WO2022084513A1

WO2022084513A1 - Cellularized structures comprising extracellular matrix protein

Info

Publication number: WO2022084513A1
Application number: PCT/EP2021/079354
Authority: WO
Inventors: Linna ZHOU; Carlos Ruiz-Puig; Hagan Bayley; Xin Lu
Original assignee: The Chancellor, Masters And Scholars Of The University Of Oxford
Priority date: 2020-10-23
Filing date: 2021-10-22
Publication date: 2022-04-28

Abstract

Provided herein are complex, cellularized structures comprising extracellular matrix proteins (EMP), methods of making said structures and method of using said structures for disease modelling and tissue implantation. In some embodiments, the complex, cellularized structures comprise collagen.

Description

CELLULARIZED STRUCTURES COMPRISING EXTRACELLULAR MATRIX PROTEIN

FIELD OF THE INVENTION

[0001] The present invention relates generally to the field of bioengineering of tissues, including of tubular, gastrointestinal (GI) tissues.

BACKGROUND

[0002] The gastrointestinal (GI) tract is essential for the intake and digestion of nutrients, drug intake, and the immune defense. Functional disorders of the GI tract can result in gastrointestinal diseases, including gastric and peptic ulcer disease, and Irritable Bowel Syndrome (IBS). In the UK, GI disorders account for around 10% of all clinical practice and IBS alone affects around 20% of the general population. Despite their widespread occurrence, the majority of GI disorders are difficult to diagnose and manage. Moreover, severe GI diseases require surgical resection, and patients may subsequently require transplantation in the event of short bowel syndrome.

[0003] Bioengineered GI tracts are highly desirable for both disease modelling and tissue implantation. Specifically, in vitro GI models allow simplified and well-controlled conditions for the study of toxicology, drug absorption, and host-microbial interaction at lower cost compared to animal models. Further, the surgical resection of the GI tracts can result in complications and a low quality of life. Accordingly, bioengineered GI tracts have the potential to repair the function of lost tissues.

[0004] However, GI tract bioengineering is particularly challenging because of the GI tract’s complex cellular composition and organization. Accordingly, it can be desirable for engineered GI tracts to comprise multiple cell types arranged in a defined pattern. Further, the most useful technology would exhibit the flexibility to generate tubular structures of different sizes and shapes using biocompatible materials, and be simple to apply.

[0005] Previous efforts in the bioengineering of GI tracts have involved scaffold materials in combination with extracellular matrix (ECM) protein, which can support cells seeded onto the scaffold. However, such scaffold materials are mechanically stiffer than ECM and are not biocompatible (i.e., cells are not able to attach to the scaffold and will digest these biologically incompatible structures).

[0006] Other strategies in GI tract bioengineering have utilized de-cellularized tissue as a scaffold. Such methods preserve some of the native EMP and the overall tissue structure, and allow re-cellularization with a patient’s own cells to reduce immunogenicity. However, the decellularized scaffolds require donor or animal tissues and are technically very laborious to prepare. Further, since this approach is not suitable for subepithelial cells embedded inside tissues, such as fibroblasts, muscle cells or neurons, the application of de-cellularized tissues is limited to seeding epithelial cells around tubular structures of tissues.

[0007] Collagen has the advantage of being fully biocompatible if it is used without additional scaffold materials. Further, collagen is known to support cell proliferation, migration, and differentiation, and can also be remodeled by cells. However, collagen derived hydrogels are generally soft and slow to reach gelation, preventing its fabrication into defined structures. While collagen has been molded into micro-patterns for the three dimensional (3D) culture of intestinal cells, these molded structures were small, not tubular, and required chemical crosslinking to increase the stiffness of the collagen for the molding process.

[0008] Polished glass molds have also been developed to support the fabrication of tubular vascular tissues. However, the use of glass molds lacks the potential for rapid production of collagen tubes with various sizes and complex shapes.

[0009] In a different approach, collagen rings were fabricated simply by adding collagen solution, seeded with mouse intestinal organoids, to the edges of the wells of 24-well plates. The contraction of the collagen rings resulted in the alignment and fusion of the organoids at the center of the collagen rings to give macroscopic epithelial tubes. However, this approach was limited to the generation of centimeter-long intestinal tubes, and the collagen rings generated were thin and irregular. Moreover, the luminal surfaces of the intestinal tubes were embedded inside the collagen rings and not exposed for further manipulation, such as infection with a pathogen, severely limiting the potential applications of this technique.

[0010] Accordingly, there is an urgent need for a simple, versatile method that allows the rapid generation of cellularized, complex 3D structures using biocompatible materials for applications in GI tract bioengineering.

SUMMARY OF THE INVENTION

[0011] Provided herein are cellularized structures comprising extracellular matrix protein (EMP), methods of making such structures, and methods of using such structures. [0012] In one aspect, provided is a method of generating a cellularized structure comprising EMP, the method comprising: (a) providing an aqueous medium comprising (i) EMP and (ii) a population of cells; (b) providing a mold comprising a hydrophobic medium comprising amphipathic molecules; (c) disposing a plurality of droplets comprising the aqueous medium into the hydrophobic medium; and (d) inducing gelation of the EMP.

[0013] In one aspect, provided is a method of generating a cellularized structure comprising EMP, the method comprising: (a) providing an aqueous medium comprising (i) EMP and (ii) a population of cells; (b) providing a mold comprising a hydrophobic medium comprising amphipathic molecules; (c) disposing a plurality of droplets comprising the aqueous medium into the hydrophobic medium, thereby generating a droplet network comprising droplets cohering through a bilayer of amphipathic molecules; and (d) inducing gelation of the EMP.

[0014] In one aspect, provided is a method of generating a cellularized structure comprising EMP, the method comprising: (a) providing an aqueous medium comprising (i) EMP and (ii) a population of cells; (b) providing a mold comprising a hydrophobic medium comprising amphipathic molecules; (c) disposing a plurality of droplets comprising the aqueous medium into the hydrophobic medium, thereby generating a droplet network comprising droplets cohering through a bilayer of amphipathic molecules; (d) reducing the concentration of the amphipathic molecules in the hydrophobic medium; and (e) inducing gelation of the EMP.

[0015] In some embodiments, the step of reducing the concentration of the amphipathic molecules in the hydrophobic medium disrupts the integrity of the bilayer.

[0016] In one aspect, provided is a method of generating a cellularized structure comprising EMP, the method comprising: (a) providing an aqueous medium comprising (i) EMP and (ii) a population of cells; (b) providing a mold comprising a hydrophobic medium comprising amphipathic molecules; (c) disposing a plurality of droplets comprising the aqueous medium into the hydrophobic medium, thereby generating a droplet network, wherein the droplet network comprises a plurality of droplets arranged in a three-dimensional structure, wherein each droplet in the three dimensional structure comprises an outer layer of amphipathic molecules around the surface of the aqueous medium; and wherein each droplet in the three dimensional structure contacts at least one other droplet in the three dimensional structure forming a bilayer of amphipathic molecules as an interface between contacting droplets; and (d) inducing gelation of the EMP. [0017] In one aspect, provided is a method of generating a cellularized structure comprising EMP, the method comprising: (a) providing an aqueous medium comprising (i) EMP and (ii) a population of cells; (b) providing a mold comprising a hydrophobic medium comprising amphipathic molecules; (c) disposing a plurality of droplets comprising the aqueous medium into the hydrophobic medium, thereby generating a droplet network, wherein: the droplet network comprises a plurality of droplets arranged in a three- dimensional structure; wherein each droplet in the three dimensional structure comprises an outer layer of amphipathic molecules around the surface of the aqueous medium; and wherein each droplet in the three dimensional structure contacts at least one other droplet in the three dimensional structure forming a bilayer of amphipathic molecules as an interface between contacting droplets; (d) reducing the concentration of the amphipathic molecules in the hydrophobic medium; and (e) inducing gelation of the EMP.

[0018] In some embodiments, the gelation of the EMP is induced by increasing the temperature. In one embodiment, the temperature is increased to about 37 °C.

[0019] In some embodiments, the step of disposing a plurality of droplets comprising the aqueous medium into the hydrophobic medium is performed at a temperature of less than about 15 °C.

[0020] In some embodiments, the EMP is collagen. In some embodiments, the collagen is collagen type 1. In some embodiments, the concentration of the EMP in the aqueous medium is from about 0.5 and about 2 mg/mL. In some embodiments, the concentration of the collagen in the aqueous medium is from about 0.5 and about 2 mg/mL.

[0021] In some embodiments, the aqueous medium comprises cell culture medium.

[0022] In some embodiments, the population of cells comprises mammalian cells. In some embodiments, the population of cells comprises mouse, rat, sheep, or human cells. In some embodiments, the population of cells comprises epithelial cells, muscle cells, fibroblasts, neuronal cells, immune cells, blood vessel cells, or gastrointestinal organoids. In some embodiments, the population of cells comprises duodenum organoids or gastric organoids. In some embodiments, the population of cells comprises enteric neurons.

[0023] In some embodiments, the aqueous medium comprises cells at a concentration from about 10² to about 10⁹ cells per mL of the aqueous medium. In some embodiments, the aqueous medium comprises cells at a concentration from about 10² to about 10⁴ cells per mL of the aqueous medium.

[0024] In some embodiments, the hydrophobic medium comprises a hydrocarbon compound and/or a silicone oil. In one embodiment, the hydrocarbon compound is undecane. In one embodiment, the silicone oil comprises polyethylmethylsiloxane. In one embodiment, the hydrophobic medium comprises a hydrocarbon compound and a silicone oil at a ratio of about 1 :4 by volume.

[0025] In some embodiments, the hydrophobic medium comprises an amphipathic compounds. In one embodiment, the amphipathic compound is a phospholipid. In one embodiment, the phospholipid is diphytanoylphosphatidylcholine. In some embodiments, the amphipathic compound is present at a concentration of about 1 mM to about 2 mM.

[0026] In some embodiments, the mold comprises acrylonitrile butadiene styrene (ABS), polylactic acid (PLA), polyethylene terephthalate (PETE), nylon, polystyrene, polyvinyl alcohol (PVA), and/or polycarbonates.

[0027] In some embodiments, the cellularized structure comprising EMP is further isolated from the hydrophobic medium. In some embodiments, the cellularized structure comprising EMP is further removed from the mold.

[0028] In some embodiments, the cellularized structure comprising EMP is a tubular structure.

[0029] In some embodiments, the method of generating a cellularized structure comprising EMP further comprises seeding the luminal surface of the structure with a second population of cells.

[0030] In one aspect, provided is a method of generating a cellularized structure comprising EMP comprising two or more layers, wherein each layer comprises a population of cells, the method comprising: (a) generating a first cellularized structure comprising EMP comprising a first population of cells in a mold; and (b) generating a second cellularized structure comprising EMP comprising a second population of cells in the mold; wherein the first and the second cellularized structures comprising EMP are layered.

[0031] In some embodiments, the density of cells in the first cellularized structure comprising EMP is different from the density of cells in the second cellularized structure comprising EMP.

[0032] In one aspect, provided is a method of generating a cellularized structure comprising EMP comprising two or more layers, the method comprising (a) providing a first aqueous medium comprising (i) EMP and (ii) a first population of cells; (b) providing a first second medium comprising (i) EMP and (ii) a second population of cells; (c) providing a mold comprising a hydrophobic medium comprising amphipathic molecules; (d) disposing a plurality of droplets comprising the first aqueous medium into the hydrophobic medium; (e) inducing gelation of the EMP in the first aqueous medium; (f) disposing a plurality of droplets comprising the second aqueous medium into the hydrophobic medium; and (g) inducing gelation of the EMP in the second aqueous medium.

[0033] In some embodiments, the concentration of cells in the first aqueous medium is different from the concentration of cells in the second aqueous medium.

[0034] In some embodiments, the first and the second populations of cells comprises cells of different cell types. In some embodiments, the first and the second populations of cells comprises cells of the same cell type. In one embodiments, at least one of the first and the second populations of cells comprises fibroblast cells.

[0035] In one aspect, provided is a cellularized structure comprising EMP comprising one or more populations of cells. In some embodiments, the cells in at least one of the one or more population of cells are selected from epithelial cells, muscle cells, fibroblasts, neuronal cells, immune cells, blood vessel cells, or gastrointestinal organoids.

[0036] In one aspect, provided is a cellularized structure comprising EMP comprising two or more layers, wherein each layer comprises a population of cells. In some embodiments, the structure comprises a first and a second layer, wherein the first layer comprises cells at a density different from the density of cells in the second layer. In some embodiments, at least one of the one or more populations of cells is a population of fibroblast cells.

[0037] In one aspect, provided is a cellularized structure comprising EMP generated using any of the methods disclosed herein.

[0038] In one aspect, provided is a method of treating a disease or condition in a patient in need thereof, the method comprising implanting a cellularized structure comprising EMP comprising one or more population of cells into the patient. In some embodiments, the cellularized structure comprising EMP replaces a gastric tissue, a colonic tissue, an intestinal tissue, or a blood vessel. In one embodiment, the disease or condition is short bowel syndrome.

[0039] In one aspect, provided is a method of treating a disease or condition in a patient in need thereof, the method comprising implanting a cellularized structure comprising EMP disclosed herein into the patient. In one aspect, provided is a method of treating a disease or condition in a patient in need thereof, the method comprising implanting a cellularized structure comprising EMP into the patient, wherein the cellularized structure comprising EMP is generated by using any of the methods disclosed herein.

[0040] In some embodiments, the EMP is collagen. In some embodiments, the collagen is collagen type 1. BRIEF DESCRIPTION OF THE FIGURES

[0041] Figs. 1A, IB, 1C, and ID illustrate the engineering of collagen tubes from droplet networks. Fig. 1A. Schematic showing the adhesion of droplets containing collagen and cells. Left to right: water-in-oil droplets spontaneously acquire a monolayer of lipids in lipid-containing oil. Upon contact, droplets form a lipid bilayer at the droplet interface. Incubation of the droplets at room temperature (RT) for 10 min allowed partial gelation of collagen. Dilution of the lipid and an increase of the temperature to 37°C led to the breakdown of the lipid bilayer and fusion of the droplets. After gelation, the structure can be transferred into culture medium. Fig. IB. Schematic for the fabrication of a collagen tube from a droplet network. Droplets were arrayed into a tube-shaped network (supported by a mold, as in Fig. 1C). The incubation steps described in Fig. 1 A produced a continuous tubular structure containing collagen and cells. Fig. 1C. An example of a 3D printed mold, which supported the generation of collagen tubes with an internal diameter of 0.25 cm, a wall thickness of 0.15 cm, and a length of 0.5 cm. Fig. ID. Cross-section and the top views of the construction process of gastric organoids and Duodenum organoids.

[0042] Figs. 2A, 2B, and 2C illustrate the construction of human intestinal tissues from fused intestinal organoids. Fig. 2A. Schematic of an intestinal tissue containing human duodenum organoids (HDOs). Fig. 2B. Schematic of the migration and fusion of HDOs in collagen tubes. Fig. 2C. Images of 0.5 cm collagen tubes containing HDOs at day 0 and day 3 post fabrication. The organoids moved to the luminal surface of the tube where they fused to form a continuous epithelial layer. The boxes indicate the areas of the higher magnification images (below).

[0043] Figs. 3A, 3B, 3C, and 3D show that fibroblast-induced contraction produces various tubular shapes. Fig. 3A. Fibroblast-induced contraction was cell density dependent. Quantification of the change of the outer diameter of the collagen tubes over 48 h. The sizes of the tubes were normalized to 1.0 at t = 0. n = 3, for all conditions. Fig. 3B. Strategy used for the generation of shaped collagen tubes by patterning with droplets containing fibroblasts at various densities. The higher the fibroblast density used, the greater the contraction induced in the collagen tubes. Fig. 3C. Images of two shaped collagen tubes. Left: Shape 1 with 5 segments containing 500 x 10³, 250 x 10³, 50 x 10³, 250 x 10³ and 500 x 10³ fibroblasts mL'¹ (top to bottom). Right: Shape 2 with 9 segments containing 500 x 10³, 250 x 10³, 100 x 10³, 250 x 10³, 500 x 10³, 250 x 10³, 50 x 10³, 250 x 10³ and 500 x 10³ fibroblasts mL'¹ (top to bottom). Fig. 3D. Fibroblast induced contraction in Shape 1. The 4 cm-long collagen tube (Shape 1, Fig. 3C) comprised five segments which contained fibroblasts at different densities. The time-lapse images show the contraction process induced by fibroblasts from Day 0 to Day 5.

DETAILED DESCRIPTION OF THE INVENTION

[0044] Provided herein are complex, cellularized structures comprising EMP, methods of making said structures and method of using said structures for disease modelling and tissue implantation.

[0045] Previous attempts of using EMP such as collagen and Matrigel to construct complex shapes such as tubular GI tracts without additional scaffold materials have faced significant obstacles. For instance, hydrogels derived from EMP, such as collagen and Matrigel, are soft and slow to attain full gelation from the liquid state. Therefore, it is difficult to fabricate these hydrogels into defined structures, due to their tendency to deform. Previous molding methods for the construction of collagen tubes relied on the use of polished glass molds, which is a less flexible approach for the fabrication of tubes of various sizes and shapes compared to the use of 3D printed plastic molds. However, attempts to generate collagen tubes by directly adding collagen to the 3D printed plastic molds frequently leads to difficulties: the collagen gel easily remains stuck to the plastic mold and the constructed tubes break frequently during removal from the molds. Further, cells/organoids are likely to sink to the bottom of the constructs due to gravity.

[0046] In contrast, provided herein are methods that utilize bilayer supported droplet networks to generate cellularized structures comprising EMP. Accordingly, the cellularized structures comprising EMP may be produced without the addition of synthetic polymers or chemical cross-linking. Oil and lipids may be used to stop the collagen gel sticking to the molds, and a compartmentalization with droplets can prevent the gravity -induced sinking of cells/organoids during the construction process. Furthermore, the methods disclosed herein allow the generation of patterns, enabling the production of a variety of shapes with a significantly higher degree of complexity as compared to simple tubes.

[0047] Methods of making cellularized structures comprising EMP

[0048] In one aspect, provided is a method of arraying droplets comprising EMP and one or more population of cells in lipid-containing oil to form droplet networks, which undergo thermal gelation to provide cellularized structures comprising EMP, including, but not limited to, continuous EMP tubes.

[0049] In the methods disclosed herein, aqueous droplets comprising EMP and one or more population of cells are dispensed in a lipid-in-oil solution and are spontaneously coated in a lipid monolayer. When monolayer-coated droplets are brought into close proximity, the droplets cohere through a lipid bilayer. Breakdown of the lipid bilayer and gelation of the EMP results in the formation of cellularized structures comprising EMP.

[0050] In one aspect, provided is a method of generating a cellularized structure comprising EMP, the method comprising:

(a) providing an aqueous medium comprising (i) EMP and (ii) a population of cells;

(b) providing a mold comprising a hydrophobic medium comprising amphipathic molecules;

(c) disposing a plurality of droplets comprising the aqueous medium into the hydrophobic medium, thereby generating a droplet network comprising droplets cohering through a bilayer of amphipathic molecules;

(d) reducing the concentration of the amphipathic molecules in the hydrophobic medium; and

(e) inducing gelation of the EMP.

[0051] In one aspect, provided is a method of generating a cellularized structure comprising EMP, the method comprising:

(c) disposing a plurality of droplets comprising the aqueous medium into the hydrophobic medium, thereby generating a droplet network, wherein: a. the droplet network comprises a plurality of droplets arranged in a three- dimensional structure; b. wherein each droplet in the three dimensional structure comprises an outer layer of amphipathic molecules around the surface of the aqueous medium; and c. wherein each droplet in the three dimensional structure contacts at least one other droplet in the three dimensional structure forming a bilayer of amphipathic molecules as an interface between contacting droplets; (d) reducing the concentration of the amphipathic molecules in the hydrophobic medium; and

(e) inducing gelation of the EMP.

[0052] A non-limiting example of a method disclosed herein is illustrated in Figs. 1A-D.

[0053] As used herein, the term “droplet” typically refers to any bound volume of a material (which may for instance be a liquid or a gel). The volume of a droplet is typically less than 1.0 mL, for instance less than 0.1 mL. For instance, a bound volume of an aqueous medium having a volume of less than 500 nL is a droplet. When first generated, a droplet may be substantially spherical in character (for instance as shown in Fig. 1A). However, once in contact with other droplets in a droplet network, a droplet may adopt a range of shapes. Typically, a droplet has a sphericity (e.g. the ratio of the surface area of a sphere of the same volume as the droplet to the actual surface area of the droplet) of greater than or equal to 0.5, for instance greater than or equal to 0.6. Thus, the greatest external dimension of a droplet (e.g. length) is typically less than or equal to 2.0 times the smallest external dimension of a droplet (e.g. width). Generating a droplet typically comprises disposing a volume of the aqueous medium within a hydrophobic medium.

[0054] As used herein, the term “droplet network” is a collection of droplets, which are typically arranged in a three dimensional pattern. Typically, in a droplet network, each droplet is in contact with at least one other droplet in the network. A droplet network produced by the methods disclosed herein may be of any size. For instance, the largest external dimension of a droplet network may be from about 0.01 mm to about 1000.0 mm. [0055] As used herein, an aqueous medium may be any liquid medium comprising water. Typically, the aqueous medium is a composition comprising greater than or equal to 80 wt % water or greater than or equal to 90 wt % water. The aqueous medium comprising a EMP compound typically comprises EMP dissolved in water. The aqueous medium may comprise other components as described herein. In some embodiments, the aqueous medium comprises phosphate buffered saline (PBS).

[0056] In some embodiments, the aqueous medium comprises cell culture medium. In one embodiment, the aqueous medium is cell culture medium. A cell culture medium is any aqueous medium suitable for culturing biological cells. Culture media are well known to the skilled person. The culture medium is typically an aqueous solution of one or more amino acids (for instance glutamine or a source thereof), one or more salts (for instance sodium chloride or sodium pyruvate), glucose, and one or more vitamins (for instance vitamins A, B, C or D). The culture medium may further comprise one or more antibiotics. Examples of antibiotics include penicillin and streptomycin. The osmolarity of the culture medium at pH 7.0 is typically from 200 to 400 mOsm, for instance from 300 to 350 mOsm.

[0057] The cellularized structure comprising EMP may comprise any type of extracellular matrix protein. In some embodiments, the cellularized structure comprising EMP comprises collagen, fibronectin, laminin, and/or Matrigel. In one embodiment, the collagen is collagen type 1. In some embodiments, the collagen is collagen type I, II, III, V, or X.

[0058] By adjusting the concentration of EMP in the aqueous medium, the mechanical stiffness of the resulting cellularized structure comprising EMP may be changed. In some embodiments, low EMP concentrations are used in the aqueous medium in the methods disclosed herein. For example, in some embodiments, the EMP concentration is between about 1 and about 1.6 mg/mL.

[0059] In some embodiments, the EMP concentration is between about 0.001 mg/L and about 100.0 mg/L. In some embodiments, the EMP concentration is between about 0.01 mg/L and about 10.0 mg/L. In some embodiments, the EMP concentration is between about 0.1 mg/L and about 1.0 mg/L. In some embodiments, the EMP concentration is between about 0.5 and about 2 mg/mL. In some embodiments, the EMP concentration is between about 1 and about 1.6 mg/mL. In one embodiment, the EMP concentration is about 1 mg/ml. In one embodiment, the EMP concentration is about 1.6 mg/mL.

[0060] In some embodiments, the collagen concentration is between about 0.001 mg/L and about 100.0 mg/L. In some embodiments, the collagen concentration is between about 0.01 mg/L and about 10.0 mg/L. In some embodiments, the collagen concentration is between about 0.1 mg/L and about 1.0 mg/L. In some embodiments, the collagen concentration is between about 0.5 and about 2 mg/mL. In some embodiments, the collagen concentration is between about 1 and about 1.6 mg/mL. In one embodiment, the collagen concentration is about 1 mg/ml. In one embodiment, the collagen concentration is about 1.6 mg/mL.

[0061] In one aspect, the aqueous medium comprises one or more population of cells. A cell typically comprises a cytoplasm (typically comprising organelles such as a nucleus or a ribosome) enclosed within a membrane. The cells used in the compositions and methods disclosed herein may be prokaryotic or eukaryotic. The cell may be naturally occurring or may be genetically (or otherwise) modified. The cell may be a mammalian cell derived from mammalian tissue, for instance mouse, rat, sheep or human tissue. The cell may be derived from primate tissue such as human or chimpanzee tissue. [0062] In some embodiments, the cellularized structure comprising EMP comprises more than one population of cells. In some embodiments, each population of cells comprises a different cell type.

[0063] Examples of cells useful for the compositions and methods disclosed herein include, but are not limited to cancer cells, epithelial cells, muscle cells, fibroblasts, neuronal cells, immune cells, and blood vessel cells. In some embodiments, the cells are human embryonic kidney (HEK) cells, osteoblast cells, chrondrocyte cells, or mesenchymal stem cells. In some embodiments, the cellularized structure comprising EMP comprises gastrointestinal organoids, including, but not limited to, duodenum organoids and gastric organoids.

[0064] Typically, the cells are disposed in the aqueous medium at a concentration of from about 10² to about 10⁹ cells per mL of the aqueous medium. In some embodiments, the cells are disposed at a concentration about 10² to about 10⁹ cells per mL. In some embodiments, the cells are disposed at a concentration of about 10², about 10³, about 10⁴, about 10⁵, about 10⁶, about 10⁷, about 10⁸, or about 10⁹ cells per mL. In some embodiments, the cells are disposed at a concentration of about 50 x 10³, 100 x 10³, about 150 x 10³, about 200 x 10³, about 250 x 10³, about 300 x 10³, about 400 x 10³, or about 500 x 10³.

[0065] In some embodiments, the hydrophobic medium comprises an organic compound, some embodiments, the hydrophobic medium comprises a hydrocarbon compound. In some embodiments, the hydrophobic medium comprises a hydrocarbon compound and/or a silicone oil.

[0066] A hydrocarbon compound is a compound comprising only carbon and hydrogen atoms. Examples of hydrogen compounds include C4 to C20 alkanes (for instance straight chain alkanes having from 6 to 18 carbon atoms) and C5 to CIO cycloalkanes (for instance cyclopentane or cyclohexane). Preferably, the hydrocarbon compound is a C8 to C16 alkane, for instance octane, nonane, decane, undecane or dodecane. Preferably, the hydrocarbon compound is undecane.

[0067] A silicone oil is an oil comprising a polymeric compound which comprises one or more siloxane groups. For instance, a silicone oil is typically a polymerized siloxane with organic side chains. For instance the silicone oil may comprise polydimethylsiloxane, polyethylmethylsiloxane or polydiethylsiloxane.

[0068] In some embodiments, the hydrophobic medium comprises a mixture of a hydrocarbon and a silicone oil in a ratio (hydrocarbon):(silicone oil) of from about 1 :5 to about 5: 1 by volume. In some embodiments, the ratio is from about 1 :4 to about 4: 1 by volume. In some embodiments, the ratio is from about 1 :1 to about 1 :8 by volume. In one embodiment, the ratio is about 1 :4 by volume. The hydrophobic medium may be a mixture of undecane and silicone oil in a ratio of from about 1 :4 to about 4: 1 by volume. In one embodiment, hydrophobic medium is a mixture of undecane and silicone oil of about 1 :4 by volume.

[0069] In one aspect, the hydrophobic medium further comprises one or more amphipathic compounds. An amphipathic compound is a compound comprising both hydrophilic groups and lipophilic groups (e.g. hydrophobic groups). Amphipathic molecules are typically able to form bilayers and micelles. Amphipathic molecules are well known to the skilled person. [0070] The amphipathic compound may be a lipid. Non-limiting examples of lipids include triglycerides, fatty acids and phospholipids. Typically, the amphipathic compound is a phospholipid. A phospholipid is compound comprising a glycerol molecule substituted with a phosphate group and one or more fatty acid groups. The amphipathic compound is preferably a phosphocholine lipid.

[0071] Examples of amphipathic molecules useful in compositions and methods disclosed herein include, but are not limited to, diphytanoylphosphatidylcholine, diphytanoylphosphatidylethanolamine, 1 ,2-didecanoyl-sn-glycero-3 -phosphocholine, 1 ,2- dierucoyl-sn-glycero-3 -phosphate, 1 ,2-dierucoyl-sn-glycero-3 -phosphocholine, 1 ,2- dierucoyl-sn-glycero-3 -phosphoethanolamine, 1 ,2-dilinoleoyl-sn-glycero-3 -phosphocholine,

1.2-dilauroyl-sn-glycero-3 -phosphate, 1 ,2-dilauroyl-sn-glycero-3 -phosphocholine, 1 ,2- dilauroyl-sn-glycero-3 -phosphoethanolamine, l,2-dilauroyl-sn-glycero-3 -phosphoserine, 1,2- dimyristoyl-sn-glycero-3-phosphate, l,2-dimyristoyl-sn-glycero-3 -phosphocholine, 1,2- dimyristoyl-sn-glycero-3-phosphoethanolamine, l,2-dimyristoyl-sn-glycero-3 -phosphoserine,

1.2-dioleoyl-sn-glycero-3 -phosphate, 1 ,2-dioleoyl-sn-glycero-3 -phosphocholine, 1 ,2- dioleoyl-sn-glycero-3 -phosphoethanolamine, 1 ,2-dioleoyl-sn-glycero-3 -phosphoserine, 1 ,2- dipalmitoyl-sn-glycero-3 -phosphate, 1 ,2-dipalmitoyl-sn-glycero-3 -phosphocholine, 1 ,2- dipalmitoyl-sn-glycero-3 -phosphoethanolamine, l,2-dipalmitoyl-sn-glycero-3 -phosphoserine,

1.2-distearoyl-sn-glycero-3-phosphate, l,2-distearoyl-sn-glycero-3 -phosphocholine, 1,2- distearoyl-sn-glycero-3 -phosphoethanolamine, l,2-distearoyl-sn-glycero-3 -phosphoserine, egg-PC, hydrogenated egg PC, hydrogenated soy PC, l-myristoyl-sn-glycero-3- phosphocholine, 1 -palmitoyl-sn-glycero-3 -phosphocholine, 1 -stearoyl-sn-glycero-3 - phosphocholine, l-myristoyl-2-palmitoyl-sn-glycero 3 -phosphocholine, l-myristoyl-2- stearoyl-sn-glycero-3-phosphocholine, l-palmitoyl-2-myristoyl-sn-glycero-3- phosphocholine, l-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, l-palmitoyl-2-oleoyl-sn- glycero-3 -phosphoethanolamine, 1 -palmitoyl-2-stearoyl-sn-glycero-3 -phosphocholine, 1 - stearoyl-2-myristoyl-sn-glycero-3 -phosphocholine, l-stearoyl-2-oleoyl-sn-glycero-3- phosphocholine and l-stearoyl-2-palmitoyl-sn-glycero-3 -phosphocholine. In one embodiment, the amphipathic molecule is DPhPC (1, 2-diphytanoyl- w-glycero-3- phosphocholine).

[0072] In some embodiments, the total concentration of the one or more amphipathic compounds in the hydrophobic medium may be from about 0.01 mM to about 100 mM. In some embodiments, the concentration is from about 0.1 mM to about 10 mM, for instance from about 0.5 mM to about 5.0 mM. In some embodiments, the concentration is from about 1 mM to about 2 mM. In some embodiments, the concentration is from about 4 mM to about 5 mM. In one embodiment, the concentration of the amphipathic molecule is about 1.2 mM. In one embodiment, the concentration of the amphipathic molecule is about 4.4 mM. In some embodiment, the hydrophobic medium may comprise diphytanoylphosphatidylcholine or diphytanoylphosphatidylethanolamine at a concentration of from about 0.5 mM to about 5.0 mM. In some embodiments, the hydrophobic medium may comprise diphytanoylphosphatidylcholine at a concentration of from about 1 mM to about 2 mM.

[0073] In some embodiments, the step of reducing the concentration of the amphipathic molecules in the hydrophobic medium comprises replacing at least a part of the hydrophobic medium with a hydrophobic medium containing no lipids. In some embodiments, the concentration of the amphipathic molecules in the hydrophobic medium is reduced by replacing at least a part of the hydrophobic medium with undecane.

[0074] In some embodiments, one or more of the steps of (1) disposing a plurality of droplets comprising the aqueous medium into the hydrophobic medium and (2) reducing the concentration of the amphipathic molecules in the hydrophobic medium are performed at a temperature of less than about 15 °C, less than about 10 °C, or less than about 5 °C.

[0075] In some embodiments, the step of inducing gelation of the EMP is achieved by increasing the temperature. In some embodiments, the temperature is increased to more than about 15 °C, more than about 20 °C, more than about 25 °C, more than about 35 °C, or more than about 40 °C. In some embodiments, the step of inducing gelation of the EMP is achieved by increasing the temperature to about 37 °C.

[0076] In some embodiments, the molds are sterilized molds. In some embodiment, the molds are submerged in the hydrophobic medium. The mold may be any shape, including, but not limited to tubular, branch tubular shapes, and tubular shapes with villi structure at the luminal surface. In some embodiments, the mold is produced with a 3D printer. In some embodiments, the mold comprises acrylonitrile butadiene styrene (ABS), polylactic acid (PLA), polyethylene terephthalate (PETE), nylon, polystyrene, polyvinyl alcohol (PVA), and/or polycarbonates. A non-limiting example of a mold is provided in Fig. 1C.

[0077] In some embodiments, the cellularized structures comprising EMP are tubular structures with an exposed lumen. In some embodiments, the tubular structure is at least about 0.5 cm, at least about 1 cm, at least about 2 cm, at least about 3 cm, at least about 4 cm, at least about 5 cm, at least about 10 cm, at least about 15 cm, at least about 20 cm, at least about 25 cm, or at least about 50 cm long.

[0078] In other embodiments, more varied EMP tubes can generated by varying the design of the 3D printed molds. For example, molds with branched shapes can be used to generate branched tubes for the formation of vascular tissues. In other embodiments, patterned lumen with villi-like structures can be produced by using patterned droplet networks or changing the shape of the molds.

[0079] Often, the methods disclosed herein further comprise isolating the cellularized structure comprising EMP from the bulk hydrophobic medium and/or removing the cellularized structure comprising EMP from the mold. Removal of the cellularized structure comprising EMP from the hydrophobic medium may be done by physically removing the cellularized structure comprising EMP from the hydrophobic medium and or alternatively by removing the hydrophobic medium (e.g., by allowing it to drain).

[0080] Generation of tubular GI tissues with both epithelial and sub-epithelial structures

[0081] The methods disclosed herein are useful for generating cellularized, tubular EMP structures that mimic - for example - intestinal, gastric, or colonic tissues. In one embodiment, the cellularized structure comprising EMP is a gastric tube. In one embodiment, the cellularized structure comprising EMP is a colonic tube. In one embodiment, the cellularized structure comprising EMP is an intestinal tube.

[0082] Provided herein is a method of generating a cellularized, tubular EMP structure, the method further comprising seeding the luminal surface of the cellularized, tubular EMP structure with one or more population of cells. Accordingly, provided is a biocompatible technique to build layered tubular GI tissues.

[0083] The bioengineered EMP tubes disclosed herein may comprise epithelial layers at their luminal surfaces and subepithelial fibroblasts embedded in the EMP wall, mimicking the layered structure of GI tracts. Other subepithelial types, such as muscle cells, immune cells and enteric neurons, may also be embedded in the EMP, for example to study their interaction with the epithelial cells under both normal and disease conditions. Further, multiple subepithelial layers can be generated by automating the 3D droplet printing process. In some embodiments, the structures disclosed herein comprise multiple subepithelial layers, each containing different cell types, in the EMP wall of the tubes. These multiple layered GI tissues closely resemble the complex cellular architecture of GI tissues and are suitable to be implanted into a patient to treat short bowel syndrome or to make humanized models in rodents. In some embodiments, the structures comprises one or more of a mucosa, submucosa, muscular with longitudinal fibers, muscular transverse fibers and adventitia.

[0084] In some embodiments, the cellularized structures comprising EMP disclosed herein comprise intestinal organoids. In some embodiments, the intestinal organoids have migrated to the luminal surfaces and fused to form a continuous epithelial layer, mimicking aspects of intestinal tissue structure.

[0085] In other embodiments, cellularized structures comprising EMP can be used to mimic blood vessels or other tubular tissues in human (such as a lung). As a non-limiting example, the bioengineering of blood vessels can achieved by seeding smooth muscle cells in the EMP wall and blood vessel endothelial cells at the luminal surface.

[0086] Production of multi-layered cellularized structures comprising EMP

[0087] In one aspect, provided is a method of generating a cellularized structure comprising EMP comprising two or more layers, wherein each layer comprises a population of cells, the method comprising:

(a) generating a first cellularized structure comprising EMP comprising a first population of cells in a mold;

(b) generating a second cellularized structure comprising EMP comprising a second population of cells in the mold; wherein the first and the second cellularized structures comprising EMP are layered.

[0088] In one aspect, provided is a method of generating a cellularized structure comprising EMP comprising two or more layers, the method comprising :

(a) providing a first aqueous medium comprising (i) EMP and (ii) a first population of cells;

(b) providing a first second medium comprising (i) EMP and (ii) a second population of cells; (c) providing a mold comprising a hydrophobic medium comprising amphipathic molecules;

(d) disposing a plurality of droplets comprising the first aqueous medium into the hydrophobic medium;

(e) inducing gelation of the EMP in the first aqueous medium;

(f) disposing a plurality of droplets comprising the second aqueous medium into the hydrophobic medium; and

(g) inducing gelation of the EMP in the second aqueous medium.

[0089] In some embodiments, the first and the second population of cells comprise cells of the same cell type. In some embodiments, the first and the second population of cells comprise cells of a different cell type. Also provided herein are cellularized structures comprising EMP comprising three, four, five, six, seven, eight, or more layers, wherein at least two of the layers comprises cells of a different cell type.

[0090] In some embodiments, the cellularized structure comprising EMP comprises two or more layers, wherein at least two of the two of or more layers comprise cells at different densities. In some embodiments, the cellularized structure comprising EMP comprises two or more layers, wherein each layer comprises cells at a different density.

[0091] In GI tracts, fibroblasts underlie the epithelial layer and contribute to epithelial cell homeostasis, inflammation and wound healing. Cellularized structures comprising EMPs, which comprise fibroblasts, can contract over time, wherein the rate and the extent of the contraction increases with fibroblast density. Accordingly, provided is a method of generating a complex cellularized structure comprising EMP by inducing fibroblast-mediated contraction of the EMP structure.

[0092] In some embodiments, provided is a cellularized structure comprising EMP comprising two or more segments. The terms layer and segment are used interchangeably herein. In some embodiments, the cellularized structure comprising EMP comprises 2, 3, 4, 5, 6, 7, 8, 9, or 10 segments. In some embodiments, the cellularized structure comprising EMP comprises at least 3, at least 5, at least 10, at least 15, at least 20, at least 50, or at least 100 segments.

[0093] In some embodiments, at least two of the two or more segments in the cellularized structure comprising EMP comprise fibroblast cells at different densities. In some embodiments, each segment of the cellularized structure comprising EMP comprises fibroblast cells at a different density. In some embodiments, some of the segments in the cellularized structure comprising EMP comprise fibroblast cells the same density. In some embodiments, a segment of the comprises fibroblast cells at a density of about 50 x 10³, 100 x 10³, about 150 x 10³, about200 x 10³, about 250 x 10³, about 300 x 10³, about 400 x 10³, about 500 x 10³, about 600 x 10³, about 700 x 10³, about 800 x 10³, or about 900 x 10³.

[0094] In some embodiments, the cellularized structure comprising EMP comprises a series of two or more segments, wherein the density of fibroblast cells is decreasing from one segment to the next segment in the series. In some embodiments, the cellularized structure comprising EMP comprises a series of two or more segments, wherein the density of fibroblast cells is increasing from one segment to the next segment in the series.

[0095] In some embodiments, a high fibroblast density (for example 500 x 10³ mL'¹) is used to generate more narrow segments (for example, at the two ends of the EMP tube). In some embodiments, a lower fibroblast density (for example, 50 x 10³ mL'¹) is used to generate wider segments (for example, in the middle portion of the tube). In some embodiments, a medium fibroblast density (for example, 250 x 10³ mL'¹) used to smooth the transition between the narrower and wider segments.

[0096] In one embodiment, a tubular structure is generated with different segments of different diameters.

[0097] One non-limiting example of a cellularized structure comprising EMP comprising multiple segments is a structure comprising a series of five segments comprising 500 x 10³, 250 x 10³, 50 x 10³, 250 x 10³, and 500 x 10³ fibroblasts mL'¹, respectively. Another nonlimiting example of a cellularized structure comprising EMP comprising multiple segments is a structure comprising a series of nine segments 500 x 10³, 250 x 10³, 100 x 10³, 250 x 10³, 500 x 10³, 250 x 10³, 50 x 10³, 250 x 10³ and 500 x 10³ fibroblasts mL'¹, respectively. Nonlimiting examples of cellularized structures comprising EMP comprising different segments with different fibroblast densities are shown in Figs. 3B and 3C.

[0098] In some embodiments, the fibroblasts are allowed to contract for 1, 2, 3, 4, 5, 6, 7, 8, 9, 11, or 12 days. In some embodiments, the fibroblasts are allowed to contract for at least

I, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least

I I, or at least 12 days.

[0099] Methods of using the cellularized structures disclosed herein

[0100] The cellularized structures comprising EMP disclosed herein are further useful for disease modelling. Specifically, in vitro GI models comprising the cellularized structures comprising EMP disclosed herein allow simplified and well-controlled conditions for the study of toxicology, drug absorption, and host-microbial interaction at lower cost compared to animal models.

[0101] In some embodiments, fluidic devices are attached to the cellularized structures comprising EMP to model infection, acid reflux, cancer cell attachment, metastasis, and also the interaction between immune and neural cell and epithelial cells under infection or cancer invasion conditions in GI tract.

[0102] In some embodiments, using fluidics, air-liquid interfaces can be generated in the cellularized structure comprising EMP and the related biology can be investigated. For example, both columnar and squamous cells can be seeded to the cellularized structure comprising EMP and exposed to air-liquid interfaces, which can promote differentiation of the cells.

[0103] In some embodiments, the cellularized structures comprising EMP disclosed herein are susceptible to infection with bacterial or viral pathogens. In some embodiments, the cellularized structures comprising EMP disclosed herein are tubular structures susceptible to infection with bacterial or viral pathogens presented within the lumen. In one embodiment, the cellularized structures comprising EMP are susceptible to infection with Helicobacter pylori, which can cause gastritis, peptic ulcers and gastric cancer. Accordingly, provided is a method of infecting a cellularized structure comprising EMP disclosed herein with a pathogen, the method comprising incubating the cellularized structure comprising EMP with the pathogen, wherein the cellularized structure comprising EMP comprises a population of cells capable of infection by the pathogen.

[0104] Treatment/implantation

[0105] Surgical resection of the GI tracts can result in complications and low quality of life. Bioengineered GI tracts comprising the cellularized structures comprising EMP disclosed herein are suitable to repair the function of lost tissues.

[0106] In one aspect, provided is a method of treating a disease or condition in a patient in need thereof, the method comprising implanting a cellularized structure comprising EMP disclosed herein into the patient. In some embodiments, the cellularized structures comprising EMP disclosed herein are used to replace tissue in a patient. In some embodiments, the cellularized structures comprising EMP replace tissue that was removed due to injury, infection, or cancer. In some embodiments, the tissue replaced is a colonic tissue, a gastric tissue, an intestinal tissue, or a blood vessel. [0107] In some embodiments, the patient has a gastrointestinal disorder. In some embodiments, the patient has gastric and peptic ulcer disease or Irritable Bowel Syndrome (IBS).

[0108] The compositions provided herein are further useful for the treatment of short bowel syndrome (SBS). SBS is a devastating disease associated with mortality rates exceeding 30%. It is a condition where the small intestinal length is far less than required for proper nutrient absorption. This condition can occur in pediatric and adult populations, and may be due to congenital processes, or acquired through the loss of large amounts of small intestine due to inflammatory conditions or ischemic events. The syndrome prevents a self- sustaining absorption of nutrients from the intestine, and supplemental parenteral nutrition is required. An estimated 40,000 patients with intestinal dysfunction from small bowel syndrome require parenteral nutrition. Several long-term effects due to parenteral nutrition have been found to be harmful, such as sepsis, liver disease, and bowel bacterial overgrowth; therefore, this method can only be used as a short-term solution. Accordingly, provided herein is a method of treating SBS in a patient in need thereof, the method comprising implanting into the patient a cellularized structure comprising EMP disclosed herein.

[0109] It is to be understood that this disclosure is not limited to the particular molecules, compositions, methodologies, or protocols described, as these may vary. Any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the disclosure. It is further to be understood that the disclosure of the invention in this specification includes all possible combinations of such particular features. For example, where a particular feature is disclosed in the context of a particular aspect or embodiment of the invention, or a particular claim, that feature can also be used, to the extent possible, in combination with and/or in the context of other particular aspects and embodiments of the invention, and in the invention generally.

[0110] Where reference is made herein to a method comprising two or more defined steps, the defined steps can be carried out in any order or simultaneously (except where the context excludes that possibility), and the method can include one or more other steps which are carried out before any of the defined steps, between two of the defined steps, or after all the defined steps (except where the context excludes those possibilities).

[OHl] All other referenced patents and applications are incorporated herein by reference in their entirety. Furthermore, where a definition or use of a term in a reference, which is incorporated by reference herein is inconsistent or contrary to the definition of that term provided herein, the definition of that term provided herein applies and the definition of that term in the reference does not apply.

[0112] To facilitate a better understanding of the disclosure, the following examples of specific embodiments are given. The following examples should not be read to limit or define the entire scope of the invention.

EXAMPLES

[0113] Material and methods used for Examples 1-5

[0114] 3D printed supporting molds

[0115] The supporting molds were computer designed using Fusion 360 software. The designs were then rendered for printing using Cura software. The printing was performed by an Ultimaker 2+ 3D printer using polymer acrylonitrile butadiene styrene.

[0116] Fabrication o f collagen tubes

[0117] Collagen type I (Coming/354249) solution was diluted and neutralized to pH 7.3 according to manufacturer’s instructions, with a final concentration of 1 or 1.6 mg mL'¹. Notably, cell culture medium was used during the dilution to replace the water and the corresponding amount of 10 * Phosphate Buffered Saline (PBS) from the manufacturer’s protocol. The prepared collagen was kept on ice prior to use.

[0118] To construct the collagen tubes from arrayed droplet networks, sterilized molds were submerged in an oil bath, which consisted of 4 mg mL'¹ DPhPC (1, 2-diphytanoyl- w- glycero-3 -phosphocholine, Avanti, 850356) in a mixture of undecane and silicone oil AR20 (both from Sigma Aldrich; v:v, 1 :4). Collagen droplets, ~ 2 pL in size, were pipetted into the molds to form droplet networks. The networks were left to settle for 10 min. Half of the lipid containing oil was then removed and replaced with undecane to dilute the lipid concentration and ensure the subsequent breakdown of the lipid bilayers. The collagen tubes were allowed to gelate at 37 °C for 20, 40 or 60 min for 0.5, 4 or 10 cm long collagen tubes respectively. The oil was subsequently removed and two washes with 1 * PBS were performed. Using sterile forceps, the supporting molds were carefully removed and a further two washes with PBS were conducted to give the self-supporting collagen tubes.

[0119] Duodenum organoid derivation and maintenance

[0120] Duodenum biopsies were collected from outpatients during endoscopy. Healthy biopsies from Barrett’s Oesophagus patients were collected in Advanced DMEM/F12 supplemented with 1% 2 mM L-Glutamine, 2 pl/ml IM (4-(2 -hydroxy ethyl)- 1- piperazineethanesulfonic acid (HEPES) and 200 U/ml penicillin/streptomycin and kept on ice prior to processing.

[0121] To derive human duodenum organoids, the tissue was cut into small (~5 mm) pieces and repeatedly washed with ice-cold 1 x PBS until the supernatant was clear. The tissue fragments were then incubated with cold 5 mM ethylenediaminetetraacetic acid (EDTA)-PBS and placed on a roller at 4 °C for 15 min. To produce further dissociation, the tissue fragments were washed twice with ice-cold PBS and incubated in TripLE (a mixture of recombinant cell-dissociation enzymes) at 37 °C for 30 min. Following a further two washes with ice-cold PBS, the sedimented tissue fragments were vigorously resuspended in ice-cold PBS and allowed to settle under gravity. The supernatant, now enriched with cells released from the tissue fragments, was collected, passed through a 35 pm cell strainer and centrifuged at 200 g for 5 min. The pellets were then embedded in Matrigel (an ECM-based hydrogel) and seeded in 24-well plates (~30 pL of Matrigel/well). The Matrigel was allowed to gelate at 37 °C for 5 min and 500 pL of human organoid medium was then overlaid and replaced every two days. To prevent anoikis (a form of programmed cell death that occurs in anchorage-dependent cells when they detach from the surrounding extracellular matrix), Rho- associated protein kinase (ROCK) inhibitor (Y-27632) and GSK-3 inhibitor (CHIR99021) were added to the medium for the first two days after derivation. Organoids were maintained at 37 °C in an atmosphere of 5% CO2 and 95% humidity.

[0122] Organoids were passaged every 7-10 days at a ratio of 1 :4. Using ice-cold PBS, the organoids were retrieved from the Matrigel and briefly microcentrifuged. To aid organoid dissociation, the pellet was vigorously resuspended in TripLE and incubated at RT for 5 min. The organoid fragments were then briefly microcentrifuged, embedded in Matrigel and seeded in 24-well plates (~30 pL of Matrigel/well). The Matrigel was allowed to gelate at 37 °C for 5 min and 500 pL of human organoid medium was then overlaid and replaced every two days.

[0123] Human organoid conditioned medium

[0124] HEK293T Rspol-Fc cells, HEK293T Nog-Fc cells and L Wnt3A cells were used to generate R-Spondin-, Noggin-, and Wnt3 A conditioned medium respectively. Cells were maintained in the appropriate specified media at 37 °C in an atmosphere of 5% CO2 and 95% humidity.

[0125] To generate the conditioned medium, the stably transfected cells were initially grown in the relevant selectable medium (see Table 1) and then expanded considerably in the relevant non-selectable medium in order to produce sufficient quantities of conditioned medium. Cells were cultured for one week when the medium was collected, sterile filtered (0.45 pm) and stored at -80 °C prior to use.

Table 1 Organoid conditioned medium

[0126] A HEK293T Wnt3 A luciferase reporter cell line was used to test the quality of the R-Spondin and Wnt3 A conditioned medium. Freshly made conditioned medium was tested against previously made batches and was only used for organoid culture if the same or better quality (detected as an increased luciferase reading and therefore Wnt3 A promoter activity) was observed. In order to test the quality of the Noggin conditioned medium, organoids were cultured in human organoid medium containing the newly made Noggin conditioned medium and the growth monitored for 7-10 days. If the organoids began to die or grow at a slower rate, the conditioned medium batch was discarded.

[0127] Cell culture of AGS, Caco-2 and fibroblast cells

[0128] The human stomach adenocarcinoma cell line, AGS (CRL-1739™) and the human colorectal adenocarcinoma cell line, Caco-2 (HTB-37™) were maintained in complete RPMI 1640 medium and the human foetal fibroblast cell line, HFF-1 was maintained in complete DMEM. Cells were cultured at 37 °C in an atmosphere of 5% CO2 and 95% humidity.

[0129] Construction of intestinal tubes with HDOs

[0130] Harvested HDOs were washed with ice-cold PBS three times before being resuspended in the prepared 1 mg mL'¹ collagen. HDOs from each confluent well of a 24- well plate were mixed with 200 pL collagen solution and 160 pL of the mixture was used to construct one 0.5 cm long collagen tube. The collagen tubes were incubated at 37 °C for 20 min and the construction process described above in the ‘Fabrication of collagen tubes’ was subsequently followed. The constructed intestinal tubes were cultured in 600 pL of human organoid medium in a 24-well plate and the medium was replaced with fresh medium every second day.

[0131] Construction of fibroblasts containing collagen tubes

[0132] In general, harvested fibroblasts were resuspended in 1.6 mg mL'¹ collagen to give the desired fibroblast densities. The construction process described in the ‘Fabrication of collagen tubes’ was followed. For collagen tubes patterned with segments containing different fibroblast densities, an extra 5 min of incubation at RT were performed between the construction of different segments to allow sufficient time for the droplets to settle before the adding of droplets with a different fibroblast density. The 0.5, 4 and 10 cm long fibroblast containing collagen tubes were incubated at 37 °C for 20 min, 40 min and 60 min respectively.

[0133] To construct complex shape 1, a high fibroblast density, 500 x 10³ mL'¹, was used for the narrow segments at the two ends of the collagen tube, whereas a lower fibroblast density, 50 x 10³ mL'¹, was used for the wider segment at the middle of the tube. A medium fibroblast density, 250 x 10³ mL'¹, was used to smooth the transition between the narrow and wide segments. Similarly, a longer tube, complex shape 2, with nine segments was constructed to give a more complex structure.

[0134] Seeding epithelial cells into fibroblast-containing collagen tubes

[0135] To seed epithelial cells to the luminal surface of the fibroblast-containing collagen tubes, one end of the tube was securely sealed with a sterile string. Then 1 x 10⁶ AGS or Caco-2 cells suspended in 100 pL complete RPMI 1640 medium were introduced into the vertically held tube through the open end, which was subsequently also sealed. The tubes were placed in a 10 cm cell culture dish containing 10 mL of fibroblast medium and incubated at 37 °C in an atmosphere of 5% CO2 and 95% humidity. To prevent an uneven distribution of cells, the tube was turned over after 3 h. After 24 h, one end of the tube was reopened and the inner surfaced rinsed with the appropriate medium before processing.

[0136] Helicobacter pylori infection of engineered tubular gastric tissues

[0137] H. pylori (G27 strain) was cultured as described in L. Buti et al., Proc Natl Acad Sci U S A 108(22) (2011) 9238-43. In order to infect AGS cells lining at the luminal surface of the gastric tissues, the open end of a tube was filled with 1 x 10⁹ H. pylori bacterium suspended in 100 pL of AGS medium without antibiotics. The open end was then re-sealed. The tubes were placed in a 10 cm cell culture dish containing 10 mL of fibroblast medium (without antibiotics) and incubated for 7 h at 37 °C in an atmosphere of 5% CO2 and 95% humidity. After the incubation, the luminal surface of the tubes were thoroughly rinsed, and the tubes were prepared for processing.

[0138] Sample processing

[0139] To prepare samples for staining, the collagen tubes were washed twice with PBS and fixed in 4% Paraformaldehyde at 4 °C overnight. Samples were then processed in an automated tissue processor (ThermoFisher Scientific/Excelsior™ AS) and embedded in paraffin wax. The 0.5 cm long tubes were embedded in HistoGel™ (an aqueous gel composition useful in processing histological and cytological specimens) prior to tissue processing and tubes longer than 0.5 cm were cut longitudinally prior to paraffin embedding and individually processed. A microtome was used to cut 0.4 pm sections, which were mounted onto glass slides and dried at 37 °C. Before immunostaining, slides were incubated at 60 °C for 1 hr and then processed accordingly to remove the paraffin wax. Further hydration of the fixed samples was achieved by submerging them in Histoclear, a clearing agent, (2 x 5 min), 100% Ethanol (1 x 1 min), 90% Ethanol (1 x 1 min) and 70% Ethanol (l x 4 min) consecutively.

[0140] Immunofluorescence staining

[0141] After processing, samples were boiled in pre-warmed IX Tris-EDTA buffer (10 mM Tris Base, 1 mM EDTA, 0.05% Tween 20, pH 8.0) for 10 min and placed in a humidifying chamber. Samples were washed twice, permeabilised in 0.1% Triton-X for 15 minutes and blocked using Image-iT™ Fx Signal Enhancer (ThermoFisher Scientific/136933) for 1 hr. Samples were incubated in primary antibody (against ASSP2, CagA, Collagen Type 1, DAPI, E-Cadherin, Cytokeratin 8, or Villin-1) diluted in background reducing antibody diluent (Agilent Dako/S3022) at 4 °C overnight. Samples were then washed in PBS (3 x 10 min) and incubated in the appropriate secondary antibody diluted in antibody diluent (Agilent Dako/S0809) for 2 hours in the dark. Finally, samples were washed 3 times with PBS and mounted using 200 pL of Prolong Gold™ Antifade mountant (ThermoFisher Scientific/P36930). The immunostained samples were imaged on a Zeiss 710MP confocal microscope.

[0142] Haematoxylin and Eosin Staining [0143] For Haematoxylin and Eosin staining, processed samples were submerged in Haematoxylin (1 x 5 min), H2O (1 x 2 min), Acidic Alcohol (1 x 5 sec), H2O (1 x 2 min), Scott’s Water (1 x 30 sec), H2O (1 x 2 min), Eosin (1 x 5 min), H2O (1 x 1 min), 70% Ethanol (1 x 30 sec), 90% Ethanol (1 x 30 sec), 100% Ethanol (1 x 1 min), 100% Ethanol (1 x 2 min) and Histoclear (2 x 5 min) consecutively. Slides were then mounted using VectaMount® Permanent Mounting Medium and imaged using a Hamamatsu Automated Slide Scanner. Analysis was performed using NDP.view2.

[0144] Time-lapse microscopy for fibroblast induced contraction

[0145] Immediately after construction, time-lapse images of the fibroblast containing collagen tubes were acquired using a Nikon TE 2000-E inverted microscope (Photometries Prime camera) using a 2X objective. The tubes were maintained at 37 °C in an atmosphere of 5% CO2 and images were acquired every hour for 48 h. Image analysis was performed using the MetaMorph software (Molecular Devices, LLC.) and processed into videos using Fiji ImageJ.

[0146] Example 1: Engineered collagen tubes from droplet networks

[0147] Collagen tubes with centimeter dimensions were produced using a droplet-based method that utilizes molds to support large and hollow droplet networks for the production of.

[0148] As part of the general protocol for generating template shapes, droplets, containing cells and collagen solution, were generated in lipid-containing oil. These droplets spontaneously acquired a monolayer of lipids (Fig. 1A). When droplets were in contact, lipid bilayers were formed at their interfaces. Partial gelation of the collagen occurred when the droplets were incubated at room temperature (RT) for 10 min. Dilution of the lipids and further incubation at 37°C led to the breakdown of the droplet-interface bilayers, and the collagen gelated further to form a continuous structure. After gelation at 37°C for 20 min, the templated shapes were transferred into cell culture medium.

[0149] To fabricate collagen tubes, a hundred to over a thousand droplets were generated, each of ~2 pL, to form droplet networks inside supporting molds of various sizes, 0.5 to 10 cm in length (Figs. IB, 1C, and ID). The molds were 3D printed based on design from polymer, acrylonitrile butadiene styrene. After 10 min at RT, lipid dilution and then 20 to 60 min at 37 °C , continuous tubular structures were formed, which were removed from the molds to give free standing tubular structures with defined dimensions. [0150] Example 2: Engineered intestinal tissues from fused organoids

[0151] Next, human intestinal tubes with defined structures and exposed lumens were generated that contained human duodenum organoids (HDOs).

[0152] Intestinal organoids are self-organized, microscopic structures that recapitulate the signaling and architecture relevant to the natural gut epithelium. Organoids are useful for studying intestinal epithelium development, stem cell biology, and disease. To generate human intestinal tubes with defined structures and exposed lumens, human duodenum organoids (HDOs), derived from patients, were embedded in 0.5 cm-long collagen tubes (Fig. 2A). Using this method, the organoids migrated towards the luminal surface of the collagen tube, instead of the center of the collagen wall (Figs. 2B and 2C). Fusion of the organoids occurred in the collagen rings after around 3 days of culture.

[0153] Further, contraction of the collagen tubes was observed, which was considered to be the force required to bring the organoids into close proximity and induce their fusion. Organoid migration, alignment, and fusion were not observed when the HDOs were embedded in Matrigel derived tubes, indicating the importance of the contractility of the collagen.

[0154] Immunofluorescence staining of sectioned tubes showed an epithelial layer as a continuous monolayer at the luminal surface. Like intact HDOs, the epithelial layer expressed the cell-cell junction protein E-cadherin and the intestinal marker villin. Immunostaining using different markers further confirmed the existence of different cell types: Muc2 (Goblet cells), Chromogranin A (enteroendocrine cells), Lysozyme (Paneth cells), and Sox9 (stem cells, nuclear marker). Therefore, the engineered intestinal tissues have a defined tubular shape and an exposed intestinal epithelial layer at the luminal surface, which resemble aspects of human intestinal structures.

[0155] Example 3: Fibroblast-induced contraction for the production of complex tubular shapes

[0156] In GI tracts, fibroblasts underlie the epithelial layer and contribute to epithelial cell homeostasis, inflammation and wound healing. Fibroblasts are able to synthesize ECM and induce contraction in collagen gels, and the latter has been used to study wound healing for decades. Accordingly, droplet network was utilized to construct complex tubular structures capable of fibroblast-induced contraction were generated. [0157] 0.5 cm-long collagen tubes containing fibroblasts at different cell densities were constructed: 0, 50, 250 and 500 x 10³ mL'¹ (Fig. 3A). In the absence of fibroblasts, no contraction was observed over 48 h. By contrast, the collagen tubes contracted over time when fibroblasts were present, and both the rate and extent of the contraction increased with fibroblast density (Fig 3A). With 500 x 10³ mL'¹ fibroblasts, the outer diameter of the tubes had decreased by over 80% after 48 h. The contraction of a 4 cm-long tube was demonstrated as well. Immunofluorescence staining confirmed the presence of collagen I, which was used to form the tubes.

[0158] To construct complex tubular structures with segments of different diameter, droplet networks were patterned by using droplets containing fibroblasts at different densities to form the various segments (Fig. 3B). Immediately after formation, the patterned collagen tubes were of uniform internal diameter. However, after 48 h of culture, the segments with higher fibroblast density had narrowed, compared to those of lower fibroblast density, showing that the internal diameter of the tubes could be tuned by adjusting the fibroblast density. Two shapes, Shape 1 and Shape 2, were produced (Fig. 3C). Shape 1 started from a 4-cm long tube and contained five segments, which resulted a stomach-like structure (Fig. 3D). Shape 2 started from a 10-cm long tube and contained nine segments. These examples demonstrate a simple method, combining the droplet network technique and fibroblast- induced contraction, to produce complex tubular structures.

[0159] Example 4: Engineered gastrointestinal tissues using fibroblast-containing collagen tubes

[0160] Next, different types of epithelial cells were seeded onto the luminal surface of the fibroblast-containing collagen. Firstly, gastric tissues were constructed by using Shape 1 (see Example 3) to mimic the stomach. After 48 h in culture, the fibroblast-containing tubes were sufficiently robust for further manipulation. The tubes were sealed at one end and then dissociated gastric adenocarcinoma epithelial cells (AGS cells) were injected to the lumen, followed by sealing of the other end of the tube. After 24 h, unattached AGS cells were washed out with fresh medium. The engineered gastric tissues were then fixed and sectioned. Haemotoxylin and eosin (H&E) staining revealed the formation of a monolayer of AGS cells around the luminal surface, while collagen I immunostaining showed the collagen used for the tube construction. Staining for the epithelial marker cytokeratin 8 and the cell-cell junction marker E-cadherin further confirmed the monolayer structure and its epithelial identity.

[0161] To demonstrate the versatility of the technique, colonic tissues were constructed by seeding human colon carcinoma epithelial cells (Caco-2 cells) into collagen tubes containing 500 x 10³ mL'¹ fibroblasts. H&E staining showed that the epithelial layer consisted of a multilayer of Caco-2 cells), consistent with the behaviour of these cells in confluent 2D culture. 4',6-Diamidino-2-phenylindole (DAPI) staining and collagen I immunostaining confirmed the multilayer epithelial structure and its location at the luminal surface of the collagen tube. The epithelial cells also expressed the epithelial marker cytokeratin 8 and the cell-cell junction marker E-cadherin.

[0162] Example 5: Infection of bioengineered GI tracts with H. pylori

[0163] To demonstrate that the bioengineered GI tracts are suitable to conduct infection experiments with H. pylori, which can cause gastritis, peptic ulcers and gastric cancer, H. pylori suspended in AGS cell culture medium were added to the lumen of the gastric tubes. After 6 h, the tubes were washed and examined by immunofluorescence. Staining for cytotoxin-associated gene A (CagA), which is expressed by H. pylori, confirmed that the pathogen was able to infect the epithelial cells in the gastric tubes. Further, the co-localisation of CagA and the apoptosis-stimulating protein of p53 2 (ASPP2) indicated the recruitment of ASPP2 by CagA during infection.

Claims

We claim:

1. A method of generating a cellularized structure comprising extracellular matrix protein (EMP), the method comprising:

(c) disposing a plurality of droplets comprising the aqueous medium into the hydrophobic medium; and

(d) inducing gelation of the EMP.

2. The method of claim 1, wherein the step of disposing a plurality of droplets generates a droplet network comprising droplets cohering through a bilayer of amphipathic molecules.

3. The method of claim 1 or 2, the method further comprising a step of reducing the concentration of the amphipathic molecules in the hydrophobic medium before inducing gelation of the EMP.

4. The method of claim 3, wherein the step of reducing the concentration of the amphipathic molecules in the hydrophobic medium disrupts the integrity of the bilayer.

5. A method of generating a cellularized structure comprising EMP, the method comprising:

(c) disposing a plurality of droplets comprising the aqueous medium into the hydrophobic medium, thereby generating a droplet network, wherein: a. the droplet network comprises a plurality of droplets arranged in a three- dimensional structure; b. each droplet in the three dimensional structure comprises an outer layer of amphipathic molecules around the surface of the aqueous medium; and c. each droplet in the three dimensional structure contacts at least one other droplet in the three dimensional structure forming a bilayer of amphipathic molecules as an interface between contacting droplets; and

(d) inducing gelation of the EMP. The method of claim 5, the method further comprising a step of reducing the concentration of the amphipathic molecules in the hydrophobic medium before inducing gelation of the EMP. The method of any one of the preceding claims, wherein the gelation of the EMP is induced by increasing the temperature. The method of claim 7, wherein the temperature is increased to about 37 °C. The method of any one of the preceding claims, wherein the step of disposing a plurality of droplets comprising the aqueous medium into the hydrophobic medium is performed at a temperature of less than about 15 °C. The method of any one of the preceding claims, wherein the EMP is collagen. The method of claim 10, wherein the collagen is collagen type 1. The method of claim 10 or 11, wherein the concentration of the EMP in the aqueous medium is from about 0.5 and about 2 mg/mL. The method of any one of the preceding claims, wherein the aqueous medium comprises cell culture medium. The method of any one of the preceding claims, wherein the population of cells comprises mammalian cells. The method of any one of the preceding claims, wherein the population of cells comprises mouse, rat, sheep, or human cells. The method of any one of the preceding claims, wherein the population of cells comprises epithelial cells, muscle cells, neuronal cells, immune cells, fibroblasts, blood vessel cells, or gastrointestinal organoids. The method of any one of the preceding claims, wherein the aqueous medium comprises cells at a concentration from about 10² to about 10⁹ cells per mL of the aqueous medium. The method of any one of the preceding claims, wherein the aqueous medium comprises cells at a concentration from about 10² to about 10⁴ cells per mL of the aqueous medium. The method of any one of the preceding claims, wherein the hydrophobic medium comprises a hydrocarbon compound and/or a silicone oil. The method of claim 19, wherein the hydrocarbon compound is undecane. The method of claims 19 or 20, wherein the silicone oil comprises polyethylmethylsiloxane. The method of claim 19, wherein the hydrophobic medium comprises a hydrocarbon compound and a silicone oil at a ratio of about 1 :4 by volume. The method of any one of the preceding claims, wherein the hydrophobic medium comprises an amphipathic compound. The method of claim 23, wherein the amphipathic compound is a phospholipid. The method of claim 24, wherein the phospholipid is diphytanoylphosphatidylcholine. The method of any one of claims 23-25, wherein the amphipathic compound is present at a concentration of about 1 mM to about 2 mM. The method of any one of the preceding claims, wherein the mold comprises one or more of acrylonitrile butadiene styrene (ABS), polylactic acid (PLA), polyethylene terephthalate (PETE), nylon, polystyrene, polyvinyl alcohol (PVA), and polycarbonates. The method of any one of the preceding claims, wherein the cellularized structure comprising EMP is further isolated from the hydrophobic medium. The method of any one of the preceding claims, wherein the cellularized structure comprising EMP is further removed from the mold. The method of any one of the preceding claims, wherein the structure is a tubular structure. The method of claim 30, wherein the method further comprising seeding the luminal surface of the structure with a second population of cells. A method of generating a cellularized structure comprising EMP comprising two or more layers, wherein each layer comprises a population of cells, the method comprising:

(a) generating a first cellularized structure comprising EMP comprising a first population of cells in a mold; and

(b) generating a second cellularized structure comprising EMP comprising a second population of cells in the mold; wherein the first and the second cellularized structure comprising EMP are layered. The method of claim 32, wherein the density of cells in the first cellularized structure comprising EMP is different from the density of cells in the second cellularized structure comprising EMP. A method of generating a cellularized structure comprising EMP comprising two or more layers, the method comprising (a) providing a first aqueous medium comprising (i) EMP and (ii) a first population of cells;

(b) providing a first second medium comprising (i) EMP and (ii) a second population of cells;

(c) providing a mold comprising a hydrophobic medium comprising amphipathic molecules;

(e) inducing gelation of the EMP in the first aqueous medium;

(g) inducing gelation of the EMP in the second aqueous medium. The method of claim 34, wherein the concentration of cells in the first aqueous medium is different from the concentration of cells in the second aqueous medium. The method of any of claims 32-35, wherein the first and the second populations of cells comprises cells of different cell types. The method of any of claims 32-35, wherein the first and the second populations of cells comprises cells of the same cell type. The method of claim 37, wherein at least one of the first and the second populations of cells comprises fibroblast cells. A cellularized structure comprising EMP comprising one or more populations of cells. The cellularized structure comprising EMP of claim 39, wherein the cells in at least one of the one or more population of cells are selected from epithelial cells, muscle cells, neuronal cells, immune cells, fibroblasts, blood vessel cells, or gastrointestinal organoids. A cellularized structure comprising EMP comprising two or more layers, wherein: a. each layer comprises a population of cells, wherein the structure comprises a first and a second layer; and b. the first layer comprises cells at a density different from the density of cells in the second layer. The cellularized structure comprising EMP of claim 41, wherein at least one of the one or more populations of cells is a population of fibroblast cells. The cellularized structure comprising EMP, wherein cellularized structure comprising EMP is generated using the method of any one of claims 1-38. A method of treating a disease or condition in a patient in need thereof, the method comprising implanting a cellularized structure comprising EMP comprising one or more population of cells into the patient. The method of claim 44, wherein the cellularized structure comprising EMP replaces a gastric tissue, a colonic tissue, an intestinal tissue, or a blood vessel. The method of claim 44, wherein the disease or condition is short bowel syndrome. The method of claim 44, wherein the cellularized structure comprising EMP is generated according to the method of any one of claims 1-38. The method of claim 44, wherein the cellularized structure comprising EMP is the cellularized structure comprising EMP of any one of claims 39-42. The method of any one of claims 32-38 or 44-48 or the cellularized structure comprising the EMP of any of claims 39-43, wherein the EMP is collagen. The method of claim 49, wherein the collagen is collagen type 1.