WO2021080516A1

WO2021080516A1 - Method of forming a vasculature structure and a vasculature structure thereof

Info

Publication number: WO2021080516A1
Application number: PCT/SG2020/050612
Authority: WO
Inventors: Tsz Him Terry CHING; Michinao HASHIMOTO; Yi-Chin Toh
Original assignee: Singapore University Of Technology And Design; National University Of Singapore
Priority date: 2019-10-23
Filing date: 2020-10-23
Publication date: 2021-04-29

Abstract

There is provided a method of forming a vasculature structure. The method includes: providing a microfluidic mold comprising a microchannel network made of a hydrogel matrix; forming a layer of hydrogel on an inner wall of the microchannel network of the microfluidic mold to form a perfusable hydrogel network; and removing the perfusable hydrogel network from the microfluidic mold to form the vasculature structure, the vasculature structure being free-standing. There is also provided a corresponding vasculature structure formed according to the method.

Description

METHOD OF FORMING A VASCULATURE STRUCTURE AND A VASCULATURE STRUCTURE THEREOF

CROSS-REFERENCE TO RELATED APPLICATION [0001] This application claims the benefit of priority of Singapore Patent Application No. 10201909912W, filed on 23 October 2019, the content of which being hereby incorporated by reference in its entirety for all purposes.

TECHNICAL FIELD

[0002] The present invention generally relates to a method of forming a vasculature structure, and a vasculature structure formed by the method.

BACKGROUND

[0003] Design and fabrication of effective biomimetic vasculature constitutes a biologically relevant and yet unsolved challenge. At present, fabrication of biomimetic vasculatures mostly revolves around extrusion-based 3D printers, where attainable scaffolds often lack the intricate branching geometries of native microvasculature. Direct ink writing (DIW) 3D printers allows direct printing of structural materials and cell-laden materials, however, the rheology of the printing materials often limits the attainable structures.

[0004] The main challenges in fabricating organs or tissues through using 3D printing technology is to achieve mechanical, chemical, and morphological properties similar to real organs and tissues. Stereolithography (SLA), for example, is a 3D printing technique which may offer avenues to engineer biomimetic vasculatures as it provides better print resolutions and less topological restrictions than extrusion-based 3D printers. However, there has been limited demonstration to fabricate biomimetic vasculature via SLA due to the lack of suitable photocurable biomaterials with adequate mechanical and biological properties. For example, poly(ethylene glycol) diacrylate (PEGDA) and gelatin methacrylamine (GelMA) are hydrogels which may be used in SLA. Although PEGDA possesses suitable mechanical properties to support SLA printed structures, it does not favor cell adhesion and cell motility, limiting applications in bioengineering. For example, PEGDA forms a synthetic hydrogel which do not offer cells with binding sites and chemistry that more accurately mimic the in vivo environment. GelMA, on the other hand, is a natural polymer which has ideal biological properties, however, its curing kinetics and mechanical properties (e.g., support its own weight) are not favorable to SLA printing of intricate structures (vascular networks). Several groups bypass the limitation by fine tuning the concentration of synthetic and natural polymers to print intricate structures that are also favorable for cells. In other studies, 3D printing of vascular constructs containing endothelium and smooth muscle cells have been shown. However, these methods do not permit the fabrication of bifurcating channels. Also, it is cumbersome to connect these fragile hydrogel constructs to pumps for perfusion cultures.

[0005] A need therefore exists for a method of forming a vasculature structure that seek to overcome, or at least ameliorate, one or more of the above-mentioned deficiencies in conventional methods of forming a vasculature structure. It is against this background that the present invention has been developed.

SUMMARY

[0006] According to a first aspect of the present invention, there is provided a method of forming a vasculature structure, the method comprising: providing a microfluidic mold comprising a microchannel network made of a hydrogel matrix; forming a layer of hydrogel on an inner wall of the microchannel network of the microfluidic mold to form a perfusable hydrogel network; and removing the perfusable hydrogel network from the microfluidic mold to form the vasculature structure, the vasculature structure being free-standing.

[0007] According to a second aspect of the present invention, there is provided a vasculature structure comprising a perfusable hydrogel network, the vasculature structure being free-standing and formed by a method comprising: providing a microfluidic mold comprising a microchannel network made of a hydrogel matrix; forming a layer of hydrogel on an inner wall of the microchannel network of the microfluidic mold to form a perfusable hydrogel network; and removing the perfusable hydrogel network from the microfluidic mold to form the vasculature structure, the vasculature structure being free-standing.

BRIEF DESCRIPTION OF THE DRAWINGS [0008] Embodiments of the present invention will be better understood and readily apparent to one of ordinary skill in the art from the following written description, by way of example only, and in conjunction with the drawings, in which:

FIG. 1 depicts a schematic flow diagram of a method of forming a vasculature structure according to various embodiments of the present invention;

FIG. 2A depicts an exploded schematic of microfluidic molds comprising a microchannel network respectively, according to various embodiments of the present invention;

FIG. 2B illustrates a schematic for forming a layer of hydrogel on an inner wall of the microchannel network of the microfluidic mold according to various embodiments of the present invention;

FIG. 3A illustrates a schematic for forming a vasculature structure, the vasculature structure being free-standing according to various embodiments of the present invention;

FIG. 3B shows cross-sectional views illustrating the mechanism behind the polymerization of the hydrogel inside the microfluidic mold according to various embodiments of the present invention;

FIG. 3C shows cross-sectional micrographs of hydrogel tubes with different incubation time according to various example embodiments of the present invention;

FIG. 3D shows an exemplary graph showing the relationship between wall thickness and incubation time according to various example embodiments of the present invention;

FIG. 3E shows images illustrating various steps in the fabrication process for forming the vasculature structure according to various embodiments of the present invention;

FIG. 3F illustrates another conceptual image for forming a vasculature structure inside the microfluidic mold according to various example embodiments of the present invention; FIGS. 3G-3H illustrate perfusion of a fibrinogen-containing precursor into the microchannel network of the microfluidic mold, and a fibrin layer formed, respectively, according to various embodiments of the present invention;

FIG. 4A shows an image of an exemplary microfluidic mold comprising the microchannel network made of the hydrogel matrix, an image illustrating a cross-section of the planar biomimetic vasculature device, fluorescence micrographs illustrating bifurcation points of the planar device, and fluorescence micrographs illustrating channel cross-section of the microfluidic mold, according to various example embodiments of the present invention;

FIG. 4B shows images of a free standing, perfusable hydrogel network according to various example embodiments of the present invention;

FIG. 5A shows an image illustrating algorithmic generation of a 3D biomimetic vasculature using a CAD software according to various example embodiments of the present invention;

FIG. 5B shows images of the 3D biomimetic vasculature with hierarchical branching according to various example embodiments of the present invention;

FIG. 5C shows fluorescence images illustrating the cross-sections of the microchannel after alginate coating according to various example embodiments of the present invention;

FIGS. 6A-6C show images illustrating exemplary free-standing vasculature structures fabricated according to various example embodiments of the present invention;

FIG. 6D shows an image of a multi-branched hydrogel network perfused with particles suspended in mineral oil for particle image velocimetry (PIV) according to various example embodiments of the present invention;

FIG. 6E and FIG. 6F illustrate PIV summary showing vectors and velocity magnitude of top branching point and bottom branching point of the multi-branched hydrogel network of FIG. 6D, respectively;

FIG. 6G shows images of a hydrogel tubing being inflated with an angioplasty balloon according to various example embodiments of the present invention;

FIG. 6H shows images demonstrating the effects of an inflated balloon on the hydrogel tubing according to various example embodiments of the present invention; FIG. 61 shows time lapse images illustrating the deployment of a stent in hydrogel tubing according to various example embodiments of the present invention;

FIG. 7 A shows an exploded view of a microfluidic device according to various example embodiments of the present invention;

FIG. 7B shows a top view schematic of the microfluidic device and particle image velocimetry (PIV) analysis of a precursor solution when perfused into the microfluidic device, according to various example embodiments of the present invention;

FIG. 7C shows exemplary graphs summarizing the results of the PIV analysis, according to various example embodiments of the present invention;

FIG. 8A shows a setup illustrating a vessel construct under active perfusion culture using a peristaltic pump according to various example embodiments of the present invention;

FIG. 8B shows a schematic demonstrating the steps of encapsulating and seeding multiple layers of cells on the hydrogel tubing (vasculature structure), according to various example embodiments;

FIG. 8C illustrates the 2D seeding of HUVECs in the vessel lumen, according to various example embodiments;

FIG. 8D illustrate the encapsulation of SMCs within the vessel construct, according to various example embodiments;

FIG. 8E shows a confocal microscopy image of straight vessel construct seeded with HUVECs, according to various example embodiments;

FIG. 8F shows a cross-sectional confocal microscopy image of the straight vessel construct seeded with HUVECs, according to various example embodiments;

FIG. 8G shows a cross-sectional confocal microscopy image of the vessel construct encapsulated with SMCs, according to various example embodiments;

FIG. 8H shows a microscopy image of bifurcating vessel construct with four cores as depicted by the encapsulated fluorescent beads, and an image illustrating a cross- sectional microscopy image highlighting the four cores, according to various example embodiments;

FIG. 81 shows an image of a setup for continuous perfusion in an incubator, according to various example embodiments; FIG. 9 illustrates exemplary 2D and 3D microfluidic devices fabricated according to various example embodiments of the present invention;

FIG. 10A and FIG. 10B illustrates the fabrication of a three -branched network and a biomimetic branching network, respectively, of a microfluidic device according to various example embodiments of the present invention;

FIG. IOC illustrates a schematic for fabricating the microfluidic mold using 3D printing according to various example embodiments of the present invention;

FIG. 10D illustrates printed sheets of the microfluidic mold being laid out on a build plate according to various example embodiments of the present invention;

FIG. 10E and FIG. 10F illustrate silicone oil with colored dye was perfused into the channels of the three -branched network and the biomimetic branching network of the microfluidic mold for visualization according to various example embodiments of the present invention;

FIG. 11A illustrates another example microfluidic mold according to various example embodiments of the present invention;

FIG. 11B shows an image of a multi-layer planar fluidic network according to various example embodiments of the present invention;

FIG. llC illustrates images of the individual layers of the fluidic network of FIG. 11 A according to various example embodiments of the present invention;

FIG. 12A illustrates a microfluidic mold comprising a 3D fluidic network having hierarchical branching according to various example embodiments of the present invention;

FIG. 12B illustrates an exemplary assembled fluidic network according to various example embodiments of the present invention;

FIG. 12C illustrates images of hierarchical branching networks at varying channel diameter according to various example embodiments of the present invention;

FIG. 12D illustrates a plot describing the relationship between channel diameter and gap width with respect to channel diameter according to various example embodiments of the present invention;

FIG. 12E illustrates a mold member of the plurality of mold members being printed by SLA according to various example embodiments of the present invention; FIG. 12F illustrates an image of a vasculature-inspired 3D network according to various example embodiments of the present invention;

FIG. 13 illustrates exemplary microfluidic molds according to various example embodiments of the present invention; and

FIG. 14 depicts an exploded schematic of another exemplary microfluidic mold comprising a microchannel network prior to assembly, and a schematic of the microfluidic mold after assembly, according to various embodiments of the present invention.

DETAILED DESCRIPTION

[0009] V arious embodiments of the present invention provide a method of forming a vasculature structure (may also be referred to herein as a vessel structure, tubular structure, and so on) and a vasculature structure formed by the method. FIG. 1 depicts a schematic flow diagram of a method 100 of forming a vasculature structure according to various embodiments of the present invention. The method 100 comprises providing (at 102) a microfluidic mold comprising a microchannel network made of a hydrogel matrix; forming (at 104) a layer of hydrogel on an inner wall of the microchannel network of the microfluidic mold to form a perfusable hydrogel network; and removing (at 106) the perfusable hydrogel network from the microfluidic mold to form the vasculature structure, the vasculature structure being free-standing.

[0010] In various embodiments, the layer of hydrogel may be a layer formed of a natural polymer.

[0011] In various embodiments, the layer of hydrogel may comprise a polymer which is ionic, biocompatible (i.e., support cell growth), has low toxicity, sufficient mechanical properties, and having mechanical properties which is easy to tune. The polymer may be an anionic polymer or a cationic polymer. In various embodiments, the layer of hydrogel comprises alginate polymer. For example, the layer of hydrogel comprises cross-linked alginate. In various embodiments, the layer of hydrogel may be a layer comprising fibrin. In various other embodiments, the layer of hydrogel may be a layer comprising chitosan- polylysine or chitosan-TPP (e.g., which may be subjected to physical crosslinking by ionic interaction). [0012] In various embodiments, the layer of hydrogel may further comprise a bioactive material such as gelatin. The gelatin may be methacrylated gelatin (GelMA) in a non limiting example. In other embodiments, the layer of hydrogel may further comprise collagen (ColMA) or hyaluronic acid (HAMA).

[0013] In various embodiments, the above-mentioned forming a layer of hydrogel on an inner wall of the microchannel network of the microfluidic mold comprises perfusing a precursor solution comprising a first precursor into the microchannel network of the microfluidic mold, the first precursor comprising alginate (i.e., alginate macromers), and causing cross-linking of alginate by a stimulating (or polymerizing) agent on the inner wall of the microchannel network to form the perfusable hydrogel network. For example, the stimulating agent may cause ionic cross-linking of the alginate. For example, the precursor solution may be a sodium alginate solution or alginic acid sodium salt solution.

[0014] In various other embodiments, the above-mentioned forming a layer of hydrogel on an inner wall of the microchannel network of the microfluidic mold comprises perfusing a precursor solution comprising a first precursor into the microchannel network of the microfluidic mold, the first precursor comprising fibrinogen, and causing cross- linking of the fibrinogen by a stimulating agent on the inner wall of the microchannel network to form the perfusable hydrogel network. For example, fibrinogen may be employed as the first precursor to form the layer of hydrogel of the perfusable hydrogel network, the layer of hydrogel comprising fibrin. In various embodiments, the stimulating agent may be thrombin, a naturally occurring enzyme that converts fibrinogen into fibrin. [0015] In various embodiments, the method may further comprise diffusing the stimulating agent into the hydrogel matrix of the microfluidic mold for the hydrogel matrix to releasably store the stimulating agent prior to perfusing the precursor solution. The stimulating agent may be diffused into the hydrogel matrix of the microfluidic mold for the hydrogel matrix to releasably store the stimulating agent prior to perfusing the precursor solution. The hydrogel matrix may be porous such that the stimulating agent may have sufficient mobility into the hydrogel matrix by diffusion.

[0016] In various embodiments, the stimulating agent may be diffused into the hydrogel matrix of the microfluidic mold prior to assembling the microfluidic mold. For example, the hydrogel matrix of the microfluidic mold may be soaked in a solution comprising the stimulating agent (e.g., calcium chloride CaCb solution) to allow the stimulating agent (e.g., calcium ions) to diffuse into the hydrogel matrix. The microfluidic mold may be assembled thereafter.

[0017] In various embodiments, the stimulating agent comprises divalent cations in the case the layer of hydrogel comprises anionic polymer. In various embodiments, the stimulating agent comprises calcium (Ca²⁺⁾ ions. For example, when an alginate-containing precursor solution is perfused into the microchannel network of the microfluidic mold, the calcium ions within the hydrogel matrix may diffuse into the alginate-containing precursor solution, prompting ionic cross-linking starting from the interface between the precursor solution and hydrogel matrix. In other embodiments, the stimulating agent may comprise magnesium (Mg²⁺), barium (Ba²⁺), strontium (Sr²⁺) ions, or combinations thereof.

[0018] In various embodiments, the method may further comprise incubating the precursor solution in the microchannel network of the microfluidic mold for a predetermined period of time below a threshold duration of time to form the perfusable hydrogel network.

[0019] In various embodiments, the precursor solution further comprises a bioactive material; and the method may further comprise exposing the perfusable hydrogel network to ultraviolet light after removal from the microfluidic mold for cross-linking the bioactive material with the alginate polymer of the perfusable hydrogel network. In various embodiments, the bioactive material may be gelatin. In various embodiments, the bioactive material comprises gelatin methacrylate. In various embodiments, the bioactive material comprises fibronectin.

[0020] In various embodiments, the precursor solution further comprises cells, and the above-mentioned perfusing a precursor solution into the microchannel network of the microfluidic mold comprises perfusing the cells into the microchannel network of the microfluidic mold cells to encapsulate the cells within a hydrogel matrix of the perfusable hydrogel network. In other words, the method may further comprise encapsulating cells within the hydrogel matrix of the perfusable hydrogel network such that the perfusable hydrogel network of the vasculature structure is cell-laden. For example, the precursor solution (e.g., including the first precursor and/or the bioactive material) may be mixed with cells prior to perfusing the precursor solution into the microchannel network of the microfluidic mold, resulting in cells encapsulated within the hydrogel matrix (e.g., the cells may be smooth muscle cells (SMC)s which may be encapsulated within the hydrogel matrix).

[0021] In various embodiments, the method may further comprise perfusing a subsequent solution of cells suspended in cell culture media into the perfusable hydrogel network of the vasculature structure for lining an inner wall of the perfusable hydrogel network of the vasculature structure with cells, such that the cells adhere to the inner wall of the perfusable hydrogel network. For example, cells may be deposited (attached or lined) on a surface of the inner wall of the perfusable hydrogel network of the vasculature structure. For example, cells such as endothelial cells (ECs) may be seeded on the surface of the inner wall of the perfusable hydrogel network of the vasculature structure. That is, a monolayer of cells is attached on the surface of the lumen.

[0022] In various embodiments, the precursor solution may further comprise a second stimulating agent for causing cross-linking of a second precursor subsequently perfused into the microchannel network of the microfluidic mold. In a non-limiting example, the second stimulating agent may be thrombin. For example, thrombin may prompt isopeptide cross-linking of the second precursor.

[0023] In various embodiments, the method may further comprise perfusing a subsequent precursor solution comprising the second precursor into the microchannel network of the microfluidic mold, and causing cross-linking of the second precursor by the second stimulating agent. In various embodiments, the second precursor comprises an enzymatic cross-linkable material. In a non-limiting example, the second precursor comprises fibrinogen. In various embodiments, the second stimulating agent comprises thrombin such that the fibrinogen is polymerized by thrombin into a second layer of hydrogel of the perfusable hydrogel network, the second layer of hydrogel comprising fibrin.

[0024] In various embodiments, the hydrogel matrix of the microfluidic mold is formed by stereolithography. The hydrogel matrix of the microfluidic mold may be formed using material having a high spatial specificity so as to produce the microfluidic mold comprising the microchannel network with high accuracy. In other words, the material for forming the microfluidic mold is a hydrogel (porosity required for the mobility of the stimulating agent), with good spatial specificity, and is photo-crosslinkable (for printing using a stereolithography printer).

[0025] In various embodiments, the hydrogel matrix of the microfluidic mold is formed of a synthetic polymer. In various embodiments, the hydrogel matrix of the microfluidic mold is a poly( ethylene glycol) diacrylate (PEGDA) hydrogel. For example, a synthetic hydrogel, such as PEGDA, possesses properties desired for SLA printing, such as fast cure kinetics and gel times and good mechanical properties. The hydrogel matrix may comprise a concentration of PEGDA which provides good print specificity. In a non-limiting example, the hydrogel matrix of the microfluidic mold may comprise PEGDA at a concentration of about 14 %. In various other embodiments, the hydrogel matrix of the microfluidic mold may be formed of other PEG derivatives such as, but not limited to, poly(ethylene glycol) methacrylate (PEGMA), poly(ethylene glycol) dimethacrylate (PEGDMA).

[0026] In various embodiments, the microfluidic mold comprises a plurality of mold members formed of the hydrogel matrix, the plurality of mold members conformally contact one another to form the microchannel network when assembled or held together. In various embodiments, the microchannel network may comprise channel with multiple branches (e.g., bifurcating channels). The microfluidic mold may be formed using separate mold members or parts to enable easy removal of the perfusable hydrogel network, forming the vasculature structure. In various embodiments, the plurality of mold members may comprise two members (e.g., two-part mold).

[0027] The microfluidic mold may further comprise at least two rigid shells which encapsulates the plurality of mold members when assembled to ensure conformal contact between the plurality of mold members. The rigid shells may be outer shells. The plurality of mold members and at least two rigid shells may be assembled and secured using a mechanical fastener, in a non-limiting example.

[0028] In various embodiments, the above-mentioned removing the perfusable hydrogel network from the microfluidic mold further comprises releasing the plurality of mold members of the microfluidic mold.

[0029] In various embodiments, the method may further comprise perfusing a subsequent precursor solution comprising cells into the perfusable hydrogel network of the vasculature structure such that the vasculature structure is incorporated with cells. For example, the cells may be deposited (attached or lined) on a surface of the inner wall of the perfusable hydrogel network of the vasculature structure. For example, endothelial cells (ECs) may be seeded on the surface of the inner wall of the perfusable hydrogel network of the vasculature structure. That is, a monolayer of cells is attached on the surface of the lumen. The cells may be seeded, by injecting the cells (suspended in cell culture media) into the channel, and allowing the cells to settle and then attach to the surface by itself. For example, the cells may be left for about more than an hour so that the cells may settle to the base and attach itself to the surface. In various embodiments, the method may further comprise encapsulating cells within a hydrogel matrix of the perfusable hydrogel network such that the perfusable hydrogel network of the vasculature structure is cell-laden. For example, the cells may be included in the precursor solution. In other words, the precursor may be mixed with cells prior to perfusing the precursor solution into the microchannel network of the microfluidic mold, resulting in cells encapsulated within the hydrogel matrix (e.g., smooth muscle cells (SMC)s may encapsulated within the hydrogel matrix). [0030] The fabricated vasculature structure comprising the perfusable hydrogel network may be an anatomically accurate model, that is freestanding, includes multiple layers of cells, and having multiple branching.

[0031] Various embodiments provide a method of forming complex, hierarchically branching biomimetic vasculature in biologically relevant materials using a 3D printed (e.g., using SLA) mold or template. The 3D printed mold may comprise a microchannel network (or fluidic networks) corresponding to the desired vasculature structure. As described, a novel two-step method of fabricating complex biomimetic vasculatures is provided. The method comprises providing the microfluidic mold comprising the microchannel network as a template having the desired biomimetic network corresponding to the vasculature structure to be fabricated, and forming a perfusable hydrogel network (e.g., perfusable alginate hydrogels) using the microfluidic mold. The perfusable hydrogel network may be then removed from the microfluidic mold to form the vasculature structure, the vasculature structure being free-standing.

[0032] The developed technology enables fabrication of complex, biomimetic vasculatures in hydrogel capable of hosting cells. The vasculature structure may be incorporated with relevant vascular cells. Accordingly, various embodiments advantageously provide a method to engineer perfusable biomimetic vasculatures in cell laden matrices, while solving the material limitation for forming biomimetic vasculature structures using SLA printers. For example, the method as described enables fabrication of free-form, cell-laden vascular models for various applications in tissue engineering, regenerative medicine, drug screening and fundamental studies in vascular biology. The fabricated vasculature structure may adequately mimic both the anatomy and physiological function of blood vessels found in-vivo, in comparison to conventional 2D cell culture models as well as animal models which do not adequately mimic the architecture found in vivo, and limits the efficacy to gain insights into the pathophysiology of diseases such as cardiovascular diseases (CVDs).

[0033] V arious embodiments provide a method of fabricating vasculature models with high biomimetic accuracy in a fast, high-throughput way. Accordingly, an anatomically accurate microchannel network may be formed by using 3D printing of the microfluidic mold to correspond to the desired vasculature structure, and a perfusable hydrogel network formed of materials suitable for cell growth, spreading and motility may be formed using the microfluidic mold as template. The vasculature structure fabricated according to various embodiment may advantageously adequately mimic both the anatomy and physiological function of blood vessels found in-vivo. The fabricated vasculature structure may be easily interfaced into a chip, for example, for further studies.

[0034] It will be appreciated by a person skilled in the art that the terminology used herein is for the purpose of describing various embodiments only and is not intended to be limiting of the present invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms "comprises" and/or "comprising", or the like such as “includes” and/or “including”, when used in this specification, specify the presence of stated features, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof.

[0035] In order that the present invention may be readily understood and put into practical effect, various example embodiments of the present invention will be described hereinafter by way of examples only and not limitations. It will be appreciated by a person skilled in the art that the present invention may, however, be embodied in various different forms or configurations and should not be construed as limited to the example embodiments set forth hereinafter. Rather, these example embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the present invention to those skilled in the art.

[0036] In particular, for better understanding of the present invention and without limitation or loss of generality, various example embodiments of the present invention will now be described with respect to forming a vasculature structure in an alginate-containing hydrogel using the SLA-printed microfluidic mold as a template, however, it will be appreciated by a person skilled in the art that other types of perfusable hydrogel network may be used.

[0037] Molding or casting is a manufacturing process that involves shaping a liquid or molten material by using a mold. A mold comprises a hollowed-out cavity that represents the desired shape. The liquid material hardens or cures inside the mold and adopts the shape of the cavity. In order to cast a tubular structure that takes the shape of the mold, the ionic cross-linking of alginate-containing precursor by the calcium ions present in the mold may be employed. As such, the microfluidic mold may be fabricated primarily with poly(ethylene glycol) diacrylate (PEGDA) hydrogel, a porous hydrogel that permits the mobility of calcium ions via diffusion into the mold cavity.

[0038] Referring to FIG. 2A, an exploded schematic 200 of a microfluidic mold (also referred to as a microfluidic template herein) 202a and a microfluidic mold 202b (also referred to as a microfluidic template herein) comprising a microchannel network according to various example embodiments of the present invention is illustrated. The microfluidic mold 202a may define a microchannel network 210a corresponding to a two- dimensional (2D) vasculature structure, while the microfluidic mold 202b may define a microchannel network 210b corresponding to a three-dimensional (3D) vasculature structure. For example, the microfluidic mold 202a may be referred to as a planar biomimetic vasculature device herein, while the 3D microfluidic mold 202b may be referred to as a 3D biomimetic vasculature device. [0039] In various example embodiments, the microchannel network of the microfluidic mold may be made of a hydrogel matrix. In other words, the microfluidic mold may be a porous mold. The microfluidic mold may comprise a plurality of mold members 220 formed of the hydrogel matrix. The plurality of mold members conformally contact one another to form the microchannel network 210 when assembled. In various example embodiments, the hydrogel matrix forming the plurality of mold members 220 of the microfluidic mold may comprise PEGDA monomers. In other words, the hydrogel matrix may be a PEGDA hydrogel. The plurality of mold members 220 of the microfluidic mold may be formed by 3D printing. In various example embodiments, the plurality of mold members 220 may be printed using SLA. The microchannel network 210 of the microfluidic mold may be formed accurately as a template to correspond to the desired vasculature structure by using the SLA printing.

[0040] In various example embodiments, the microfluidic mold may further comprise at least two rigid shells 230 which encapsulates the plurality of mold members 220 when assembled to ensure conformal contact between the plurality of mold members. The rigid shells may be outer shells. In various example embodiments, rigid shells may be patterned with a laser cutter. For example, the rigid shell may be formed of poly(methyl methacrylate) (PMMA) sheets. The PMMA sheets may be patterned with a laser cutter to form the rigid enclosure. A cover slip 240 may be included in the microfluidic mold. [0041] FIG. 2B illustrates a schematic 250 for forming a layer of hydrogel on an inner wall of the microchannel network of the microfluidic mold according to various example embodiments of the present invention. The scale bars in FIG. 2B may be 10 mm, in a non limiting example. In various example embodiments, the layer of hydrogel may be a layer of GelMA-alginate (GM-alg) hydrogel. In various example embodiments, the GM-alg hydrogel may be formed or casted by first perfusing a calcium chloride (CaCb) solution into the microchannel network. The calcium (Ca²⁺) ions may be allowed to diffuse into the hydrogel matrix (e.g., PEGDA matrix) of the microfluidic mold for a predetermined period of time. In other words, the hydrogel matrix may be diffused with a stimulating agent, such as calcium ions in a non-limiting example, for causing cross-linking (or polymerization) (e.g., by ionic interaction) of a subsequently perfused precursor solution. The microchannel network of the microfluidic mold may be rinsed with a buffer solution. After rinsing the microchannel network, a solution of sodium GM-alg mixture may be perfused into the microchannel network of the microfluidic mold. The calcium ions within the hydrogel matrix may cause or prompt ionic cross-linking of the alginate-containing solution, forming the layer of hydrogel on the inner wall of the microchannel network of the microfluidic mold. In various example embodiments, the layer of hydrogel may be a thin layer having a thickness of about 200 pm, in a non-limiting example. The layer of hydrogel on the inner wall of the microchannel network of the microfluidic mold may form a perfusable hydrogel network. The perfusable hydrogel network may correspond to the desired vasculature structure. The perfusable hydrogel network, for example, may be formed on a chip.

Synthesis ofPEGDA precursor

[0042] In various example embodiments, 700 MW PEGDA (14 % v/v) may be mixed with HEPES buffer solution. Ruthenium (Ru)/sodium persulfate (0.2/2 mM) may be added as photoinitator. Quinoline Yellow (0.5 mg/mL) may be added as photoabsorber.

Preparation ofPEGDA mold

[0043] In various example embodiments, computer-aided design (CAD) files may be designed in RHINOCEROS ® (Robert McNeel & Associates, WA, USA). Asiga Pico 2HD (Sydney, Australia) may be employed for all 3D printed parts (corresponding to the mold members and/or rigid shells of the microfluidic mold as described hereinbefore). In various example embodiments, the PEGDA elements or parts (mold members) may be formed of PEGDA using the PEGDA precursor. The PEGDA elements may be printed using 3D printing (e.g., SLA) and may be soaked in deionized (DI) water overnight to leech out the remaining photoabsorber. Before assembling the microfluidic mold, the PEGDA parts may be sterilized by soaking in about 70 % ethanol for about an hour, and subsequently soaked HEPES buffer solution for about an hour. Prior to use, the PEGDA parts may be soaked in about 0.2 M CaCb solution for about an hour. The PEGDA elements may be then fitted into the rigid shell. In various example embodiments, mechanical fasteners were used to maintain conformal contact between the PEGDA elements to prevent leakage. In various example embodiments, a photoresin such as, Dental SG resin (Formlabs, MA, USA) in a non-limiting example, may be employed to print the rigid components of the microfluidic mold using 3D printing (e.g., SLA).

[0044] In various other example embodiments, the rigid shells may be formed of PMMA. For example, PMMA sheets (DAMA Trading Pte. Ltd, Singapore) of 1.5, 2 and 3 mm were patterned with a laser cutter (GCC LaserPro Spirit GLS, GCC innovation, California, USA) and used as rigid elements. Tubing employed may be PTFE thin wall tubing (#20 AWG, Cole Parmer, Illinois, USA). The tolerance hole for the connection on tubing on the PMMA sheets were created using hand drill (Bosch, Stuttgart, Germany) that is fitted with a twist drill bit (1.5 mm diameter, Dremel, Illinois, USA). The tubing may be press fitted into the tolerance hole, in a non-limiting example. Stainless steel socket head cap screws (ISO Metric size, M2) may be used as mechanical fasteners.

Preparation of Alg-GM precursor

[0045] In various example embodiments, the precursor solution may comprise Gelatin Methacrylate macromer (gelatin functionalized with methacrylate groups) and alginate macromer (also referred to herein as Alg-GM precursor). In various example embodiments, the freeze dried GelMA macromer (Cellink, Boston, USA) (e.g., at a concentration of about 6.7 wt %,, low viscosity alginate (Sigma Aldrich, St. Louis, Missouri, United States) (e.g., at a concentration of about 2 wt %) and a photoinitiator (e.g., about 0.25 wt % Lithium phenyl-2, 4, 6-trimethylbenzoylphosphinate (Sigma Aldrich, St. Louis, Missouri, United States) may be mixed into a buffer solution (e.g., 10 mM HEPES buffer solution) under constant stirring (e.g., at about 60 °C for about one hour). The mixture may be kept at a temperature of about 37 °C until further use. The photoinitiator may initiate the cross- linking of GelMA macromers when exposed to UV light. In other example embodiments, the photoinitiator may be about 0.5 % (w/v) 2 -hydroxy- l-(4-(hydroxyethoxy) phenyl)-2- methyl- 1 -propanone (Irgacure 2959, Sigma Aldrich, St. Louis, Missouri, United States). In yet other example embodiments, the photoinitiator may be a combination of ruthenium (Ru) and sodium persulfate (SPS).

[0046] In various example embodiments, to fabricate SMCs-laden vessel constructs, fibronectin may be added to the Alg-GM precursor (e.g., at a concentration of about 50 mg/mL). The cells may be mixed with the Alg-GM precursor prior to fabrication of the freestanding vessel constructs.

[0047] FIG. 3A illustrates a schematic 300 for forming a vasculature structure, the vasculature structure being free-standing according to various embodiments of the present invention. At 310 and 320, the microfluidic mold comprising the microchannel network made of a hydrogel matrix may be provided. As illustrated, the microfluidic mold comprises a plurality of mold members 322. The plurality of mold members 322 may be formed of the hydrogel matrix. The plurality of mold members conformally contact one another to form microchannel network when assembled. In various example embodiments, the plurality of mold members 322 may be a two-part PEGDA mold which may be assembled to form a fluidic channel (corresponding to the microchannel network as described hereinbefore). In various example embodiments, one or more tubings (e.g., a pair of PTFE tubes) 325 may be provided when assembling the plurality of mold members 322. The tubings may be connected to the subsequently formed vasculature structure 345. For example, the tubings may facilitate handling of the subsequently formed vasculature structure 345. The tubings 325 (e.g., having outer diameter of about 0.77 mm) may be fitted with O-rings (e.g., having a dimension of 1.4 mm x 0.6 mm x 0.4 mm), in a non-limiting example. In various example embodiments, the tubings 325 may be aligned with grooves present in the microfluidic mold as illustrated in FIG. 3A. For example, the grooves may be formed in one or more of the plurality of mold members 322 and/or in one or more of the rigid shells 327. The mold members 322 in the microfluidic mold may be clamped in place using a binder clip (not shown in FIG. 3A), in a non-limiting example.

[0048] At 330, the Alg-GM precursor (corresponding to the precursor solution as described hereinbefore) may be perfused into the microfluidic mold (e.g., at a flowrate of about 100 pF-min ¹) to completely fill the mold cavity (corresponding to the microchannel network of the microfluidic mold as described hereinbefore). Upon filling the cavity, the flowrate may be changed e.g., to about 40 pF-min ¹) and may be held at that flowrate for about one minute. Uncrossed-linked Alg-GM precursor may be removed to form a layer of hydrogel on an inner wall of the mold cavity (microchannel network). In a non-limiting example, HEPES buffer may be perfused through the microfluidic mold to remove the uncrossed-linked Alg-GM precursor, leaving the layer of hydrogel in the microchannel network. The layer of hydrogel forms a perfusable hydrogel network in the microchannel network of the microfluidic mold. At 340, the perfusable hydrogel network may be removed from the microfluidic mold to form the vasculature structure 345, the vasculature structure being free-standing. The freestanding hydrogel network (vasculature structure) may be exposed to UV light to crosslink the embedded Gelatin Methacrylate macromer. In various example embodiments, the freestanding hydrogel network (vasculature structure) may be cured inside the Anycubic cure machine (Shenzhen, China) for about 25 seconds. For example, the freestanding hydrogel network (vasculature structure) may be exposed to a 405nm LED light source for about 25 seconds. In other example embodiments, the freestanding hydrogel network may be placed at about 8 cm away from the light source and exposed to 1.5 W/cm² UV light (320-500 nm) for about 60 seconds.

[0049] FIG. 3B shows cross-sectional views 350 illustrating the mechanism behind the polymerization of the hydrogel inside the microfluidic mold (e.g., PEGDA mold) according to various example embodiments of the present invention. The cross-sectional views may be along A-A of the schematic 300 in FIG. 3A. As illustrated, at 352, a two- part PEGDA mold is assembled to form the fluidic channel (the microchannel network of the microfluidic mold) 353. At 354, the alginate-containing precursor may be perfused through the PEGDA mold cavity. Due to a concentration gradient, the calcium ions within the porous PEGDA mold may diffuse into the fluidic channel and simultaneously prompt ionic crosslinking of the alginate-containing precursor, starting from the interface between the precursor and the PEGDA mold. Given time, the calcium ions may diffuse radially inwards, growing the layer of cured alginate-containing hydrogel that outlines the inner wall of the PEGDA mold, as illustrated at 356. When sufficient alginate has been cured, the remaining uncured precursor may be removed by perfusing HEPES buffer solution through the microchannel network, as illustrated at 358. At 360, the cured tubular construct formed of the Alg-GM hydrogel network (corresponding to the perfusable hydrogel network as described hereinbefore) may then be removed by disassembling the microfluidic mold. At 362, the perfusable hydrogel network may be exposed to UV light to crosslink the Gelatin Methacrylate present in the matrix. In other words, the Gelatin Methacrylate macromer entrapped within the matrix of the perfusable hydrogel network may be cured by exposing it under UV light. [0050] The thickness of the wall of the perfusable hydrogel network may be controlled by varying the incubation time (IT), that is, the time precursor is left in the microchannel network of the microfluidic mold after the cavity is fully filled. In various example embodiments, the mold cavity may be rapidly filled with the precursor with a flow rate of about 100 pL/min. Once the mold cavity is fully filled, the flow rate may be changed to about 40 pF/min. Incubation time may be defined by the period where the cavity is filled to the time where HEPES buffer solution is perfused into the microchannel network of the microfluidic mold. By varying the incubation time, the thickness of the wall of the GM-alg hydrogel network (or vasculature structure) may be controlled. FIG. 3C shows cross- sectional micrographs 370 of hydrogel tube with different incubation time. The scale bar of the micrographs is 500 pm. It was observed that by varying incubation time from 0 second to about 1 minute, the wall thickness may be varied from about 200 pm to about 550 pm. When incubation time was increased to 1 minute and 30 seconds, the channels of the hydrogel network were observed to be clogged. FIG. 3D shows an exemplary graph 380 illustrating the relationship between wall thickness and incubation time. When using no IT, the vessel wall was observed to be too thin (e.g., having a thickness of about 200 pm) and the vessel collapsed under its own weight. The mechanical properties of the perfusable hydrogel network may be tuned by varying the concentration of alginate and GelMA. However, to effectively perfuse the precursor into the microfluidic mold, a precursor with lower viscosity may be used.

[0051] FIG. 3E shows images 390 illustrating various steps in fabrication process for forming the vasculature structure according to various embodiments of the present invention. To ensure conformal contact between the two-part mold, the rigid shell 327 was fabricated. In various example embodiments, grooves may be provided in the mold member and the rigid shell so that one or more tubings (e.g., rigid tubings) may be integrated into the tubular constructs (perfusable hydrogel network) in one single step. In various example embodiments, the rigid tubings may be provided with O-ring to prevent leakages during perfusion.

[0052] FIG. 3F illustrates another conceptual image for forming a vasculature structure inside the microfluidic mold (e.g., PEGDA mold) according to various example embodiments of the present invention. In various example embodiments, the Alg-GM precursor may further comprise a second stimulating agent. The second stimulating agent may be thrombin, in a non-limiting example. A subsequent precursor solution may be perfused into the microchannel network of the microfluidic mold. In various example embodiments, the subsequent precursor solution may comprise a second precursor. In various example embodiments, the second precursor may be fibrinogen. For example, fibrinogen is an enzymatic cross-linkable material which may be polymerized by thrombin. The second stimulating agent may prompt polymerization of the fibrinogen to form a fibrin hydrogel. The uncured fibrinogen-containing precursor may be removed by flushing the microchannel network with Hepes buffer solution in a non-limiting example, leaving the perfusable hydrogel network of the vasculature structure in the microfluidic mold. The perfusable hydrogel network of the vasculature structure may be then removed from the microfluidic mold. The perfusable hydrogel network of the vasculature structure may be exposed to UV light to photopolymerize the GelMA in the perfusable hydrogel network. FIGS. 3G-3H illustrate perfusion of the fibrinogen-containing precursor into the microchannel network of the microfluidic mold, and a fibrin layer formed, respectively. [0053] To visualize the formed microchannels of the perfusable hydrogel network, fluorescence beads (e.g., having a diameter D of about 10 pm) may be added to the solution of GM-alg. Fluorescence microscopy shows that the branched network (microchannel network of the microfluidic mold) was coated with a uniform layer of GM-alg hydrogel encapsulating the fluorescence beads. FIG. 4A shows an image 410 of an exemplary microfluidic mold comprising the microchannel network made of the hydrogel matrix (e.g., a fabricated planar biomimetic vasculature device), an image 420 illustrating a cross- section of the planar biomimetic vasculature device, fluorescence micrographs 430 illustrating bifurcation points of the planar device, and fluorescence micrographs 440 illustrating channel cross-section of the microfluidic mold. Scale bars of images 410 and 420 are 10 mm, while scale bars of images 430 and 440 are 200 pm. Fluorescence micrographs 440 includes white arrows indicating the boundary of the layer of hydrogel on the inner wall of the microchannel network (e.g., alginate boundary) which forms the perfusable hydrogel network.

[0054] The perfusable hydrogel network (e.g., GM-alg network) may be then removed from the microfluidic mold or template to form a free-standing perfusable hydrogel network. FIG. 4B shows images 450 and 460 of a free standing, perfusable hydrogel network (e.g., perfusable alginate network). Scale bars of images 450 and 460 are 10 mm. [0055] In various example embodiments, an algorithm may be developed for the design of a 3D biomimetic vasculature using a software application such as Grasshopper® to generate the 3D models (Fig. 3 A). FIG. 5 A shows an image 510 illustrating algorithmic generation of a 3D biomimetic vasculature using a CAD software. The same procedure as described above may be employed to coat the inner wall of the microchannel network of the microfluidic mold with beads-laden alginate. FIG. 5B shows images 520, 530 and 540 of the 3D biomimetic vasculature with hierarchical branching. The image 540 illustrates a magnified view of the 3D biomimetic vasculature after alginate coating. Scale bars of images 520 and 530 are 10 mm, while the scale bar of image 540 is 1 mm. FIG. 5C shows fluorescence images 550 and 560 illustrating the cross-sections of the microchannel after alginate coating. Scale bars of images 550 and 560 are 200 pm. The developed method circumvents the availability of photocurable biomaterials in SLA. The biocompatibility of the alginate hydrogels may be tuned by blending with other bioactive polymers.

[0056] FIGS. 6A-6C show images illustrating exemplary free-standing vasculature structures fabricated according to various example embodiments of the present invention. More particularly, hydrogel hollow tubings of the vasculature structures exhibit the following structure: single straight vessel (FIG. 6A), bifurcating vessel (FIG. 6B), and multiple branching vessel (FIG. 6C). As illustrated, according to various embodiments, anatomical accuracy (e.g., multiple branching) may be incorporated to the vessel models. [0057] The vessel constructs (corresponding to the vasculature structure) may be applied for the assessment of mechanisms and therapeutic interventions in the blood vessel. For instant, blood flow in complex vascular geometries (such as, stenosis, aneurisms and bifurcations) induces changes in haemodynamic forces (such as, pressure, flow and shear stress) that are known to govern endothelial activation and SMC proliferation. By using an alginate-GelMA mixture, a transparent vessel constructs amenable for particle image velocimetry (PIV) analysis was fabricated. The fabricated vessels may be fairly transparent and may be perfused with particles to visualize flow within the vessel. For example, PIV may be applied to the multi-branched vessel to gain better insights on the flow vector field which may then be correlated to models incorporated with cells to understand structural- functional relationship. FIGS. 6D-6F illustrate a multiple branching network which was fabricated and perfused with particles to visualize the dynamics of fluid flow across the multiple branching network. More particularly, FIG. 6D shows a micrograph image 610 of a multi-branched hydrogel network perfused with particles suspended in mineral oil for PIV. FIG. 6E and FIG. 6F illustrate PIV summary showing vectors and velocity magnitude of top branching point and bottom branching point of the multi-branched hydrogel network of FIG. 6D, respectively.

[0058] By designing the mold to incorporate a constriction in the channel, a vessel construct with stenosis was fabricated, as illustrated in FIGS. 6G-6H. The model was subjected to angioplasty balloon to enlarge the stenosis. More particularly, FIG. 6G shows micrograph images of a hydrogel tubing being inflated with a 2 mm angioplasty balloon. FIG. 6H shows micrograph images demonstrating the effects of the inflated balloon on a stenosis vessel construct. The stenosis model was also subjected to the insertion of a stent at the location of the constriction. FIG. 61 shows micrograph time lapse images illustrating the deployment of a stent in the stenosis vessel construct. These experiments highlighted an important use case for this model to assess therapeutic intervention such as drug eluting balloons or stents in an anatomically accurate model incorporated with ECs and SMCs. [0059] A simplified microfluidic experiment was performed to gain a better understanding on the parameters that would affect the rate of ionic curing of alginate when perfused through the molds. Various parameters such as, concentration of CaCF, wt % of alginate present in the precusor solution, flowrate of the precursor in microfluidic mold, and v/v % of PEGDA concentration used to fabricate the mold were considered. FIG. 7A shows an exploded view 710 of a microfluidic device used in a material studies. First, in the example experiment, a PEGDA hydrogel block 710 was 3D printed with different concentrations of PEGDA. It was then soaked in varying concentration of CaCF solution before fitted into a slip 715 having a fluidic channel, and the rigid shell 720 in the example experiment. The slip 715 was fabricated using polydimethylsiloxane (PDMS) by first casting a thin film in a petri dish, and then cutting out the desired pattern. Precursor solutions with varying alginate concentration was perfused into the fluidic channel at varying flowrates. To help visualize the curing of the precursor within the microfluidic device, fluorescent tracing beads were mixed in with the precursor solution. FIG. 7B shows a top view schematic 720 of the microfluidic device and particle image velocimetry (PIV) analysis 730 of the precursor solution when perfused into the microfluidic device. When the precursor was perfused into the channel (x-direction), it was observed that the fluorescent beads closest to the PEGDA interface were instantly immobililized upon contact, indicating that they were trapped in the cross-linked alginate matrix. As time progresses, it was observed that the layer of immobilized fluorescent beads grew thicker in the y-direction. The PIV analysis 730 of the fluorescent beads at a specific time point highlighted a layer of stationary beads at the interface of the PEGDA. Results of the study is summarized in FIG. 7C. More particularly, FIG. 7C shows exemplary graphs 740 summarizing the results of the PIV analysis. The triangular scatter plot in all the graphs represented the thickness (y-direction) versus time trend when using the following parameters: PEGDA blocks soaked in 0.2 M CaCb, precursor with 2 wt % alginate, flowrate at 11 pL/mi n, and PEGDA block printed with 18 v/v % PEGDA.

[0060] According to Fick’s law of diffusion, a simplified equation to model the diffusion distance of particle across a concentration gradient may be expressed with the following power function model: y = 2 Dt)^~ Equation (1) where y is the diffusion distance of the calcium ions, D is the diffusion coefficient [m-s ¹], and t is time. As expected, the plots representing the diffusion of calcium ions across the channel length (y) can be fitted (average R² value of 0.995) to a power function model. It is noted that the key parameters to vary the rate of diffusion distance was the concentration of CaCb and percentage of alginate. Higher concentration of CaCb led to a more rapid rate of diffusion distance, presumably because of a higher concentration gradient of calcium ions. Lower percentage of alginate led to a more rapid rate of diffusion distance, presumably because a higher percentage alginate led to a denser matrix that obstructed the diffusion of calcium ions. It is noted that varying the flowrate did not result in a noticeable change in the rate of diffusion. This may be due to the no-slip condition in fluid dynamics where the fluid velocity at the fluid-solid boundaries is equal to zero. Lastly, v/v % of the PEGDA block resulted in a small change in the rate of diffusion distance. [0061] Accordingly, a simple way to efficiently control the thickness of the wall is by varying the incubation time of the precursor inside the mold (duration between the steps 354 to 358 as described with respect to FIG. 3B). When the desired thickness is formed, the uncured alginate precursor may be rapidly flush by perfusing HEPES buffer solution to stop the construct from growing thicker.

Cell culture and Maintenance

[0062] In various example embodiments, Human Umblical Vein Endothelial Cells (HUVEC) (Lonza, Basel, Switzerland) were cultured in an incubator maintained at a temperature of about 37°C with about 5% CO2. The (EC) cells were supplemented with EGM™-2 endothelial cell growth media (Lonza, Basel, Switzerland). Passage was conducted at about 70% to about 80% confluency and only cells with passage numbers between 3 and 10 were used for experiments, according to various example embodiments. In various example embodiments, Human Aortic smooth muscle cells (Lonza, Basel, Switzerland) were cultured in an incubator maintained at about 37°C, with about 5% CO2. The (SMC) cells were supplemented with SmGM™ growth media (Lonza, Basel, Switzerland). Passage was conducted at about 70 % to about 80 % confluency and only cells with passage numbers between 3 and 10 were used for experiments.

Cell seeding

[0063] In various example embodiments, the HUVECs were seeded into the vessel constructs (perfusable hydrogel network of the vasculature structure) by first filling a silicone tubing with about 20 pL to about 30 pL of cell suspension (e.g., 5 x 10⁶ cells per mL) using a syringe (e.g., 1 ml, syringe). The vessel construct was incubated for about 30 mins at about 37 °C to facilitate cell adhesion. After about 30 mins, the vessel construct was flipped 180°, where another batch of cell suspension (e.g., about 20 pL to about 30 pL of suspension, 5 x 10⁶ cells per mL) were perfused through the channel with a 1 mL syringe. The vessel construct was incubated overnight before commencing active perfusion inside the incubator (e.g., at about 4.5 pL-min ¹). LIG. 8A shows a setup illustrating a vessel construct under active perfusion culture according to various example embodiments of the present invention. For example, an active perfusion culture may be performed using a peristaltic pump.

[0064] FIG. 8B shows a schematic 810 demonstrating the steps of encapsulating and seeding multiple layers of cells on the hydrogel tubing (vasculature structure), according to various example embodiments. Multiple layers of cells, such as endothelial cells (ECs) and smooth muscle cells (SMCs) may be provided in the vasculature structure. Two different techniques may be employed to incorporate cells in the vasculature structure, (1) the direct 2D seeding of ECs on the surface of the vessel lumen (corresponding to the inner wall of the perfusable hydrogel network of the vasculature structure), as illustrated in FIG. 8C, and (2) the encapsulation of SMCs within the vessel matrix (corresponding to the matrix of the perfusable hydrogel network of the vasculature structure), as illustrated in FIG. 8D. More particularly, FIG. 8C illustrates the 2D seeding of HUVECs in the vessel lumen, and FIG. 8D illustrate the encapsulation of SMCs within the vessel construct. [0065] In various example embodiments, using an alginate-GelMA blend to fabricate the vascular constructs, a 2D seeding of human umbilical vascular ECs (HUVECs) was able to be performed in the vessel lumen, as illustrated in FIGS. 8E-8F. More particularly, FIG. 8E shows a confocal microscopy image of straight vessel construct seeded with HUVECs at day 3. For example, Sytox and F-Actin were used as the fluorescent stains in the immunofluorescent (IF) staining employed. The scale bar of the image is 500 pm. By day 3, the HUVECs were able to form a confluent layer lining the lumen, as illustrated in FIG. 8F. More particularly, FIG. 8F shows a cross-sectional confocal microscopy image of the straight vessel construct seeded with HUVECs at day 3. The scale bar of the image is 200 pm. Next, to demonstrate the ability to encapsulate SMCs within the vessel constructs, SMCs were mixed with the precursor solution prior to the fabrication of the tubular constructs which resulted in a SMC-laden vascular structure. The encapsulated SMCs was able to be maintained for 21 days under active perfusion and those SMCs maintained its phenotype characterized by the expression of a-smooth muscle actin (a-SMA). FIG. 8G shows a cross-sectional confocal microscopy image of the vessel construct encapsulated with SMCs. The scale bar of the image is 500 pm.

[0066] To demonstrate the possibility of fabricating vessel constructs having multiple cores, a bifurcating vessel construct with four-cores was molded by sequential perfusion of four different precursors into the mold. This was visualized by the fluorescent beads of different color encapsulated within the different cores (e.g., layer 2, 3, 4) in FIG. 8H. More particularly, FIG. 8H shows a microscopy image 880 of bifurcating vessel construct with four cores as depicted by the encapsulated fluorescent beads of different colors, and image 885 illustrating a cross-sectional microscopy image highlighting the four cores. The scale bar of the images 880 and 885 are 1 mm and 500 pm, respectively. This strategy may be employed to mimic the multilayer vessel architecture found in-vivo (i.e., multiple layers consisting of fibroblasts, SMCs and ECs). Accordingly, various example embodiments provide a fabrication technique capable of fabricating vessel constructs that is (1) freestanding, (2) bifurcating, and (3) having up to four-cores.

[0067] FIG. 81 shows an image of a setup for continuous perfusion in an incubator. Accordingly, the fabrication of freestanding, biomimetic vessels with alginate-containing materials is demonstrated according to various example embodiments. As described above, straight vessel, bifurcating vessel, and multiple branching vessel were fabricated with alginate-containing precursor. One advantage of using the approach as described according to various example embodiments is the ability to incorporate other bioactive materials. While alginate is a biocompatible material, it does not support the attachment, spreading, and motility of cells. As such, other bioactive materials such as GelMA and/or fibrinogen may be included in the precursor to form the hydrogel comprising GelMA and/or Fibrin. The possibility of incorporating relevant vascular cells in the biomimetic vessel construct has been explored above. Native blood vessel found in-vivo composed of a multilayer architecture composing of endothelial cells (ECs), smooth muscle cells (SMCs), fibroblasts, and extracellular matrix (ECM) may be included in the vasculature structure.

Fabrication of micro fluidic mold

[0068] According to various example embodiments, a matrix encapsulating fluidic (or microchannel) networks may be deconstructed into readily printable pieces and assembled together to form the fluidic (or microchannel) networks. The developed method may be compatible with existing 3D printers such as SLA printers and applicable to fabricate complex 3D microfluidic constructs in polymers and hydrogels. In various example embodiments, SLA may be used to fabricate microchannels with higher resolution (e.g., about 20 mih) than other 3D printing techniques (e.g., about 350 pm in fused deposition modelling (FDM) and about 300 pm in polymer jetting (PJ)). The finished surfaces of the models created by SLA is smoother than those by FDM, which is advantageous to fabricate fluidic channels with controlled dimensions.

[0069] According to various example embodiments, the entire fluidic device may be deconstructed into 3D printable elements (a plurality of mold members), instead of printing a monolithic device in a single step of 3D printing. The individual elements (mold members) may be printed as independent parts using hydrogels, and subsequently assembled to form the fluidic networks. The deconstructed parts may be printed with a rigid material and/or an elastomeric material. The rigid parts may be used as an external enclosure and provided structural support for the entire assembly of the microfluidic mold. The elastomeric parts encapsulated the design of microchannels. The elasticity of the material allowed forming conformal contacts with other elements to create leak-free microchannels. 2D and 3D fluidic networks may be fabricated with high anatomical accuracy, which is difficult to achieve when printed as monolithic devices.

[0070] Various embodiments of the present invention design and fabricate building blocks of microchannels each of which are printable by SLA. By design, a matrix encapsulating fluidic networks may be deconstructed into simple and printable pieces (plurality of mold members). The pieces may be independently and subsequently assembled together to form the fluidic (or microchannel) networks. A rigid material may be used to print pieces for external enclosure, and an elastomeric material to print pieces containing microchannels. The elasticity of the channel-containing pieces allowed forming conformal contacts to avoid leakage of the flow.

[0071] Referring to FIG. 9, 2D and 3D microfluidic devices may be fabricated according to various example embodiments of the present invention. In various example embodiments, for the fabrication of 2D fluidic network devices 910, the microfluidic devices may be deconstructed into four segments: two elastomeric sheets containing the intended geometry of microchannels, and two rigid sheets located at the top and bottom of the elastomer. On the elastomeric sheet, gutters bearing the pattern of the microchannels may be included. The void spaces of the gutter formed the fluidic channels. The elastomeric layers were made to form conformal contacts between them and with the surfaces of the rigid enclosure. Conformal contact was ensured using mechanical fasteners and prevented the leakage of the fluid from the channels. It is noted that deconstructing the fluidic channel into separate sheets ensured the fabrication of the channel. The layers with the gutters were the terminal layer to be printed, and it was not necessary to print overhang layers below the gutters.

[0072] In various example embodiments, for the fabrication of 3D fluidic (microchannel) network devices, a first approach 920 to fabricate 3D fluidic networks may be by stacking sliced elastomeric sheets, while a second approach 930 to fabricate 3D fluidic networks may be by assembling elastomeric blocks with chamfers. In the first approach 920, complex 3D fluidic networks may be deconstructed by slicing the matrix and void spaces of microchannels to thin and printable sheets of elastomers. To illustrate the principles, a network of microchannels forming hierarchical branches was designed. In the cross-sectional view, the fluidic network was deconstructed into a pair of rigid shells and a set of elastomeric sheets containing apertures. The CAD of the entire device (i.e. the matrix and the channels) was redrawn into the thin sheets of equal thickness (e.g., thickness of about 2 mm), each of which contained different patterns of microchannels as apertures of gutters. All sheets were printed in an elastomer and assembled in a rigid enclosure. The channels embedded in the elastomeric sheets were aligned to form a network of channels (microchannel network). Deconstruction of the entire matrix into the printable slices may similarly alleviate the challenges due to inadvertent polymerization of the elastomers and the difficulty to evacuate uncured elastomers. Dividing the rectilinear block into the sheets offered an advantage to reduce the time required for SLA printing.

[0073] In the second approach 930, microchannels may be defined by the chamfered edges of 3D printed blocks (mold members). To illustrate the principle, a rectilinear lattice network was designed. The entire microchannels may be deconstructed into a pair of rigid shells and a set of eight elastomeric cubes with chamfered edges, in a non-limiting example. These cubes were interfaced with the rigid enclosures, and the void spaces were formed in the regions with the chamfered edges. When the geometry permitted, the rigid shells were patterned with the gutters to ensure the formation of microchannels.

[0074] As described, the approaches may be based on the deconstruction of the microfluidic device into simple and printable parts or mold members (e.g., sheets with simple geometries of apertures, and cubes with chamfered edges). The printability of the simple parts may circumvent the difficulty to fabricate microchannels by SLA 3D printing of monolithic matrices.

[0075] In various example embodiments, deconstruction of monolithic microfluidic devices allowed fabricating 2D microchannels with different level of complexity. FIG. 10A and FIG. 10B illustrates the fabrication of a three -branched network and a biomimetic branching network, respectively, of a microfluidic device. The scale bar of the images is 10mm. The microfluidic device may be first deconstructed into a combination of elastomeric and rigid elements. In various example embodiments, the elastomeric sheets may be printed using a SLA printer while poly( methyl methacrylate) (PMMA) sheets may be patterned with a laser cutter to form the rigid enclosure. A cover slip may be included in the assembly for microscopy. As a result, a three -branched network and a biomimetic branching network is fabricated. The elastomeric sheet (e.g., bottom elastomeric sheet) with gutters may be printed with two exposure with each layer height set to about 0.60 mm, as illustrated in FIG. IOC. FIG. 10D illustrates both sheets being laid out on the build plate. The scale bar of the image is 10mm. FIG. 10E and FIG. 10F illustrate silicone oil with colored dye was perfused into the channels of the three -branched network and the biomimetic branching network for visualization. The scale bar of the images is 10mm. The time lapse images suggested the differing rate of perfusion across different branches. The fluid occupied the short and wide branches before the long and narrow branches. Such observation illustrates the reason for the difficulty to evacuate photoresins from the multi- branched network in conventional fabrication technique where the purging fluid favored the path of the least resistance. The channels exhibited sharp and well-defined boundaries, verifying that the network was well formed by the conformal contact between the elastomeric sheets.

[0076] A benefit of this approach is that it does not require the material to possess light attenuating properties due to the elimination of all overhanging structures. In an exemplary demonstration, photoresin with no addition of photoabsorbers was employed. This also meant that each layer thickness (as determined by the distance between build plate and the container film (e.g., (Fluorinated ethylene propylene) that contains the photoresin) may be increased substantially to increase the speed of print. In the demonstration, the sheet with gutters had a design height of 1.20 mm. By setting each layer thickness to 0.6 mm, only two exposure was required. The increase in layer thickness also resulted in a reduction of printing time. The time required to print both top and bottom sheets were less than one minute. Another benefit of this approach is the improvement in attainable channel dimensions. For example, the three-branched network with 500 pm x 500 pm cross-section was not successfully fabricated when printed as a monolithic entity. By deconstructing the devices into printable pieces, the attainable channel dimensions were also substantially reduced. Microchannels with design width and height of 100 pm and 600 pm, respectively, was able to be printed. The measured lateral width of the three -branch channel after assembly was about 192 pm, according to various example embodiments. The design dimensions of the biomimetic vasculature had width ranging from about 300 pm to about 100 pm and designed height of about 600 pm. The measured width of the channels ranged from about 389 to about 120 pm. The discrepancy between design width and measure width may be attributed to the elastomeric properties of the material. However, various example embodiments advantageously provide a quick alternative to fabricate planar, multiple branching networks of microchannels with a substantial improvement to the attainable channel dimensions. The deconstruction of the devices into printable parts obviates the need for light-dose optimization and use of photoabsorbers. Existing SLA printer may be readily used for 3D printing of various 2D microchannels.

[0077] As described, multi-layer planar fluidic networks may be fabricated. Referring to FIG. 11A, the fluidic network may be deconstructed into sliced segments with equal thickness along the same direction, encapsulated by an external rigid shell that provides compressive stress onto the elastomeric slices. FIG. 1 IB shows an image of the multi-layer planar fluidic network. An Aqueous solution containing red dyes may be perfused into the network for visualization. FIG. 11C illustrates images of the individual layers of the fluidic network of FIG. 11A. The rigid elements maintained conformal contacts between the slices to prevent leakage.

[0078] As described, 3D fluidic networks may be fabricated. The hierarchical branching was designed to have one channel with the diameter of about 1000 pm, four channels with the diameter of about 800 pm and 16 channels with the diameter of about 300 pm. Referring to FIG. 12A, using the elastomeric nature of the channel, the dimension of the channel may be manipulated after assembly. More particularly, FIG. 12A illustrates a microfluidic mold comprising a 3D fluidic (or microchannel) network having hierarchical branching. FIG. 12B illustrates an assembled fluidic network. FIG. 12C illustrates images of varying channel width of the hierarchical branching networks with respect to varying the distance between the jaws as illustrated in FIG. 12A. For example, varying the distance between the jaws (wj) allows the channel width ( ny) to be manipulated (FIG. 12A-C). FIG. 12D illustrates a plot describing the relationship between channel diameter d and gap width with respect to d. The channel width may be reduced as much as two-fold (e.g., 1.08 to 0.50 mm; 0.90 to 0.40 mm; 0.46 to 0.33 mm). Dynamic manipulation of the dimension of microchannels may find applications for dynamic cell cultures that would require real-time actuation of the device.

[0079] The same print strategy was applied for the fabrication of random, multi-scale networks. An algorithm may be used to model a randomly branched network to fabricate a vasculature-inspired network of microchannels. The algorithm was programmed to create random branching from one single start point to an end point within a confined lobe -like space, as illustrated in FIG. 5A. The generated model was sliced, 3D printed and assembled. The vasculature inspired microchannels exhibited multi-scale branching, where a channel of a larger diameter (e.g., about 2 mm) branched into a series of smaller channels diameter (e.g., about 500 pm). The demonstration to 3D print complex fluidic networks validated the merits of the 3D printing approach according to various embodiments of the present invention.

[0080] FIG. 12E illustrates a mold member of the plurality of mold members being printed by SLA. FIG. 12F illustrates an image of a vasculature-inspired 3D network. [0081] The use of elastomers with chamfers to fabricate a series of 3D fluidic networks was investigated. This approach involves the exclusion of specific topologies in a 3D objects to form channel voids. Referring to FIG. 13, a rectilinear lattice of microchannels, a network of curved microchannels, a helical microchannel network and a serpentine mixer were fabricated. For a rectilinear lattice fluidic network, eight elastomeric cubes with chamfered edges and a pair of rigid shell were printed. A separate elastomeric gasket was printed to facilitate in the sealing between the two rigid shells. The assembly of the device was achieved with mechanical fasteners. The resulting network had a total of 54 branches and 36 nodes (circles). Well-defined boundary lines of the channels may be observed, indicating that the elastomeric cubes formed conformal contact with the rigid shell and prevented the leakage of fluids. A similar design was considered to fabricate a fluidic network consisting of curved 3D microchannels. In various example embodiments, twelve wedge-shaped elastomeric elements, an elastomeric gasket and two dome-shaped rigid shells were printed. The network of microchannels having 26 branches and 9 nodes was fabricated. The same principle was applied to fabricate devices such as helical channels and 3D serpentine mixer. The helical channel may comprise an elastomeric cylinder (designed with grooves that spiral around its circumference), a rigid cylindrical shell, two printed elastomeric gasket and two patterned PMMA sheets. A 3D serpentine mixer may comprise six elastomeric blocks with chamfered sides at selected edges, a pair of rigid shell and an elastomeric gasket. Colored aqueous solutions were perfused into the device and the mixing of the solutions were observed. As these 3D geometries of microchannels have been fabricated using respective methods, the versatility of the proposed approach to attain diverse range of complex fluidic networks was demonstrated.

[0082] Accordingly, 3D printing of complex fluidic network is demonstrated using SLA printers. A complex network of microchannels were deconstructed into simple and printable elements, 3D printed, and assembled to provide the fluidic device as the microfluidic mold, according to various embodiments. Various embodiments overcome limitations of attainable complexity and channel dimensions when channels were printed as a monolithic entity; namely, (1) the undesired polymerization leading to the occlusion of channel void and (2) the challenge in evacuating multiple -branch networks.

[0083] FIG. 14 depicts an exploded schematic of another exemplary microfluidic mold comprising a microchannel network prior to assembly, and a schematic of the microfluidic mold after assembly, according to various embodiments of the present invention.

[0084] While embodiments of the invention have been particularly shown and described with reference to specific embodiments, it should be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the scope of the invention as defined by the appended claims. The scope of the invention is thus indicated by the appended claims and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced.

Claims

CLAIMS What is claimed is:

1. A method of forming a vasculature structure, the method comprising: providing a microfluidic mold comprising a microchannel network made of a hydrogel matrix; forming a layer of hydrogel on an inner wall of the microchannel network of the microfluidic mold to form a perfusable hydrogel network; and removing the perfusable hydrogel network from the microfluidic mold to form the vasculature structure, the vasculature structure being free-standing.

2. The method of claim 1, wherein the layer of hydrogel is formed of a natural polymer.

3. The method of claim 1 or 2, wherein the layer of hydrogel comprises alginate polymer.

4. The method of claim 3, wherein said forming a layer of hydrogel comprises perfusing a precursor solution comprising a first precursor into the microchannel network of the microfluidic mold, the first precursor comprising alginate, and causing cross-linking of alginate by a stimulating agent on the inner wall of the microchannel network to form the perfusable hydrogel network.

5. The method of claim 4, further comprising diffusing the stimulating agent into the hydrogel matrix of the microfluidic mold for the hydrogel matrix to releasably store the stimulating agent prior to perfusing the precursor solution.

6. The method of claim 4 or 5, wherein the stimulating agent comprises divalent cations.

7. The method of any one of claims 4 to 6, wherein the stimulating agent comprises calcium ions.

8. The method of claim 4, further comprising incubating the precursor solution in the microchannel network of the microfluidic mold for a predetermined period of time below a threshold duration of time to form the perfusable hydrogel network.

9. The method of any one of claims 4 to 8, wherein the precursor solution further comprises a bioactive material, and the method further comprising exposing the perfusable hydrogel network to ultraviolet light after removal from the microfluidic mold for crosslinking the bioactive material with the alginate polymer of the perfusable hydrogel network.

10. The method of claim 9, wherein the bioactive material comprises gelatin.

11. The method of claim 9, wherein the bioactive material comprises gelatin methacrylate.

12. The method of claim 9, wherein the bioactive material comprises fibronectin.

13. The method of any one of claims 4 to 8, wherein the precursor solution further comprises cells, and said perfusing a precursor solution into the microchannel network of the microfluidic mold comprises perfusing the cells into the microchannel network of the microfluidic mold cells to encapsulate the cells within a hydrogel matrix of the perfusable hydrogel network.

14. The method of claim 4, further comprising perfusing a subsequent solution of cells suspended in cell culture media into the perfusable hydrogel network of the vasculature structure for lining an inner wall of the perfusable hydrogel network of the vasculature structure with cells.

15. The method of claim 4, wherein the precursor solution further comprises a second stimulating agent for causing cross-linking of a second precursor subsequently perfused into the microchannel network of the microfluidic mold.

16. The method of claim 15, further comprising perfusing a subsequent precursor solution comprising the second precursor into the microchannel network of the microfluidic mold, and causing cross-linking of the second precursor by the second stimulating agent.

17. The method of claim 14 or 16, wherein the second precursor comprises an enzymatic cross-linkable material.

18. The method of claim 17, wherein the second precursor comprises fibrinogen.

19. The method of claim 17, wherein the second stimulating agent comprises thrombin such that the fibrinogen is polymerized by thrombin into a second layer of hydrogel of the perfusable hydrogel network, the second layer of hydrogel comprising fibrin.

20. The method of any one of claims 1 to 19, wherein the hydrogel matrix of the microfluidic mold is formed by stereolithography.

21. The method of any one of claims 1 to 20, wherein the hydrogel matrix of the microfluidic mold is formed of a synthetic polymer.

22. The method of any one of claims 1 to 21, wherein the hydrogel matrix of the microfluidic mold is a poly( ethylene glycol) diacrylate (PEGDA) hydrogel.

23. The method of any one of claims 1 to 22, wherein the microfluidic mold comprises a plurality of mold members formed of the hydrogel matrix, the plurality of mold members conformally contact one another to form the microchannel network when assembled together.

24. The method of claim 23, wherein said removing the perfusable hydrogel network from the microfluidic mold further comprises releasing the plurality of mold members of the microfluidic mold.

25. A vasculature structure comprising a perfusable hydrogel network, the vasculature structure being free-standing and formed according to the method of any one of claims 1 to 24.