A continuous fibrous support barrier for engineered vascular networks

Abstract

Disclosed herein are devices including one or more conduits defining a luminal space, the luminal space being defined by an inner wall of a fibrous basement membrane material, and methods of making and using the same. By utilizing electrospinning and suitable sacrificial materials, vascular networks surrounded by a continuous fibrous membrane can be produced. Such vascular networks are highly advantageous because they can withstand higher pressures than other artificial vascular networks, making them suitable for in vivo applications where high pressures are present. Furthermore, such vascular networks can be designed to reduce or eliminate the immunogenicity of cells implanted within the network. Furthermore, such vascular networks allow the use of lower density surrounding materials that better mimic certain body tissues, and in certain applications, may be susceptible to remodeling by the host.

Description

[Technical field]

[0001] (Related Applications) This application claims the benefit of U.S. Provisional Application No. 63 / 333,107, filed April 20, 2022, and U.S. Provisional Application No. 63 / 333,533, filed April 21, 2022, the contents of both of which are incorporated by reference in their entireties herein. [Background technology]

[0002] The bioengineering of human-scale tissue and organ grafts has the potential to alleviate the shortage of donor organs and provide novel tissues for the treatment of conditions where transplantation is not readily available. However, realization of this goal will require the recapitulation of native endothelial architecture, in particular a continuous basement membrane.

[0003] Current methods for fabricating artificial vascular networks do not allow the formation of a continuous basement membrane structure surrounding the ducts. This structure is important for proper vascular function because it provides a stable substrate for the endothelial cell layer while at the same time providing a robust physical barrier against cell infiltration, aneurysm, and pressure-induced rupture. For artificial grafts in contact with blood, used for extracorporeal perfusion or transplantation via vascular anastomosis, a continuous artificial basement membrane structure may provide a means to modulate or inhibit the interaction of the innate immune system with the artificial tissue graft.

[0004] There is a need to create artificial vascular networks with defined architecture that incorporate a continuous basement membrane structure within the graft. Summary of the Invention [Means for solving the problem]

[0005] Applicant has unexpectedly discovered that by utilizing electrospinning and suitable sacrificial materials, it is possible to fabricate vascular networks surrounded by a continuous fibrous membrane. Such vascular networks are highly advantageous because they can withstand higher pressures than other artificial vascular networks, making them suitable for in vivo applications where high pressures are present. Furthermore, such vascular networks can be engineered to reduce or eliminate the immunogenicity of cells implanted within the network. Furthermore, such vascular networks allow the use of lower density surrounding materials that better mimic certain body tissues (e.g., epithelial tissues, soft tissues) and may be susceptible to remodeling by the host in certain applications.

[0006] Some aspects of the invention relate to a device comprising one or more conduits defining a luminal space, the luminal space being defined by an inner wall of a fibrous basement membrane material. In some embodiments, the one or more conduits form a vascular network. In some embodiments, the vascular network has a first end configured for fluid communication with a fluid supply and a second end configured for fluid communication with a fluid outlet.

[0007] In some embodiments, the device further comprises a scaffold material in contact with the outer wall of the fibrous basement membrane material. In some embodiments, the scaffold material optionally comprises one or more additional vascular networks defined by the inner wall of the fibrous basement membrane material. In some embodiments, the scaffold material comprises cells or bulk tissue. In some embodiments, the scaffold material has insufficient mechanical strength to define a luminal space in the absence of a fibrous basement membrane material support.

[0008] In some embodiments, the inner wall of the fibrous basement membrane material, when implanted in a subject, inhibits or prevents an immunological response by the subject to cells and other substances within the luminal space.

[0009] In some embodiments, the fibrous basement membrane material comprises pores of a size sufficient to allow diffusion of one or more biologically relevant molecules. In some embodiments, the one or more channels can withstand an internal pressure of at least 60 mmHg. In some embodiments, the vascular network can withstand an internal pressure of at least 60 mmHg.

[0010] In some embodiments, the fibrous basement membrane material comprises one or more of gelatin, gelatin composite, collagen, fibrin, chitosan, nitrocellulose, polylactic acid, polycaprolactone, polyethylene glycol, polyethylene glycol diacrylate, or other biopolymers, polymers, or liquefied or homogenized decellularized tissue or extracellular matrix.

[0011] In some embodiments, the fibrous basement membrane material comprises electrospun fibers. In some embodiments, the fibrous basement membrane material comprises electrospun fibers comprising a first component selected from the group consisting of polycaprolactone, polyethylene glycol, and polyethylene glycol diacrylate, and a second component selected from the group consisting of gelatin, collagen, and fibrin, and the thickness of the fibrous basement membrane is between 0.5 and 30 micrometers.

[0012] In some embodiments, the fibrous basement membrane material has been subjected to one or more post-manufacturing treatments selected from compression, annealing, chemical crosslinking, stretching, drawing, heat treatment, and solvent bonding, thereby imparting enhanced mechanical properties to the treated fibrous basement membrane material compared to a fibrous basement membrane material that has not been subjected to one or more of the post-manufacturing treatments. In some embodiments, the enhanced mechanical properties are selected from the group consisting of enhanced tensile strength, enhanced tensile modulus, enhanced abrasion resistance, enhanced thermal stability, enhanced elongation at break, enhanced hardness, enhanced crystallinity, and combinations thereof. In some embodiments, the post-manufacturing treatment comprises solvent bonding. In some embodiments, the solvent bonding is performed in the presence of pressure applied by opposing support substrates. In some embodiments, the post-manufacturing treatment comprises heat treatment combined with pressure applied by opposing support substrates.

[0013] In some embodiments, the device is implanted within the subject's body. In some embodiments, the device is external to the subject's body.

[0014] In some embodiments, the luminal space comprises one or more cells selected from endothelial cells, epithelial cells, mesenchymal cells, cells derived from induced pluripotent stem cells, endocrine cells, and stromal cells.

[0015] In some embodiments, the device is configured to function as an artificial kidney, pancreas, lung, cardiac muscle, liver, spleen, small intestine, large intestine, nervous tissue, skeletal muscle, composite tissue, adipose tissue, bone tissue, or skin.

[0016] The patent or application file contains at least one color drawing. Copies of this patent or patent application publication containing color drawing(s) will be provided by the Office upon request and payment of the necessary fee. [Brief description of the drawings]

[0017] [Figure 1]13 is an image of a sacrificial material (water-soluble polyvinyl alcohol (PVA)) formed as a hierarchical vascular pattern surrounded by electrospun nanofibers of PCL and gelatin.

[0018] [Diagram 2] 13 is an SEM image (cross section) of a nanofibrous membrane deposited around a printed Pluronic® F127 vascular pattern.

[0019] [Diagram 3] The left panel is an image of the printed sacrificial material being encased during the electrospinning process, and the right panel is an image of the construct embedded in bulk gelatin material and perfused with dye.

[0020] [Figure 4] Graph showing burst pressure of vascular networks with and without fibrous supports, N=3.

[0021] [Diagram 5] Figure 1 shows the properties of electrospun fibrous membrane wrappings saturated with pure acetone and compressed for 5 minutes. a) shows a scaffold formed by compression of acetone-saturated fibrous membrane on a sacrificial substrate, compression lasted for 5 minutes. b) SEM image of the cross section of the fibrous membrane after treatment. c) SEM image of the fibrous membrane surface after treatment. d) The same scaffold after treatment with perfused ducts with dye.

[0022] [Figure 6] Figure 2 shows the properties of fibrous membrane wrappings saturated with pure acetone and compressed for 1 min. a, shows a scaffold formed by compression of acetone-saturated fibrous membrane on a sacrificial substrate, compression lasted for 5 min. b, SEM image of the cross section of the fibrous membrane after treatment. c, SEM image of the fibrous membrane surface after treatment. d, shows the same scaffold after treatment with perfusion of the ducts with dye.

[0023] [Figure 7]Figure 2 shows the characterization of fibrous membrane wrapping sprayed with a small amount of pure acetone and compressed for 5 minutes. a) Shows a scaffold formed by compression of a sacrificial substrate overlaid with a fibrous membrane sprayed with a small amount of pure acetone, compression lasted for 5 minutes. b) SEM image of the cross section of the fibrous membrane after treatment. c) SEM image of the fibrous membrane surface after treatment. d) Shows the same scaffold after treatment with perfusion of the ducts with dye.

[0024] [Figure 8] Figure 1 shows the sequential assembly of large-scale tissue scaffolds using water-soluble polyurethane as a sacrificial material. a) Large-scale tissue scaffolds containing membranes printed with water-soluble polyurethane ductal patterns undergoing proximity electrospinning of membrane wrapping. b) Electrospun fibers accumulating on the printed water-soluble polyurethane patterns, where the fiber accumulation position is influenced by the collector electrode. c and d are SEM images of the cross-sections of the membrane and water-soluble polyurethane patterns after proximity electrospinning at 160x and 120x magnification, respectively.

[0025] [Figure 9] Figure 1 shows the properties of the membrane wrapping after heat treatment involving heating to about 55 °C for 18 hours plus light compression of about 3-8 psi. a, SEM image taken at 55x magnification of a cross section of the heat-treated electrospun membrane surrounding a water-soluble polyurethane pattern. b, SEM image taken at 180x magnification of a cross section of the heat-treated electrospun membrane surrounding a water-soluble polyurethane pattern. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0026] Provided herein are devices and compositions that provide a continuous barrier layer of controlled composition between the vascular network and the artificial tissue material or graft. Some embodiments relate to artificial vascular networks made from sacrificial materials that serve as a substrate for fabricating a basement membrane layer that is continuous, surrounds (e.g., completely) the vascular network, and provides isolation from any additional tissue material that is placed outside the vascular lumen. This membrane is fibrous, porous, and mechanically strong, allowing fluids and molecules of a certain size to pass through the membrane while preventing the passage of molecules, particles, cells, and other materials larger than a desired size.

[0027] Applicant has unexpectedly discovered that by utilizing electrospinning and suitable sacrificial materials, it is possible to fabricate vascular networks surrounded by a continuous fibrous membrane. Such vascular networks are highly advantageous because they can withstand higher pressures than other artificial vascular networks, making them suitable for in vivo applications where high pressures are present. Furthermore, such vascular networks can be engineered to reduce or eliminate the immunogenicity of cells implanted (or migrated) within the network. Such vascular networks also allow the use of low density surrounding materials to better mimic certain body tissues (e.g., epithelial tissues, soft tissues) and are susceptible to remodeling by the host in certain applications.

[0028] Some aspects of the invention relate to devices that include a tissue scaffold that includes a vascular network surrounded by a continuous, biocompatible, fibrous basement membrane layer, which in some embodiments provides an integral barrier that prevents cells inside the vascular network (e.g., blood cells) from contacting cells outside the network.

[0029] Another aspect of the invention relates to a device having one or more conduits defining a luminal space. The luminal space may be defined by an inner wall of a fibrous basement membrane material. The cross-sectional and longitudinal profiles of the one or more conduits may encompass a variety of shapes and configurations. In one embodiment, the cross-sectional profile of the one or more conduits has a cylindrical, approximately cylindrical, or partially flattened cylindrical shape. The longitudinal profile of the one or more conduits may be linear, non-linear, or include a combination of linear and non-linear configurations, conforming to a branched, unbranched, or networked configuration. In a preferred embodiment, the longitudinal profile of the one or more conduits is non-linear and includes a curved configuration, such as an S-shaped configuration. Additionally, the cross-sectional profile of the one or more conduits defines a diameter of about 6 μm to 10 cm, more preferably about 15 μm to 5 cm, and most preferably about 60 μm to 1.5 cm. The cross-sectional diameter of the one or more conduits may be constant or may vary over a portion or the entire longitudinal axis of the one or more conduits.

[0030] In a preferred embodiment, the combination of the cross-sectional profile and the longitudinal profile results in one or more conduits configured to mimic lumens naturally found in a mammal. For example, the one or more conduits are configured to mimic a substructure or combination of substructures of the mammalian cardiovascular system. Thus, in one embodiment, the one or more conduits can be configured to mimic an aorta, an artery, an arteriole, an arteriovenous anastomosis, a capillary, a metateriole, a capillary bed, a venule, a vein, or a combination thereof. In a particularly preferred embodiment, the one or more conduits include at least two conduits configured to form a vascular network.

[0031] In some embodiments, the vascular network has a first end configured to be in fluid communication with a fluid supply and a second end configured to be in fluid communication with a fluid outlet. In such a configuration, the vascular network can be used ex vivo in a laboratory, hospital, or other research and clinical environment, or in vivo and directly implanted in a subject. When the vascular network is configured for ex vivo use, the fluid supply, the fluid outlet, or both the fluid supply and the fluid outlet include or are in fluid communication with one or more pumps that initiate or sustain the movement of fluid within the vascular network. By way of non-limiting example, pumps for use in the present invention include pumps commonly used in laboratory or clinical environments, including piston pumps, diaphragm pumps, peristaltic pumps, syringe pumps, pneumatic pumps, microfluidic pumps, infusion pumps, vacuum pumps, and combinations thereof. Additionally, the fluid supply, the fluid outlet, or both the fluid supply and the fluid outlet include or are in fluid communication with one or more valves for regulating the flow rate of fluid within the vascular network. By way of non-limiting examples, valves encompassed by the present invention are those commonly used in laboratory or clinical environments and include isolation valves, flow control valves, throttling valves, check valves, and special purpose valves, each of which may be categorized as gate valves, ball valves, pinch valves, diaphragm valves, needle valves, butterfly valves, plug valves, or other types of valves. Optionally, the present invention may include one or more sensors for determining flow rate, temperature, pressure, and the like.

[0032] In some embodiments, the device has a scaffold material in contact with the outer wall of the fibrous basement membrane material. In some embodiments, the scaffold material optionally includes one or more additional vascular channels networks defined by the inner wall of the fibrous basement membrane material. In some embodiments, the scaffold material may include multiple layers of material, each of which is solid, semi-solid, or has one or more channels or channels networks defined therein. In some embodiments, at least one channel or channel network of at least one scaffold layer interfaces with at least one other channel or channel network of at least one other scaffold layer or channel defined by the outer wall of the fibrous basement membrane material. In preferred embodiments, the interface between channels of different scaffold layers is by direct fluid communication or via a fibrous membrane.

[0033] In some embodiments, the scaffold material comprises cells or bulk tissue. The type of cells and bulk tissue seeded and placed on or within the scaffold is not particularly limited and includes any of the cell or bulk tissue types described herein. In preferred embodiments, the cells include stem cells, endothelial cells, cardiomyocytes, parietal epithelial cells, hepatocytes, bile duct epithelial cells, stellate cells, adipocytes, osteoblasts, osteoclasts, enterocytes, goblet cells, enteroendocrine cells, Paneth cells, microfold cells, cup cells, brush cells, or other cells present in the kidney, pancreas, lung, cardiac muscle, liver, spleen, small intestine, large intestine, nervous tissue, skeletal muscle, composite tissue, adipose tissue, bone tissue, or skin.

[0034] In some embodiments, the scaffolding material has insufficient mechanical strength to define a luminal space in the absence of fibrous basement membrane material support.

[0035] In some embodiments, the inner wall of the fibrous basement membrane material, when implanted in a subject, inhibits or prevents an immunological response by the subject to cells and other substances within the luminal space.

[0036] In some embodiments, the fibrous basement membrane material comprises pores of a size sufficient to allow the diffusion of one or more biologically relevant molecules. The pores of the nanofibrous membrane may have any suitable pore size, without limitation. In one embodiment, the average or median pore diameter is about 0.05 to about 0.6 μm. In another embodiment, the average or median pore diameter is about 0.05 μm, 0.1 μm, 0.15 μm, 0.2 μm, 0.25 μm, 0.3 μm, 0.35 μm, 0.4 μm, 0.45 μm, 0.5 μm, 0.55 μm, or about 0.6 μm. The volume of the voids (V v ) is the measured total volume of the nanofiber membrane (V Tvm ) is obtained by dividing by (P=V v / V Tvm × 100%), porosity of the nanofiber membrane (P nm ) can be any suitable porosity, including but not limited to. In some embodiments, the porosity is at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more. In other embodiments, the porosity is 20-80%, 30-70%, or 40-60%.

[0037] In some embodiments, the one or more conduits can withstand an internal pressure of at least 60 mmHg, hi other embodiments, the one or more conduits can withstand an internal pressure of at least 80 mmHg, 100 mmHg, 150 mmHg, 200 mmHg, 250 mmHg, 300 mmHg, 350 mmHg, 400 mmHg, 450 mmHg, 500 mmHg, 550 mmHg, or at least 600 mmH.

[0038] In some embodiments, the vascular network can withstand an internal pressure of at least 60 mmHg. In other embodiments, the vascular network can withstand an internal pressure of at least 80 mmHg, 100 mmHg, 150 mmHg, 200 mmHg, 250 mmHg, 300 mmHg, 350 mmHg, 400 mmHg, 450 mmHg, 500 mmHg, 550 mmHg, or at least 600 mmH.

[0039] In some embodiments, the fibrous basement membrane material comprises one or more of gelatin, gelatin composite, collagen, fibrin, chitosan, nitrocellulose, polylactic acid, polycaprolactone, polyethylene glycol, polyethylene glycol diacrylate, biopolymers, polymers, or liquefied or homogenized decellularized tissue or extracellular matrix.

[0040] In some embodiments, the fibrous basement membrane material comprises electrospun fibers formed from a binary, ternary, quaternary, or quinary mixture of materials. Particularly preferred mixtures include a binary mixture of collagen and polycaprolactone. In one particularly preferred embodiment, the collagen has the form of bovine, porcine, or fish gelatin having a molecular weight of 15-400 kDa. In some embodiments, the gelatin has a Bloom value of 30-300, a Bloom value of 40-100, a Bloom value of 100-200, or a Bloom value of 200-280. Additionally, the gelatin is crosslinked. In another preferred embodiment, the polycaprolactone utilized to form the electrospun fibers into the fibrous basement membrane has a molecular weight of 10-100 kDa, 25-80 kDa, or 30-60 kDa. The individual materials used in the binary mixture are present in a ratio ranging from 1:10 to 10:1, preferably from 1:4 to 4:1, or more preferably from 1:2 to 2:1. In a particularly preferred embodiment, the individual materials in the binary mixture are present in a ratio of 1:1.

[0041] The thickness of the fibrous basement membrane is not particularly limited and varies based on various factors including the end use of the device. In some embodiments, the fibrous basement membrane has a thickness of 0.5 to 30 μm, has a thickness of 3 to 25 μm, or has a thickness of 10 to 20 μm. When the fibrous basement membrane is designed for use under high pressure, the fibrous basement membrane is configured to have a large thickness and has a thickness of 10 to 30 μm. Conversely, when the fibrous basement membrane is configured for use under low or medium pressure, or when high membrane elasticity is desired, the fibrous basement membrane is configured to have a smaller thickness and has a thickness of 0.5 to 20 μm.

[0042] In some embodiments, the device is implanted within the subject's body. In some embodiments, the device is configured to facilitate surgical anastomosis with native tissue that defines an opening or luminal space. In preferred embodiments, the device is attached in fluid communication with an artery, vein, renal collecting duct, bronchiole, endocrine duct, small intestine, or large intestine. In another embodiment, the implanted device functions as an artificial organ.

[0043] In some embodiments, the device is placed outside the subject's body. When the device is configured for external use, the device is attached to the subject's body in direct or indirect fluid communication with natural tissue within the subject's body that defines an opening or luminal space. In another embodiment, the device is supported or held by the external surface of the subject's body. In yet another embodiment, the device is free-standing and placed on a horizontal surface or otherwise fixedly or removably attached to an external support near the subject. In another embodiment, the implanted device functions as an external artificial organ.

[0044] In some embodiments, the luminal space has one or more cells. These cells are not particularly limited and can include any of the cells described herein. In one embodiment, the one or more cells are endothelial cells, epithelial cells, mesenchymal cells, cells derived from induced pluripotent stem cells, endocrine cells, or stromal cells. In a preferred embodiment, the luminal space includes two different cells, three different cells, four different cells, or five or more different cells.

[0045] In some embodiments, the device is configured to function as an artificial mammalian organ, hi preferred embodiments, the device is configured to function as an artificial kidney, pancreas, lung, cardiac muscle, liver, spleen, small intestine, large intestine, nervous tissue, skeletal muscle, composite tissue, adipose tissue, bone tissue, or skin.

[0046] In some embodiments, this biocompatible fibrous basement membrane layer serves as a boundary between the endothelial region of the tissue scaffold and other regions that may have their own lumen and ductal network (such as kidney, lung) or may be simple bulk tissue (such as muscle). This layer also provides mechanical support to the vascular network, preventing undesirable events such as aneurysms or ruptures due to excessive pressure, which is a major challenge for artificial vascular networks. In some embodiments, in addition to the mechanical support provided by the biocompatible fibrous basement membrane layer, this layer also provides a controlled interface between vascular fluids and the surrounding tissue. Thus, in some embodiments, by controlling the porosity and composition of the biocompatible fibrous basement membrane layer, it is possible to regulate or prevent undesirable interactions or effects such as immune responses. This is essentially an encapsulation strategy for artificial vascular networks, providing a barrier between the host and graft tissue with the vascular basement membrane.

[0047] In some embodiments, a hypoimmunogenic endothelium is provided inside the tissue scaffold, while the remaining tissue within the scaffold is isolated from the immune system via a biocompatible fibrous basement membrane layer, resulting in a hypoimmune tissue graft or extracorporeal device.

[0048] Some aspects of the present disclosure relate to the manufacture of a single continuous membrane or multiple fibrous membranes surrounding a sacrificial material in the form of a vascular network. These membranes are made from fibers, preferably nanofibers, produced by electrospinning, a process known to those skilled in the art (see Xue et al., Chem. Rev. 2019, 119, 8, 5298-5415, and Teo et al., Nanotechnology 17 (2006) R89-R106, both of which are incorporated herein by reference). The fibers are preferably produced by direct electrospinning onto the sacrificial material to form a porous fibrous membrane along the entire sacrificial material that defines the vascular network, or by production and subsequent deposition or embedding steps.

[0049] In some embodiments, electrospinning is performed in close proximity to a substrate / collector plate that is to be coated with electrospun fibers. In some embodiments, the distance between the tip and the collector plate is less than 15 cm, less than 12 cm, less than 10 cm, less than 9 cm, less than 8 cm, less than 7 cm, less than 6 cm, less than 5 cm, less than 4 cm, less than 3 cm, or less than 2 cm. In some embodiments, smaller diameter fibers are produced by reducing the diameter of the inner opening of the tip, the feed rate of the fiber precursor solution or melt, and / or the concentration of polymer in the precursor solute or melt. Additionally, the applied voltage can be adjusted to reduce the diameter of the fibers formed, such as by significantly stretching or drawing the injected precursor solution or melt prior to deposition on the collector substrate. In some embodiments, the volatility of any solvent used to dissolve the fiber precursor material is carefully considered along with other spinning parameters to obtain proper fiber formation and deposition in the finished fibrous membrane. In certain embodiments, the fiber diameter is reduced to allow for evaporation of solvent in the precursor solution or cooling and hardening of the precursor melt before arriving as a solid fiber on the sacrificial substrate / collector plate. This can be particularly important when the distance between the tip and the sacrificial substrate / collector plate is reduced or small, as required in certain embodiments of the present invention. In alternative embodiments, the feed rate, needle opening size, and applied voltage are selected to provide semi-solid fibers that anneal to each other upon deposition on the substrate / collector plate, thereby reducing the time of post-fabrication processing steps or even eliminating post-fabrication processing altogether.

[0050] In some embodiments, the diameter of the fibers is less than 10 μm, less than 9 μm, less than 8 μm, less than 7 μm, less than 6 μm, less than 5 μm, less than 4 μm, less than 3 μm, less than 2 μm, or less than 1 μm. In some embodiments, the majority of the fibers present in the fibrous membrane are nanofibers having a diameter of 950 nm or less, 800 nm or less, 600 nm or less, 450 nm or less, or 200 nm or less. In some embodiments, the majority of the fibers in the fibrous membrane are nanofibers having a diameter of about 100 nm to 750 nm, about 100 nm to 500 nm, or about 250 nm to 800 nm. In some embodiments, the fibrous membrane comprises fibers with a diameter of more than 950 nm, more than 2 μm, more than 3 μm, more than 4 μm, more than 5 μm, or more. In some embodiments, the diameter of the electrospun fibers is at least 5 μm, at least 6 μm, at least 7 μm, at least 8 μm, at least 9 μm, or more.

[0051] In some embodiments, the manufacture of the devices disclosed herein does not involve forming a porous fibrous membrane directly on a sacrificial substrate, either by electrospinning or any other means. Instead, in some embodiments, a porous fibrous membrane is formed and then manipulated, alone or with other fibrous membranes, to form one or more conduits that define a luminal space. In some embodiments, the one or more porous fibrous membranes are manipulated to form a vascular network by contacting the one or more fibrous membranes with one or more conduit templates. In preferred embodiments, the contacting is performed under conditions suitable for inducing a permanent morphological change in the one or more porous fibrous membranes that mimics the shape of the one or more templates. Conditions suitable for inducing a permanent morphological change in the one or more porous fibrous membranes include contacting in the presence of one or more of elevated temperature, compressive force, and one or more solvents. In some embodiments, the conditions include application of a compressive force sufficient to stretch or expand the fibrous membrane around the one or more templates. In preferred embodiments, at least two conditions are simultaneously present to induce a permanent morphological change in the one or more porous fibrous membranes upon contact with the one or more templates. In preferred embodiments, one or more of the molds include a sacrificial substrate deposited directly onto the fibrous membrane. In some embodiments, the sacrificial substrate is deposited onto a substrate suitable for releasing the sacrificial substrate once the porous fibrous membrane is in contact with or applied to the substrate.

[0052] In some embodiments, the initial production of the electrospun fibrous membrane is followed by post-production treatments, including, but not limited to, one or more of compression, annealing, chemical crosslinking, stretching / orientation, and solvent bonding.

[0053] In some embodiments, post-manufacturing processing can occur at temperatures between 20° C. and 22° C. or greater than 22° C. In some embodiments, the post-manufacturing process occurs at a temperature below the glass transition temperature (Tg) of one, two, or all of the materials used to form the fibrous membrane, e.g., at least 5° C., 10° C., 15° C., 20° C., 30° C., or more below the glass transition temperature.

[0054] In certain embodiments, the post-fabrication step is annealing, which is performed at a temperature approximately equal to the glass transition temperature of the material used to form the fibrous membrane. For purposes of this application, approximately equal to the glass transition temperature (Tg) is defined as ±5°C of the published glass transition temperature of the material, or ±5% of the published Tg, whichever provides the smaller temperature range. In some embodiments, the post-fabrication step is performed at a temperature between the glass transition temperature and the melting temperature of the material used to form the fibrous membrane. Those skilled in the art will recognize that care must be taken to select an appropriate post-fabrication treatment time so that the porosity of the membrane is not destroyed by significant melting of the fibrous membrane as the temperature is increased above the glass transition temperature.

[0055] In some embodiments, the fibrous membrane is subjected to a post-fabrication process at a temperature of 25° C., 30° C., 35° C., 40° C., 45° C., 50° C., 55° C., 60° C., 65° C., 70° C., 75° C., 80° C., 85° C., 90° C., 100° C., 125° C., 150° C., 160° C., 170° C., 180° C. or higher. In some embodiments, the post-fabrication process is carried out for 1 second, 5 seconds, 10 seconds, 15 seconds, 30 seconds, 45 seconds, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 20 minutes, 30 minutes, 45 minutes, or 1 hour. In certain embodiments, post-production processing may occur for longer periods of time, including 1.5 hours, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 25 hours, 1 day, 2 days, 3 days or more.

[0056] In some embodiments, the post-fabrication annealing step includes applying compression to facilitate the envelopment of the sacrificial substrate by the fibrous membrane, as described herein. In some embodiments, applying compression at elevated temperatures promotes better or more rapid bonding or annealing of the fibers. In some embodiments, the post-fabrication annealing and compression steps are performed at temperatures of 120-180° C., 40-70° C., 45-65° C., 50-60° C., or 53-57° C. for a period of 11-25 hours, 13-22 hours, 15-20 hours, or 17-19 hours. In certain preferred embodiments, the compression and annealing steps are performed at about 55° C. for about 18 hours.

[0057] The amount of pressure used in the compression step is not particularly limited, as long as it is sufficient to promote fiber-to-fiber bonding or annealing at the fiber crossing locations, while protecting the fibrous membrane or substrate on which the pressure is applied from damage.In some embodiments, compression is applied by placing the fibrous membrane and the substrate on which the fibrous membrane is manufactured between two opposing release substrates of a size, shape, and hardness that allows the application of sufficient force to induce fiber-to-fiber bonding or adhesion without damaging the fibrous membrane or substrate on which compression is applied.In some embodiments, the release substrate is a soft silicone pad.In certain preferred embodiments, the compression step is performed by placing the fibrous membrane and the sacrificial substrate between two silicone pads placed on a solid surface, and the only compression is caused by the weight of the silicone pads placed on top.

[0058] In some embodiments, additional compressive force may be applied using pressure applied by placing objects of increasing weight on the upper silicone pad. In some embodiments, the pressure applied to the fibrous membrane and sacrificial substrate during the compression process is less than 25 psi, less than 20 psi, less than 18 psi, less than 15 psi, less than 12 psi, less than 10 psi, less than 8 psi, less than 7 psi, less than 6 psi, less than 5 psi, less than 4 psi, less than 3 psi, less than 2 psi, less than 1 psi, less than 0.5 psi, less than 0.4 psi, less than 3 psi, less than 2 psi, or less than 0.1 psi. In some preferred embodiments, when pressure is applied under heating, the pressure is 0.05 psi to 2 psi, 0.05 psi to 1 psi, or 0.1 to 0.8 psi. In some further embodiments, the pressure applied during the compression process is 0.5 to 5 psi, 2 to 8 psi, 5 to 14 psi, 3 to 7 psi, or 18 to 25 psi.

[0059] In some embodiments, post-production treatment includes exposing the fibrous membrane to a solvent to promote adhesion at the intersections of the partially softened or swollen fibers. The solvent used for solvent bonding should be capable of at least partially softening or swelling the fibers of the fibrous membrane material in a reasonable time to promote adhesion between the fibers. In some embodiments, the solvent is substantially non-toxic. In other embodiments, the solvent has a Hildebrand solubility parameter similar to that of the material used to form the electrospun fibrous membrane. In situations where a particular concentrated solvent may rapidly dissolve and destroy the morphology of the fibers of the fibrous membrane, the solvent may be mixed with one or more non-solvents to provide a dilute solvent formulation that can provide controlled swelling of the fibers and adhesion between the fibers without destroying the morphology of these fibers. In some embodiments, the solvent is selected from one or more of acetone, methyl ethyl ketone, dimethylacetamide, ethyl acetate, methyl acetate, N-methylpyrrolidone, propylene carbonate, lactate, diethyl ether, dichloromethane, tetrahydrofuran, ethanol, and methanol. In some embodiments, the solvent is acetone in concentrated form, e.g., a formulation close to 100% pure (undiluted). In some embodiments, the acetone is diluted with a polar non-solvent, such as isopropanol, to obtain a formulation containing 20-80% acetone. In certain embodiments, the acetone is diluted to a concentration of 50% or 25%.

[0060] The method of applying the solvent to the fibrous membrane is not particularly limited, so long as the solvent is applied relatively uniformly to at least one surface of the fibrous membrane. In some embodiments, the solvent can be applied to the fibrous membrane before the membrane is contacted with the sacrificial substrate. In some embodiments, the fibrous membrane is saturated with acetone. Saturation of the fibrous membrane with the solvent can be done by permeation, immersion, or spraying, in combination with a length of time to allow for saturation. In some embodiments, it is not desirable to saturate the fibrous membrane with the solvent. In these circumstances, the solvent can be applied to the surface of the fibrous membrane in limited amounts, such as by spraying. In some embodiments, the solvent can be applied to a 100 cm 2 surface of the fibrous membrane in a sacrificial substrate. 2In some embodiments, 0.5 to 10 mL of solvent is applied per 100 cm of fibrous membrane. 2 Per injection, 0.5-1.5 mL, 1.0-2.5 mL, 2.0-4.0 mL, 3.0-4.5 mL, 4.0-5.5 mL, 5.0-6.5 mL, 6.0-7.5 mL, 7.0-8.5, or 8.0-9.5 mL of solvent is applied.

[0061] In some embodiments, the solvent bonding is accompanied by the application of compression to further induce morphological changes in the fibrous membrane when applied to the sacrificial substrate and / or to strengthen the adhesion between the fibers of the fibrous membrane. The specific pressure used in the compression step to induce morphological changes in the membrane and adhesion between the fibers can be any pressure disclosed herein and is not particularly limited, so long as the pressure does not cause the substrate to which the fibrous membrane is applied to collapse. In some embodiments, compression is used in combination with the solvent bonding and includes a compression force of at least 0.1 to 25 Newtons, which is generated by compressing opposing release substrates that sandwich the fibrous membrane and the sacrificial substrate to which the fibrous membrane is applied.

[0062] In some embodiments, the application of compression during the post-production solvent bonding process reduces the time required to achieve strong bonds between the fibers. The specific time required to provide high strength bonds under compression is not particularly limited and includes those times disclosed herein. In some embodiments, the fibrous membrane is exposed to pressure and / or solvent for at least 30 seconds, 45 seconds, 60 seconds, 75 seconds, 90 seconds, 105 seconds, 120 seconds, 2.5 minutes, 3 minutes, 3.5 minutes, 4 minutes, 4.5 minutes, 5 minutes, 5.5 minutes, 6 minutes, 6.5 minutes, 7 minutes, 7.5 minutes, 8 minutes, 8.5 minutes, 9 minutes, 9.5 minutes, 10 minutes or more.

[0063] In some embodiments, post-manufacturing treatments include physical or chemical cross-linking of the fibers. In some embodiments, cross-linking is achieved by exposure to ultraviolet light, gamma radiation, or plasma, or by adding one or more cross-linking agents, or a combination thereof. Cross-linking agents that can be used to promote cross-linking of fibers include, but are not limited to, those that induce the formation of covalent bonds between compounds found in adjacent fibers. In some cases, cross-linking agents include glyoxal, isocyanates, glutaraldehyde, formaldehyde, carbodiimides, epoxides, citric acid, tannin, ferulic acid, glyceraldehyde, genepin, or transglutaminase.

[0064] In some embodiments, post-manufacturing treatments change the morphology of the fibrous membrane and / or improve the mechanical properties of the fibrous membrane. Improved mechanical properties may include, but are not limited to, one or more of: improved tensile strength, improved tensile modulus, improved abrasion resistance, improved thermal stability, improved elongation at break, improved hardness, and improved crystallinity. Changes in morphology include, but are not limited to, one or more of: increased fiber diameter, decreased fiber diameter, increased porosity, decreased porosity, increased pore tortuosity, and decreased pore tortuosity.

[0065] In some embodiments, once the sacrificial material is completely surrounded by the fibrous membrane, the device can be further modified with additional ductal networks or other tissue materials that may or may not contain cells. The vascular network is then formed by removing the sacrificial material, forming a device consisting of a hollow lumen that may or may not contain a hierarchical ductal network(s) (1) completely surrounded by a fibrous basement membrane that separates the vascular network (2) from the adjacent bulk tissue or ductal network (3).

[0066] The fibrous membrane comprises fibers, preferably nanofibers, produced, preferably by electrospinning, either randomly aligned as in a nonwoven fabric or in a specific orientation.

[0067] In some embodiments, the fibers comprise a single material or a blend of materials, such as gelatin, gelatin composite, collagen, fibrin, chitosan, nitrocellulose, polylactic acid, polycaprolactone, polyethylene glycol, polyethylene glycol diacrylate, or other biopolymers, polymers, or liquefied or homogenized decellularized tissue or extracellular matrix. In a preferred embodiment, the fibers comprise a combination of gelatin and polycaprolactone.

[0068] When fabricated into nanofibers, these materials can exhibit highly desirable mechanical properties and are strong enough to form thin films capable of withstanding physiological pressures. The composition of nanofiber membranes also allows for highly controlled porosity that can be used for filtration, diffusion, or other such transport of certain molecules while excluding others based on size. This allows for modularity of various functions, including immune sensing and response.

[0069] In some embodiments, the fibrous membrane is formed by electrospinning fibers directly onto the sacrificial material or template that will form the vascular network. This can be done in a uniform manner by varying the electric field of the system, including adjusting the voltage, current, and polarity of the nozzle, collector, sacrificial material, and auxiliary guides. Similar results can also be achieved by dip-coating, spraying, or otherwise depositing a layer of fibers suspended in a solution / solvent onto the sacrificial material or template that will form the vascular network.

[0070] Further examples of the present invention include devices in which the sacrificial material is PVA, BVOH, poloxomer P407, sucrose, or other water-soluble sacrificial materials. In particular, the water-soluble sacrificial material includes water-soluble polyurethanes. Particularly contemplated are water-soluble polyurethanes that are non-toxic to human kidney fibroblasts and can be rapidly dissolved in water at room temperature or 37° C. under neutral pH without significant swelling.

[0071] Further examples of the invention include devices in which the tissue material is gelatin methacryol, fibrin, collagen, methylcellulose, or homogenized extracellular matrix, and the like.

[0072] Further examples of the present invention include devices in which multiple structures or components are assembled and operatively bonded to one another by deposition of additional membrane material, ultrasonic bonding, solvent bonding, adhesives, or other techniques to form a continuous basement membrane throughout all of the structures or components.

[0073] Further examples of the present invention include membranes as described in the previous examples, including hydrogels, polymers, and composites of material that have been modified by the addition of reinforcing agents or compounds to provide tunable mechanical and biological properties.

[0074] Further examples of the present invention include membranes, as described in the previous examples, that are manufactured in a multi-step process to form membranes of mixed composition or architecture.

[0075] Further examples of the present invention include membranes, as described in the previous examples, that contain hydrogels and polymers that are encapsulated or loaded with biological factors that promote cell and tissue growth.

[0076] Those skilled in the art will readily recognize that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The details and examples described herein are representative of certain embodiments, are illustrative, and are not intended as limitations on the scope of the invention. Modifications and other uses herein will occur to those skilled in the art. These modifications are encompassed within the spirit of the invention. It will be readily apparent to those skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention.

[0077] The articles "a" and "an" as used herein should be understood to include plural referents unless clearly indicated to the contrary in the specification and claims. A claim or description containing "or" between one or more members of a group is deemed satisfied if one, more than one, or all of the members of the group are present, used, or otherwise relevant in a given product or process, unless indicated to the contrary or otherwise clear from the context. The invention includes embodiments in which exactly one member of a group is present, used, or otherwise relevant in a given product or process. The invention also includes embodiments in which more than one member or all members of a group are present, used, or otherwise relevant in a given product or process. Moreover, it should be understood that the present invention provides for all variations, combinations, and permutations, wherein one or more limitations, elements, clauses, descriptive terms, etc. from one or more of the listed claims are introduced into another claim, relying on the same base claim (or any other claim, if relevant), unless otherwise indicated or unless it is obvious to one skilled in the art that a contradiction or inconsistency would result. All embodiments described herein are contemplated as applicable to all different aspects of the present invention, where appropriate. It is also contemplated that any of the embodiments or aspects may be freely combined with one or more other such embodiments or aspects, where appropriate. When elements are presented as a list, for example in a Markush group or similar format, it should be understood that each subgroup of the elements is also disclosed and that any element(s) may be removed from the group. In general, when the invention, or aspects of the invention, are referred to as including certain elements, features, etc., it should be understood that a certain embodiment of the invention or aspect of the invention consists of or consists essentially of such elements, features, etc. For purposes of simplicity, these embodiments have not in all instances been specifically described in great detail herein.It should also be understood that any embodiment or aspect of the invention may be explicitly excluded from the claims, regardless of whether a specific exclusion is recited in the specification. For example, any one or more active agents, additives, ingredients, optional agents, organism types, disorders, subjects, or combinations thereof may be excluded.

[0078] Where a claim or description is directed to a composition of matter, it should be understood that methods of making or using the subject compositions according to any of the methods disclosed herein, and methods of using the subject compositions for any of the purposes disclosed herein, are aspects of the invention, unless otherwise indicated or it is apparent to one of ordinary skill in the art that a contradiction or inconsistency would arise. Where a claim or description is directed to a method, it should be understood that methods of making compositions useful, for example, for carrying out the method, and products produced according to the method, are aspects of the invention, unless otherwise indicated or it is apparent to one of ordinary skill in the art that a contradiction or inconsistency would arise.

[0079] When ranges are given herein, the invention encompasses embodiments in which the endpoints are included, in which both endpoints are excluded, and in which one endpoint is included and the other endpoint is excluded. It should be assumed that both endpoints are included unless otherwise indicated. Furthermore, unless otherwise indicated or otherwise evident from the context and the understanding of one of ordinary skill in the art, values ​​expressed as ranges should be understood to include any particular value or subrange within the range set forth in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise. It is also understood that when a series of numerical values ​​is set forth herein, the invention encompasses embodiments related to any intervening value or range defined by any two values ​​in the series, where the minimum value may be considered as the minimum and the maximum value may be considered as the maximum. Numeric values, as used herein, include values ​​expressed as a percentage. For any embodiment of the invention in which a numerical value is prefaced by "about" or "approximately," the invention encompasses the embodiment in which the exact value is recited. For any embodiment of the invention where a numerical value is not prefaced by "about" or "approximately," the invention includes embodiments where the value is prefaced by "about" or "approximately."

[0080] As used herein, "A and / or B," where A and B are different claim terms, generally means at least one of A, B, or both A and B. For example, a sequence that is complementary and / or hybridizes to another sequence includes (i) a sequence that is complementary to the other sequence, even if the one sequence does not necessarily hybridize to the other sequence under all conditions, (ii) a sequence that hybridizes to the other sequence, even if the one sequence is not fully complementary to the other sequence, and (iii) a sequence that is both complementary and hybridizes to the other sequence.

[0081] "Approximately" or "about" generally includes numbers that fall within 1% of a number, or in some embodiments, within 5% of a number, or in some embodiments, within 10% of a number, in either direction (greater or less than that number), unless otherwise stated or otherwise clear from the context (unless such number unacceptably exceeds 100% of the possible values). Unless expressly indicated to the contrary, in any method claimed herein that includes two or more acts, the order of the method acts is not necessarily limited to the order in which the method acts are recited, but it should be understood that the invention includes embodiments in which the order is so limited. It should also be understood that any product or composition described herein may be deemed to be "isolated" unless otherwise indicated or clear from the context.

[0082] As used herein, the terms "comprising" or "comprises" are used in reference to compositions, methods, and their respective component(s) that are essential to the invention, but are open to the inclusion of unspecified elements, whether essential or not.

[0083] As used herein, the term "consisting essentially of" refers to elements required for a given embodiment. The term permits the presence of additional elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment of the invention.

[0084] The term "consisting of" refers to compositions, methods, and each component thereof described herein that do not include any element not recited in that description of an embodiment. EXAMPLES

[0085] Example 1 Manufacture of fibrous basement membrane

[0086] Using sacrificial materials, vascular structures with the desired hierarchical branching vascular channel architecture were fabricated from water-soluble polyurethane by extrusion 3D printing.

[0087] Next, a mixture of gelatin and polycaprolactone (PCL) (1:1 weight ratio) was dissolved in a solvent containing 8 parts acetic acid and 1 part formic acid to give a final concentration of 9 wt% gelatin and 9 wt% PCL.

[0088] The vascular structure made from the sacrificial material was then mounted in an electrospinning apparatus on a motorized stage. The gelatin and PCL solution was loaded into the system and fibers were produced using standard electrospinning conditions with a voltage range of 8000-20000 volts and a current of 0.1-50 mA. Fluid flow rates varied with the number and configuration of nozzles. Transverse scanning, rotation, and reorientation of the sacrificial material of the vascular channel in combination with an electrospinning configuration were used to produce a complete and continuous membrane coating. Further manipulation of the electric field by using opposing oppositely charged nozzles was used to obtain a complete circumferential coating of the sacrificial material in a method called "bipolar electrospinning." Once the sacrificial material was completely coated, the structure was removed from the electrospinning system. The completed structure had an exposed outer membrane surface and an inner vascular surface in contact with the sacrificial material.

[0089] Additional sacrificial material was then deposited onto the outer membrane surface of the membrane material to form an epithelial ductal network or space.

[0090] A second solution of 17% by weight gelatin in phosphate buffered saline was then prepared.

[0091] This second solution was deposited into the mold, imparting the vascular pattern and associated membrane to the surface of the solution, and a second amount of solution was added to the mold, completely encapsulating the vascular pattern and membrane material with the tissue solution.

[0092] The tissue solution was then crosslinked by the addition of transglutaminase (10 U / g gelatin) and allowed to harden for 24 h at 4°C to form a single tissue construct containing a hierarchical vascular network surrounded by an epithelial ductal network, with the entire interface between the two defined by a basement membrane.

[0093] Example 2 The experiment was carried out to generate tube structures of known diameter with and without supports based on the following steps:

[0094] A 3 mm inner diameter mandrel is placed into the mold with or without the fiber support tube in place.

[0095] The mold is then filled with a 12.5% ​​by weight gelatin solution and allowed to solidify at 4° C. for 1 hour.

[0096] The mandrel along with the gelatin structure is then removed from the mold.

[0097] The gelatin structure is then removed from the mandrel and placed in a solution containing 10 U of transglutaminase for 1 hour at room temperature.

[0098] The gelatin construct is attached to a barbed wire connector and held in place with silk sutures.

[0099] One end of the gelatin tube is capped and the other end is connected to a pressure sensor and a syringe.

[0100] The syringe barrel is depressed at a rate equivalent to 50 ml / min until rupture occurs and data is collected by a pressure sensor. [Table 1]

Claims

1. An apparatus comprising one or more conduits defining a lumen space, wherein the lumen space is defined by the inner wall of a fibrous basement membrane material containing electrospun fibers.

2. The apparatus according to claim 1, wherein the one or more conduits form a vascular network.

3. The apparatus according to claim 2, wherein the vascular network has a first end configured to communicate fluidly with a fluid supply unit and a second end configured to communicate fluidly with a fluid outlet.

4. The apparatus according to claim 1, further comprising a scaffolding material that contacts the outer wall of the fibrous basement membrane material.

5. The apparatus according to claim 4, wherein the scaffolding material includes one or more further vascular pathway networks, which may be defined by the inner wall of a fibrous basement membrane material.

6. The apparatus according to claim 4, wherein the scaffolding material includes cells or bulk tissue.

7. The apparatus according to claim 5, wherein the scaffolding material has insufficient mechanical strength to define the lumen space in the absence of the fibrous basement membrane material support.

8. The apparatus according to claim 1, wherein the inner wall of the fibrous basement membrane material, when embedded in the object, inhibits or prevents an immunological reaction by the object against cells and other substances in the tubular space.

9. The apparatus according to claim 1, wherein the fibrous basement membrane material includes pores of a sufficient diameter to allow the diffusion of one or more bio-related molecules.

10. The apparatus according to claim 1, wherein one or more of the conduits can withstand an internal pressure of at least 60 mmHg.

11. The apparatus according to claim 1, wherein the fibrous basement membrane material comprises one or more of gelatin, gelatin complex, collagen, fibrin, chitosan, nitrocellulose, polylactic acid, polycaprolactone, polyethylene glycol, polyethylene glycol diacrylate, or other biopolymers, polymers, or liquefied or homogenized decellularized tissue or extracellular matrix.

12. The apparatus according to claim 1, wherein the electrospun fiber comprises a first component selected from the group consisting of polycaprolactone, polyethylene glycol, and polyethylene glycol diacrylate, and a second component selected from the group consisting of gelatin, collagen, and fibrin, and the fibrous basement membrane has a thickness of 0.5 to 30 micrometers.

13. The apparatus according to claim 1, wherein the fibrous basement membrane material is subjected to one or more post-processing treatments selected from compression, annealing, chemical crosslinking, elongation, stretching, heat treatment, and solvent bonding, thereby imparting enhanced mechanical properties to the treated fibrous basement membrane material compared to a fibrous basement membrane material that has not undergone one or more of these post-processing treatments.

14. The apparatus according to claim 13, wherein the enhanced mechanical properties are selected from the group consisting of enhanced tensile strength, enhanced tensile modulus, enhanced wear resistance, enhanced thermal stability, enhanced elongation at break, enhanced hardness, enhanced crystallinity, and combinations thereof.

15. The apparatus according to claim 13, wherein the post-manufacturing treatment includes solvent bonding.

16. The apparatus according to claim 15, wherein the solvent bonding is performed in the presence of pressure applied by an opposing support substrate.

17. The apparatus according to claim 13, wherein the post-manufacturing treatment includes a heat treatment combined with pressure applied by an opposing support substrate.

18. The apparatus according to claim 3, wherein the apparatus is embedded in the target or placed outside the body of the target.

19. The apparatus according to claim 1, wherein the tubular space contains one or more cells selected from endothelial cells, epithelial cells, mesenchymal cells, induced pluripotent stem cell-derived cells, endocrine cells, and stromal cells.

20. The apparatus according to claim 1, wherein the apparatus is configured to function as an artificial kidney, pancreas, lung, myocardium, liver, spleen, small intestine, large intestine, nerve tissue, skeletal muscle, composite tissue, adipose tissue, bone tissue, or skin.

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