Intravascular artificial pancreas

Abstract

Disclosed herein are endovascular artificial pancreas devices and methods for their manufacture. The endovascular artificial pancreas device includes a first vascular layer comprising a plurality of first vascular channels, each having a first end and a second end, where each of the first ends of the plurality of vascular channels connects to a first input conduit and each of the second ends of the plurality of vascular channels connects to a first output conduit, thereby forming a first vascular channel network; a pancreatic islet layer comprising pancreatic islets and / or beta cells disposed within at least one islet chamber, where the pancreatic islets and / or beta cells are further embedded within the islet chamber matrix; and a first thin electrospun membrane disposed as a biomolecule- and gas-permeable interface between the plurality of vascular channels and a first side of the at least one islet chamber.

Description

[Background technology]

[0001] (Related Applications) This application claims the benefit of U.S. Provisional Application No. 63 / 483,316, filed February 6, 2023, the entire teachings of which are incorporated herein by reference.

[0002] BACKGROUND OF THE INVENTION One hundred years after the discovery of insulin, insulin-dependent diabetes mellitus (IDDM or type 1 diabetes (T1D)) has become a chronic disease affecting at least 1.6 million Americans. Still, despite daily management of the disease, most patients fail to achieve glycemic control targets. Poor glucose control is linked to the development of complications, including vascular damage, which manifests as blindness, kidney failure, limb amputation, and more. Cell solutions for insulin replacement can provide tight glucose control without patient involvement. [1] Pancreas transplantation can restore glucose control in diabetic patients, at least for a period of time; however, the need for immunosuppression and the limited supply of donor organs limit this treatment. In the United States alone, more than 110,000 people are waiting for some type of organ. [2] Bioartificial organs could have a significant impact on organ waiting lists by doubling the number of patients a single organ can serve and could act as a platform for methods to avoid the need for immunosuppression.

[0003] An attractive alternative to pancreas transplantation, requiring less tissue volume and adaptable to various transplantation techniques, is pancreatic islet transplantation. Pancreatic islets are the endocrine portion of the pancreas, comprising approximately 10% of the pancreatic mass. Transplantation of pancreatic islets into the portal venous system has resulted in reduced exogenous insulin requirements and prevention of severe hypoglycemic events in clinical trials [3, 4]. However, complete insulin independence has proven difficult to achieve. Identified factors contributing to reduced function include immediate blood-borne immune responses (IBMIR), alloimmune and autoimmune responses, lack of vascular regeneration [5], immunosuppressive drug toxicity, and hypoxia at the core of cell clusters [6]. Promising methods to address these issues have been extensively scrutinized and investigated, including macroencapsulation, microencapsulation, nanoencapsulation, 3D-printed scaffolds, and others [7-10] [11-13]. Careful selection of materials and additives can increase vascular growth and oxygen tension

[14] , decrease inflammation, enable cell recovery, and provide a platform for monitoring the graft [15, 16].

[0004] Pancreatic islet blood flow is one of the highest in the human body. It approximates 5–6 mL / min / g of islet mass

[17] , corresponding to an estimated arteriolar blood flow of 10–20 nL / min per islet. Clinically used sites, the liver (approximately 1 mL / min / g) and subcutaneous fat (approximately 0.03 mL / min / g), are less perfused than native islets (Figure 1), demonstrating the need for tissue-engineered vasculature that can provide the required flow rate. Several approaches to compensate for the lower blood flow, such as oxygenation and microvascular navigation, have been explored [18, 19]. Described herein is a tissue-engineered vasculature designed to provide the required blood flow immediately after surgical implantation and anastomosis, enabling superior graft survival and function. Summary of the Invention [Means for solving the problem]

[0005] (Summary of the Invention) In some aspects, the invention is directed to an endovascular artificial pancreas device capable of producing insulin, the endovascular artificial pancreas device comprising: a first vascular layer comprising a plurality of first vascular channels each having a first end and a second end, wherein each of the first ends of the plurality of vascular channels connects to a first input conduit and each of the second ends of the plurality of vascular channels connects to a first output conduit, thereby forming a first vascular channel network; and a pancreatic islet layer comprising pancreatic islets and / or beta cells disposed within at least one islet chamber, wherein the pancreatic islets and beta cells are and / or beta cells further comprise a pancreatic islet layer embedded within the pancreatic islet chamber matrix; and a first thin electrospun membrane disposed as a biomolecule and gas permeable interface between a plurality of vascular channel layers of a first blood vessel and a first side of the at least one islet chamber, wherein the plurality of vascular channels and the first side of the at least one islet chamber are juxtaposed opposite each other across the first thin electrospun membrane, thereby enabling exchange of biomolecules and gases between the pancreatic islets and / or beta cells disposed within the at least one islet chamber and the plurality of vascular channels in the first vascular layer.

[0006] In some embodiments, the at least one islet chamber comprises a plurality of islet chambers configured as a plurality of islet channels, wherein at least two of the plurality of vascular channels are in interfacial contact with and juxtaposed across a first side of each islet channel through a first thin electrospun membrane.

[0007] In some embodiments, the endovascular artificial pancreas device as disclosed herein further comprises a second vascular layer comprising a plurality of vascular channels, each having a first end and a second end, wherein each of the first ends of the plurality of vascular channels connects to a second input conduit and each of the second ends of the plurality of vascular channels connects to a second output conduit, thereby forming a second vascular channel network; and a second thin electrospun membrane disposed as a biomolecule- and gas-permeable interface between the plurality of vascular channels of the second vascular layer and a second side of the at least one islet chamber, thereby enabling exchange of biomolecules and gases between pancreatic islets and / or beta cells disposed in the at least one islet chamber and the plurality of vascular channels in the second vascular layer.

[0008] In some embodiments, the at least one islet chamber comprises a plurality of islet chambers configured as a plurality of islet channels, wherein at least two of the plurality of vascular channels are in opposing juxtaposition across and in interfacial contact with a second side of each islet channel through a second thin electrospun membrane.

[0009] In some embodiments, the plurality of vascular channels of the first vascular layer and / or the second vascular layer are lined with endothelial cells. In some embodiments, the endothelial cells are glomerular microvascular endothelial cells or human umbilical vein endothelial cells. In some embodiments, the beta cells are hypoimmunogenic (B2M- / -, CIITA- / -) and / or derived from induced pluripotent stem cells (iPSCs).

[0010] In some embodiments, the endovascular artificial pancreas device is glucose responsive and produces an amount of insulin proportional to the amount of glucose within the device.

[0011] In some embodiments, the plurality of vascular channels of the first vascular and / or second vascular layers are microchannels that form the first and / or second microvascular networks.

[0012] In some embodiments, at least one islet chamber comprises elongated first and / or second sides, allowing an increased surface area to interfacially contact one or more vascular channels across the first and / or second thin electrospun membranes.

[0013] In some embodiments, the amount of islets and / or beta cells present in the device comprises at least 500,000 islet equivalents, hi some embodiments, at least one islet chamber is capable of accommodating about 660,000 islet equivalents.

[0014] In some embodiments, the pancreatic islet chamber matrix comprises collagen. In some embodiments, the pancreatic islet chamber matrix comprises an in situ polymerized matrix.

[0015] In some embodiments, the thin electrospun membrane comprises polycaprolactone and gelatin. In some embodiments, the first and / or second sides of the at least one islet chamber interface with the plurality of vascular channels of the first and / or second vascular layers through the first and / or second thin electrospun membrane at a distance of about 10-30 micrometers (μm).

[0016] In some embodiments, the pancreatic islet layer and / or the first and / or second vascular layers further comprise an encapsulation matrix encapsulating the first and / or second vascular channel network and the at least one islet chamber.

[0017] In some embodiments, the first and / or second inlet conduits and / or outlet conduits are tapered to minimize shear stress applied across the first and / or second vascular networks. In some embodiments, the first and second inlet conduits are fluidly connected to a first end of a fluid supply conduit, and the first and second outlet conduits are fluidly connected to a first end of a fluid exit conduit. In some embodiments, the fluid supply conduit and the fluid exit conduit have second ends that are adjacent to each other and / or on the same side of the device.

[0018] In some embodiments, the first vascular layer, the pancreatic islet layer, the first thin electrospun membrane, and optionally the second vascular layer and the second thin electrospun membrane form a first device unit, and the intravascular artificial pancreas device further comprises a second device unit comprising a first vascular layer, a pancreatic islet layer, a thin electrospun membrane, and optionally a second vascular layer and a second thin electrospun membrane that are the same as the first vascular layer, the pancreatic islet layer, the first thin electrospun membrane, and the optional second vascular layer and the second thin electrospun membrane of the first device unit, and the inlet and outlet conduits of the first device unit are fluidly connected to the respective inlet and outlet conduits of the second device unit.

[0019] In another aspect, the invention is directed to a process for fabricating an intravascular artificial pancreas, the process comprising the steps of: (A) generating a first thin nanofibrous membrane by electrospinning a polymer-containing solution; (B) depositing a sacrificial substrate in the form of at least one pancreatic islet chamber on a first side of the thin nanofibrous membrane; and (C) depositing a plurality of sacrificial substrates in the form of at least a plurality of first vascular channels on a second side of the thin nanofibrous membrane, each sacrificial substrate in the form of a first plurality of vascular channels having a first end and a second end, the first end connected to a first input conduit. (D) encapsulating at least the thin nanofibrous membrane having the sacrificial substrate of steps (B) and (C) deposited thereon in an encapsulation matrix; (E) removing the sacrificial substrate of steps (B) and (C) to provide a first vascular network and at least one islet chamber; and (F) filling at least one islet chamber with an islet chamber matrix comprising islets and / or beta cells capable of producing proinsulin peptide and / or glucagon.

[0020] In some embodiments, the process further comprises: (C)(i) generating a second thin nanofibrous membrane by electrospinning a polymer-containing solution over a sacrificial substrate in the form of at least one islet chamber; and (C)(ii) depositing a plurality of sacrificial substrates in the form of a plurality of second vascular channels on a side of the second thin nanofibrous membrane opposite the side in contact with the sacrificial substrate in the form of the at least one islet chamber, each sacrificial substrate in the form of a second vascular channel having a first end and and a second end, the first end connected to a second inlet conduit and the second end connected to a second outlet conduit; step (D) comprises encapsulating the second nanofibrous membrane, sacrificial substrate, second inlet conduit, and second outlet conduit of (C)(i)-(ii) together with the first thin nanofibrous membrane, sacrificial substrate, first inlet conduit, and first outlet conduit of (A)-(C) in an encapsulation matrix; and step (E) further comprises removing the sacrificial substrate of (C)(ii) to provide a second vascular network.

[0021] In some embodiments, the process further comprises (G) introducing a suspension of endothelial cells into the first vascular network and / or the second vascular network and, after a period of time, inverting the device 180 degrees.

[0022] In some embodiments, the process further comprises step (C)(iii) generating a third thin nanofibrous membrane by electrospinning the polymer-containing solution over the plurality of sacrificial substrates formed in step (C), and step (D) further comprises encapsulating the third nanofibrous membrane, second nanofibrous membrane, sacrificial substrate, second inlet conduit, and second outlet conduit of (C)(i)-(ii) together with the first thin nanofibrous membrane, sacrificial substrate, first inlet conduit, and first outlet conduit of (A)-(C) in an encapsulation matrix. [Brief explanation of the drawings]

[0023] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0024] [Figure 1] FIG. 1 depicts the blood flow rate per tissue weight for native islets as well as common clinical transplant sites.

[0025] [Figure 2] Figures 2A-2E depict designs for pancreatic scaffolds. Figure 2A depicts a scaled-down device containing a single islet channel with a single vascular channel. Figure 2B depicts a single-layer full-pattern device containing 50 vascular channels fed by a tapered distribution network. Figure 2C depicts a cross-section of a full-scale design with a two-tiered bilayer structure containing a total of 200 channels. Figure 2D is an image of a full-scale device with a two-tiered bilayer structure. Figure 2E depicts the vascular and islet layers of a full-scale device with a two-tiered bilayer structure.

[0026] [Figure 3] Figures 3A-3C depict a computational model and implant cell seeding of the pancreatic scaffold. Figure 3A depicts a computational model of shear stress and fluid pressure as cell culture medium or blood is passed through the blood vessel and islet layers. Figure 3B is an overview of the pancreatic scaffold, with a cross-section of the scaffold shown in Figure 3C. Figure 3C depicts a cross-sectional view of the blood vessel and islet layers after two weeks of in vitro perfusion, demonstrating good cell distribution and durability of the endothelial layer within the vascular channels.

[0027] [Figure 4] Figures 4A-4C depict pancreatic scaffold glucose responsiveness of rat islets. Figure 4A demonstrates that rat islets loaded into the pancreatic scaffold were glucose responsive on day 1 and positive for insulin and glucagon on day 4. Figure 4B depicts the stimulation index of the pancreatic scaffold, while Figure 4C depicts the stimulation index of conventional suspension culture.

[0028] [Figure 5-1] 5A-5H depict various aspects of the development of iPS-derived insulin-producing cells within the scaffold in vitro. [Figure 5-2] 5A-5H depict various aspects of the development of iPS-derived insulin-producing cells within the scaffold in vitro.

[0029] [Figure 6] 6A-6D depict the results of experiments conducted in pigs by implanting a one-layer endovascular artificial pancreas device by connecting it to a central venous catheter and perfusing it with pig blood.

[0030] [Figure 7-1] 7A-7H depict results from in vitro experiments involving a two-tiered bilayer intravascular artificial pancreas device and luciferase-secreting cells. [Figure 7-2] 7A-7H depict results from in vitro experiments involving a two-tiered bilayer intravascular artificial pancreas device and luciferase-secreting cells. DETAILED DESCRIPTION OF THE INVENTION

[0031] (Detailed Description of the Invention) Pancreatic islets are one of the most highly perfused organs in the human body; however, exoportal transplantation sites and encapsulation devices often prevent high levels of vascularization from occurring in transplanted islets. Described herein is an endovascular prosthetic device designed to cross mechanical and endothelial-based immune barriers to support glucose sensing and insulin secretion, provide immediate blood supply at physiological levels after implantation, and thereby support beta cell engraftment and long-term function. The scaffold design is flexible, allowing the device to be tailored to a size useful for experimental or prospective use with a specific patient. The pattern is designed to provide the shear rate required for healthy endothelium, using different flow rates for media and blood to account for differences in viscosity. Rat islets demonstrated glucose responsiveness in the scaffold in vitro. Acute and initial survival pig anastomosis experiments demonstrated proof-of-concept for the functional capabilities of embodiments of this device. Finally, experiments with HEK-Lucia cells as model cells, which secrete a product with a similar molecular weight to insulin in response to a soluble signal, demonstrated that full-scale scaffolds as described herein support highly proliferative cell types in vitro.

[0032] The estimated total islet dose in humans is often cited as 500,000 islet equivalents (IEQ, approximately 1,000-2,000 cells, approximately 150 μm diameter). Although this may not necessarily be clinically required in the future, we have designed a full-scale device to accommodate approximately 660,000 IEQ. Assuming monolayer spreading and square filling, this number of islet equivalents would be at least 145 cm. 2 Therefore, the inventors set out to design a compact device that would store a large dose of islets / beta cells without sacrificing perfusion of the islets / beta cells contained therein.

[0033] In one aspect, the present invention is directed to a compact device capable of producing an amount of insulin sufficient to control glucose in a human subject. In some embodiments, the device comprises a plurality of first vascular channels, each having a first end and a second end, where each of the first ends of the plurality of vascular channels connects to a first input conduit and each of the second ends of the plurality of vascular channels connects to a first output conduit, thereby forming a first vascular channel network; pancreatic islets and / or beta cells disposed within at least one islet chamber; and a thin membrane disposed as a biomolecule- and gas-permeable interface between the plurality of vascular channels and a first side of the at least one islet chamber, where the plurality of vascular channels and the first side of the at least one islet chamber are juxtaposed across the thin membrane. This arrangement allows for the exchange of biomolecules and gases between the pancreatic islets and / or beta cells disposed within the at least one islet chamber and the plurality of vascular channels. In some embodiments, the device contains a total of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250, 275, 300, 350, 400, 450, 500, 750, 1,000, 2,000, or 10,000 channels. In some embodiments, the device contains 10-1,000, 15-500, 20-300, 25-200, 30-160, or 40-80 vascular channels. In some embodiments, the device contains between 2 and 200, between 4 and 150, between 10 and 80, between 20 and 60, between 60 and 180, or between 30 and 80 islet channels.

[0034] The diameter and cross-sectional profile of the vascular channel and at least one islet chamber are not particularly limited and may comprise any diameter and cross-sectional profile known to those skilled in the art or as specifically described herein. In some embodiments, the islet chamber and vascular channel have a cross-sectional profile that is round, oval, square, rectangular, or concave, wide, or flattened on one or more sides. In some embodiments, the at least one islet chamber comprises a plurality of islet chambers, and the islet chambers are formed as an islet channel. In some embodiments, the islet channel and / or vascular channel have a wide cross-sectional profile, and the profile is elongated horizontally. In some embodiments, the islet channel and / or vascular channel have a flattened cross-sectional profile, and the profile is elongated horizontally but reduced vertically. When the pancreatic islets and / or vascular channels have a widened or flattened cross-sectional profile, it is beneficial to juxtapose the elongated side of the pancreatic islets and / or vascular channels across the membrane opposite their corresponding channels to provide a larger surface area for the exchange of biomolecules and gases across the membrane.

[0035] In some embodiments, the diameter of the islet channel is greater than the diameter of the vascular channel. In some embodiments, the diameter of the islet channel is equal in size to the diameter of the vascular channel. In other embodiments, the diameter of the islet channel is smaller than the diameter of the vascular channel. For purposes of measuring the inner diameter of a non-circular vascular or islet channel, the diameter is the longest distance that can be measured between any two opposing points on the channel wall. In some embodiments, the diameter can include diameters of 100 mm, 50 mm, 10 mm, 5 mm, 1 mm, 500 μm, 50 μm, 10 μm, 5 μm, 3 μm, 1 μm, 0.5 μm, 0.1 μm, 0.05 μm, 0.02 μm, or 0.01 μm, respectively. In certain embodiments, the islet channel and / or vascular channel is a microchannel with a diameter of 900 μm, 800 μm, 750 μm, 700 μm, 650 μm, 625 μm, 600 μm, 575 μm, 550 μm, 525 μm, 500 μm, 475 μm, 450 μm, 425 μm, 400 μm, 375 μm, 350 μm, 325 μm, 300 μm, 275 μm, 250 μm, 225 μm, 200 μm, 175 μm, 150 μm, 125 μm, 100 μm, 75 μm, 50 μm, or 25 μm. In some embodiments, the shortest distance, which can be measured between any two opposing points on the channel wall, is about 10 μm, 15 μm, 17 μm, 20 μm, 22 μm, 25 μm, 27 μm, 30 μm, 33 μm, 35 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm, 110 μm, 120 μm, 130 μm, 140 μm, 150 μm, 160 μm, 170 μm, 180 μm, 190 μm, 200 μm, 210 μm, 220 μm, 250 μm, 270 μm, 280 μm, 300 μm, 330 μm, 350 μm, 400 μm, 500 μm, 600 μm, 700 μm, 800 μm, 900 μm, 1000 μm, 1100 μm, 1200 μm, 1300 μm, 1400 μm, 1500 μm, 1600 μm, 1700 μm, 1800 μm, 1900 μm, 2000 μm, 2100 μm, 2200 μm, 2500 μm, 2700 μm, 3000 μm, 3300 μm, 3500 μm, 4000 μm, 5000 μm, 6000 μm, 7000 μm, 8000 μm, 9000 μm, 1000 μm, 1100 μm, 1200 μm m, 130 μm, 140 μm, 150 μm, 160 μm, 170 μm, 180 μm, 190 μm, 200 μm, 210 μm, 220 μm, 230 μm, 240 μm, 250 μm, 260 μm, 270 μm, 280 μm, 290 μm, 300 μm, 400 μm, 500 μm, 600 μm, or 700 μm.

[0036] In some embodiments, at least two vascular channels interface with and are juxtaposed across opposite sides of the islet chamber and / or channel through a thin membrane. In some embodiments, three vascular channels interface with and are juxtaposed across opposite sides of the islet chamber and / or channel through one or more thin membranes. In some embodiments, four vascular channels interface with and are juxtaposed across opposite sides of the islet chamber and / or channel through one or more thin membranes. In some embodiments, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, thirty, forty, fifty, or more vascular channels interface with and are juxtaposed across opposite sides of the islet chamber and / or channel through one or more thin membranes. In some embodiments, each islet channel is covered by one or more vascular channels over a majority of the space surrounding it, thereby participating in productive diffusion. In some embodiments, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or substantially all of the surface area of ​​the vascular channel is in interfacial contact with the vascular channel through one or more thin films.

[0037] In some embodiments, it is beneficial to provide a flattened vascular channel, conserving space within the device while providing an increased surface area for interfacing with one or more vascular channels. Thus, in some embodiments, the vascular channel is flattened and multiple vascular channels interface with the elongated top side of the vascular channel through a thin film. In some embodiments, the vascular channel is flattened and multiple vascular channels interface with the elongated bottom side of the vascular channel through a thin film. In some embodiments, multiple vascular channels interface with the elongated top side of the vascular channel through one or more thin films and multiple vascular channels interface with the elongated bottom side of the vascular channel.

[0038] In some embodiments, it is beneficial to provide a vascular channel that is flattened and can accommodate only a small number of vertically stacked islet / beta cell clusters within it. This ensures that each islet / beta cell is in close proximity to the membrane, facilitating rapid diffusion or exchange of glucose, insulin, oxygen, and other biomolecules across the membrane to each neighboring islet / beta cell. In some embodiments, the vascular channel is flattened and its height is capable of fitting only two, three, four, five, or six vertically stacked islet / beta cell clusters within it. In some embodiments, the height of the vascular channel is capable of fitting only two vertically stacked islet / beta cell clusters within it. In some embodiments, the height of the vascular channel is approximately 20-300 μm, 25-250 μm, 30-225 μm, 25-150 μm, or 75-300 μm.

[0039] In another aspect, the present invention is directed to a device comprising one or more thin films or films as a basement membrane. In some embodiments, the one or more thin films or films form the walls of each of a plurality of vascular channels, islet chambers, or islet channels. In some embodiments, the plurality of vascular channels are juxtaposed opposite the islet chamber or islet channel across the thin film or film. In some embodiments, the thin film or film serves as a biomolecule and / or gas permeable interface between the plurality of vascular channels and at least one islet chamber or islet channel.

[0040] The composition of the thin film or film is not particularly limited and may be any composition suitable for forming a thin film or film with the desired porosity and mechanical strength necessary to withstand the shear stresses and pressures imparted during the manufacturing process and in vivo use of the device, including the flow of biological fluids therethrough under normal hemodynamic pressures, such as a pressure of at least 60 mmHg. In some embodiments, each of the multiple membranes is generated by chemical or physical thin film deposition, powdering, spraying, electrospinning, dip coating, or gelling a solution comprising decellularized tissue, gelatin, gelatin composite, collagen, fibrin, hydrogel, hydrogel composite, chitosan, nitrocellulose, polylactic acid, polycaprolactone, liquefied or homogenized extracellular matrix, or a mixture thereof in a thin film layer, followed by curing, crosslinking, polymerizing, drying, or gelling the solution to form the membrane layer. In some embodiments, the thin film or film has a thickness of 0.5 to 30, 1 to 20, 4 to 15, 7 to 25, 8 to 13, 9 to 20, or 10 to 14 micrometers. In some embodiments, the thin film or film has a thickness of about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 micrometers.

[0041] In some embodiments, the membrane solution further comprises a porogen homogeneously mixed therein. In some embodiments, the porogen is a self-assembling triblock copolymer. In some embodiments, the self-assembling triblock copolymer is a poloxamer formulation, preferably Pluronic® F127, at a concentration of 1-40% by weight.

[0042] In some embodiments, the membrane solution further comprises one or more agents that modify the mechanical or biological properties of one or more membranes. In some embodiments, the one or more agents are selected from glycerin, sorbitol, propylene glycol, plasticizers, fibers or other longitudinal elements, and encapsulated growth factors. In some embodiments, the membrane solution further comprises fibers, nanotubes, or other longitudinally oriented materials to provide improved mechanical properties. These fibers can be mixed into the membrane solution prior to processing to uniformly distribute the fibers throughout the membrane. Alternatively, these fibers can be deposited or integrated onto the membrane after processing through techniques such as electrospinning, 3D printing, or other techniques. The fibers may be uniformly distributed throughout the membrane or distributed in an organized manner to provide heterogeneous mechanical properties for the membrane. In some embodiments, this method of generating a thin film layer is repeated one or more times to generate a membrane or membranes having two or more membrane layers. In some embodiments, the two or more layers are generated from solutions having different components, agents, and / or concentrations. In some embodiments, at least one of the plurality of films is treated to remove the porogen, thereby forming pores within the film.

[0043] In some embodiments, the membrane solution comprises 3-35% by weight gelatin or gelatin-polymer composite. In some embodiments, the thin film layer is cross-linked with a solution comprising glutaraldehyde, transglutaminase, or other cross-linking enzymes or molecules known to those of skill in the art or described herein.

[0044] In some embodiments, the thin film is a fibrous membrane comprising electrospun fibers. In some embodiments, the fibrous membrane material comprises electrospun fibers comprising a binary, ternary, quaternary, or pentary mixture of materials. In some embodiments, the fibrous 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. The ratio of the first component to the second component includes a ratio of 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 10:95, 1:10, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 95:10, or 10:1. In some embodiments, the first and second components are polycaprolactone / gelatin, polycaprolactone / collagen, polycaprolactone / fibrin, polyethylene glycol diacrylate / gelatin, polyethylene glycol diacrylate / collagen, polyethylene glycol diacrylate / fibrin, polyethylene glycol / gelatin, polyethylene glycol / collagen, or polyethylene glycol / fibrin. Particularly preferred mixtures include binary mixtures of collagen and polycaprolactone. In one particularly preferred embodiment, the collagen takes the form of bovine, porcine, or fish gelatin with a molecular weight of 15 to 400 kDa. In some embodiments, the gelatin will have a Bloom value of 30 to 300, a Bloom value of 40 to 100, a Bloom value of 100 to 200, or a Bloom value of 200 to 280. Additionally, the gelatin is crosslinked. In another preferred embodiment, the polycaprolactone utilized to form the electrospun fibers in the thin film has a molecular weight of 10-100 kDa, 25-80 kDa, or 30-60 kDa. In some embodiments, the film is formed from a mixture of polycaprolactone and gelatin in a 1:1 ratio. In some embodiments, the fiber electrospun film has a thickness of 0.5-30, 1-20, 4-15, 7-25, 8-13, 9-20, or 10-14 micrometers.In some embodiments, the fiber electrospun membrane has a thickness of about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 micrometers.

[0045] In some embodiments, the thin fiber electrospun membrane material is subjected to one or more post-processing treatments selected from annealing, chemical crosslinking, stretching, drawing, heat treatment, and solvent welding, thereby imparting improved mechanical properties to the treated fiber electrospun membrane material compared to a fiber electrospun membrane material that has not been subjected to one or more of the post-processing treatments. In some embodiments, the improved mechanical properties are selected from the group consisting of improved tensile strength, improved tensile modulus, improved abrasion resistance, improved thermal stability, improved elongation at break, improved hardness, improved crystallinity, and combinations thereof. In some embodiments, the post-processing treatment comprises solvent welding. In some embodiments, the solvent welding is performed in the presence of pressure applied by opposing supporting substrates. In some embodiments, the post-processing treatment comprises heat treatment in combination with pressure applied by opposing supporting substrates.

[0046] In some embodiments, electrospinning is performed in close proximity to the substrate / current collector to be coated with the electrospun fibers. In some embodiments, the distance between the tip and the current collector 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, 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 are reduced to produce smaller diameter fibers. In addition, the applied voltage may be adjusted to reduce the diameter of the formed fibers, such as by causing increased stretching or elongation of the expelled precursor solution or melt prior to deposition onto the current collecting substrate. In some embodiments, the volatility of any solvents used in dissolving the fiber precursor materials is carefully considered along with other spinning parameters to ensure proper fiber formation and deposition within the finished fiber film. In some embodiments, reducing the fiber diameter allows for evaporation of solvents in the precursor solution or cooling and hardening of the precursor melt before arriving as a solid fiber on the sacrificial substrate / current collector. This can be important, especially when the distance between the tip and the sacrificial substrate / current collector is reduced or short, as required in some embodiments of the present invention. In alternative embodiments, the feed rate, needle aperture size, and applied voltage are selected to provide semi-solid fibers that anneal to each other upon deposition on the substrate / current collector, thus reducing the duration of post-processing processing steps or even allowing for the complete elimination of post-processing processing.

[0047] In some embodiments, the fiber diameter 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, with diameters 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, with diameters 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 diameters greater than 950 nm, greater than 2 μm, greater than 3 μm, greater than 4 μm, greater than 5 μm, or greater. 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 greater.

[0048] In some embodiments, the thin film or membrane comprises pores of a size sufficient to allow diffusion of one or more biologically relevant molecules. The pore size of the pores in the membrane or film may be any suitable size and is not limited. In one embodiment, the mean or median pore size diameter is about 0.05 to about 0.6 μm. In another embodiment, the mean or median pore size 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 porosity (Pnm) of the nanofibrous membrane, obtained by dividing the void volume (Vv) by the measured total volume of the membrane or film (VTvm) (P=Vv / VTvm × 100%), may be any suitable porosity and is not limited. In some embodiments, the porosity is at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more, while in other embodiments, the porosity is 20-80%, 30-70%, or 40-60%.

[0049] In another aspect, the present invention is directed to providing a compact device that does not sacrifice perfusion of a large number of pancreatic islets. Thus, in some embodiments, the device comprises multiple functional units stacked together. In some embodiments, the functional units of the device comprise a vascular layer comprising multiple vascular channels, an islet layer comprising at least one islet chamber or multiple islet channels, and a thin film or membrane that serves as a biomolecule- and gas-permeable interface between the multiple vascular channels of the vascular layer and the islet chambers or multiple islet channels of the islet layer. In some embodiments, the functional units comprise first and second vascular layers, each comprising multiple vascular channels, disposed on either side of the islet layer comprising at least one islet chamber or multiple islet channels, with the thin film or membrane positioned between the first and second vascular layers and the opposing sides of the islet layer to provide a dual-vascular layer functional unit. In some embodiments, a device is provided in which two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, or more functional units are combined to enable perfusion of large numbers of pancreatic islets / beta cells. Extrapolating data from approximately 15k Hypo1-SCβC IEQs (acute single-layer anastomosis, Figure 6) to a human dose of 500,000 IEQs, it is estimated that approximately 100 units of insulin would be produced, more than sufficient to control glucose in patients. Thus, in some embodiments, the device is a two-unit, dual-layer vascular device capable of storing 500,000 IEQs. Therefore, the device has the potential to function for pre-IND experiments in its current form if it stores the number of cells that a two-unit, dual-layer vascular device can accommodate. The device design is enabled by layering to achieve the required functional membrane area within a reasonable footprint.

[0050] In some embodiments, the current device has a footprint similar to the size of a smartphone (about 10 cm wide, about 6 cm long, and about 1 cm thick). In some embodiments, the width of the device is about 4-20 cm, 8-15 cm, 6-13 cm, or 8-12 cm. In some embodiments, the length of the device is about 3-18 cm, 4-15 cm, 5-8 cm, or 6-12 cm. In some embodiments, the thickness of the device is about 0.25-3 cm, 2-6 cm, 0.5-2 cm, 0.75-3 cm, or 0.25-1 cm. Broadly speaking, layering techniques connect layer inputs to a common input and layer outputs to a common output.

[0051] In one aspect, the device is designed to allow functional maturation before implantation, an important feature when stem cell-derived insulin-producing cells are used. Final function can be measured and adjusted before the device is implanted.

[0052] Thrombogenesis is a major concern with any blood contacting material and is also a concern with IVAP. The primary defense against clot formation is endothelialization of the IVAP vascular channel. No acute thrombogenic response was observed in short-term (<4 hours) porcine anastomosis studies.

[0053] Those skilled in the art will readily appreciate that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned and inherent therein. The details of the description and examples herein represent certain embodiments and are exemplary and are not intended as limitations on the scope of the invention. Modifications therein and other uses will also 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 various substitutions and modifications may be made to the present invention without departing from the scope and spirit of the invention disclosed herein.

[0054] The articles "a" and "an," as used in the specification and claims, should be understood to include plural reference unless clearly indicated otherwise. A claim or description including "or" between one or more members of a group is considered to be satisfied when one, more than one, or all of the members of the group are present in, employed in, or otherwise relevant to a given product or process, unless indicated otherwise or otherwise apparent from the context. The invention includes embodiments in which exactly one member of a group is present in, employed in, or otherwise relevant to a given product or process. The invention also includes embodiments in which more than one or all of the group members are present in, employed in, or otherwise relevant to a given product or process. Furthermore, it should be understood that the present invention provides for all variations, combinations, and permutations in which one or more limitations, elements, clauses, descriptive terms, etc. from one or more of the claims recited therein are introduced into another claim (or, by association, any other claim) relying on the same base claim, unless otherwise indicated or unless it is apparent to one skilled in the art that a contradiction or inconsistency would result. All embodiments described herein are contemplated as being applicable to any different aspect of the invention, as appropriate. It is also contemplated that any of the embodiments or aspects can be freely combined, whenever appropriate, with one or more other such embodiments or aspects. When elements are presented as lists, for example, in a Markush group or similar format, it is understood that each subgroup of the elements is also disclosed and that any element can be removed from the group. In general, when the invention or aspects of the invention are referred to as comprising particular elements, features, etc., it should be understood that a given embodiment or aspect of the invention consists of, or consists essentially of, such elements, features, etc. For purposes of brevity, those embodiments will not in all instances be described with such specificity herein. It is also understood that any embodiment or aspect of the invention may be explicitly excluded from the claims, regardless of whether a specific exclusion is recited herein.For example, any one or more active substances, additives, ingredients, optional agents, organism types, disorders, subjects, or combinations thereof can be excluded.

[0055] Where a claim or description relates to a composition of matter, it is understood that methods of making or using the composition of matter according to any of the methods disclosed herein, and methods of using the composition of matter 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. For example, where a claim or description relates to a method, it is understood that methods of making compositions useful for practicing 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.

[0056] When ranges are given herein, the invention includes embodiments in which the endpoints are included, embodiments in which both endpoints are excluded, and embodiments in which one endpoint is included and the other endpoint is excluded. Both endpoints are to be assumed to be inclusive unless otherwise indicated. Furthermore, unless otherwise indicated or otherwise apparent from the context and the understanding of one of ordinary skill in the art, values ​​expressed as ranges are to be understood to contemplate any specific value or subrange within the stated range to the nearest tenth of the lower limit of the range, unless clearly dictated otherwise by context. Also, when a series of numerical values ​​is set forth herein, the invention includes embodiments relating to any intervening value or range defined by any two values ​​within the series, and it is to be understood that the lowest value may be taken as a minimum value and the highest value may be taken as a maximum value. Numerical values ​​as used herein include values ​​expressed as percentages. For any embodiment of the invention in which a numerical value is preceded by "about" or "approximately," the invention includes embodiments in which the exact value is recited. For any embodiment of the invention in which a numerical value is preceded by "about" or "approximately," the invention includes embodiments in which the value is preceded by "about" or "approximately."

[0057] As used herein, "A and / or B" (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 to 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 perfectly complementary to the other sequence, and (iii) a sequence that is both complementary to and hybridizes to the other sequence.

[0058] "Approximately" or "about" generally includes numbers that are within 1% of a number, or in some embodiments, within 5%, or in some embodiments, within 10% of a number, in both directions (greater than or less than that number), unless otherwise stated or clear from the context (except where such number would unacceptably exceed 100% of the possible values). Unless expressly indicated otherwise, in any method claimed herein that includes more than one act, the order of the acts of the method is not necessarily limited to the order in which the acts of the method are recited, but it should be understood that the invention also includes embodiments in which the order is so limited. It should also be understood that, unless otherwise indicated or clear from the context, any product or composition described herein can be considered to be "isolated."

[0059] As used herein, the terms "comprising" or "comprises" are used in reference to compositions, methods, and individual components thereof that are essential to the invention but allow for the inclusion of non-specified elements, whether essential or not.

[0060] As used herein, the term "consisting essentially of" refers to those 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 characteristics of that embodiment of the invention.

[0061] The term "consisting of" refers to compositions, methods, and individual components thereof, as described herein, excluding any element not recited within that description of an embodiment.

[0062] (material and method) (Scaffolding design) Pancreatic scaffolds were fabricated in three categories: scaled-down single-channel devices, single-layered full-pattern devices, and multilayered full-scale devices. The scaled-down devices evolved through several iterations, all containing a single islet channel with a single vascular channel (Figure 2A), facilitating preliminary testing. The single-layered full-pattern devices contained 50 vascular channels fed by a tapered distribution network, with the islet compartment having separate access (Figure 2B). Finally, to reach an IEQ capacity of >500k, based on findings from loading multilayered implants, the multilayered full-scale devices contained a two-tiered bilayer, with the islet layer able to accommodate two vertically stacked islets within the width of the channel, with vascular layers above and below to supply adjacent cells (Figures 2C and 2D). The vascular and islet segments were accessed by conduits connecting all layers of that type. Two islet conduits were present on opposite corners. For the vascular conduit, a circumferential channel was designed to bring both the arterial and venous conduits to the same side for surgical anastomosis. To facilitate cell seeding, a third vascular conduit at the opposite corner was used, which was closed after seeding. This design can hold an IEQ of approximately 660k.

[0063] (Electrospinning of membranes) Polycaprolactone (80 kDa, Sigma 440744 or Polysciences 26290) and gelatin (Type A, 300 Bloom, Sigma G2500) were dissolved in a mixture of acetic acid (AA) and formic acid (FA) (8:1, AA:FA) at a concentration of 9% (w / v) (18% total polymer). Membranes were electrospun in a custom bipolar electrospinning setup equipped with environmental control, automated stage movement, custom voltage control, and dual syringe pumps, all housed within a ventilated enclosure. The following parameters were used: 12 kV, 15 mA, reverse polarity relative to the opposing nozzle, approximately 0.5–1.5 μL / min, 30–40% relative humidity, 24–26 °C, and a duration of approximately 10–15 min adjusted to achieve a film thickness of approximately 10–14 μm. Membranes were either used directly or stored dry until use.

[0064] (Building a Scaffold) The overall workflow, with adjustments for specific scaffold types, was as follows: PCL-gelatin hybrid membranes were electrospun onto PLA 3D printing frames as detailed above. Blood vessel and islet patterns were printed onto the membrane using Pluronic® F127 hydrogel (28%), and conduits were placed and manually connected to the pattern using F127. The processed pattern layer was sterilized with ETO. Inside a biosafety cabinet (BSC), the mold containing the membrane was filled with a warm T-gaze gelatin mixture (gelatin (12.25%) and transglutaminase (25%) in a 1:10 ratio), and scaffold assembly proceeded by alternating gelatin and prepared membrane as needed. After initial thermal solidification as the cast gelatin cooled, the scaffold with the frame was immersed in phosphate-buffered saline (PBS) supplemented with antibiotics and sealed in a sterile container, which was stored at 4°C. After 24-48 hours, the PLA mold and frame were removed using sterile technique in the BSC, and the scaffolds, still in PBS with antibiotics, were placed in a 37°C incubator to allow gelatin shrinkage to occur. After 24-48 hours, the scaffolds were returned to 4°C until cell loading.

[0065] (HEK cells) Human embryonic kidney (HEK) cells (hkl-null, Invivogen, San Diego, CA, USA), which secrete soluble luciferin (Lucia) in response to IFN in the culture medium, were used as a surrogate for insulin-producing cells in the scaffold studies. After growing in monolayer as described by Invivogen, the cells were aggregated in non-adherent cell culture dishes using orbital rotation at approximately 50–100 rpm and allowed to form aggregates for approximately 7 days before loading into scaffolds. The HEK aggregates were then treated like pancreatic islets for loading into the scaffolds.

[0066] (stem cell-derived insulin-producing cells) Insulin-producing cells were differentiated according to the Millman Lab's adherent culture protocol [29,30] and modifications suggested in published literature to improve differentiation for specific cell lines, with one exception. The pH of the enriched serum-free medium (ESFM) was adjusted to more closely resemble pH 7, resulting in beta cell insulin content and glucose responsiveness comparable to other modifications assayed. The starting induced pluripotent stem cells (iPSCs) were a double knockout line (B2M- / -, CIITA- / -) lacking HLA class I and II (Hypo1) provided by our collaborator, Pancella (Toronto, Ontario).

[0067] (Isolation of rat pancreatic islets) The isolation procedure was similar to published methods. Briefly, rats were individually euthanized by carbon dioxide inhalation and placed in a supine position. A V-shaped incision was made from the costal arch through the lower abdomen to the opposite costal arch, exposing the peritoneal cavity. The common bile duct was ligated where it enters the intestine, and collagenase solution was injected into the common bile duct in the area adjacent to the liver. After distending with the enzyme, the pancreas was carefully removed and placed on ice in a centrifuge tube. Once all animals had been processed, digestion proceeded in a 37°C water bath, and the centrifuge tube was vigorously shaken for 10 seconds. The tube was immediately placed on ice, and the digested tissue was repeatedly washed and purified on a two-layer Histopaque gradient, after which it was transferred to a plate.

[0068] (Loading of pancreatic islets or cell clusters) The prepared scaffold was repeatedly flushed with sterile PBS using all conduits to remove any remaining traces of F127. The PBS was then drained from the scaffold. The cell clusters in the islet chamber were embedded in a collagen-based matrix (Islet Viability Matrix (IVM)

[31] ). Cold "IVM base" was mixed with cold type 1 collagen to generate the IVM. The cell clusters were carefully resuspended in the IVM. The cell cluster suspension was immediately loaded into the islet chamber of the scaffold, the vascular channel was flushed with PBS, and the scaffold was placed in a 37°C incubator in a sealed container or bioreactor for 2 hours to allow the IVM to polymerize. (Note: In a monolayer design, it is desirable to have the islets at the membrane; therefore, the scaffold was kept cold for 1 hour to allow the islets to settle to the membrane by gravity, after which it was polymerized at 37°C.)

[0069] Seeding of endothelial cells and scaffold cultures After the IVM / cluster suspension was polymerized within the scaffold, a vascular conduit was attached to a port on the bioreactor, allowing a roller pump-driven flow loop to perfuse the scaffold during culture. A stopcock was mounted on the outside of the bioreactor to allow interaction with the flow channel. The endothelial cell suspension was pushed through the stopcock into the vascular channel, and the closed bioreactor was then inverted and placed in a 37°C incubator for 1 hour (designated as Seeding 1). Seeding 2 proceeded similarly, except the bioreactor was placed in its "home position" for the 1 hour incubation. In this way, cells were seeded on both sides of the channel. After both seedings were completed, flow was initiated with medium (single-channel scaffold: 0.15 mL / min, full monolayer: 5 mL / min, two-stage bilayer: 20 mL / min).

[0070] We endothelialized two-tiered bilayer scaffolds with glomerular microvascular endothelial cells (GMECs, for in vitro experiments) and human vascular endothelial cells (HUVECs, for in vivo experiments). Based on the calculated surface area, we measured cell numbers above confluent monolayer (approximately 2 × 10 cells per total channel length). 6cells were used), reducing the time it took to seed and reach confluence.

[0071] (Computational Fluid Dynamics) A validated computational fluid dynamics (CFD) model was required to verify and iteratively improve the vascular pattern and increase the velocity. For the medium, a Newtonian fluid model (assumed to be equivalent to water) was utilized. For the blood, a pressure-driven flow with a non-Newtonian Carreau fluid model

[32] was used. The model was implemented in Ansys Fluent 2022 R1, assuming laminar flow.

[0072] (GSIS Scaffolding Protocol) After scaffold seeding and overnight perfusion culture in maturation medium, the device will be perfused with Krebs-Ringer buffer in the following order: 1) low glucose (4 hours), 2) low glucose (2 hours), 3) high glucose (2 hours), and 4) low glucose (4 hours). Samples will be collected hourly throughout the 12-hour experiment and frozen for assay using human insulin or C-peptide enzyme-linked immunosorbent assay (ELISA, Mercodia). The stimulation index (SI) will be calculated by dividing the insulin secreted in the high glucose condition by the insulin secreted in the low glucose condition.

[0073] (Large animal research) Porcine subjects were anesthetized and administered heparin to maintain an extended clotting time (ACT) at the CBSET facility in Lexington, MA. For this terminal experiment, a central venous line was placed and blood flow was controlled by an external pump through the implant within the bioreactor. Blood samples were collected into EDTA-coated tubes and kept on ice until centrifugation to isolate serum could be performed. Serum samples were then frozen at -20°C until quantification by ELISA (as described in GSIS above).

[0074] (Histology and Analysis) Scaffolds were fixed by immersion in buffered formalin (Electron Microscopy Sciences), washed three times with PBS, and stored at 4°C. Sections of interest were immersed in sucrose, embedded in gelatin / sucrose, and mounted on chucks for frozen sectioning. For large scaffolds (single layer or two-tiered bilayer), six sections were made at three locations along the length of the channel. (Note: The scaffold width is twice that of a standard histology cassette, so there are six sections instead of three.) Samples were stained with DAPI or HOESCHT to identify nuclei. Immunostaining for C-peptide / insulin (NBP1-05433, Novus Biologicals, Centennial, CO, USA; 1:10 dilution) and glucagon (259A-18, Sigma-Aldrich, undiluted) was completed on rat islet scaffold sections. TUNEL staining was completed according to the manufacturer's instructions (Roche 11684795910).

[0075] (result:) (Computational model and implant cell seeding) After verifying that the computationally predicted flow rates occurred on the benchtop for water, we used in silico modeling to calculate the flow rates required to reach shear rates within the range required to maintain endothelial integrity. The modeling predicted a uniform distribution of flow (Figure 3A). Our findings suggested good cell distribution when pattern fidelity was high. Note that only a single islet channel (asterisk) was not seeded; the majority of the vascular channels still had an endothelial layer after 2 weeks of in vitro perfusion (Figures 3B and 3C).

[0076] (Demonstration of scaffold glucose responsiveness) Rat islets loaded into scaffolds using the best compact device design were positive for insulin and glucagon by day 4 of in vitro culture after demonstrating glucose responsiveness on day 1 (Figure 4A). The best scaffolds reached stimulation indices similar to conventionally cultured islets, suggesting that variability in cell loading or islets, rather than material incompatibility, prevented all three from demonstrating clear glucose responsiveness (Figures 4B, 4C).

[0077] (Development of iPS-derived insulin-producing cells) We adapted the Millman Lab's adherence protocol [29,30] to immunoreactive iPS cells (Hypo1) using suggested additions to the chemical cocktail applied during certain stages of differentiation. Thus far, cells have shown inconsistent glucose responsiveness (Figure 5A), with 80% PDX1+ on Matrigel and 90%+ on vitronectin (however, vitronectin did not support cell attachment long-term) (Figure 5B), but better than the ATCC-derived control stem cell line (Figure 5C). Substituting PDBU for TPPB, adding bFGF at S1 of the protocol, and adding betabetacellulin at S5 resulted in modest functional improvements for Hypo1 SCβCs at 2 weeks of maturation (Figure 5D). Early mRNA expression analysis from Hypo1 cells showed the continued presence of OCT4 cells throughout the differentiation stages, delayed and short-term expression of FOXA2, and temporally rational expression of NKX6.1 ( Figure 5E ).

[0078] (iPS beta cells in scaffolds in vitro and ex vivo) Although cells from one batch were loaded into the microscaffolds, they did not exhibit glucose responsiveness (Figure 5F), but maintained an aggregated morphology within the scaffolds over a prolonged period (Figures 5G and 5H). In a series of non-survival large animal experiments, two monolayer implants were connected to a central venous catheter in porcine subjects. When the implants contained Hypo1 SCβC, the vascular pattern was endothelialized with HUVECs, and the implants were perfused with porcine blood, we first detected human C-peptide in the device output and were able to detect human C-peptide in the porcine circulation at 45 minutes (Figure 6A). In the second implant, recombinant C-peptide also peaked at 45 minutes (Figure 6B), demonstrating that the device can deliver larger doses, reaching the human fasting concentration of C-peptide at 0.9 ng / mL. The insulin delivery rate with Hypo1 SCβC was 1.4 IU / hour, compared with 78 IU / hour when recombinant C-peptide was loaded into the IVAP. A cell covering layer in the vascular channel (Fig. 6C) and Hypo1 SCβC were still evident in the islet channel following blood flow (Fig. 6D).

[0079] In vitro two-tier bilayer IVAP cell viability and function In a series of experiments using luciferase-secreting cells (luciferase ~24 kDa vs. insulin ~5.8 kDa), diffusion to the outflow was demonstrated in in vitro perfusion cultures in two-tiered bilayer scaffolds on days 2 and 7 after IFN stimulation in the inflow. Three two-tiered bilayer scaffolds were loaded with HEK aggregates impregnated with at least 1 mL of cell pellet. All three scaffolds produced luciferase on day 2, and then increased in amount on day 7 in response to a second stimulation (Figure 7A). Luciferase continued to be produced by the scaffolds (data not shown). Furthermore, the viability of the perfusion cultures after 2 weeks was approximately 80%, indicating a high degree of cellularity (Figures 7B-7F).

[0080] (Two-Stage Double-Layer IVAP In Vivo Anastomosis and Implantation) Two HUVEC-endothelialized, two-tiered, bilayered IVAPs with vascular conduits were anastomosed to the aorta and vena cava (Figure 7G). Blood flowed freely within the vascular pattern upon physical inspection of the graft at the end point. Scaffold architectural fidelity remained high even after excision (Figure 7H; the presence of blood within the channels on the excised pieces does not fully represent perfusion, as blood drained from the channels when the excised pieces were removed from the scaffold). Both animals survived to the designated end points on days 1 and 7, demonstrating proof-of-concept for surgical model and implant construction.

[0081] In summary, the device disclosed herein provides permanent insulin independence and freedom from the risk of diabetic complications by enabling complete maturation before implantation and immediate perfusion after implantation. The use of hypoimmunogenic iPSC-derived endothelial cells provides biological immune protection and prevents rejection by the recipient. As a better treatment for diabetes, IVAP will reduce the burden on the healthcare system and decrease the number of people who die off organ transplant waiting lists.

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Claims

1. An intravascular artificial pancreas device capable of producing insulin, comprising: a first vascular layer comprising a plurality of first vascular channels, each having a first end and a second end, wherein each of the first ends of the plurality of vascular channels connects to a first input conduit and each of the second ends of the plurality of vascular channels connects to a first output conduit, thereby forming a first vascular channel network; a pancreatic islet layer comprising pancreatic islets and / or beta cells disposed within at least one islet chamber, wherein the pancreatic islets and / or beta cells are further embedded within an islet chamber matrix; a first thin electrospun membrane disposed as a biomolecule and gas permeable interface between the plurality of vascular channels and a first side of the at least one islet chamber, the plurality of vascular channels and the first side of the at least one islet chamber being juxtaposed opposite one another across the first thin electrospun membrane, thereby enabling exchange of biomolecules and gases between the pancreatic islets and / or beta cells disposed in the at least one islet chamber of the islet layer and the plurality of vascular channels in the first vascular layer; An intravascular artificial pancreas device comprising:

2. 2. The endovascular artificial pancreas device of claim 1, wherein the at least one islet chamber comprises a plurality of islet chambers configured as a plurality of islet channels, and wherein at least two vascular channels of the plurality of vascular channels are in opposing juxtaposition across and interfacing with a first side of each islet channel through a first thin electrospun membrane.

3. a second vascular layer comprising a plurality of vascular channels, each having a first end and a second end, wherein each of the first ends of the plurality of vascular channels connects to a second input conduit and each of the second ends of the plurality of vascular channels connects to a second output conduit, thereby forming a second vascular channel network; a second thin electrospun membrane disposed as a biomolecule and gas permeable interface between the plurality of vascular channels of the second vascular layer and a second side of the at least one pancreatic islet chamber, thereby enabling exchange of biomolecules and gases between the pancreatic islets and / or beta cells disposed in the at least one pancreatic islet chamber and the plurality of vascular channels in the second vascular layer; 10. The endovascular artificial pancreas device of claim 1, further comprising:

4. 4. The endovascular artificial pancreas device of claim 3, wherein the at least one islet chamber comprises a plurality of islet chambers configured as a plurality of islet channels, and wherein at least two of the plurality of vascular channels of the second vascular layer are in opposing juxtaposition across and interfacing with a second side of each islet channel through a second thin electrospun membrane.

5. The endovascular artificial pancreas device of claim 1 or 3, wherein the plurality of vascular channels of the first vascular layer and / or the second vascular layer are lined with endothelial cells.

6. The endovascular artificial pancreas device of claim 1 or 3, wherein the beta cells are hypoimmunogenic (B2M- / -, CIITA- / -) and / or derived from induced pluripotent stem cells (iPSCs).

7. The intravascular artificial pancreas device of claim 5 , wherein the endothelial cells are glomerular microvascular endothelial cells or human umbilical vein endothelial cells.

8. 10. The endovascular artificial pancreas device of claim 1, wherein the endovascular artificial pancreas device is glucose responsive and produces an amount of insulin proportional to the amount of glucose within the device.

9. The endovascular artificial pancreas device of claim 1 or 3, wherein the plurality of vascular channels of the first blood vessel and / or the second blood vessel layer are microchannels that form a first and / or second microvascular channel network.

10. The endovascular artificial pancreas device of any one of claims 2 and 4-9, wherein the at least one islet chamber comprises an elongated first and / or second side, allowing an increased surface area to interfacially contact one or more vascular channels across the first and / or second thin electrospun membranes.

11. The endovascular artificial pancreas device of any one of claims 1-10, wherein the amount of pancreatic islets and / or beta cells present within the device comprises at least 500,000 pancreatic islet equivalents.

12. The endovascular artificial pancreas device of any one of claims 1-11, wherein the at least one islet chamber is capable of accommodating approximately 660,000 islet equivalents.

13. The endovascular artificial pancreas device of any one of claims 1-12, wherein the islet chamber matrix comprises collagen.

14. The endovascular artificial pancreas device of any one of claims 1-13, wherein the pancreatic islet chamber matrix comprises an in situ polymerized matrix.

15. The endovascular artificial pancreas device of any one of claims 1-14, wherein the first and / or second thin electrospun membranes comprise polycaprolactone and gelatin.

16. 4. The endovascular artificial pancreas device of claim 1, wherein the first and / or second sides of the at least one pancreatic islet chamber interface with the plurality of vascular channels of the first and / or second vascular layers through the first and / or second thin electrospun membranes at a distance of about 10 to 30 micrometers (μm).

17. The endovascular artificial pancreas device of any one of claims 1 to 16, wherein the pancreatic islet layer and / or the first and / or second vascular layers further comprise an encapsulation matrix encapsulating the first and / or second vascular channel network and the at least one pancreatic islet chamber.

18. The endovascular artificial pancreas device of any one of claims 2-17, wherein the first and / or second inlet conduits and / or outlet conduits are tapered to minimize shear stress applied across the first and / or second vascular networks.

19. The endovascular artificial pancreas device of claims 2-18, wherein the first and second inlet conduits are fluidly connected to a first end of a fluid supply conduit and the first and second outlet conduits are fluidly connected to a first end of a fluid exit conduit.

20. 20. The endovascular artificial pancreas device of claim 19, wherein the fluid supply conduit and fluid exit conduit have second ends that are adjacent to each other and / or on the same side of the device.

21. The endovascular artificial pancreas device of any one of claims 1-20, wherein the first vascular layer, the pancreatic islet layer, the first thin electrospun membrane, and optionally the second vascular layer and second thin electrospun membrane form a first device unit, and the endovascular artificial pancreas device further comprises a second device unit comprising the first vascular layer, the pancreatic islet layer, the thin electrospun membrane, and optionally the second vascular layer and second thin electrospun membrane, and the inlet and outlet conduits of the first device unit are fluidly connected to the respective inlet and outlet conduits of the second device unit.

22. 1. A process for manufacturing an intravascular artificial pancreas, comprising: (A) generating a first thin nanofibrous membrane by electrospinning a polymer-containing solution; (B) depositing a sacrificial substrate in the form of at least one pancreatic islet chamber on a first side of the thin nanofibrous membrane; (C) depositing a plurality of sacrificial substrates in the form of at least a plurality of first vascular channels on the second side of the thin nanofiber membrane, each of the sacrificial substrates in the form of first vascular channels having a first end and a second end, the first end connected to a first input conduit and the second end connected to a first outlet conduit; (D) encapsulating at least the thin nanofiber membrane in an encapsulation matrix having the sacrificial substrate of steps (B) and (C) deposited thereon; (E) removing the sacrificial substrate of steps (B) and (C) to provide a first vascular network and at least one pancreatic islet chamber; (F) filling said at least one islet chamber with an islet chamber matrix comprising pancreatic islets and / or beta cells capable of producing proinsulin peptides and / or glucagon; The process includes:

23. (C)(i) generating a second thin nanofibrous membrane by electrospinning a polymer-containing solution over said sacrificial substrate in the form of at least one islet chamber; (C)(ii) depositing a plurality of sacrificial substrates in the form of a plurality of second vascular channels on a side of the second thin nanofiber membrane opposite the side in contact with the sacrificial substrate in the form of at least one pancreatic islet chamber, each of the sacrificial substrates in the form of second vascular channels having a first end and a second end, the first end connected to a second inlet conduit and the second end connected to a second outlet conduit; further comprising step (D) comprises encapsulating the second nanofiber membrane, sacrificial substrate, second inlet conduit, and second outlet conduit of (C)(i)-(ii) together with the first thin nanofiber membrane, sacrificial substrate, first inlet conduit, and first outlet conduit of (A)-(C) in an encapsulation matrix; 23. The process of claim 22, wherein step (E) further comprises removing the sacrificial substrate of (C)(ii) to provide a second vascular network.

24. 24. The process of claim 22 or 23, further comprising (G) introducing a suspension of endothelial cells into the first vascular network and / or the second vascular network, and after a period of time, inverting the device 180 degrees.

25. (C)(iii) generating a third thin nanofibrous membrane by electrospinning a polymer-containing solution over the plurality of sacrificial substrates formed in step (C); 24. The process of claim 23, wherein step (D) further comprises encapsulating the third nanofiber membrane, second nanofiber membrane, sacrificial substrate, second inlet conduit, and second outlet conduit of (C)(i)-(ii) together with the first thin nanofiber membrane, sacrificial substrate, first inlet conduit, and first outlet conduit of (A)-(C) in an encapsulation matrix.

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