WO2008100226A1 - Method for formation of tubules - Google Patents

Abstract

There is provided a method for forming tubules. The method involves providing a gel body enclosing one or more channels with each channel enclosed entirely within the gel body, the gel body comprising basement membrane components and growth medium. A suspension of cells, for example endothelial cells or epithelial cells, is introduced to one or more of the channels and the gel body containing the introduced cells is incubated under conditions sufficient to permit formation of a tubule, the tubule having an interior lumen.

Description

METHOD FOR FORMATION OF TUBULES

FIELD OF THE INVENTION

[0001] The present invention relates generally to methods for forming tubules.

BACKGROUND OF THE INVENTION

[0002] The histo-architecture and consecutive function of various epithelial organs, such as the lung and the kidney, are characterized by a system of tubular structures. In order to engineer such organs, it would be desirable to control the formation of tubules with regard to their shape, spatial orientation and interconnectedness.

[0003] Considerable research has been done on tubules during embryonic development or tubulogenesis. During tubulogenesis, two key events occur: tubule elongation and branching morphogenesis (1-3).

[0004] The underlying mechanisms and molecules during the branching morphogenesis of the ureteric bud in embryonic kidney development have been studied intensively. Such development leads to the complex formation of the collecting duct system (4, 5). In particular, it has been shown that the action and regulation of matrix metalloproteinases (MMPs) play a critical role since MMPs are capable of digesting the surrounding extracellular matrix (ECM) to open up space, and hence enable further tubular growth (6, 7).

[0005] At the cellular level, it has been shown that epithelial cells assemble into polarized tubular structures by surface-detecting mechanisms via integrin receptors, which in turn activate intracellular signaling pathways leading to cell polarization (8- 10). For example, it has been shown that Madin-Darby Canine Kidney (MDCK) epithelial cells cultured in a liquid suspension form lumen-containing epithelial structures in the form of cysts, where the luminal surfaces of the epithelial cells are in contact with the surrounding suspension liquid. In contrast, when epithelial cells are exposed to the surrounding ECM in a 3D culture set-up, the luminal surfaces are found at the inner side of the resulting epithelial structure (11). Hence, the nature of the exposed material sensed by the epithelial cell surface determines in which direction the cells polarize. [0006] Tissue engineering approaches aim to engineer cell or tissue constructs via a combination of cells and biomaterials in vitro, which are subsequently implanted to restore lost tissue in the body (12, 16). In addition, tissue engineering approaches can be applied to develop in vitro systems that allow the investigation of biological processes, such as the self-assembly of cells, in order to gain insights into the process of tissue formation during embiyonic development (12, 17).

[0007] Until recently, the development of tubular structures has been mostly achieved by seeding cells or cell aggregates into scaffold structures or hydrogel sponges with limited control of tubule formation (18, 19.) However, researchers have recently discovered the potential of applying embryonic principles to tissue engineering approaches for the formation of tubular structures (20-22).

[0008] Tubules had been successfully generated by culturing embryonic explants on polyester fleece, with tubules forming at the interface of the explants and fleece (21). However, this approach requires access to a ready supply of embryonic tissue and thus is not practical for large-scale production of engineered tubules.

[0009] Recently, micromolding of a photocrosslinkable hydrogel was used in tissue engineering approaches to form co-culture systems (25, 26) or spheroid micro- arrays (25). However, such an approach can have a negative effect on biological materials due to the required UV-crosslinking.

[0010] Photopolymerizable hydrogels and collagen-based grooved membranes (30) have been used for tissue engineering, but they have not been applied to form 3D lumen-carrying tubules or to orient tubules with the formation of complex patterns and interconnectedness.

[0011] Epithelial tubular cells are situated in vivo on natural meshes of nanoporous membranes in the form of basement membranes. In principle, techniques such as electrospinning of nanofibers could allow for fabrication of an appropriate support scaffold for tubule formation. However, it remains very challenging to mold electrospun nanofibers into small tubular scaffolds (36-39).

[0012] Rapid prototyping techniques to fabricate scaffolds from synthetic materials do allow the design of complex micro-architectures, but they are associated with a limited printing resolution (18, 40, 41)that fails to provide for nanoporous superstructures.

[0013] Organ printing approaches have attracted much attention as they allow for the printing of gel-entrapped cell aggregates in the desired shapes and geometry (12, 22). The printed cell aggregates tend to fuse into ring-like structures after a certain period. The challenge of such approaches lies in the difficulty of scaling-up to 3D structures of significant dimensions. The wall of ring-like structures is composed of fused non-polarized cell aggregates.

[0014] Accordingly, there is a need for development of additional approaches to tissue engineering including engineering of tubules, particularly approaches that allow for formation of complex tissue structures from multicellular systems.

SUMMARY OF THE INVENTION

[0015] The present invention provides a new tissue engineering approach that results in formation of tubular structures. The approach is based on providing space and three dimensional exposure of developing structures to a gel support that contains basement membrane components. Combining these factors has resulted in a successful new method for creating biological tubules for various applications.

[0016] Using the present method, it is possible to generate various tubule structures, including kidney epithelial tubules and endothelial tubules of predetermined size and shape. The ability to generate small renal tubular structures is critical for kidney tissue engineering, as this allows for a large tubule surface area within a given volume.

[0017] The method presented may also be applied to reveal new insights into developmental and tissue formation processes in vitro. The assembly of individual cells and cell-based building blocks into tissue structures in pre-formed spaces in transparent gels can be easily performed, thus providing an in vitro model system. Therefore, the method can be employed in the optimization of in vitro tissue formation processes, as well as, for example, in the in vivo engraftment of tissue- engineered renal epithelial tubules on injured kidneys or organs with impaired micro vessel systems. [0018] Thus, in one aspect there is provided a method of forming tubules, the method comprising providing a gel body enclosing one or more channels with each channel enclosed entirely within the gel body, the gel body comprising basement membrane components and growth medium; introducing a suspension of endothelial cells or epithelial cells to one or more of the channels; and incubating the gel body containing the introduced endothelial cells or epithelial cells under conditions sufficient to permit formation of a tubule, the tubule having an interior lumen.

[0019] In another aspect, there is provided a kit comprising a gel body and instructions for forming a tubule according to the method described herein, the gel body enclosing one or more channels with each channel enclosed entirely within the gel body, the gel body comprising basement membrane components and growth medium.

[0020] Other aspects and features of the present invention will become apparent to those of ordinary skill in the art upon review of the following description of specific embodiments of the invention in conjunction with the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In the figures, which illustrate, by way of example only, embodiments of the present invention,

[0022] FIG. 1 (A) is a schematic representation of 3D Gel molding: grooves are created on the surface of the Gel using microfabricated grooved glass templates; the grooves are filled with cells; a second layer of Gel is then added, exposing the cells to 3D ECM environment and inducing the formation of tubular structures; (B) is light microscopy image showing MDCK cells added to the grooved Gel surface; (C) is light microscopy image of subsequently formed tubular networks;

[0023] FIG. 2 (A) is a diagram of a pre-formed channel in an ECM-gel; (B) a schematic representation of the procedure of filling the channel; (C)-(F) are transmission light microscopy images of (C) a formed channel; (D) cells assemble into tubule-like structures after 2 to 3 days; (E) parallel system of 1-cm long tubules; and (F) interconnected tubules;

[0024] FIG. 3 is light microscopy images of 3-D culture of MDCK cells in (A) collagen gel; (B) collagen/ MATRIGEL™; and (C) MATRIGEL™ reveal no tubule formation; formation of cell-to-cell contact was observed in the channels made in (D) collagen; (E) collagen/ MATRIGEL™; and (F) MATRIGEL™; histological cross- sections demonstrated no lumen formation in the (G) collagen gel but there was lumen formation in the (H) collagen/ MATRIGEL™ and in (I) MATRIGEL™;

[0025] FIG. 4 fluorescence microscopy images (A₅D₃G) DAPI staining of a cross- sectioned tubule, β-catenin expression (arrows in B and C) and PNA staining (arrows in E and F) at the baso-lateral cell show epithelial cell polarization; (H₅I) no smooth muscle expression was observed in the epithelial cells;

[0026] FIG. 5 histological sections reveal that cells in the center of the tubule that had no direct contact to the ECM (A, D) undergo cell death, which can be recognized (arrows in B, black dotted cell nuclei in E) by pycnotic cells; eventually, one lumen is formed, as seen in (C₅F);

[0027] FIG. 6 light and fluorescence microscopy images show that the tubules released from gels (A₅B) possess structural integrity; cells generated from cellular cells can be recognized by DAPI staining (C); after release, few cells were non- viable (red-stained cells in D), while most of the cells remained viable (green-stained cells in E); and

[0028] FIG. 7 light microscopy images demonstrate (A) organization of HMEC into endothelial tubules having a vessel stem with lumen formation (inset, arrow) and a few short branches after 6-8 days; (B) the number and length of branches increased substantially after 11 days.

DETAILED DESCRIPTION

[0029] The present method is based on the finding that individual epithelial and endothelial cells can be used to develop tubules having fully formed lumens, the tubules being of pre-determined length, interconnectedness and geometry. The method involves providing suspended cells with contact with a gel constructed of ECM basement membrane components and growth medium at a density that allows for cell-to-cell contact, in a tubular channel to direct tubule growth. The cells in the tubular channel contact the gel around the perimeter of the channel along the longitudinal axis of the tubular channel; the cells then grow into tubule formation, including having an interior lumen.

[0030] The present method allows for the formation of tubules, including tubules of 1 cm in length or greater, having a tubule wall that consists of polarized cells which surround an interior lumen. In some embodiments, the lumen comprises a monolayer of polarized cells. Depending on the dimensions of the tubular channels, the tubules may range in cross-sectional width from about 10 microns to about 400 microns.

[0031] The approach presented herein is adapted to use cell suspensions rather than relying on embryonic explants. When the cells are injected into the pre-formed tubular channels, they re-establish cell-to-cell contact, and subsequently assemble into tubular structures, typically within 24-36 h, with lumen formation typically taking approximately 8-11 days.

[0032] The present method allows for creation of interconnected tubules, thus providing a mechanism for producing higher-ordered tubule structures. The tubules, including the interconnected structures, are suitable for use in engineered tissues, and can be used in the creation of engineered organs or can be implanted in a patient.

[0033] Thus, in one aspect, there is provided a method for forming tubules, which results in the tubules having an interior lumen. A cell suspension is placed in a tubular channel pre-formed in a gel body, with the cell suspension in the channel having contact with the gel around the perimeter of the channel along the length of the longitudinal axis of the channel, under conditions that allow for cell growth and tubule formation.

[0034] The term "cell" as used herein refers to a single cell and is also intended to include reference to a plurality of cells, including a population, culture or suspension of cells, unless otherwise indicated. Similarly, the term "cells" refers to a plurality of cells, such as a population of cells, a cell culture or a suspension of cells, but is also intended to include reference to a single cell, unless otherwise indicated. A cell suspension refers to a liquid or semi-solid culture of cells, and a continuous cell suspension refers to a cell suspension of sufficient density to allow for cell-to-cell contact once the cells are deposited on a solid support. [0035] The cells used in the present method may be any cells that are capable of forming tubules, including epithelial and endothelial cells, including epithelial and endothelial cells derived from embryonic stem cells. The cells may be primary cells, explanted cells or cells from a cultured cell line. The cells may be precursor cells, including embryonic stem cells, adult stem cells or not fully differentiated cells. The cells may also be transgenic cells that have been transfected to include one or more transgenes.

[0036] In a particular embodiment, the cells are lung cells. In another particular embodiment the cells are kidney cells, and may be kidney epithelial cells or kidney endothelial cells. Particularly, the cells may be MDCK cells or human microvessel endothelial cells (H-MEC-I).

[0037] A support or scaffold for the tubules is formed from a gel body. A gel body, as used herein, refers to a three-dimensional portion of gel, the gel comprising one or more basement membrane components and a cell growth medium.

[0038] As will be understood, basement membrane refers to an extracellular structure that anchors a cell layer, such as an epithelial layer, in vivo, and which may include a basal lamina secreted by epithelial cells and a reticular lamina secreted by other cells. Thus, in vivo, the basement membrane is an acellular support material for endothelial, epithelial, and some mesenchymal cells and is made up of a complex mix of extracellular matrix proteins, including laminins, collagens including collagen I, III and IV, entactin, perlecan, proteoglycans, as well as heparan sulphate and other glycosaminoglycans. The basement membrane often includes various growth factors.

[0039] Thus, the gel used in the present method as a support comprises one or more basement membrane components, for example, one or more of laminin, collagen I, III and/or IV, entactin, or perlecan. The gel contains sufficient number and amounts of basement membrane components to allow the cells within the tubule to polarize so as to form an interior lumen, having a luminal surface of the cells forming the surface of lumen and a baso-lateral surface of the cells on the exterior of the tubule, as is described herein. A skilled person can readily test various compositions of gel body to determine which components are required to result in formation of a tubule having a fully formed lumen, based on the present method. For example, a skilled person could make various gel body compositions, adding or deleting various basement membrane components for example laminin, collagen IV, various heparan sulfate proteoglycans and entactin.

[0040] The basement membrane components may further include one or more growth factors, for example epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), nerve growth factor (NGF), platelet-derived growth factor (PDGF), insulin-like growth factor 1 (IGF-I), transforming growth factor-betal (TGFbetal), activin, bone morphogenic growth factor-2 or -7 (BMP-2, BMP-7), glial cell line- derived neurotrophic factor (GDNF), hepatocyte growth factor (HGF), neurturin or persephin.

[0041] Basement membrane component preparations are known and are commercially available. In a particular embodiment, the gel used in the present method comprises MATRIGEL™ Matrix, available from BD Biosciences. MATRI GEL™ polymerizes at room temperature, and contains various basement membrane components and bound growth factors that are known to promote the establishment of epithelial tissues (27).

[0042] The gel body may include additional extracellular matrix components other than basement membrane components, or may be enriched for particular basement components above the levels naturally seen in basement membranes. For example, the ECM components may include collagen other than types I, III or IV collagen, or may include greater levels of type IV collagen than naturally found in basement membranes.

[0043] The gel body may also contain a peptide or glyco-peptide that comprises an ECM-mimicking sequence, for example the sequence RGD, which peptides and glyco-peptides are known to act as basement membrane substitutes and to promote the establishment of tissues, in particular epithelial tissues.

[0044] As will be understood, extracellular matrix or ECM refers to material produced by cells and secreted into the surrounding medium, which forms the non- cellular portion of animal tissues. Thus, ECM refers to the material outside cells which provides support and structure to tissue. [0045] The proportion of basement membrane components included in the gel body may vary. Typically, the concentration of basement membrane components may be from about 5% v/v (volume /volume) to about 95% v/v, from about 10% v/v to about 90% v/v, or from about 20% v/v to about 80% v/v. In particular embodiments, the concentration of basement membrane components is about 5 % v/v or greater, about 10% v/v or greater, or about 20% v/v or greater. In other particular embodiments, the concentration of basement membrane components is about 95% v/v or less, about 90% v/v or less or about 80% v/v or less.

[0046] Additional ECM components included in the gel body may range in concentration from about 0% v/v to about 90% v/v, from about 5% v/v to about 85% v/v, or from about 15% v/v to about 75% v/v. In particular embodiments, the concentration of basement membrane components is about 0 % v/v or greater, about 5% v/v or greater, or about 15% v/v or greater. In other particular embodiments, the concentration of basement membrane components is about 90% v/v or less, about 85% v/v or less or about 75% v/v or less.

[0047] In addition to basement membrane components and optionally other ECM components, the gel body also comprises cell growth medium. The cell growth medium is any medium that typically supports the growth of the cells used in the method, including specific medium for epithelial cells or for endothelial cells. Cell growth media are known in the art, and typically include various components required by the cell, such as amino acids, an energy source for example glucose and may include vitamins and hormones such as growth factors. In one particular embodiment, the cell growth medium is Dulbecco's modified Eagle's medium (DMEM).

[0048] The proportion of growth medium included in the gel body may also vary. Typically, the concentration of growth medium components may be from about 5% v/v to about 30% v/v, or from about 10% v/v to about 25% v/v.

[0049] The gel body used as a support in the present method should gel or polymerize so as to be sufficiently solid to allow for formation of the pre-formed tubular channels. Many basement membrane components, particularly proteins, such as collagen, tend to gel at room temperature. Thus, the gel body may gel without the addition of a gelling agent, depending on the concentration of particular basement membrane components such as proteins in the gel.

[0050] However, if necessary, a gelling agent may be added to the gel body to assist in gelation of the gel. Gelling agents are generally known and any gelling agent that does not interfere with cell growth and tubule formation may be used, for example gelatin, agarose, hyaluronic acid, alginate or PEG.

[0051] The gel body may include various other components if desired, provided that such components do not interfere with tubule formation. For example, the gel may include additional growth factors, enzymes or other components that promote tubule formation and lumen formation, or which may modify cell growth, differentiation or expression. For example, enzyme-conjugated hydrogels are known (28, 25, 26, 29), and such hydrogels may be included in the gel body used in the present method. For example, a hydrogel having conjugated horseradish peroxidase may be used.

[0052] The gel body is formed so as to contain channels within the gel body for tubule formation. The channels are formed so as to be encased within the gel body, such that the channel is fully encased along its length by the gel body. That is, the channel will have a longitudinal axis and will be encased around the perimeter of the channel along the longitudinal axis of the channel.

[0053] Thus, the gel body encloses one or more channels, with each channel extending entirely within the gel body.

[0054] If desired, an access passage may be included at the exterior of the gel body to permit access to one or more channels enclosed within the gel body, the access passage sized to admit a cell suspension into the channel. Thus, for example, one or both ends of the channel may be open to the exterior of the gel body, providing that the channel is enclosed within the gel body along the length of the longitudinal axis of the channel.

[0055] The channels may be of any length, shape and diameter so as to produce tubules of the desired dimensions. Generally, the channels are elongate or tubular, meaning that the channels have a larger dimension along a longitudinal axis than a cross-sectional axis. The channels may be cylindrical or substantially cylindrical, or the channels may be oval, square, rectangular, triangular or irregular in cross-section. For example, the channels may be in the range of millimetres or centimetres in length.

[0056] The channels may be in the range of about 5 microns to about 1000 microns in diameter or cross-sectional width, about 10 microns to about 1000 microns in diameter or cross-sectional width, about 15 microns to about 1000 microns in diameter or cross-sectional width, about 20 microns to about 1000 microns in diameter or cross-sectional width, about 30 microns to about 1000 microns in diameter or cross-sectional width, about 10 microns to about 500 microns in diameter or cross-sectional width, about 15 microns to about 500 microns in diameter or cross- sectional width, about 20 microns to about 500 microns in diameter or cross-sectional width, or about 30 microns to about 500 microns in diameter or cross-sectional width. It will be appreciated that the diameter or cross-sectional width is limited by the dimensions of the cells that will comprise the tubule, and so the channel will typically have a cross-sectional width that is at least as wide as one cell. A cell may typically have dimensions of about 5 to 15 microns, for example. "Cross-sectional width" refers to the smallest dimension of the cross-section of the channel or tubule, for example, when the channel is measured in a dimension that is substantially perpendicular to the longitudinal axis. If the channel is circular in cross-section, the cross-sectional width will be equivalent to the diameter.

[0057] The channels may be straight, curved, bent, spiralled or have any shape along the longitudinal axis as desired. The channels may be discrete channels or may interconnect with other channels or be branched.

[0058] The gel body may be formed in any suitable manner so as to create a channel within the gel body. Typically, the gel body is formed around a mold when the gel is in liquid form and is allowed to gel around the mold, thus forming channels in the gel body.

[0059] For example, the gel body may be formed by pouring a liquid gel into a mold, and while the gel is still liquid, a cylindrical or tubular form, for example the barrel of a needle, may be inserted so it is encased within the liquid gel. The liquid gel is then allowed to gel or polymerize with the cylindrical or tubular form in place, and once set, the cylindrical or tubular form is then removed by withdrawing it from the gel.

[0060] It will be understood that the length and cross-sectional dimensions of the cylindrical or tubular form is chosen so as to provide a tubule having the desired dimensions. For example, the form may be a cylindrical form and may be about 0.1 mm to about 0.5 mm in diameter and may be about 5 mm to about 15 mm in length.

[0061] This method of forming the channels in the gel body allows for formation of straight, unbent channels in the gel body, and thus provides for formation of tubules having a straight stem, although fine branches may form along the stem, as discussed below. Alternatively, the form may be slightly flexible, which may allow for formation of channels having a slight bend or curve, providing that the form can deform for removal from the solidified or gelled gel body without affecting the shape of the channel formed within the gel body.

[0062] Alternatively, it may be desirous to form tubule arrangements or structures having two or more interconnected stems. To achieve such structures, the gel body may be made in two parts, for example as two slabs of gel. For example, the liquid gel may be poured into a tray-shaped mold to form a slab. While the gel is still liquid, a top form having raised sections in the desired configuration to form the interconnected channels is placed on the top of the tray of liquid gel. The liquid gel is then allowed to gel or polymerize around the top form with raised sections, thus forming a slab with pre-formed indented grooves in an interconnected arrangement. A second slab formed from the liquid gel can be placed over top of the slab having the indented grooves, so as to form a channel or channels that are surrounded by the gel body on all sides of the channel. It will be appreciated that this method can also be used to form channels having a straight stem.

[0063] Once a gel body is formed having the enclosed pre-formed channels of the desired length, diameter and interconnected geometry, the cells are introduced to the channels so as to allow for tubule formation. It will be appreciated that if the approach using two slabs to form the gel body as described above is used, the cells may be added to the grooves prior to positioning of the second slab, and that the cells are thus contained within the channels once the channels are formed by the placement of the second slab. [0064] The cells are introduced to the channels as a cell suspension, with the cell suspension being of sufficient density to allow for formation of a continuous, uninterrupted tubule structure, meaning a structure that does not have gaps along its length. For example, the cells may be introduced at a sufficient density to allow for cell-to-cell contact once the cells have been deposited in the channels. "Cell-to-cell contact" means that each cell in the suspension is in physical contact with at least one other cell, so as to promote formation of a complete, continuous tubule structure.

[0065] For example, the cells may be suspended in growth media and injected or pipetted into the channel (or the groove in a slab of the gel body prior to formation of the channel by placement of the second slab) so as to fill the channel at a density that allows for the cells to form a continuous, uninterrupted tubule structure.

[0066] The gel body having the introduced cells in the enclosed channel is then incubated at conditions that allow for growth of the cells, allowing the cells to establish tubule structures. For example, the cells may be incubated at an appropriate temperature, for example 37⁰C, and at a humidity that promotes cell growth. The gel body is designed to incorporate cell growth medium, which provides the necessary growth factors and nutrients to the cells. Appropriate growth conditions for culturing various cell types, including endothelial and epithelial cells, are known in the art.

[0067] Due to the manner in which the channels are formed, so that the channels are completely surrounded or encased by the gel body containing the basement membrane components, the cells on the exterior of the tubule formation are in contact with the basement membrane components present in the gel body. This contact contributes to the polarization of the cells within the tubule, orienting the cells so that a monolayer of cells in contact with the basement membrane components survive and cells on the interior of the tubule structure undergo cell death. Thus, a tubule is formed having an interior lumen, the tubule being formed from polarized cells having an interior luminal surface and an exterior baso-lateral cell surface.

[0068] The luminal surface of the cells forming the tubule can be identified by the presence of known luminal surface markers, for example peanut agglutinin binding markers. Similarly, the baso-lateral surface can be identified by detection of the presence of known markers such as β-catenin. [0069] The present method results in a tubule having a shape determined by the channels in the gel body. However, fine branches may form or grow from the tubules, such fine branches not defined or restricted by the shape of the channels in the gel body. For example, the tubules formed from endothelial cells may readily form branches in response to the basement membrane components in the gel body, including various growth factors. In some instances, the small branches may form within 5 to 10 days of deposition of the cells.

[0070] Tubules formed from epithelial cells may also form fine branches, although such tubules may require the addition of certain growth factors to the gel body to encourage the formation of branches. For example, growth factors such as epidermal growth factor or hepatocyte growth factor may be added to the gel body to encourage branching. The fine branches may grow from the tubule stem, penetrating into the surrounding gel body.

[0071] The tubules formed according to the present method may be released from the gel body using gentle manual manipulation, and may be soaked in a recovery solution to reduce cell death due to the release procedure. The recovery solution may be an isotonic solution and may comprise a buffer in which the cells are viable.

[0072] Tubules formed by this method may be formed to natural shape and dimensions, and may be used in engineered tissues and organs. Due to the ability to control the shape, spatial orientation and interconnectedness of the tubules, it is possible to develop tubule structures and arrangements that closely mimic the tubule systems in natural organs, for example lung and kidney.

[0073] It will be appreciated that it is possible combine the present method with other tissue engineering techniques known in the art. For example, it is possible to include cells of another type other than the cells used to form the tubule in the gel body itself, for example fibroblasts or muscle cells, producing a final gel body which is formed or molded by a template which can be then be used in this method to include tubules having a lumen, thus forming an engineered tissue replacement containing various cell types and including tubules of the desired geometry. The present method may also be combined with techniques involving the use of polymer- free or polymer-containing cell sheet or cell layer forming techniques and may allow flowing nutrients in between cell sheets to improve mass transfer and survival of the cells. The present method may also be combined with bio-printing techniques which serve the same aim to improve mass transfer in between printed layers.

[0074] Also contemplated herein are kits and commercial packages for forming tubules according to the present method. The kits and commercial packages comprise a gel body enclosing one or more channels, each channel extending entirely within the gel body as described herein, such that the channel is surrounded by the gel body along the length of the longitudinal axis of the channel, and instructions for using the gel body to form tubules from endothelial or epithelial cells, in accordance with the described method.

[0075] The invention is further exemplified by the following non-limiting examples.

EXAMPLES

[0076] EXAMPLE 1

[0077] In this work, channels were formed in extracellular matrix (ECM) gels, which were subsequently filled with Madin-Darby canine kidney epithelial cells (MDCK) or human endothelial microvessel cells (HMEC- 1). These systems were used to promote the development of tubular structures originating from individual kidney epithelial cell or endothelial cell suspensions. 3 to 5 days after filling the channels with MDCK cells, light microscopy studies showed the first signs of assembly of the cell suspension into tubular structures with a diameter of 120 μm and a length of up to 1 cm. After 10 days, histological cross-sections showed the formation of a lumen lined by a monolayer of polarized epithelial cells. Similar results were obtained with endothelial cells. In addition, the endothelial tubules displayed multiple fine branches. Interconnected pre-formed channels led to the generation of simple tubular networks. A more complex pattern of tubular network was achieved by molding ECM gels through microfabricated grooved templates. Thus, these methods allow for generation of epithelial and endothelial tubules of significant length and regular anatomical shape.

[0078] Material and Methods [0079] Cells and Cell Culture

[0080] Madin-Darby canine kidney (MDCK) cells were obtained from American Type Culture Collection (Rockville, USA). MDCK cells were cultured in DMEM (Invitrogen Singapore Pte Ltd, Singapore) with 10% fetal calf serum (FCS) (Invitrogen Singapore Pte Ltd, Singapore) and 1% antibiotic-antimycotic solution (Invitrogen Singapore Pte Ltd. Singapore). Human microvessel endothelial cells (HMEC- 1) were kindly provided by Dr. E. Ades and Dr. F. Candal from the Centers of Disease Control and Prevention (Atlanta, GA, USA), and cultured in the endothelial growth medium EGM-2 (Cambrex, Walkersville, MD, USA) under 5% C0₂ at 37°C.

[0081] ECM gel Casting and Channel Formation

[0082] A plastic mold (10 mm x 10 mm x 5 mm) was pierced with a 27 G x ¹A (0.40 mm x 12 mm) needle. 500 μl of an ECM gel solution was mixed from: 400 μl of collagen solution (Inamed, Fremont, CA₅ USA) and 100 μl DMEM culture medium; 200 μl of collagen, 200 μl of MATRIGEL™ (BD Biosciences, Franklin Lakes, NJ, USA) and 100 μl of DMEM; or 400 μl of MATRIGEL™ and 100 μl of DMEM; and was subsequently dispensed into the plastic mold. Needles were left in place to form channels in the ECM gel solution once solidified. After the gel has set in the mold, the needles were retracted, and renal epithelial cells were injected. The seeded ECM gel molds were then placed in the CO₂ incubator. The biological tubules formed were released by means of a cell recovery solution according to the manufacturer's instructions (BD Biosciences, Franklin Lakes, NJ, USA).

[0083] Molding of ECM gel Surfaces Using Microfabricated Grooved Glass Templates

[0084] Molds for the channels were formed by partial cuts into 500 μm-thick glass wafers, using a DISCO DAD 3350 dicing saw with a Resinoid blade (00777- 3030-003-kup) from Advance Dicing Technologies®. Each cut was 250 μm deep and 100 μm wide (at the surface of the wafer), with a spacing of 180 μm between adjacent cuts. The sidewall angle for the grooves was measured by scanning electron microscopy (SEM) to be 8°. After the grooves were cut, the glass wafer was diced into 8 mm x 8 mm squares. A transparent container was filled with the ECM gel solution consisting of 200 μl of collagen, 200 μl of MATRIGEL™, DMEM with 10% FCS, and 1% antibiotic-antimycotic solution. The grooved template was then placed on top of the ECM gel solution (see Figure 1(A)). After gelation of the ECM gel solution, the template was removed and the cell suspension was added. Upon cell adherence, a second layer of ECM gel was introduced. After gelation for 1 h, the samples were placed in a 12-well plate, which contained DMEM and 10% FBS.

[0085] Viability/Toxicity Test

[0086] The Viability/Cytotoxicity-Kit was obtained from Invitrogen, and performed according to the manufacturer's instructions (Carlsbad, CA, USA). In brief, 0.5 μl of the stock solution was diluted with 500 μl of the respective culture medium consisting of calcein and EthD- 1 to cover the sample for 45 ruin. The specimens were investigated using an 1X71 Olympus microscope (Tokyo, Japan).

[0087] Cryosectioning, Cell Staining and Immunohistochemistry

[0088] The cells were labeled by fixing them in ice-cold ethanol for 10 min. After several rinses with phosphate-buffered saline (PBS), the samples were incubated with blocking solution containing PBS, 10% FCS and 1% bovine serum albumin (BSA) for 30 ruin. The β-catenin antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) was diluted at a ratio of 1 :100 and incubated for 2 h. After washing several times, the specimens were incubated for 45 min with the donkey-anti-mouse-IgG-FITC- conjugated secondary antibody (Jackson Immunoresearch Laboratories, West Grove, USA), which had been diluted at 1:200 in PBS containing 1% BSA. FITC-peanut agglutinin (PNA) and DAPI were obtained from Sigma- Aldrich (Singapore). The specimens were then analyzed using an 1X71 Olympus microscope. Images were taken with a digital camera, and processed using Photoshop 5.5 (Adobe Systems, San Jose, CA, USA).

[0089] Results

[0090] Formation of Epithelial Tubules in ECM gel Channels

[0091] After setting the ECM gels, the needle inserted was carefully retracted, and light microscopy revealed the formation of channels in the cast gel (Figs. 2(A)-(C)). To form tubules, the pre-formed channels were filled with epithelial cells as the needle was retracted, so that a continuous line of suspended cells was visible (Fig. 2(B)). After successful cell injection, no leakage into the surrounding gel was observed. Discontinuous suspension led to incomplete or absence of tubule formation (results not shown). After 3-5 days of culture, the MDCK cells assembled into tubule- like structures with a diameter of -100 μm (Fig. 2(D)). The diameter of the tubules did not changed significantly over the culture period of up to 14 days (Fig. 2(E)). The tubule-like structures were formed over the complete length of the pre-formed channels, and could be easily expanded into a parallel tubular system (Fig. 2(E)) or as interconnected systems (Fig. 2(F)).

[0092] To find out whether the tubule formation was determined by the ECM gel used, a pure collagen type 1 gel (Figs. 3(A), (D) and (G)), a 1:1 volume mixture of collagen type I and MATRIGEL™ (Figs. 3(B), (E) and (H)), and a pure MATRIGEL™ (Figs. 3(C), (F) and (I)) were examined. Figs. 3(A)-(C) show that in the classical 3D culture set-up, whereby cells were mixed with the gel in the absence of pre-formed channels, no tubule formation or interconnection were observed for all three gel types. When cells were injected into the pre-formed channels, confined tubule-like structures were observed in all three gel types (Figs. 3(D)-(F)). However, tubule of a larger diameter was observed in the collagen gel (Fig. 3(D)), compared to the mixed collagen- MATRIGEL™ system (Fig. 3(E)) and pure Matrigel (Fig. 3(F)). To investigate if the tubules consisted of lumen structures, histological cross-sections of the samples were examined. No regular lumen formation was observed in the collagen gel after 10 days (Fig. 3(G)), while a lumen lined by an almost single cell monolayer was found in both the mixed collagen- MATRIGEL™ system (Fig. 3(H)) and pure MATRIGEL™ (Fig. 3(D).

[0093] Cellular Organization and Proposed Mechanism Underlying Tubule Formation

[0094] Kidney epithelial cells are characterized by their polarization, which is represented by the establishment of baso-lateral and luminal surfaces. The separation between the two surfaces can be shown by the histochemical localization of specific proteins found on each surface, β-catenin is a cell adhesion protein localized at the baso-lateral cell surface, which bridges E-cadherin with the actin-cytoskeleton. Peanut agglutinin (PNA) binding is known to be found at the luminal surface of MDCK cells. We investigated the expression of these markers in the tubules obtained to assess the polarization of the cell lining (Figs. 4(A)-(F)). β-catenin expression was found in the baso-lateral surface of the epithelial cells, indicating a normal polarization behavior (Figs. 4(B) and (C)). This finding was underscored by the exclusive PNA staining at the luminal surface of the MDCK cells (Figs. 4(E) and (F)). Other studies have shown that the expression of smooth muscle actin might play an important role during tubule formation. However, no smooth muscle actin expression was detected during the late phases of tubule formation in this case (Figs. 4(H) and (I)).

[0095] One key event underlying the formation of lumen during tubulogenesis is apoptosis. Cells found in the lumen without any contact to ECM would undergo apoptosis, leading to the formation of a lumen, and hence, to tubule formation. Histological studies at different time points strongly indicated that apoptosis played a role in the formation of tubules in this approach. After 1-3 days, aggregated cells without any lumen were found (Figs. 5(A)). After 5 days, multiple, smaller lumens were observed (Fig. 5(B)). At that stage of tubulogenesis, numerous pycnotic cells were found in the center of the tubule. In contrast, the cells that were in contact with the ECM revealed normal cell morphology (Fig. 5(B₅E)). After 10 days, one major lumen was created (Fig. 5(C)).

[0096] Releasing Tubular Structures from ECM gels

[0097] Tubules were released from the ECM gel to enable them to be used in biological analysis or implantation procedures, as well as to demonstrate their integrity (Fig. 6). Tubules were exposed to a cell recovery solution, and were released by gentle manipulation with fine tweezers (Figs. 6(A) and (B)). This showed that the tubules were structurally robust. DAPI staining showed that the tubules were formed from cellular elements (Fig. 6(C)). The viability assay showed that only a few dead cells were observed after the release procedure (Fig. 6(D)). Most of the cells remained viable (Fig. 6(E)).

[0098] Formation of Endothelial Tubules in ECM gel Channels

[0099] Endothelial cells are known to form capillary networks in vitro when grown on 2-dimensional (2D) surfaces of ECM gels, such as MATRIGEL™. We wanted to investigate whether beside the tubule formation from epithelial cells, the formation of endothelial tubules could be achieved in the pre-formed channels. It was found that HMEC-I cells assembled at the linear oriented tissue stems 2-4 days after they were injected into the pre-formed channels. Simultaneously, the tissue stems began to develop small branches (Fig. 7(A)). Histological crosssections revealed the formation of well-defined lumens (with inner diameter of 40-50 μm) with lining endothelial cells from which cellular strands were originated (Fig. 7(A) inset). After 6-8 days, the structures generated were characterized by many small branches, while the tissue stem remained recognizable (Fig. 7(B)). The formation of such small branches was not observed in the case of MDCK cells.

[00100] Formation of Tubular Networks through ECM gel Molding

[00101] Beside the formation of tubular structures in the ECM gel channels, we wanted to investigate whether tubular structures made from MDCK cells could be formed in grooved ECM gel, such as a mixed MATRIGEL™ and collagen system. Tubules were not formed when the cells were just seeded and attached to the grooved structures (data not shown). However, when the cells were attached to the grooved surface and a second layer of an ECM gel was added (Fig. 1(B)), tubule formation was observed along the grooves (Fig. 1(C)). Some tubular bridges were formed between the linearly oriented tubules, most probably due to overgrowth beyond the grooved surface (Fig. l(B) and (C)).

[00102] As can be understood by one skilled in the art, many modifications to the exemplary embodiments described herein are possible. The invention, rather, is intended to encompass all such modification within its scope, as defined by the claims.

[00103] All documents referred to herein are fully incorporated by reference.

[00104] Although various embodiments of the invention are disclosed herein, many adaptations and modifications may be made within the scope of the invention in accordance with the common general knowledge of those skilled in this art. Such modifications include the substitution of known equivalents for any aspect of the invention in order to achieve the same result in substantially the same way. All technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art of this invention, unless defined otherwise.

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Claims

WHAT IS CLAIMED IS:

1. A method of forming tubules, the method comprising:

providing a gel body enclosing one or more channels with each channel enclosed entirely within the gel body, the gel body comprising basement membrane components and growth medium;

introducing a suspension of endothelial cells or epithelial cells to one or more of said channels; and

incubating the gel body containing the introduced endothelial cells or epithelial cells under conditions sufficient to permit formation of a tubule, the tubule having an interior lumen.

2. The method of claim 1 wherein the gel body further comprises an access passage extending between an outer surface of the gel body to at least one of the channels, the access passage sized to admit a cell suspension.

3. The method of claim 1 or claim 2 wherein the endothelial cells or the epithelial cells are kidney cells.

4. The method of claim 3 wherein the kidney cells are Madin-Darby Canine Kidney epithelial cells (MDCK cells) or human microvessel endothelial cells (H- MEC-I).

5. The method of any one of claims 1 to 4 wherein the basement membrane components comprise one or more of laminin, collagen I, collagen III, collagen IV, entactin, perlecan, and a heparan sulphate proteoglycan.

6. The method of any one of claims 1 to 5 wherein the gel body comprises about 5% v/v or greater or about 95% v/v or less basement membrane components.

7. The method of any one of claims 1 to 6 wherein the gel body comprises about 0% v/v to about 90% v/v extracellular matrix components in addition to the basement membrane components.

8. The method of any one of claims 1 to 7 wherein the gel body comprises from about 5% v/v to about 30% v/v growth medium.

9. The method of any one of claims 1 to 8 wherein the growth medium is Dubelcco's modified Eagle's medium.

10. The method of any one of claims 1 to 9 wherein the gel body encloses two or more interconnected channels.

11. The method of any one of claims 1 to 10 wherein the gel body further comprises at least one additional growth factor.

12. The method of claim 11 wherein the additional growth factor is epidermal growth factor, basic fibroblast growth factor, nerve growth factor, platelet-derived growth factor, insulin-like growth factor 1, transforming growth factor-beta 1, activin, bone morphogenic growth factor-2, bone morphogenic growth factor-7, glial cell line- derived neurotrophic factor, hepatocyte growth factor, neurturin or persephin.

13. The method of any one of claims 1 to 12 wherein the gel body further comprises a peptide or glyco-peptide comprising a ECM-mimicking sequence.

14. The method of any one of claims 1 to 13 wherein the gel body further comprises additional cells, wherein the additional cells are of a different cell type than the introduced endothelial or epithelial cells.

15. The method of any one of claims 1 to 14 wherein one or more of the tubules has a length of at least about 1 cm.

16. The method of any one of claims 1 to 15 wherein one or more of the tubules has a cross-sectional width of about 15 microns to about 500 microns.

17. The method of any one of claims 1 to 16 further comprising the step of removing the tubule from the gel body.

18. The method of claim 17 further comprising the step of soaking the tubule in a recovery solution.

19. A kit comprising a gel body and instructions for forming a tubule according to the method of any one of claims 1 to 18, the gel body enclosing one or more channels with each channel enclosed entirely within the gel body, the gel body comprising basement membrane components and growth medium.