness, tissue fibrosis and tissue inflammation, and 2) promotion of tissue progenitor proliferation.
  • a method for obtaining a three-dimensional (3D) cellular model of tissue fibrosis comprising: a) culturing epithelial cells in a 3D hydrogel in conditions suitable for organoid formation, wherein the cells comprise one or more of cells, tissue fragments and organoids or fragments thereof, and wherein the hydrogel comprises a crosslinked hydrophilic polymer having a first shear modulus which mimics physiological conditions; and b) culturing said organoids in a 3D hydrogel in conditions suitable for three- dimensional cellular cluster formation, wherein the hydrogel comprises a crosslinked hydrophilic polymer having a second, higher, shear modulus which mimics a fibrotic condition.
  • Figure 3 (a) Effect of matrix stiffness on organoid formation efficiency (b) Liver progenitor cells 3 days after embedding in PEG-RGD hydrogels of increasing stiffness (c) Gene expression was analyzed by qPCR in liver organoids 6 days after embedding in PEG-RGD hydrogels with indicated stiffness (d) Gene expression was analyzed by qPCR in liver organoids 6 days after embedding in PEG-RGD hydrogels with indicated stiffness (e) Schematic representation of cellular mechano-signaling pathways. Inhibitors of key elements are depicted in red.
  • Figure 4 (a) Representative images of cell proliferation assay EdU (Click-iT EdU Alexa Fluor 647) staining of liver organoids (following manufacturer’s instructions), 3 days after embedding in PEG-RGD hydrogels with indicated stiffness (b) Gene expression was analyzed by qPCR in liver organoids embedded in soft (0.3 kPa) or physiologically-stiff (1.3 kPa) PEG-RGD hydrogels and maintained in DM.
  • EdU Click-iT EdU Alexa Fluor 647
  • a hydrogel is a matrix comprising a network of hydrophilic polymer chains.
  • a biofunctional hydrogel is a hydrogel that contains bio-adhesive (or bioactive) molecules, and/or cell signalling molecules that interact with living cells to promote cell viability and a desired cellular phenotype.
  • Biofunctional hydrogels may also be referred to as bioactive.
  • bio-adhesive molecules include, but are not limited to, fibronectin, vitronectin, bone sialoprotein, laminin, collagen and elastin. These molecules contain cell adhesive peptides that govern their interaction with cells.
  • cell adhesion peptide sequences include, but are not limited to fibronectin-derived RGD, KQAGDV, REDV and PHSRN, laminin-derived YIGSR, LGTIPG, IKVAV, PDGSR, LRE, LRGDN and IKLLI, collagen-derived DGEA and GFOGER, and elastin-derived VAPG.
  • Such peptide sequences themselves may be considered bio-adhesive molecules.
  • Bio-adhesive (or biofunctional, or bioactive) molecules that interact with epithelial cells to promote epithelial cell viability have been previously described.
  • Bio-adhesive molecules that render a hydrogel biofunctional include, but are not limited to, fibronectin or functional variants thereof, for example FF III1-C fragment, FNIII9-10 fragment, and FNII112-14, or RGD containing peptides, for example RGD, RGDS, RGDSP, RGDSPK, RGDTP and RGDSPASSKP .
  • Functional variants of bioactive molecules are molecules having the same or similar biological or biochemical function and a similar sequence or composition - for example, truncated molecules, or fragments of such molecules.
  • a biocompatible hydrogel is a crosslinked polymer network that is not significantly toxic to living tissue and/or cells, and does not elicit an immunopathogenic response in healthy individuals.
  • a biocompatible active mechanism is a process that is not toxic to particular cells or tissues, for example a temperature increase within the physiological temperature range of tissues, or that is applied briefly enough so as not to cause significant toxicity.
  • Cross-linkable by cell-compatible reaction(s) means that molecules are cross-linkable by reactions which are not significantly toxic to living tissue and/or cells. Such reactions may include (i) permanent covalent bond formation, chosen from the group consisting of a) enzymatically catalyzed reactions, preferably depending on activated transglutaminase such as factor Xllla or type 2 tissue TG (tTGases), or sortases, such as sortase A ; and b) not- enzymatically catalyzed and/or uncatalyzed reactions, preferably a Michael addition reaction; and/or ii) reversible covalent bond formation, chosen from the group consisting of Schiff base (imine) bonds, reversible hydrazone bonds, oxime bonds, disulfide bonds and bonds formed by reversible Diels-Alder reactions; and/or iii) non-covalent (i.e.
  • a cross-linked hydrophilic polymer functionalized with RGD- containing peptide refers to the incorporation of a peptide containing the amino acid sequence“RGD” into a hydrogel by crosslinking between the hydrophilic polymer of the hydrogel and the RGD-containing peptide.
  • Culturing cells refers to the process of keeping cells in conditions appropriate for maintenance and/or growth, where conditions refers to, for example, the temperature, nutrient availability, atmospheric CO2 content and cell density in which the cells are kept.
  • Cells can be cultured in vivo or in vitro.
  • the appropriate culturing conditions for maintaining, proliferating, expanding and differentiating different types of epithelial cells are well-known in the art.
  • the conditions suitable for organoid formation are those that facilitate or permit cell differentiation and the formation of multicellular structures.
  • “conditions suitable for three-dimensional cellular cluster formation” will be understood in the same way. See Materials and Methods for details of culturing conditions suitable according to the present invention.
  • Matrigel is a commercial product widely used in both 2D and 3D models of cell culture. It comprises a solubilized basement membrane preparation extracted from an ECM rich mouse tumour.
  • Organoids are three-dimensional culture systems of organ-specific cell types that develop from stem cells and self-organize (or self-pattern) through cell sorting and spatially restricted lineage commitment in a manner similar to the situation in vivo.
  • An organoid therefore represents the native physiology of the cells and it has a cellular composition (including both remaining stem cells, a near-physiological niche, as well as specialized cell types) and anatomy that emulate the native situation.
  • Stem cells may be isolated from tissue or organoid fragments.
  • the cells from which an organoid is generated differentiate to form an organ-like tissue exhibiting multiple cell types that self-organize to form a structure very similar to the organ in vivo.
  • Organoids are therefore excellent models for studying human organs and human organ development in a system very similar to development in vivo.
  • Epithelial cell organoids are organoids containing epithelial cells.
  • a three-dimensional cellular cluster is a group or ensemble of cell types growing in a three-dimensional fashion under culture conditions impeding or otherwise altering full differentiation and/or self-organization of the cells, in a way as to eventually impeding or otherwise altering the formation of an organoid. Therefore, a three-dimensional cellular cluster according to the present disclosure represents a cellular structure which does not reproduce the physiological, native situation exhibited by an organoid, but rather a patho physiological status thereof, reproducing or otherwise mimicking an organ-specific tissue and/or organ in diseased conditions such as preferably tissue and/or organ fibrosis.
  • RGD or RGD sequence refers to a minimal bioactive RGD sequence, which is Arginine-Glycine-Aspartic Acid (RGD) sequence, and which is the smallest (minimal) fibronectin-derived amino acid sequence that is sufficient to mimic cell binding to fibronectin and/or to promote adhesion of the anchorage-dependent cells.
  • RGD Arginine-Glycine-Aspartic Acid
  • the shear modulus of a hydrogel is equivalent to the modulus of rigidity, G, elastic modulus or elasticity of a hydrogel.
  • the shear modulus is defined as the ratio of shear stress to the shear strain.
  • the shear modulus of a hydrogel can be measured using a rheometer.
  • the present invention provides hydrogels suitable for obtaining a three-dimensional cellular model of tissue fibrosis, as well as methods for producing thereof.
  • the technical properties of 3D hydrogels can be adjusted (according to the culturing method for which the hydrogel is required) by varying the hydrophilic polymer content in the hydrogel, as well as the molecular weight and/or functionality (number of sites available for crosslinking) of the polymeric hydrogel precursors as described in the Examples.
  • the invention is based upon studies that demonstrated the effect of aberrant mechanical properties of microenvironment on liver progenitor cell proliferation as well as inflammation and fibrosis markers.
  • the invention is based upon the discovery that a mechanically tunable hydrogel can be used to generate an organotypic 3D model of liver fibrosis.
  • the invention allows for assaying test compounds for use in the treatment of a variety of chronic diseases or conditions, clinically characterized by increased liver stiffness (or other epithelial fibrotic conditions).
  • the conditions that should respond to such treatment include, among others, various stages of Non-alcoholic Steatohepatitis (NASH), viral hepatitis and liver cancer.
  • NASH Non-alcoholic Steatohepatitis
  • viral hepatitis liver cancer
  • Other epithelial fibrotic conditions of relevance may include pulmonary fibrosis, including cystic fibrosis, idiopathic pulmonary fibrosis and radiation- induced lung injury; endomyocardial fibrosis, old myocardial infarction, glial scar, arterial stiffness, arthrofibrosis, Crohn's disease, mediastinal fibrosis, retroperitoneal fibrosis, and scleroderma/systemic sclerosis.
  • pulmonary fibrosis including cystic fibrosis, idiopathic pulmonary fibrosis and radiation- induced lung injury
  • endomyocardial fibrosis old myocardial infarction, glial scar, arterial stiffness, arthrofibrosis, Crohn's disease, mediastinal fibrosis, retroperitoneal fibrosis, and scleroderma/systemic sclerosis.
  • the method involves incubating liver-derived 3D cellular clusters according to the invention in a 3D mechanically tunable hydrogel which models liver fibrosis, in the presence of a test compound, measuring the amount of cell proliferation and/or the gene expression of inflammation and fibrosis markers in the incubated cells, and then comparing this result with cells assayed under similar conditions but in the absence of the test compound.
  • test compound should be useful in the treatment methods if the amount of cell proliferation and/or the decrease in gene expression of inflammation and/or fibrosis markers, in liver progenitor cells embedded in high stiffness hydrogels (e.g. 4 kPa) is higher in the presence of the test compound than in its absence.
  • methods that are standard in the art can be used (for instance, qPCR to assess mRNA levels and ELISA to assess inflammatory cytokine levels).
  • the inventors optimized a method to monitor, in a fast and automatic manner, the BEC (biliary epithelial cell) number when grown as organoids in 3D hydrogels.
  • the invention further provides the key microenvironmental components that govern distinct stages of epithelial stem cell-driven organoid formation, including liver stem cell self-renewal, differentiation and morphogenesis.
  • This insight was used to create fully defined three- dimensional culture systems for the expansion of liver stem cells and organoids, comprising poly(ethylene glycol)-based hydrogels of precise mechanical properties and short synthetic peptide sequences that mimic adhesion to the extracellular matrix.
  • These systems offer a fully defined, reproducible environment that can be subjected to controlled biophysical and biochemical modifications, while also offering the possibility for the large-scale production of liver cells and tissues that model patho-physiological conditions found in liver disorders characterized by a high stiffness of the extra-cellular matrix.
  • the invention provides two fully defined three-dimensional hydrogel systems for liver cell culture -one supporting the high-purity expansion of liver-derived or biliary stem cells, supporting a subsequent organoid formation, and the other allowing the creation of liver-derived cell clusters mimicking pathological liver conditions, by recreating high stiffness environments retrieved in fibrotic tissues.
  • these hydrogel systems comprising synthetic PEG-based hydrogels of modular physicochemical properties, the key biophysical and biochemical parameters have been identified, including types and abundance of adhesion ligands and a precisely defined range of mechanical properties, which govern the distinct stages of liver-derived cell clusters formation.
  • the three-dimensional hydrogel system of the invention might be adapted for the culture of other types of adult epithelial stem cells and organoids, normal or tumor-derived.
  • the methods and hydrogels according to the invention can be used and adapted, mutatis mutandis, to the study of pathological conditions characterized by an increase of the stiffness of the extracellular matrix, as in the case of fibrosis, that can be typically retrieved in the stroma or parenchyma of several diseased organs such as lungs, kidneys, cartilages, brain or skin, to cite some.
  • the methods and hydrogels according to the invention can be used as three-dimensional patho-physiological models of diseases selected from a non-exhaustive group comprising pulmonary fibrosis, including cystic fibrosis, idiopathic pulmonary fibrosis and radiation- induced lung injury; endomyocardial fibrosis, old myocardial infarction, glial scar, arterial stiffness, arthrofibrosis, Crohn's disease, mediastinal fibrosis, retroperitoneal fibrosis, and scleroderma/systemic sclerosis.
  • pulmonary fibrosis including cystic fibrosis, idiopathic pulmonary fibrosis and radiation- induced lung injury
  • endomyocardial fibrosis old myocardial infarction, glial scar, arterial stiffness, arthrofibrosis, Crohn's disease, mediastinal fibrosis, retroperitoneal fibrosis, and scleroderma/systemic sclerosis.
  • the three-dimensional hydrogel system of the invention can find wide applications as basic research tools for studying epithelial tissue development, physiology and disease, but also as platforms for pharmacologic screens in a chemically defined and reproducible environment.
  • An aspect of the invention provides a three-dimensional hydrogel for culturing adult epithelial, such as biliary or hepatic, stem cells to form an organotypic three-dimensional model of liver fibrosis comprising a cross-linked hydrophilic polymer functionalized with an RGD containing peptide, wherein the concentration of the RGD-containing peptide is of at least 0.05% w/v (0.5 mM), and wherein the hydrogel has a shear modulus (stiffness) of at least 4 kPa, such as between 4 and 10 kPa.
  • adult epithelial such as biliary or hepatic
  • the hydrophilic polymer is selected from the group comprising poly(ethylene glycol), polyoxazoline, polyaliphatic polyurethanes, polyether polyurethanes, polyester polyurethanes, polyethylene copolymers, polyamides, polyvinyl alcohols, poly(ethylene oxide), polypropylene oxide, polypropylene glycol, polytetramethylene oxide, polyvinyl pyrrolidone, polyacrylamide, poly(hydroxy ethyl acrylate), poly(hydroxyethyl methacrylate), or mixtures or co-polymers thereof.
  • the hydrogels used which are obtained by cross-linking hydrogel precursor molecules, are preferably composed of hydrophilic polymers such as poly(ethylene glycol) (PEG)-based polymers, most preferably multiarm (i.e. branched) PEG-based polymers that are crosslinked by cell-compatible crosslinking reactions.
  • hydrophilic polymers such as poly(ethylene glycol) (PEG)-based polymers, most preferably multiarm (i.e. branched) PEG-based polymers that are crosslinked by cell-compatible crosslinking reactions.
  • Hydrogel precursors can be selected from a group comprising linear PEG molecules, or multiarm PEG hydrogel precursor molecules, preferably those bearing 4-arms or 8-arms. Hydrogel precursors can be further selected from a group comprising PEG hydrogel precursor molecules with molecular weight of 10-40 kDa.
  • the hydrophilic polymer content of the hydrogels, swollen to equilibrium in a buffer can range between 1 and 10% w/v, with preferred ranges of 2.0 to 4.0%% w/v and 2.5 to 3.5% w/v, optimized for the expansion of liver-derived cells.
  • PEG-based precursor molecules are chosen such as to be cross- linkable using either thrombin-activated Factor Xllla under physiological conditions or by another enzymatic crosslinking mechanism known in the art, or via Michael addition or by another mild chemical crosslinking mechanism known in the art.
  • one of at least two hydrogel precursor molecules is functionalized with a lysine-bearing peptide sequence, whereas the other is functionalized with a glutaminebearing peptide sequence.
  • PEG-VS-GIn and PEG-VS-Lys are used.
  • one of the two hydrogel precursor molecules is a multiarm PEG end functionalized with a nucleophilic group, most preferably a thiol, whereas the other is a multiarm PEG end-functionalized with an electrophilic group, most preferably a vinylsulfone or a maleimide.
  • Cross-linking of the hydrogel precursor molecules can be done in the presence of cell types to be cultured within the hydrogel, in such a way that the cells or cell aggregates are encapsulated by the forming hydrogel matrix, i.e. are residing in a distinct cell culture microenvironment.
  • the RGD-containing peptide is a peptide containing RGD binding motif selected from the group comprising fibronectin, fibronectin analogue or a fibronectin-derived fragment, Ac-GRCGRGDSPG-NH2 and H-NQEQVSPLRGDSPG-NH2.
  • the fibronectin-derived fragment or fibronectin analogue may be a peptide selected from the group comprising RGD, RGDS, RGDSP, RGDSPK, RGDTP, RGDSPASSKP or a fragment selected from the group comprising 1111 -C fragment, FNIII9-10 fragment, and FNII112-14 fragment.
  • the concentration of the RGD-containing peptide is within the range of 0.05%-1% w/v (0.5-10 mM).
  • the presence of fibronectin, fibronectin analogue or a fibronectin-derived fragment in a quantity sufficient to provide a concentration of RGD sequence of at least 0.05% (0.5 mM), preferably within the range of 0.05%-1% w/v (0.5-10 mM) is important for the survival and proliferation of adult epithelial stem cells.
  • Mechanical properties, i.e. stiffness, of the three-dimensional hydrogels according to the invention can be changed by varying the hydrophilic polymer content in hydrogel, as well as the molecular weight and/or functionality (number of sites available for crosslinking) of the polymeric hydrogel precursors.
  • liver- derived cells such as liver stem cells or BECs
  • shear modulus shear modulus
  • the desired initial stiffness range is achieved by fixing the polymer (PEG) content within the hydrogel to 2.0-4.0% w/v.
  • the three-dimensional hydrogel of the invention has hydrophilic polymer content within a range of 2.0 - 4.0% w/v, the concentration of RGD within a range of 0.05%-1.0% w/v, and the hydrogel has a shear modulus of 0.5 to 1.3 kPa.
  • these settings relate to conditions suitable for organoid formation; in order to obtain a three-dimensional cellular model of liver fibrosis, the shear modulus of the 3D hydrogel should be raised up to values of at least 4 kPa, such as between about 4 and about 10 kPa.
  • the change in the shear modulus which can be obtained by modulation of the hydrogel’s crosslinking density, solid content or molecular weight and functionality of the pre-polymers, causes the activation of mechanically-driven cellular pathways, including triggering of inflammatory and fibrotic responses, expression of markers of inflammation, cytoskeletal cellular remodeling and the like, that eventually lead to the formation of 3D aberrant cellular clusters reproducing organotypic 3D model of tissue fibrosis.
  • tissue-derived cells comprise one of cells, tissue fragments and organoids or fragments thereof.
  • the tissue-derived cells are intestinal, colonic, gastric, pancreatic, rectal, mammary, renal, brain, skin or lung stem cells.
  • liver-derived cells are preferred, said cells comprising one of 1) isolated liver tissue, 2) liver organoid fragments, 3) differentiated hepatocytes, 4) liver stem cells, 5) clusters of liver-derived cells, 6) bile ducts cells or fragments as well as any combinations of the foregoing.
  • the method comprises a step of augmenting the shear modulus of the 3D hydrogel from conditions suitable for organoid formation up to at least 4 kPa, such as between about 4 and about 10 kPa, such that conditions are suitable for three- dimensional cellular cluster formation, thereby obtaining a three-dimensional cellular model of tissue, such as liver, fibrosis.
  • the three-dimensional hydrogels used to culture epithelial cells in conditions suitable for organoid formation have an initial shear modulus (stiffness) of between 0.05 and 3.1 kPa, such as between 0.5 and 2.5 kPa, preferably between 1 and 2 kPa, and most preferably between 1.3 kPa and 2kPa, and a final shear modulus (stiffness) of at least 4 kPa, such as between about 4 and about 10 kPa in conditions suitable for cellular cluster formation.
  • the kinetics (time) of the hydrogel hardening should occur within a time window after cell differentiation, epithelial budding and organoid formation.
  • the three-dimensional hydrogels of the invention harden to shear modulus values beyond those retrievable in physiological conditions, i.e. at least 4 kPa and above.
  • the change in stiffness (hardening) is crucial to the reproduction of the mechanical inputs as those found in fibrotic conditions.
  • the hardening, i.e. stiffness increase, of the three-dimensional hydrogel of the invention can be achieved by various strategies known to persons skilled in the art, such as for instance via photopolymerization of cross-linkable sites by the application of a suitable actinic light. Suitable techniques will depend on the nature of the hydrogel polymer, and the skilled person will be aware how to select an appropriate polymer and an appropriate technique.
  • the monitoring steps can be carried out by methods known to the person skilled in the art, such as for instance ELISA assays, rheological tests, qPCR, colony test formations and the like.
  • Example 1 effect of hydrogel stiffness on hepatocyte phenotype
  • liver organoids grown in PEG-RGD hydrogels with stiffness values comparable to that of fibrotic livers might serve as a model to investigate how stem cells translate aberrant mechanical inputs into coherent biological responses.
  • the inventors tuned hydrogel stiffness from values far below the normal mouse liver (0.3 kPa) to values progressively reaching in vivo levels (1.3 kPa) and beyond in fibrotic liver (4 kPa).
  • organoid formation and proliferation was profoundly affected by ECM stiffness, with physiological liver rigidity (1.3 kPa) being optimal (Figs. 3a, b and Fig. 4a).
  • Example 2 effect of hydrogel stiffness on hepatocyte markers expression
  • Integrins are plasma membrane mechano-transducers essential for liver regeneration.
  • the Hippo pathway nuclear effector, Yes associate protein (YAP) is activated by physical cues downstream of integrin signaling and is required for liver progenitor proliferation and plasticity.
  • YAP induction was assessed in organoids cultured in soft (0.3 kPa) or physiologically-stiff (1.3 kPa) matrices by monitoring both the expression of canonical YAP target genes and YAP subcellular localization.
  • YAP targets and YAP nuclear accumulation were increased compared to soft matrices (Fig. 3d and Fig.
  • Example 3 PEG hydrogels can be tuned to model fibrotic liver mechanics
  • liver disease progression is strongly associated with abnormal tissue architecture and mechanotransduction [14-16]
  • tissue stiffness increases in time and severely compromises its function [17-19]
  • the changes in liver stiffness associated with disease are used for diagnostics based on longitudinal non-invasive monitoring [20]
  • liver organoids grown in defined hydrogels recapitulating the stiffness of fibrotic liver could serve as physiologically relevant 3D model to investigate how stem cells translate aberrant mechanical inputs into disease relevant phenotypes.
  • liver organoids showed a reduction in the expression of hepatic progenitor markers (Fig 5c) and upregulation of genes involved in cellular response to hepatic injury (Fig 5d, e), indicating impaired sternness potential and concomitant induction of a stress response.
  • fibrosis-mimicking hydrogels led to an increase in the expression of matrix metalloproteases (Fig 5f), a compensatory phenomenon known to be induced in response to increased ECM stiffness [22-24]
  • Fig 5f matrix metalloproteases
  • YAP activation was not affected (Fig 5g), suggesting the existence of other pathways controlling liver progenitor growth in response to increased stiffness.
  • engineered mouse BEC-derived progenitor cells which constitutively express luciferase, are used.
  • the luciferase signal is used as a proxy of cell number.
  • the luciferase signal is used as a proxy of cell number.
  • Mouse liver organoids are established from biliary duct fragments. Briefly, liver tissues are digested in digestion solution (Collagenase type XI 0.012%, dispase 0.012%, FBS 1 % in DMEM medium) for 2 hours. When digestion is complete, bile ducts are pelleted by mild centrifugation (200 rpm for 5 min) and washed with PBS. Isolated ducts are then resuspended either in Matrigel (BD Bioscience) or PEG precursor solution and cast in 10 pi droplets in the center of the wells in a 48-well plate. After the gels are formed, 250 mI of isolation medium is added to each well.
  • digestion solution Collagenase type XI 0.012%, dispase 0.012%, FBS 1 % in DMEM medium
  • Isolation medium is composed of AdDMEM/F12 (Invitrogen) supplemented with B27 and N2 (both GIBCO), 1.25 mM N-acetylcysteine (Sigma- Aldrich), 10 nM gastrin (Sigma-Aldrich) and the following growth factors: 50 ng ml -1 EGF (Peprotech), 1 pg ml -1 Rspol (produced in-house), 100 ng ml -1 Fgf10 (Peprotech), 10 mM nicotinamide (Sigma-Aldrich), 50 ng ml -1 HGF (Peprotech), Noggin (100 ng ml -1 produced in- house), Wnt 3a (1 pg ml 1 , Peprotech) and Y-27632 (10 mM, Sigma). After the first 4 days, isolation medium is changed with expansion medium (EM) which consists of isolation medium without Noggin, Wnt and Y-27632. Passaging
  • Ultrasound (US)-guided needle biopsies are obtained with a coaxial liver biopsy technique. One biopsy cylinder is fixed in formalin and paraffin-embedded for histopathological diagnosis. Additional cylinders are collected in advanced DMEM/F-12 (GIBCO) for organoid generation. Human liver organoids are generated as follows: biopsies are placed in advanced DMEM/F-12 (GIBCO) on ice for further processing.
  • Liver samples are then digested to small-cell clusters in basal medium containing 2.5 mg/mL collagenase IV (Sigma) and 0.1 mg/mL DNase (Sigma) at 37°C.
  • Cell clusters are embedded in Matrigel or PEG gels, cast and after the gels were formed, human isolation medium (HIM) is added.
  • HIM human isolation medium
  • HIM is composed of advanced DMEM/F-12 (GIBCO) supplemented with B-27 (GIBCO), N-2 (GIBCO), 10 mM nicotinamide (Sigma), 1.25 mM N-acetyl-L-cysteine (Sigma), 10 nM [Leu15]-gastrin (Sigma), 10 mM forskolin (Tocris), 5 mM A83-01 (Tocris), 50 ng/mL EGF (PeproTech), 100 ng/mL FGF10 (PeproTech), 25 ng/ml HGF (PeproTech), 1 pg ml -1 Rspd (produced in-house), Wnt3a (1 pg ml 1 , Peprotech), Y-27632 (10 pM, Sigma) and Blebbistatin (10 pM StemCell Technologies). After the first 4 days, isolation medium is changed with human expansion medium (HEM) which consists of HIM without Noggin, Wnt
  • PEG hydrogels are used. Additionally, to mimic the micro-environment, key minimal extracellular matrix (ECM) components such as RGD are incorporated in the gels. For liver fibrosis model, the PEG hydrogel stiffness is tuned to 4 kPa (fibrotic liver) from 1.3 kPa (in vivo level). Finally, liver progenitor cells, which are obtained from BEC-derived liver organoids are cultured in 4 kPa PEG hydrogels with isolation medium for the 3-day drug screening study.
  • ECM extracellular matrix
  • Hydrogel precursors are synthesized as follows: vinylsulfone functionalized 8-arm PEG (PEG-VS) is purchased from NOF.
  • the transglutaminase (TG) factor XIII (FXIIIa) substrate peptides Ac- FKGGGPQGIWGQ-ERCG-NH2 with matrix metalloproteinases (MMPs) sensitive sequence, Ac-FKGG-GDQGIAGF-ERCG-NH2, H-NQEQVSPLERCGNH2 and the RGD-presenting adhesion peptide H-NQEQVSPLRGDSPG-NH2 are purchased from GL Biochem.
  • MMPs matrix metalloproteinases
  • FXIIIa substrate peptides and 8-arm PEG-VS are dissolved in triethanolamine (0.3 M, pH 8.0) and mixed at 1.2 stoichiometric excess (peptide-to-VS group) and allowed to react for 2 h under inert atmosphere.
  • the reaction solution is dialysed (Snake Skin, MWCO 10K, PIERCE) against ultrapure water for 3 days at 4 °C, after which the products are lyophilized and dissolved in ultra-pure water to make 13.33% w/v stock solutions.
  • Vinylsulfone-functionalized 8-arm PEG is purchased from NOF.
  • the peptide Ac-GCRE-GPQGIWGQ-ERCG-NH2 (mol wt 1773.97 g/mol) with matrix metalloproteinases (MMPs) sensitive sequence was obtained from Biomatik.
  • MMPs matrix metalloproteinases
  • the adhesion peptide Ac-GRCGRGDSPG-NH2 (mol wt 1002.04 g/mol) is purchased from GL Biochem.
  • the reaction solution is dialyzed (Snake Skin, MWCO 10K, PIERCE) against ultrapure water (pH ⁇ 7) for 4 days at 4°C, and the final product is lyophilized.
  • the lyophilized product is dissolved in water to make 10% precursor solutions.
  • Enzymatically crosslinked hydrogels are formed by mixing PEG precursor solutions in stoichiometrically balanced ratios and adding thrombin-activated FXIIIa (10 U ml 1 ; Galexis) in the presence of Tris-buffered saline (TBS; 50 mM, pH 7.6) and 50 mM CaCh.
  • Chemically crosslinked hydrogels are formed by Michael type addition of octa-thiol containing PEG precursors onto 8-arm PEG-VS.
  • Gels cast on PDMS-coated 24 well- plate are allowed to crosslink by incubation at 37°C for 10 min.
  • gels are detached from the bottom of the plates using a tip of a metal spatula and transferred to 15-ml Falcon tube containing 1 ml of Dispase (1 mg/ml, Thermo Fisher Scientific). After 10 minutes enzymatic digestion, the reaction is quenched using 10% FBS containing 1 mM EDTA, washed with cold basal medium and centrifuged for 3 min at 1000 rpm.
  • Elastic modulus (G 1 ) of hydrogels is measured by performing small-strain oscillatory shear measurements on a Bohlin CVO 120 rheometer with plate-plate geometry. Briefly, 1-1.4 mm thick hydrogel discs are prepared and allowed to swell in water for 24 hrs. The mechanical response of the hydrogels sandwiched between the parallel plates of the rheometer is recorded by performing frequency sweep (0.1-10 Hz) measurements in a constant strain (0.05) mode at 25 °C.
  • the invention includes assay methods that can be used to screen for compounds that are likely to be useful in treating the diseases and conditions, in which liver stiffness increases.
  • One way to accomplish this is to determine whether a compound promotes liver progenitor proliferation in a model of liver fibrosis.
  • Another way to screen for compounds that are likely to be useful in treating the diseases and conditions described above is to determine whether a compound decreases inflammation and fibrosis in liver stem cells investigated by gene expression and protein expression.
  • the compound being tested is incubated with the cells for 3 days; cell proliferation is measured with a luminometer and gene and protein expression are measured following protocols that are standard in the art. The results obtained are compared with those from control incubations carried out in a similar manner but in the absence of the test compound.
  • Compounds that cause an increase in cell proliferation and decrease in inflammation and fibrosis relative to controls have potential as therapeutic agents.
  • Engineered liver organoids with constitutive luciferase expression are dissociated into single cells using TrypLE express (Gibco) and filtered twice. After counting the cell number with a hemocytometer, several cell densities are plated in a 96-well plate.
  • the number of cells to be seeded in order to generate a calibration curve are the following: No cells, 10000 cells/well, 20000 cells/well, 30000 cells/well, 40000 cells/well, 60000 cells/well, 80000 cells/well, 100000 cells/well and 200000 cells/well. Cells are seeded per well in 5 pi of hydrogel in the center of the wells in a 96-well plate.
  • luciferase signal was read using Bright-GloTM Luciferase Assay System kit (Promega) and VICTORTM X4 2030 multilabel reader (Perkin Elmer). Correlation between the number of cells seeded and the luciferase signal is determined by linear regression and quality of fit was determined by R square value. A plateau in luciferase signal was observed starting with 80000 cells/well. Thus, the final cell number was chosen as 35000 cells/well as the luciferase rested in linear increase range.
  • Engineered liver organoids with constitutive luciferase expression are dissociated into single cells using TrypLE express (Gibco) and filtered twice. After counting the cell number with a hemocytometer, several cell densities are plated in a 96-well plate.
  • the number of cells to be seeded in order to generate a calibration curve are the following: No cells, 1000 cells/well, 2000 cells/well, 3000 cells/well, 4000 cells/well, 5000 cells/well, 10000 cells/well, 12000 cells/well and 20000 cells/well. Cells are seeded per well in 5 pi of hydrogel in the center of the wells in a 96-well plate. For each cell concentration, six replicates are used.
  • luciferase signal was read using Bright-GloTM Luciferase Assay System kit (Promega) and VICTORTM X4 2030 multilabel reader (Perkin Elmer). Correlation between the number of cells seeded and the luciferase signal is determined by linear regression and quality of fit was determined by R square value.
  • Drug screening validation for liver fibrosis model Engineered liver organoids with constitutive luciferase expression are dissociated into single cells using TrypLE express (Gibco) and filtered twice. After counting the cell number with a hemocytometer, 35000 cells are seeded per well in 5 mI of hydrogel (4 kPa) in the center of the wells in a 96-well plate. For each treatment at least three replicates are used. 250 mI of isolation medium with drugs is added to each well. Drug concentrations are as follows: DMSO (0.1%), A769662 (100 mM), Olaparib (1 mM), AICAR (500 mM ), ACMSD (500 nM) and TC100 (50 mM).
  • Unbiased drug screening for liver fibrosis model To investigate the effect of compounds and biomolecules on liver progenitor cell proliferation, engineered liver organoids with constitutive luciferase expression are dissociated into single cells using TrypLE express (Gibco) and filtered twice. After cell number count, 35000 cells are seeded per well in 2.5 mI of hydrogel (4 kPa) in the center of the wells in a 384-well plate via robot-based automation. For each treatment at least sixteen replicates are used. 50 mI of isolation medium with drug library is added to each well via robot-based automation. After 3 days, culture media is removed and 50 mI of basal media is added per well.
  • TrypLE express Gibco

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Abstract

L'invention concerne un procédé d'obtention d'un modèle cellulaire tridimensionnel (3D) de fibrose tissulaire, tel que la fibrose hépatique, comprenant la culture de cellules épithéliales dans un hydrogel 3D dans des conditions appropriées pour une formation de grappe cellulaire tridimensionnelle, l'hydrogel comprenant un polymère hydrophile réticulé ayant un module de cisaillement d'au moins 4 kPa, tel qu'entre environ 4 et environ 10 kPa.
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