Biofluid purification using biocompatible membranes

Abstract

Providing biofluid purification using biocompatible membranes. [Solution] An apparatus for integrated adaptive biological blood purification, comprising a functional unit, the functional unit comprising: a membrane having a tubular surface and a filtration surface; a vascular channel system comprising a first lumen space adhered to the vascular surface of the membrane and in fluid communication with the vascular surface of the membrane, the vascular channel system comprising a first end configured to be connected in fluid communication with a fluid supply section and a second end configured to be connected in fluid communication with a filtration fluid outlet; and a filtration channel system comprising a second lumen space adhered to the filtration surface of the membrane and in fluid communication with the filtration surface of the membrane, the filtration channel system comprising a third end configured to be connected in fluid communication with a filtrate outlet.

Description

Background Art

[0001] Cross-reference to Related Applications This application claims the benefit of U.S. Provisional Patent Application No. 62 / 812,239, filed Feb. 28, 2019, the entire contents of which are incorporated herein by reference.

[0002] (Background Art) Current methods of artificial blood purification require the use of a dialysate filter (in conventional hemodialysis), a dialysate fluid (in both conventional hemodialysis and peritoneal dialysis), a replacement fluid (in hemofiltration and hemodiafiltration), an anticoagulant to prevent activation of the coagulation cascade by filter materials (conventional hemodialysis), a blood pump to generate the flow and hydrostatic gradients necessary to enable filtration and dialysis (conventional hemodialysis), and, in some applications, the use of adsorbent materials to bind toxin molecules.

[0003] Currently, there is no technology to create a fully integrated, preferably implantable, blood purification system that can function continuously without the need for these components. Furthermore, bioengineering of human-scale tissue and organ transplants that can address the aforementioned problems requires the creation of a matrix that provides the necessary functional architecture to enable each cell to perform its specific role and generate a functional tissue construct. Tissues and organs containing one or more epithelial structures (digestive, endocrine, nervous, lymphatic, covering, reproductive, respiratory, sensory, urinary, and circulatory) rely on the presence of a thin basement membrane that enables functions such as fluid filtration (kidneys, eyes, lymphatic system, brain), gas diffusion (lungs), secretion and absorption of electrolytes and other molecules (kidneys, intestines, liver, intestinal tissue), and hormone diffusion (pancreas, pituitary gland, adrenal gland) from one lumen or compartment to another. In most cases, this basement membrane must be less than 1 µm or less than 10 µm thick to enable function (see Rayat et al., Indian Journal of Pathology & Microbiology 48, 453-458 (2005) and Kopf et al., Nature Immunology 16, 36-44 (2015)).

[0004] Currently, there are no technologies to produce membranes composed of biological and / or natural matrix materials with the adjustable micro or nanoporosity and physiological thickness necessary to enable such functions in biological tissue scaffolds, such as for artificial tissues or organs for hemodialysis. [Overview of the project] [Means for solving the problem]

[0005] The results described herein demonstrate the fabrication of biocompatible membranes with adjustable thickness and pore size.

[0006] Furthermore, the results described herein demonstrate the design, manufacture, and use of an integrated adaptive biological blood purification (IABBP) device having a biocompatible membrane. The purpose of this device is to remove toxins and excess water from the patient's circulation, replacing the body's own purification systems, such as the kidneys and liver. In contrast to currently available systems, the function of this device does not depend on the use of external dialysate fluids (as used in hemodialysis and peritoneal dialysis), replacement fluids (as used in hemofiltration and hemodiafiltration), or adsorbent materials (as used in portable dialysis and extracorporeal liver replacement). The IABBP device is connected to the patient's vascular system via direct connections to arteries and veins. The patient's cardiovascular system is used to perfuse the IABBP device with a blood flow sufficient to enable its function. This can be achieved without the use of an additional mechanical pump. However, in some cases, a pump may be used to enhance the function of the IABBP device.

[0007] Connection to the patient's vascular system is established via arteriovenous shunt or cannula insertion into the vena cava, or via direct anastomosis of the IABBP device vascular conduit to the patient's vascular structure. The IABBP device may be used extracorporeally or implanted in the patient, as well as in donor organs. The IABBP device may be used for continuous or intermittent treatment. The IABBP device produces a filtrate that is discharged into an extracorporeal collection system or connected to the patient's bladder via surgical anastomosis.

[0008] IABBP can be manufactured by creating a scaffold, which is then cultured to allow cells to be rearranged and mature so that the resulting tissue becomes functional. IABBP scaffolds are generated by combining multiple functional units. Each functional unit is generated by fabricating a biocompatible extracellular matrix membrane. This membrane can be porous or non-porous depending on the functional requirements. Sacrificial material is then printed on both sides of the membrane. The entire membrane is then embedded in the matrix material. The sacrificial material is removed, thereby obtaining two channel systems separated by the membrane. Several functional units can be stacked to generate a scaffold large enough to meet the needs of a human body. Cells can then be rearranged into both channel systems to generate functional living tissue.

[0009] Some aspects of the present disclosure relate to an apparatus for integrated adaptive biological blood purification, comprising a functional unit, the functional unit comprising a membrane having a tubular surface and a filtration surface, a vascular channel system comprising a first lumen space adhered to the vascular surface of the membrane and in fluid communication with the vascular surface of the membrane, the vascular channel system comprising a first end configured to be connected in fluid communication with a fluid supply section, and a second end configured to be connected in fluid communication with a filtration fluid outlet, and a filtration channel system comprising a second lumen space adhered to the filtration surface of the membrane and in fluid communication with the filtration surface of the membrane, the filtration channel system comprising a third end configured to be connected in fluid communication with a filtrate outlet, The present invention relates to a device comprising a vascular channel system and a filtration channel system, wherein the vascular channel system and the filtration channel system are in fluid communication with each other across a membrane, and the functional unit further comprises at least three segments, the at least three segments including at least a filtration segment configured to provide ultrafiltration for producing a primary ultrafiltration product, a tubular segment connected thereto configured to provide reabsorption for producing a secondary ultrafiltration product, and a conduit segment connected thereto configured to provide concentration for producing a tertiary ultrafiltration product, and the membrane comprises three membrane segments including at least a filtration membrane segment, a tubular membrane segment and a conduit membrane segment.

[0010] In some embodiments, the membrane comprises a biocompatible extracellular matrix membrane that separates the vascular channel system from the filtration channel system, and the biocompatible extracellular matrix membrane is embedded in the matrix material. In some embodiments, the biocompatible extracellular matrix membrane comprises a collagen membrane having a thickness of 0.1 to 10 micrometers (0.1 to 10 μm) that supports cell adhesion on both the vascular and filtration surfaces of the collagen membrane. In some embodiments, the matrix material is gelatin. In some embodiments, the biocompatible extracellular matrix membrane comprises fibers, nanofibers, or other longitudinal elements. In some embodiments, the fibers, nanofibers, or other longitudinal elements increase or regulate the mechanical strength of the membrane. In some embodiments, the fibers, nanofibers, or other longitudinal elements are uniformly distributed throughout the membrane to provide homogeneous mechanical reinforcement. In some embodiments, the fibers, nanofibers, or other longitudinal elements are heterogeneously distributed in the membrane to provide heterogeneous mechanical reinforcement.

[0011] In some embodiments, the filtration membrane segment allows for the generation of filtrate from a first lumen space in the vascular channel system to a second lumen space in the filtration channel system; the tubular membrane segment allows for the exchange and / or diffusion of solute and water between the vascular channel system and the filtration channel system; and the conduit membrane segment allows for the transfer of water and solute from the filtration channel system to the vascular channel system.

[0012] In some embodiments, the functional unit comprises at least one biofluid inflow conduit fluidly communicating with a first end of the vascular channel system and a first lumen space, and at least one biofluid outflow conduit fluidly communicating with a second end of the vascular channel system and a first lumen space; the functional unit comprises at least one filtrate outflow conduit fluidly communicating with a third end of the filtration channel system and a second lumen space; the functional unit further comprises one or more vascular segment conduits interconnecting the filtration segments, tubular segments and conduit segments of the vascular channel system and the first lumen space, and one or more filtration segment conduits interconnecting the filtration segments, tubular segments and conduit segments of the filtration channel system and the second lumen space.

[0013] In some embodiments, at least one biofluid inflow conduit is in fluid communication with an arterial conduit, at least one biofluid outflow conduit is in fluid communication with a vascular conduit, and at least one filtrate outflow conduit is in fluid communication with an outlet conduit. In some embodiments, the device uses the outlet conduit to generate an ultrafilter that is discharged into an extracorporeal collection system or, using the outlet conduit, into the patient's bladder. In some embodiments, the first and second lumen spaces are embedded in a scaffold.

[0014] In some embodiments, the filtration segment of the vascular channel system comprises a vascular channel wall lined with endothelial cells selected from primary human glomerular endothelial cells, induced pluripotent stem cell (iPSC)-derived endothelial cells, and / or human umbilical cord endothelial cells. In some embodiments, the tubular segment of the vascular channel system comprises a vascular channel wall lined with endothelial cells selected from primary human paratubular capillary endothelial cells, iPSC-derived endothelial cells, and / or human umbilical cord endothelial cells. In some embodiments, the conduit segment of the vascular channel system comprises a vascular channel wall lined with endothelial cells selected from primary human renal medullary endothelial cells, iPSC-derived endothelial cells, and / or human umbilical cord endothelial cells. In some embodiments, the filtration segment of the filtration channel system comprises a filtration channel wall lined with epithelial cells selected from primary human podocytes and / or human iPSC-derived podocytes. In some embodiments, the tubular segments of the filtration channel system comprise a filtration channel wall lined with epithelial cells selected from primary human tubular epithelial cells and / or iPSC-derived tubular epithelial cells.

[0015] In some embodiments, the tubular segment of the filtration channel system comprises a filtration channel wall lined with epithelial cells selected from primary human tubular epithelial cells and / or iPSC-derived tubular epithelial cells.

[0016] In some embodiments, the device comprises a plurality of functional units, each of which has a first end and a first lumen space of a vascular channel system fluid-communicating with at least one biofluid inflow conduit, a second end and a first lumen space of a vascular channel system fluid-communicating with at least one biofluid outflow conduit, and a third end and a second lumen space of a filtration channel system fluid-communicating with a filtrate outflow conduit. Each first end of the plurality of functional units is individually connected in parallel to one of a plurality of manifold ports of at least one biofluid inflow conduit, each second end of the plurality of functional units is individually connected in parallel to one of a plurality of manifold ports of at least one biofluid outflow conduit, and each third end of the plurality of functional units is individually connected in parallel to one of a plurality of manifold ports of at least one filtrate outflow conduit. In some embodiments, the device comprises a plurality of functional units, each of which has a plurality of functional units, each of which has a plurality of functional units, stacked in a parallel layer of functional units, and includes a plurality of functional units and additional functional units of the same configuration.

[0017] In some embodiments, at least one biofluid inflow conduit comprises a blood inlet conduit configured to transport blood inflow, and at least one biofluid outflow conduit comprises a blood outflow conduit configured to transport blood outflow, and a parallel layer of functional units configured for biological blood purification.

[0018] In some embodiments, the filtration segments of the filtration channel system are configured to provide ultrafiltration that generates primary ultrafiltration material, the tubular segments are configured to provide reabsorption that generates secondary ultrafiltration material by absorption of solute and water, and the conduit segments are configured to provide concentration that generates tertiary ultrafiltration material by absorption of water.

[0019] In some embodiments, the device is configured for extracorporeal operation in a sterile, heated enclosure. In some embodiments, the device is sized and configured for in-body placement in a human body, to replace or enhance kidney or liver function, and is placed within a capsule. In some embodiments, the membrane includes a porous membrane containing pores arranged to interconnect vascular surfaces and filtration surfaces. In some embodiments, the pores have a diameter of 1 μm to 15 μm.

[0020] Some aspects of this disclosure relate to a method for treating a patient with insufficient renal or hepatic function, comprising: fluidly connecting the apparatus described herein to the patient's circulatory system; and passing the patient's blood through the apparatus's vascular channel system from a filtration member segment to a tubular member segment, from the tubular member segment to a conduit member segment, and from the conduit member segment back into the patient's circulatory system. In some embodiments, the apparatus is implanted in the patient. In some embodiments, the ultrafilter produced by the apparatus is delivered to the patient extracorporeally. In some embodiments, the ultrafilter produced by the apparatus is delivered to the patient's bladder.

[0021] In some embodiments, the device is external to the patient.

[0022] Some aspects of the present disclosure relate to a method for manufacturing the apparatus described herein, comprising providing a plurality of membranes having sacrificial material in the form of a vascular channel network on a vascular surface and sacrificial material in the form of a filtration channel system on a filtration surface, and a method comprising immersing the plurality of membranes in a solution containing a scaffold material (i.e., a membrane solution), gelling the scaffold material, and removing the sacrificial material to thereby form luminal spaces for the vascular channel system and the filtration channel system.

[0023] In some embodiments, multiple membranes are produced by chemical or physical thin-film deposition, atomization, spraying, electrospinning, immersion coating, or gelation of a solution containing liquefied or homogenized decellularized tissue, gelatin, gelatin complex, collagen, fibrin, hydrogel, hydrogel complex, chitosan, nitrocellulose, polylactic acid, or extracellular matrix in a thin-film layer, followed by curing, crosslinking, polymerization, drying, or gelation of the solution to form a membrane layer.

[0024] In some embodiments, the membrane solution further comprises a pologen homogeneously mixed therein. In some embodiments, the pologen 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 to 40% by weight.

[0025] 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, 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 may be mixed into the membrane solution before fabrication to uniformly distribute the fibers throughout the membrane. Alternatively, these fibers may be deposited or integrated onto the membrane after fabrication through techniques such as electrospinning, 3D printing, or other techniques. The fibers may be homogeneously distributed throughout the membrane or systematically distributed to provide heterogeneous mechanical properties to the membrane. In some embodiments, the method for producing thin film layers is repeated one or more times to produce one or more membranes having two or more membrane layers. In some embodiments, the two or more layers are produced from solutions having different components, agents, and / or concentrations.

[0026] In some embodiments, at least one of the multiple membranes is treated to remove pologens, thereby forming pores in the membrane.

[0027] In some embodiments, the membrane solution contains 3 to 35% by weight of gelatin or a gelatin-polymer composite. In some embodiments, the thin film layer is crosslinked using a solution containing glutaraldehyde, transglutaminase, or other crosslinking enzymes or molecules.

[0028] In some embodiments, the sacrificial material may have thermoreversible gelling properties or be soluble in a nonpolar solvent. In some embodiments, the sacrificial material is removed using a nonpolar solvent or by thermal reversal of the gelation. In some embodiments, the sacrificial material comprises a poloxamer formulation, preferably Pluronic F127.

[0029] In some embodiments, the scaffold material is an extracellular matrix material. In some embodiments, the extracellular matrix material is gelatin. In some embodiments, the scaffold material is gelled by crosslinking with a solution containing glutaraldehyde, transglutaminase, or other crosslinking enzymes or molecules, and / or the scaffold material is thermally crosslinked.

[0030] In some embodiments, the steps of immersing a plurality of membranes in a solution containing a scaffolding material and gelling the scaffolding material include: (a) providing a bottom mold (64) having an open top reservoir and comprising a vascular channel system inflow conduit structure (63) and a vascular channel system outflow conduit structure (65), each having an internal lumen filled with sacrificial material, wherein the reservoir is partially filled with gelled scaffolding material that partially embeds the vascular channel system inflow conduit structure and the vascular channel system outflow conduit structure; (b) providing a plurality of membranes in a frame; (c) filling the open top reservoir of the bottom mold (64) with a solution containing the scaffolding material; (d) positioning the frame on the top of the bottom mold so that the membranes in the frame are in contact with the solution; (e) gelling the solution and then removing the frame from the membranes; (f) positioning a spacer (62) having an internal volume around the top of the membranes; and (g) filling the internal volume of the spacer with the solution containing the scaffolding material. (h) filling the apparatus with (i) a spacer (57) on top of the final membrane which is configured to have a filtration channel system outflow conduit structure (56) having an internal lumen filled with sacrificial material (k) filling the internal volume of the spacer (57) with a solution containing scaffolding material and gelling the solution (l) adding a shaft filled with sacrificial material to the gelled solution which fluidly connects the first ends of the plurality of membranes to the vascular channel system inflow conduit structure (63), adding a shaft filled with sacrificial material to the gelled solution which fluidly connects the second ends of the plurality of membranes to the vascular channel system outflow conduit structure (65), adding a shaft filled with sacrificial material to the gelled solution which fluidly connects the third ends of the plurality of membranes to the filtration channel system outflow conduit structure (56), and (m) removing the sacrificial material from the structure.

[0031] In some embodiments, the method for generating the device further includes adding cells to one or more segments of a vascular channel system and / or a filtration channel system. In some embodiments, the cells are added to the segment by (a) filling the vascular channel system and the filtration channel system with fluid; (b) placing the cells in a first volume of fluid approximately equal to the volume of fluid in the channel system of the target segment; (c) adding the first volume to the device through a first fluid supply or fluid outlet that is in fluid communication with the target segment; and (d) adding a second volume of fluid approximately equal to the volume of fluid contained between the target segment and the first fluid supply or fluid outlet, and / or removing a third volume of fluid approximately equal to the volume of fluid contained between the target segment and a second fluid supply or fluid outlet that is in fluid communication with the first fluid supply or fluid outlet.

[0032] In some embodiments, cells are added to each functional unit of the device. In some embodiments, cells are added to each segment of each functional unit of the device (for example, both or either of the vascular channel system and the filtration channel system located within each segment). In some embodiments, cells are added to both the vascular channel system and the filtration channel system. The cells are not limited and may be any cells described herein.

[0033] Some aspects of this disclosure relate to membranes having pores having a diameter of about 1 μM to 15 μM, comprising a biological or synthetic matrix material. In some embodiments, the biological or synthetic matrix material comprises decellularized tissue, gelatin, gelatin complex, collagen, fibrin, hydrogel, hydrogel complex, chitosan, nitrocellulose, polylactic acid, or extracellular matrix. In some embodiments, the membrane has a thickness of about 0.1 μM to 100 μM. The membrane may have any thickness described herein and is not limited thereto.

[0034] Several aspects of this disclosure relate to methods for producing the membranes described herein, comprising chemically or physically depositing, atomizing, spraying, electrospinning, immersion coating, or gelling a solution (i.e., membrane solution) containing liquefied or homogenized decellularized tissue, gelatin, gelatin complex, collagen, fibrin, hydrogel, hydrogel complex, chitosan, nitrocellulose, polylactic acid, or extracellular matrix in a thin film layer, and subsequently curing, crosslinking, polymerization, drying, or gelling the solution to form a membrane layer. In some embodiments, the membrane solution further comprises a pologen homogeneously mixed therein. The pologen is not limited and may be any pologen described herein. In some embodiments, the pologen 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 to 40% by weight in the membrane solution.

[0035] In some embodiments, the membrane solution further comprises one or more agents that modify the mechanical or biological properties of the membrane. In some embodiments, one or more agents are selected from glycerin, sorbitol, propylene glycol, plasticizers, fibers or other longitudinal elements, and growth factors (e.g., encapsulated growth factors).

[0036] In some embodiments, the method for producing a membrane further includes adding one or more additional membrane layers to a first membrane layer by a method disclosed herein in order to produce a membrane of a mixed composition or architecture. In some embodiments, two or more layers are produced from membrane solutions having different components, agents, and / or concentrations.

[0037] In some embodiments, the film is treated to remove pologens, thereby forming pores in the film. In some embodiments, the pologen material may have thermoreversible gelling properties or be soluble in a nonpolar solvent. In some embodiments, the pologen material is Pluronic F127, which is removed by treatment with a nonpolar solvent (e.g., isopropanol).

[0038] In some embodiments, the membrane solution contains 3 to 35% by weight of gelatin or a gelatin-polymer composite.

[0039] In some embodiments, the thin film layer can be dried, gelled, crosslinked, or otherwise solidified and removed from the substrate. In some embodiments, the thin film layer is crosslinked using a solution containing glutaraldehyde, transglutaminase, or other crosslinking enzymes or molecules. In some embodiments, the concentration of the crosslinking agent is about 0.01 to 5 g per 10 g of scaffolding material.

[0040] This patent or application file includes at least one drawing made in color. A copy of this patent or patent application publication containing the color drawing will be provided by the Office upon request and payment of the required fees. The present invention provides, for example, the following: (Item 1) A device for integrated adaptive biological blood purification, It is equipped with a functional unit, and the functional unit is A membrane comprising a blood vessel surface and a filtration surface, A vascular channel system comprising a first tubular space adhered to the surface of the vascular membrane and in fluid communication with the surface of the vascular membrane, the vascular channel system comprising a first end configured to be connected to a fluid supply section in fluid communication, and a second end configured to be connected to a filtration fluid outlet in fluid communication, A filtration channel system comprising a second lumen space adhered to the filtration surface of the membrane and having fluid communication with the filtration surface of the membrane, and further comprising a third end configured to be connected to the filtrate outlet in fluid communication, The vascular channel system and the filtration channel system are in fluid communication with each other across the membrane. The functional unit further comprises at least three segments, the at least three segments including a filtration segment configured to provide ultrafiltration for generating primary ultrafiltration material, a tubular segment connected thereto configured to provide reabsorption for generating secondary ultrafiltration material, and a conduit segment connected thereto configured to provide concentration for generating tertiary ultrafiltration material. The apparatus comprises three membrane segments, each including at least a filtration membrane segment, a tubular membrane segment, and a conduit-like membrane segment. (Item 2) The apparatus according to item 1, wherein the membrane includes a biocompatible extracellular matrix membrane that separates the vascular channel system from the filtration channel system, and the biocompatible extracellular matrix membrane is embedded in a matrix material. (Item 3) The apparatus according to item 2, wherein the biocompatible extracellular matrix membrane includes a collagen membrane, the collagen membrane having a thickness of 0.1 to 10 micrometers (0.1 to 10 μm) and supporting cell adhesion on both the vascular surface and the filtration surface of the collagen membrane. (Item 4) The apparatus according to items 1 to 3, wherein the membrane is configured to have a minimum shear stress of 5 dynes / cm² and / or a minimum intermembrane pressure difference of 10 mmHg. (Item 5) The filtration membrane segment enables the generation of filtrate from the first lumen space in the vascular channel system to the second lumen space of the filtration channel system. The tubular membrane segment allows for the exchange and / or diffusion of solute and water between the vascular channel system and the filtration channel system. The apparatus according to items 1 to 4, wherein the conduit-like membrane segment enables the transfer of water and solute from the filtration channel system to the vascular channel system. (Item 6) The apparatus according to items 1 to 5, wherein the functional unit comprises at least one biofluid inflow conduit that is in fluid communication with the first end and the first lumen space of the vascular channel system, and at least one biofluid outflow conduit that is in fluid communication with the second end and the first lumen space of the vascular channel system, the functional unit comprises at least one filtrate outflow conduit that is in fluid communication with the third end and the second lumen space of the filtration channel system, and the functional unit further comprises one or more vascular segment conduits that interconnect the filtration segment, the tubular segment and the conduit segment of the vascular channel system and the first lumen space, and one or more filtration segment conduits that interconnect the filtration segment, the tubular segment and the conduit segment of the filtration channel system and the second lumen space. (Item 7) The apparatus according to item 6, wherein the at least one biofluid inflow conduit is in fluid communication with an arterial conduit, the at least one biofluid outflow conduit is in fluid communication with a vascular conduit, and the at least one filtrate outflow conduit is in fluid communication with an outlet conduit. (Item 8) The apparatus according to item 7, wherein the apparatus generates an ultrafilter that is discharged into an extracorporeal collection system using an discharge conduit, or discharged into the patient's bladder using an discharge conduit. (Item 9) The apparatus according to items 1 to 8, wherein the first lumen space and the second lumen space are embedded in a scaffold. (Item 10) The apparatus according to items 1 to 9, wherein the filtering segment of the vascular channel system comprises a vascular channel wall lined with endothelial cells selected from primary human glomerular endothelial cells, induced pluripotent stem cell (iPSC)-derived endothelial cells, and / or human umbilical cord endothelial cells. (Item 11) The apparatus according to items 1 to 10, wherein the tubular segment of the vascular channel system comprises a vascular channel wall lined with endothelial cells selected from primary human paratubular capillary endothelial cells, iPSC-derived endothelial cells, and / or human umbilical cord endothelial cells. (Item 12) The apparatus according to items 1 to 11, wherein the ductile segment of the vascular channel system comprises a vascular channel wall lined with endothelial cells selected from primary human renal medullary endothelial cells, iPSC-derived endothelial cells, and / or human umbilical cord endothelial cells. (Item 13) The apparatus according to items 1 to 12, wherein the filtration segment of the filtration channel system comprises a filtration channel wall lined with epithelial cells selected from primary human podocytes and / or human iPSC-derived podocytes. (Item 14) The apparatus according to items 1 to 13, wherein the tubular segment of the filtration channel system comprises a filtration channel wall lined with epithelial cells selected from primary human tubular epithelial cells and / or iPSC-derived tubular epithelial cells. (Item 15) The apparatus according to items 1 to 14, wherein the tubular segment of the filtration channel system comprises a filtration channel wall lined with epithelial cells selected from primary human tubular epithelial cells and / or iPSC-derived tubular epithelial cells. (Item 16) The apparatus according to items 1 to 15, wherein the endothelial cells and / or epithelial cells are homogeneous or autologous to the patient using the apparatus. (Item 17) The apparatus comprises a plurality of functional units, including the functional unit and additional functional units with the same configuration, and each of the plurality of functional units is The first end and first lumen space of a vascular channel system that is in fluid communication with at least one biofluid inflow conduit, The second end and first lumen space of a vascular channel system that is in fluid communication with at least one biofluid outflow conduit, It has a third end and a second lumen space of the filtration channel system which are in fluid communication with the filtrate outflow conduit, Each first end of the plurality of functional units is individually connected in parallel to one of the plurality of manifold ports of the at least one biofluid inflow conduit. Each second end of the plurality of functional units is individually connected in parallel to one of the plurality of manifold ports of the at least one biofluid outflow conduit. The apparatus according to items 1 to 16, wherein each third end of the plurality of functional units is individually connected in parallel to one of the plurality of manifold ports of the at least one filtrate outflow conduit. (Item 18) The apparatus according to item 17, wherein the apparatus comprises a plurality of functional units, each including the functional unit and additional functional units of the same configuration, stacked in a parallel layer of functional units. (Item 19) The apparatus according to items 1 to 18, wherein the at least one biofluid inflow conduit comprises a blood inlet conduit configured to transport blood inflow, and the at least one biofluid outflow conduit comprises a blood outflow conduit configured to transport blood outflow, and a parallel layer of functional units configured for biological blood purification. (Item 20) The apparatus according to item 19, wherein the filtration segment of the filtration channel system is configured to provide ultrafiltration for generating primary ultrafiltration material, the tubular segment is configured to provide reabsorption for generating secondary ultrafiltration material by absorption of solute and water, and the conduit segment is configured to provide concentration for generating tertiary ultrafiltration material by absorption of water. (Item 21) The apparatus according to items 1 to 20, wherein the apparatus is configured for extracorporeal manipulation in a sterile, heated enclosure. (Item 22) The apparatus according to item 21, wherein blood or dialysis fluid from the abdominal cavity is delivered to the apparatus using a mechanical pump. (Item 23) The apparatus according to items 1 to 20, wherein the apparatus is placed inside a capsule and is sized and configured for placement in the human body to replace or enhance the function of a kidney or liver. (Item 24) The apparatus according to items 1 to 23, wherein the membrane includes a porous membrane having pores arranged to interconnect the blood vessel surface and the filtration surface. (Item 25) The apparatus according to item 24, wherein the pores have a diameter of 1 μm to 15 μm. (Item 26) A method for treating a patient with insufficient renal or hepatic function, comprising: fluidly connecting an apparatus described in items 1 to 25 to the patient's circulatory system; and passing the patient's blood through the vascular channel system of the apparatus, from the filtration member segment to the tubular member segment, from the tubular member segment to the conduit member segment, and from the conduit member segment back into the patient's circulatory system. (Item 27) The method according to item 26, wherein the device is implanted in the patient. (Item 28) The method according to item 27, wherein the ultrafiltered material produced by the apparatus is delivered extracorporeally to the patient. (Item 29) The method according to item 27, wherein the ultrafiltered material produced by the apparatus is delivered to the patient's bladder. (Item 30) The method according to item 26, wherein the device is outside the body relative to the patient. (Item 31) A method for manufacturing the apparatus described in items 1 to 25, To provide a plurality of membranes having sacrificial material in the form of a vascular channel network on the vascular surface and sacrificial material in the form of a filtration channel system on the filtration surface, The plurality of films are immersed in a solution containing scaffolding material, The scaffolding material is gelled, A method comprising removing the sacrificial material to thereby form the luminal space of the vascular channel system and the filtration channel system. (Item 32) The method according to item 31, wherein each of the plurality of membranes is produced by chemical or physical thin-film deposition, atomization, spraying, electrospinning, immersion coating, or gelation of a solution containing liquefied or homogenized decellularized tissue, gelatin, gelatin complex, collagen, fibrin, hydrogel, hydrogel complex, chitosan, nitrocellulose, polylactic acid, or extracellular matrix in a thin-film layer, followed by curing, crosslinking, polymerization, drying, or gelation of the solution to form a membrane layer. (Item 33) The method according to item 32, wherein the solution further comprises a pologen homogeneously mixed therein. (Item 34) The method according to item 33, wherein the pologen is a self-assembling triblock copolymer. (Item 35) The method according to item 34, wherein the self-assembling triblock copolymer is a poloxamer formulation, preferably Pluronic F127 at a concentration of 1 to 40% by weight. (Item 36) The method according to items 32-35, wherein the solution further comprises one or more agents that modify the mechanical or biological properties of the one or more membranes. (Item 37) The method according to item 36, wherein the one or more agents are selected from glycerin, sorbitol, propylene glycol, plasticizers, fibers or other longitudinal elements, and encapsulated growth factors. (Item 38) The method according to items 32-37, further comprising repeating the method according to item 32 one or more times to produce one or more films having two or more film layers. (Item 39) The method according to item 38, wherein the two or more layers are formed from solutions having different components, drugs, and / or concentrations. (Item 40) The method according to items 32-39, wherein at least one of the plurality of membranes is treated to remove the pologen, thereby forming pores in the membrane. (Item 41) The method according to items 32-40, wherein the solution comprises 3-35% by weight of gelatin or a gelatin-polymer composite. (Item 42) The method according to items 32-41, wherein the thin film layer is crosslinked using a solution containing glutaraldehyde, transglutaminase, or other crosslinking enzymes or molecules. (Item 43) The method according to items 31-42, wherein the sacrificial material has thermoreversible gelling properties or can be dissolved in a nonpolar solvent. (Item 44) The method according to items 31-43, wherein the scaffold material is an extracellular matrix material. (Item 45) The method according to item 44, wherein the extracellular matrix material is gelatin. (Item 46) The method according to items 31-45, wherein the scaffolding material is gelled by crosslinking with a solution containing glutaraldehyde, transglutaminase, or other crosslinking enzymes or molecules, and / or the scaffolding material is thermally crosslinked. (Item 47) The method according to items 31-46, wherein the sacrificial material is removed using a nonpolar solvent or by reversing the gelation with heat. (Item 48) The method according to items 31 to 47, wherein the sacrificial material comprises a poloxamer formulation, preferably Pluronic F127. (Item 49) The steps of immersing the plurality of membranes in a solution containing scaffolding material and gelling the scaffolding material are as follows: a. To provide a bottom mold (64) comprising a vascular channel system inflow conduit structure (63) and a vascular channel system outflow conduit structure (65), each having an open top reservoir and an internal lumen filled with sacrificial material, wherein the reservoir is partially filled with a gelling scaffold material that partially embeds the vascular channel system inflow conduit structure and the vascular channel system outflow conduit structure, b. Providing multiple films in the frame, c. Filling the reservoir at the top of the opening of the bottom mold (64) with the solution containing the scaffolding material, d. Positioning the frame on top of the bottom mold such that the film in the frame is in contact with the solution, e. Gelating the solution, and then removing the frame from the film, f. Arranging a spacer (62) having an internal volume around the top of the film, g. Filling the internal volume of the spacer with a solution containing the scaffolding material, h. The frame is positioned on the top of the spacer such that the film in the frame is in contact with the solution, i. Optionally, repeat steps e. to h. one or more times to add an additional film to the apparatus. j. A spacer (57) is placed on the top of the final membrane, which is configured to have an outflow conduit structure (56) of a filtration channel system having an internal lumen filled with sacrificial material, k. The internal volume of the spacer (57) is filled with a solution containing the scaffolding material, the solution is gelled, and thereby the final membrane is embedded. l. Adding a shaft filled with sacrificial material to the gelled solution that fluidly connects the first ends of the plurality of membranes to the inflow conduit structure (63) of the vascular channel system; adding a shaft filled with sacrificial material to the gelled solution that fluidly connects the second ends of the plurality of membranes to the outflow conduit structure (65) of the vascular channel system; and adding a shaft filled with sacrificial material to the gelled solution that fluidly connects the third ends of the plurality of membranes to the outflow conduit structure (56) of the filtration channel system. The method according to items 31-48, including removing the sacrificial material from the structure. (Item 50) The method according to items 31-49, further comprising adding cells to one or more segments of the vascular channel system and / or filtration channel system of the membrane. (Item 51) The aforementioned cells, a. To provide a fluid-filled vascular channel system and a filtration channel system, b. Placing the cells in a first volume of fluid approximately equal to the volume of fluid in the channel system of the target segment, c. The above-mentioned first volume is added to the apparatus through a first fluid supply unit or fluid outlet that is in fluid communication with the target segment, d. The method according to item 50, wherein fluid is added to the segment by adding a second volume of fluid approximately equal to the volume of fluid contained between the target segment and the first fluid supply or fluid outlet, and / or removing a third volume of fluid approximately equal to the volume of fluid contained between the target segment and the second fluid supply or fluid outlet which is in fluid communication with the first fluid supply or fluid outlet. (Item 52) The method according to items 50-51, wherein the cells are added to multiple segments of a vascular channel system and / or a filtration channel system. (Item 53) A membrane comprising a biological or synthetic matrix material and having pores with a diameter of approximately 1 μM to 15 μM. (Item 54) The membrane according to item 53, wherein the biological or synthetic matrix material comprises decellularized tissue, gelatin, gelatin complex, collagen, fibrin, hydrogel, hydrogel complex, chitosan, nitrocellulose, polylactic acid, or extracellular matrix. (Item 55) A film described in items 53-54, having a thickness of approximately 0.1 μM to 100 μM. (Item 56) A method for producing the membranes described in items 53 to 55, comprising chemically or physically depositing, atomizing, spraying, electrospinning, immersion coating, or gelling a solution containing liquefied or homogenized decellularized tissue, gelatin, gelatin complex, collagen, fibrin, hydrogel, hydrogel complex, chitosan, nitrocellulose, polylactic acid, or extracellular matrix in a thin film layer, and subsequently curing, crosslinking, polymerization, drying, or gelling the solution to form a membrane layer. (Item 57) The method according to item 56, wherein the solution further comprises a pologen homogeneously mixed therein. (Item 58) The method according to item 57, wherein the pologen is a self-assembling triblock copolymer. (Item 59) The method according to item 58, wherein the self-assembling triblock copolymer is a poloxamer formulation, preferably Pluronic F127 at a concentration of 1 to 40% by weight. (Item 60) The method according to items 56-59, wherein the solution further comprises one or more agents that modify the mechanical or biological properties of the membrane. (Item 61) The method according to item 60, wherein the one or more agents are selected from glycerin, sorbitol, propylene glycol, plasticizers, fibers or other longitudinal elements, and encapsulated growth factors. (Item 62) The method according to items 56-61, further comprising repeating the method according to item 56 one or more times to produce a film having two or more film layers. (Item 63) The method according to item 62, wherein the two or more layers are formed from solutions having different components, drugs, and / or concentrations. (Item 64) The method according to items 56-63, wherein the membrane is treated to remove the pologen, thereby forming pores in the membrane. (Item 65) The method according to items 56-64, wherein the solution comprises 3-35% by weight of gelatin or a gelatin-polymer composite. (Item 66) The method according to items 56-65, wherein the thin film layer is crosslinked using a solution containing glutaraldehyde, transglutaminase, or other crosslinking enzymes or molecules. [Brief explanation of the drawing]

[0041] [Figure 1] Microscopic imaging of thin films fabricated using a variable amount of sacrificial pologen material is shown to facilitate control of porosity and pore size. [Figure 2] Subsequently, microscopic imaging of thin films fabricated using sacrificial pologens removed to create porous thin films is shown. [Figure 3] This shows a porous thin film with layers of fluorescently labeled endothelial and epithelial cells seeded on the opposite side of the membrane. [Figure 4]A schematic diagram of a single-function unit of an IABBP device is shown. Blood flows from the recipient's artery into the filtration segment, where the primary filtrate is produced. The blood and filtrate then flow into the tubular segments in their respective channels. Absorption and secretion occur in the tubular segments. The blood and secondary filtrate then flow into the conduit segments in their respective channels. The secondary filtrate is concentrated and discharged through the conduit, while the blood is returned to the recipient's circulation. (1) Blood inflow, (2) Blood inflow conduit, (3) Filtration segment blood channel system, (4) Blood flow to tubular segments, (5) Tubular segment blood channel system, (6) Blood flow to conduit segments, (7) Conduit segment channel system, (8) Blood outflow conduit, (9) Blood outflow, (10) Filtration, (11) Filtration segment filtrate channel, (12) Filtrate flow to tubular segments, (13) Solute and water secretion, (14 (15) Capillary segment filtrate channel system, (16) Solute and water absorption, (17) Filtrate flow into conduit segments, (18) Concentration (water absorption), (19) Conduit segment filtrate channel system, (20) Filtrate outflow conduit, (21) Embedding matrix, (22) Capsule, (23) Filtration segment, (24) Capillary segment, (25) Conduit segment, (26) Filtration membrane, (27) Capillary membrane, (28) Conduit membrane. [Figure 5] A schematic diagram of the channel architecture of a single functional IABBP unit is shown. The corresponding segments of the nephron are shown to illustrate the sequential function of each segment: (2) blood inflow conduit, (8) blood outflow conduit, (23) filtration segment, (24) tubular segment, (25) conduit segment, and (30) corresponding segment of the human nephron. [Figure 6]A schematic diagram of each segment of the functional IABBP unit with rearranged cells is shown. Each channel system within each segment is separated by a specialized membrane and lined with specialized cells to enable a higher level of function. (26) Filtration segment membrane, (31) Filtration segment blood channel, (32) Filtration segment endothelial cell, (27) Tubular segment membrane, (33) Tubular segment blood channel, (34) Tubular segment endothelial cell, (28) Conduit segment membrane, (35) Conduit segment blood channel, (36) Conduit segment endothelial cell, (37) Filtration segment epithelial cell, (38) Filtration segment filtrate channel, (39) Tubular segment epithelial cell, (40) Tubular segment filtrate channel, (41) Conduit segment epithelial cell, (42) Conduit segment filtrate channel. [Figure 7] A three-dimensional rendering of a cross-section of a functional IABBP unit is shown. The vascular space is separated from the filtrate space by a specialized membrane. (41) Vascular side channel, (42) Matrix membrane, (43) filtrate side channel, (44) Main vessel channel. [Figure 8] A schematic diagram of the stacking of functional units in an IABBP device that are perfused and discharged in parallel is shown. (45) graft blood inflow, (46) graft blood inlet conduit, (47) stacked functional unit, (48) stacked functional unit, (49) stacked functional unit, (50) stacked functional unit, (51) graft blood outflow conduit, (52) graft blood outflow, (53) graft filtrate outflow conduit, (54) graft filtrate outflow. [Figure 9] This shows a 3D rendering of a multilayer IABBP device. Five layers of functional units are stacked and connected in parallel, perfused with blood to produce filtrate, which is discharged through filtrate discharge conduits. (46) graft blood inlet conduit, (51) graft blood outlet conduit, (53) graft filtrate outlet conduit, (47) stacked functional unit, (55) connection channel. [Figure 10]This shows a three-dimensional rendering of a manufacturing mold for generating a multilayer IABBP device. The base mold and top mold house the blood vessels and filtrate conduits. Each cassette allows for the addition of embedding material and supports the patterned membrane. (56) Notch for top conduit, (57) Top mold, (58) Spacer 1, (59) Spacer 2, (60) Spacer 3, (61) Spacer 4, (62) Spacer 5, (63) Groove for bottom conduit 1, (64) Bottom mold, (65) Groove for bottom conduit 2. [Figure 11] A membrane testing apparatus and its schematic diagram are shown. A shows a photograph of the membrane testing apparatus. B shows a schematic diagram of the membrane testing apparatus and its components. (66) Plasma or blood inflow, (67) Plasma or blood inflow port, (68) Top chamber vascular space, (69) Solute and fluid exchange, (70) Plasma or blood outflow, (71) Plasma or blood outflow port, (72) Membrane, (73) Bottom chamber, (74) Bottom mold, (75) Filtrate discharge port, (76) Filtrate discharge, (77) O-ring. [Figure 12] This shows the pore size and area distribution of a membrane prepared from a mixture of gelatin and 0.25% Pluronic F127. [Figure 13] The upper panel shows the Pluronic F127 concentration convection velocity. The lower panel shows the fluid pressure convection velocity. [Modes for carrying out the invention]

[0042] Biofluid Purification Some aspects of the present disclosure relate to an apparatus for adaptive biological blood purification (AIBBP) comprising a functional unit, the functional unit comprising: (1) a membrane having a vascular surface and a filtration surface; (2) a vascular channel system comprising a first lumen space adhering to the vascular surface of the membrane and having fluid communication with the vascular surface of the membrane, the vascular channel system comprising a first end configured to be connected to a fluid supply in fluid communication with a fluid supply and a second end configured to be connected to a filtration fluid outlet in fluid communication with a fluid outlet; and (3) a filtration channel system comprising a second lumen space adhering to the filtration surface of the membrane and having fluid communication with the filtration surface of the membrane, the filtration channel system comprising a third end configured to be connected to a filtrate outlet in fluid communication with a fluid outlet. The vascular channel system and the filtration channel system are in fluid communication with each other across the membrane. Furthermore, the functional unit further comprises at least three segments, the at least three segments including at least a filtration segment configured to provide ultrafiltration for producing a primary ultrafiltrate, a tubular segment connected thereto configured to provide reabsorption for producing a secondary ultrafiltrate, and a conduit segment connected thereto configured to provide concentration for producing a tertiary ultrafiltrate, and the membrane comprises three membrane segments including at least a filtration membrane segment, a tubular membrane segment, and a conduit membrane segment.

[0043] In some embodiments, the filtration membrane segment allows for the generation of filtrate from a first lumen space in the vascular channel system to a second lumen space in the filtration channel system. In some embodiments, the tubular membrane segment allows for the exchange and / or diffusion of solute and water between the vascular channel system and the filtration channel system. In some embodiments, the conduit membrane segment allows for the transfer of water and solute from the filtration channel system to the vascular channel system.

[0044] In some embodiments, the functional unit comprises at least one biofluid inflow conduit that fluidly communicates with a first end of the vascular channel system and a first lumen space, and at least one biofluid outflow conduit that fluidly communicates with a second end of the vascular channel system and a first lumen space. In some embodiments, the functional unit comprises at least one filtrate outflow conduit that fluidly communicates with a third end of the filtration channel system and a second lumen space. In some embodiments, the functional unit further comprises one or more vascular segment conduits interconnecting the filtration segments, tubular segments and conduit segments of the vascular channel system and the first lumen space, and one or more filtration segment conduits interconnecting the filtration segments, tubular segments and conduit segments of the filtration channel system and the second lumen space.

[0045] In some embodiments, at least one biofluid inflow conduit is in fluid communication with an arterial conduit, at least one biofluid outflow conduit is in fluid communication with a vascular conduit, and at least one filtrate outflow conduit is in fluid communication with an outlet conduit.

[0046] In some embodiments, the membrane comprises a porous membrane containing pores arranged to interconnect the vascular surface and the filtration surface. In some embodiments, the pores have diameters of approximately 15 μm, 14 μm, 13 μm, 12 μm, 11 μm, 10 μm, 9 μm, 8 μm, 7 μm, 6 μm, 5 μm, 4 μm, 3 μm, 2 μm, or less than 1 μm. In some embodiments, the pores have an average or mean diameter of approximately 15 μm, 14 μm, 13 μm, 12 μm, 11 μm, 10 μm, 9 μm, 8 μm, 7 μm, 6 μm, 5 μm, 4 μm, 3 μm, 2 μm, or 1 μm. In some embodiments, the pore-containing membrane has a flow rate of about 0.2–2.0 mL / min, about 0.5–1.5 mL / min, or about 0.8–1.2 mL / min when subjected to a fluid pressure of 40 mmHg. In some embodiments, the membrane is manufactured by a process comprising mixing an extracellular matrix material (e.g., gelatin) with a porogen (pore-forming agent). In some embodiments, the pore-forming agent is Pluronic F127. In some embodiments, the membrane is manufactured by a process disclosed herein.

[0047] In some embodiments, the membrane is as described in PCT application PCT / US2017 / 67141 (filed December 18, 2017, incorporated herein by reference in its entirety). In some embodiments, the membrane may be constructed from any biological, synthetic, or composite material suitable for thin-film deposition and capable of maintaining mechanical viability and barrier integrity between compartments. The membrane may include pores, slits, surface roughness, or other functional properties imparted during manufacturing using techniques known in the art designed to improve the functionality, biocompatibility, or other qualities of the membrane. The membrane may be manufactured from biological, synthetic, or composite materials such as collagen, gelatin, other hydrogels, cellulose, or other materials that can be deposited into a thin film and subsequently crosslinked, dried, gelled, cured, or otherwise stabilized to form an adhesive, mechanically stable membrane. The membrane may undergo further processing or manipulation to provide enhanced functionality or mechanical structure. The membrane may be uniform or varied in thickness in the range of 0.01 μm to 100 μm or more. In some embodiments, the film has a thickness of approximately 0.1 μM to approximately 100 μM, approximately 0.1 μM to approximately 100 μM, approximately 0.5 μM to approximately 50 μM, approximately 1.0 μM to approximately 40 μM, approximately 5.0 μM to approximately 30 μM, or approximately 10 μM to approximately 20 μM, or any range in between. In some embodiments, the film has a thickness of approximately 10 μM or less. In some embodiments, the film has a thickness of approximately 1 to 8 μM. In some embodiments, the film has a thickness of approximately 5 μM or less.

[0048] In some embodiments, the biocompatible extracellular matrix membrane includes fibers, nanofibers, or other longitudinal elements. In some embodiments, the fibers, nanofibers, or other longitudinal elements are bonded to the outer surface of the membrane. In some embodiments, the fibers, nanofibers, or other longitudinal elements increase or regulate the mechanical strength of the membrane. In some embodiments, the fibers, nanofibers, or other longitudinal elements form a mesh (e.g., an ordered mesh, a disordered mesh) or are oriented in substantially one direction or substantially two directions (e.g., a mesh).

[0049] In some embodiments, fibers, nanofibers, or other longitudinal elements increase the mechanical strength of the membrane or a portion of it by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or more compared to the same membrane without fibers, nanofibers, or other longitudinal elements. In some embodiments, fibers, nanofibers, or other longitudinal elements increase the mechanical strength of the membrane or a portion of it by at least about 1.1 times, 1.5 times, 2 times, 3 times, 5 times, 10 times, or more compared to the same membrane without fibers, nanofibers, or other longitudinal elements. In some embodiments, fibers, nanofibers, or other longitudinal elements are uniformly distributed throughout the membrane, providing homogeneous mechanical reinforcement. In some embodiments, fibers, nanofibers, or other longitudinal elements are heterogeneously distributed in the membrane, providing heterogeneous mechanical reinforcement. In some embodiments, fibers, nanofibers, or other longitudinal elements provide resistance to cell invasion and maintain the separation of different cell populations on each side of the membrane. In some embodiments, fibers, nanofibers, or other longitudinal elements form a mesh, providing resistance to cell invasion and maintaining the separation of different cell populations on each side of the membrane.

[0050] The fibers, nanofibers, and other longitudinal elements disclosed herein may be made from a variety of materials, including (but not limited to) Dyneema®, an extremely strong polyethylene manufactured by DSM High Performance Fibers, a subsidiary of DSM NV. The fibers may also be combined with fibers or wires of other materials, such as Nitinol (a version of a shape-memory nickel-titanium alloy), to assist in controlling the extended shape of the filter. Other viable materials for use as fibers, nanofibers, and other longitudinal elements include those known in the fiber field, such as carbon, glass, ceramics, metals and metal alloys (including the aforementioned Nitinol), natural and synthetic polymers (including ultra-high molecular weight, highly oriented polymers and silk), or combinations thereof. In some embodiments, the fibers, nanofibers, or other longitudinal elements include silk or polymers (e.g., polycarbonate). In some embodiments, the fibers, nanofibers, or other longitudinal elements form a mesh (e.g., polycarbonate mesh). Furthermore, fibers, nanofibers, and other longitudinal elements may be fabricated as monofilaments or multifilaments and may be configured to have all kinds of cross-sections and orientations. Fibers may be made as round, flat, or monofilaments or multifilaments of different shapes. In some embodiments, the fibers are non-immunogenic.

[0051] In some embodiments, the membrane includes, essentially consists of, or comprises a biocompatible extracellular matrix membrane that separates the vascular channel system from the filtration channel system. In some embodiments, the biocompatible extracellular matrix membrane includes a collagen membrane having a thickness of about 0.1 to 10 micrometers (e.g., 0.3 to 10 μm) that supports cell adhesion on both the vascular and filtration surfaces of the collagen membrane.

[0052] In some embodiments, a biocompatible extracellular matrix membrane is embedded in a matrix material (i.e., scaffold material). In some embodiments, the scaffold comprises hydrogels such as gelatin, PLA, chitosan, hydrogels or other hydrogel materials, as well as hydrogels of varying concentrations and compositions. In some embodiments, varying the concentrations and compositions of hydrogel materials and complexes allows for the tuning of the mechanical and biological properties of the scaffold, which can enhance and further specialize the tissue construct for a desired biological application. In some embodiments, the scaffold may involve the addition of glycerin, sorbitol, propylene glycol, or other plasticizers to gelatin or gelatin-complex hydrogels. The composition of the scaffold is not limited and may be any suitable scaffold material known in the art.

[0053] In some embodiments, the device comprises at least one biofluid inflow conduit that is in fluid communication with a first end of the vascular channel system and a first lumen space, and at least one biofluid outflow conduit that is in fluid communication with a second end of the vascular channel system and a first lumen space, and the functional unit comprises at least one filtrate outflow conduit that is in fluid communication with a third end of the filtration channel system and a second lumen space.

[0054] In some embodiments, the blood vessel and filtration channel system is manufactured by the method disclosed in PCT Application No. PCT / US2017 / 67141 (filed December 18, 2017, which is hereby incorporated by reference in its entirety). Briefly, a sacrificial material is layered on a membrane and then on a scaffold material, and then the sacrificial material is removed, leaving a luminal space bounded by the scaffold material and the membrane. The membrane is then inverted, and the sacrificial material is layered on the membrane and then on the scaffold material, and then the sacrificial material is removed, leaving a second luminal space bounded by the scaffold material and the membrane. In some embodiments, the blood vessel and filtration channel system is partially or completely in fluid communication across the membrane (e.g., mirror images of each other across the membrane). In some embodiments, the area of the membrane where the first and second luminal spaces are in fluid communication across the membrane is at least 30 cm 2 , at least 60 cm 2 , at least 90 cm 2 , at least 100 cm 2 , at least 150 cm 2 , at least 200 cm 2 , at least 250 cm 2 , at least 300 cm 2 , at least 450 cm 2 , at least 600 cm 2 , at least 800 cm 2 , at least 1000 cm 2 , or at least 1200 cm 2 or more. In some embodiments, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or more of the first and / or second luminal space is in fluid communication across the membrane with another luminal space.

[0055] In some embodiments, at least one biofluid inflow conduit is in fluid communication with an arterial conduit. In some embodiments, at least one biofluid outflow conduit is in fluid communication with a vascular conduit. In some embodiments, at least one filtrate outflow conduit is in fluid communication with a drainage conduit.

[0056] In some embodiments, the device generates a filtrate and / or ultrafiltrate that is drained into an extracorporeal collection system (e.g., a waste container) using a drainage conduit. In some embodiments, the filtrate and / or ultrafiltrate is discharged into a piping system or wastewater treatment system. In some embodiments, the filtrate and / or ultrafiltrate is discharged into the patient's bladder or digestive system using a discharge conduit.

[0057] In some embodiments, the vascular channel system comprises vascular channel walls lined with endothelial cells and / or epithelial cells, and / or filtration channels comprises filtration channel walls lined with endothelial cells and / or epithelial cells. In some embodiments, the cells are confluenced on the vascular channel walls and / or filtration channel walls. In some embodiments, the vascular channel walls and / or filtration channel walls comprise at least 2 × 10⁻¹⁴ cells. 6 It contains a number of cells. In some embodiments, the vascular channel wall and / or filtration channel wall is at least 1 × 10 7 It contains a number of cells. In some embodiments, the vascular channel wall and / or filtration channel wall is at least 2 × 10 7 It contains several cells. In some embodiments, the cells are at least 30 cm 2 , at least 60cm 2 , at least 90cm 2 , at least 100cm 2 , at least 150cm 2 , at least 200cm 2 , at least 250cm 2 , at least 300cm 2 , at least 450cm 2 , at least 600cm 2 , at least 800cm 2 , at least 1000cm 2 , or at least 1200cm 2 Or above the vascular channel wall and / or filtration channel wall.

[0058] In some embodiments, the epithelial cell type is selected from prostate cells, mammary gland cells, hepatocytes, islet cells including β-cells, lung epithelial cells, kidney cells, bladder cells, gastric epithelial cells, colonic and small intestinal epithelial cells, urethral epithelial cells, testicular epithelial cells, ovarian epithelial cells, cervical epithelial cells, thyroid cells, parathyroid cells, adrenal cells, thymocytes, gallbladder cells, and pituitary cells. In some embodiments, the endothelial cells are brain endothelial cells, vascular endothelial cells, primary human paratubular capillary endothelial cells, iPSC-derived endothelial cells, human umbilical cord endothelial cells, primary human renal medullary endothelial cells, human podocytes, or human iPSC-derived podocytes. In some embodiments, the cells are allogeneic to the patient using the device. In some embodiments, the cells are autologous to the patient using the device. In some embodiments, the cells are cell line-derived. In some embodiments, the cells are autologous stem cell-derived cells. In some embodiments, the autologous stem cells are derived from induced pluripotent stem cells.

[0059] In some embodiments, the filtering segment of the vascular channel system comprises a vascular channel wall lined with endothelial cells selected from primary human glomerular endothelial cells, induced pluripotent stem cell (iPSC)-derived endothelial cells, and / or human umbilical cord endothelial cells.

[0060] In some embodiments, cells are obtained from a kidney, as described below in the section titled “Isolation of Primary Cells from Discarded Kidneys” in the Examples.

[0061] In some embodiments, the tubular segments of the vascular channel system comprise a vascular channel wall (e.g., a wall including scaffolding material and membrane) lined with endothelial cells selected from primary human paratubular capillary endothelial cells, iPSC-derived endothelial cells, and / or human umbilical cord endothelial cells.

[0062] In some embodiments, the ductal segments of the vascular channel system comprise a vascular channel wall lined with endothelial cells selected from primary human renal medullary endothelial cells, iPSC-derived endothelial cells, and / or human umbilical cord endothelial cells.

[0063] In some embodiments, the filtration segment of the filtration channel system comprises a filtration channel wall lined with epithelial cells selected from primary human podocytes and / or human iPSC-derived podocytes.

[0064] In some embodiments, the tubular segments of the filtration channel system comprise a filtration channel wall (e.g., a wall including scaffolding material and membrane) lined with epithelial cells selected from primary human tubular epithelial cells and / or iPSC-derived tubular epithelial cells.

[0065] In some embodiments, the tubular segment of the filtration channel system comprises a filtration channel wall lined with epithelial cells selected from primary human tubular epithelial cells and / or iPSC-derived tubular epithelial cells.

[0066] In some embodiments, the vascular channel system includes vascular channel diameters of 1 mm to 10 μm. In some embodiments, the filtration channel system includes filtration channel diameters of 1 mm to 10 μm. In some embodiments, the channel system (e.g., the vascular channel system and / or the filtration channel system) comprises more than one channel (e.g., 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, 100, 200, 500, 750, 1000, 2000, 10000 channels). In some embodiments, the channel system comprises a branched channel network having one or more branches of decreasing diameter (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 100, 200, 500, 750, 1000, 2000, 10000 branches). In some embodiments, the channel diameter may include, but is not limited to, approximately 10 cm, 5 cm, 2 cm, 1 cm, 500 mm, 250 mm, 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. In some embodiments, the vascular channel system and the filtration channel system are embedded in a scaffold (e.g., a biocompatible matrix material).

[0067] In some embodiments, the device comprises a plurality of functional units, including a functional unit and additional functional units of the same configuration (see, for example, Figure 8), each functional unit having a first end and first lumen space of a vascular channel system in fluid communication with at least one biofluid inflow conduit, a second end and first lumen space of a vascular channel system in fluid communication with at least one biofluid outflow conduit, and a third end and second lumen space of a filtration channel system in fluid communication with a filtrate outflow conduit, wherein each first end of the plurality of functional units is individually connected in parallel to one of a plurality of manifold ports of at least one biofluid inflow conduit, each second end of the plurality of functional units is individually connected in parallel to one of a plurality of manifold ports of at least one biofluid outflow conduit, and each third end of the plurality of functional units is individually connected in parallel to one of a plurality of manifold ports of at least one filtrate outflow conduit. In some embodiments, the plurality of functional units, including a functional unit and additional functional units of the same configuration, are stacked in a parallel layer of functional units.

[0068] In some embodiments, at least one biofluid inflow conduit comprises a blood inlet conduit configured to transport blood inflow, and at least one biofluid outflow conduit comprises a blood outflow conduit configured to transport blood outflow, and a parallel layer of functional units configured for biological blood purification.

[0069] In some embodiments, the filtration segments of the filtration channel system are configured to provide ultrafiltration that generates primary ultrafiltration material, the tubular segments are configured to provide reabsorption that generates secondary ultrafiltration material by absorption of solute and water, and the conduit segments are configured to provide concentration that generates tertiary ultrafiltration material by absorption of water.

[0070] In some embodiments, the device is configured for extracorporeal operation in a sterile, heated enclosure. In some embodiments, blood is delivered to the device using a mechanical pump. In some embodiments, the device is placed in a capsule and sized and configured for in-body placement in a human body to replace or enhance the function of a tissue or organ (e.g., the function of the liver and / or kidneys).

[0071] Some aspects of this disclosure relate to a method for treating a patient with insufficient renal or hepatic function, comprising: fluidly connecting the apparatus described herein to the patient's circulatory system; and passing the patient's blood through the apparatus's vascular channel system from a filtration member segment to a tubular member segment, from the tubular member segment to a conduit member segment, and from the conduit member segment back into the patient's circulatory system. In some embodiments, the apparatus comprises a plurality of functional units described herein, and the patient's blood is passed through the plurality of functional units. In some embodiments, the patient's blood is passed through the functional units in parallel. In some embodiments, the patient's blood is passed through the functional units in series. In some embodiments, the apparatus is implanted in the patient. In some embodiments, the ultrafilter produced by the apparatus is delivered to the patient extracorporeally. In some embodiments, the ultrafilter produced by the apparatus is delivered to the patient's bladder.

[0072] In some embodiments, the device is external to the patient. For example, the device may be stationary, or it may be carried by the patient, such as in a backpack or waist pack, so that the patient can move while using the device.

[0073] In some embodiments, the device is extracorporeal to the patient and configured for use in peritoneal dialysis (e.g., as an accessory device for peritoneal dialysis). Peritoneal dialysis (PD) is a type of dialysis that uses the peritoneum in the patient's abdomen as a membrane through which fluids and dissolved substances are exchanged with blood. PD is used in individuals with renal failure to remove excess fluid, correct electrolyte problems, and remove toxins. Continuous ambulatory peritoneal dialysis (CAPD) requires the patient to have dialysate added and removed through a catheter in the abdomen multiple times per day. The patient may move around (i.e., walk around) while the dialysate is present in the abdominal cavity. However, the longer the dialysate is present in the abdominal cavity, the less effective it becomes at removing dialysate waste as the dialysate approaches equilibrium. In continuous flow peritoneal dialysis (CFPD), dialysate is continuously added and removed from the patient's abdominal cavity, usually even during sleep. CFPD requires a large dialysate reservoir and a total dialysate volume of 6-12 liters per sleep period.

[0074] In some embodiments, the device is configured to connect to the peritoneal cavity so that fluid from the peritoneal cavity circulates through the device (e.g., via a pump) and at least a portion of it returns to the peritoneal cavity. In some embodiments, the device is configured to remove a portion of the fluid from the peritoneal cavity as waste fluid. In some embodiments, the device includes a waste outlet or waste storage receptacle that can be optionally discharged or replaced (e.g., hot-swapped between operations) for a new waste storage receptacle if necessary. In some embodiments, the device is part of a peritoneal dialysis system (CAPD and / or CFPD) that includes a pump that circulates the dialysate solution from the patient's peritoneal cavity through the device and returns it to the peritoneal cavity, thereby removing toxins and / or excess fluid from the dialysate. In some embodiments, the use of the device reduces the volume of dialysate required for effective dialysis (e.g., at least 10%, 25%, 50%, or more). In some embodiments, the use of the device increases the interval between dialysate exchanges in CAFD without loss of effectiveness compared to CAFD without the device (e.g., at least 10%, 25%, 50%, or more). In some embodiments, the apparatus includes kidney cells (for example, cells from discarded kidneys, as detailed in the Examples section below).

[0075] Manufacturing method Some aspects of this disclosure relate to a method for manufacturing the apparatus disclosed herein, comprising providing a plurality of membranes having sacrificial material in the form of a vascular channel network on a vascular surface and sacrificial material in the form of a filtration channel system on a filtration surface; immersing the plurality of membranes in a solution containing a scaffold material (e.g., a sol-state scaffold material); gelling the scaffold material; and removing the sacrificial material to form the luminal spaces of the vascular channel system and the filtration channel system described herein.

[0076] In some embodiments, multiple membranes are produced by chemical or physical thin-film deposition, atomization, spraying, electrospinning, immersion coating, or gelation of a solution (membrane solution) containing liquefied or homogenized decellularized tissue, gelatin, gelatin complex, collagen, fibrin, hydrogel, hydrogel complex, chitosan, nitrocellulose, polylactic acid, or extracellular matrix in a thin-film layer on a substrate, followed by curing, crosslinking, polymerization, drying, or gelation of the solution to form a membrane layer.

[0077] In some embodiments, the membrane solution further comprises fibers, nanotubes, or other longitudinally oriented materials to provide improved mechanical properties. These fibers, nanotubes, or other longitudinally oriented materials are not limited to and may be any fibers, nanotubes, or other longitudinally oriented materials disclosed herein. These fibers, nanotubes, or other longitudinally oriented materials may be mixed into the membrane solution before fabrication to uniformly distribute the fibers throughout the membrane. Alternatively, these fibers, nanotubes, or other longitudinally oriented materials may be deposited or integrated onto the membrane after fabrication through techniques such as electrospinning, 3D printing, or other techniques. In some embodiments, the membrane may be bonded to the fibers, nanotubes, or other longitudinally oriented materials. The fibers, nanotubes, or other longitudinally oriented materials may be uniformly distributed throughout the membrane, or they may be systematically distributed to provide heterogeneous mechanical properties to the membrane.

[0078] In some embodiments, the membrane solution further comprises pologen homogeneously mixed therein. In some embodiments, the pologen is in the form of micelles in the solution (for example, the pologen is at a concentration sufficient to form micelles in the solution). In some embodiments, the pologen is incorporated into the solution by mixing or sonication. In some embodiments, the pologen is a self-assembling triblock copolymer. In some embodiments, the self-assembling triblock copolymer is a poloxamer formulation. In some embodiments, the pologen is Pluronic F127. In some embodiments, the pologen is at a concentration of 1 to 40% by weight. In such embodiments, the pologen is present in solution at concentrations of approximately 1% by weight, approximately 2% by weight, approximately 3% by weight, approximately 4% by weight, approximately 5% by weight, approximately 6% by weight, approximately 7% by weight, approximately 8% by weight, approximately 9% by weight, approximately 10% by weight, approximately 11% by weight, approximately 12% by weight, approximately 13% by weight, approximately 14% by weight, approximately 15% by weight, approximately 16% by weight, approximately 17% by weight, approximately 18% by weight, approximately 19% by weight, approximately 20% by weight, approximately 21% by weight, approximately 22% by weight, approximately 23% by weight, approximately 24% by weight, approximately 25% by weight, approximately 26% by weight, approximately 27% by weight, approximately 28% by weight, approximately 29% by weight, approximately 30% by weight, approximately 31% by weight, approximately 32% by weight, approximately 33% by weight, approximately 34% by weight, approximately 35% by weight, approximately 36% by weight, approximately 37% by weight, approximately 38% by weight, approximately 39% by weight, or approximately 40% by weight. The pore size in the resulting membrane can be controlled by using various polymers, but also by controlling micelle size and aggregation by varying the concentration, solution properties, and processing techniques. Various concentrations and compositions of sacrificial pologen materials can enable substantial opportunities for tuning mechanical and biological properties, including, but not limited to, porosity, pore size, permeability, sieving, filtration, and other functions that can enhance and further specialize the tissue construct for desired biological applications.

[0079] In some embodiments, the membrane solution further comprises one or more agents that modify the mechanical or biological properties of one or more membranes. Examples of agents include, but are not limited to, glycerin, sorbitol, propylene glycol, or other plasticizers for gelatin or gelatin-based hydrogels (see FMVanina et al., Food Hydrocolloids 19, 899-907 (2005)). In some embodiments, the agents include growth factors (e.g., encapsulated growth factors). In some embodiments, one or more agents are selected from glycerin, sorbitol, propylene glycol, plasticizers, fibers or other longitudinal elements, and encapsulated growth factors.

[0080] In some embodiments, the method for producing a membrane further includes adding one or more additional membrane layers to a first membrane layer by a method disclosed herein in order to produce a membrane of a mixed composition or architecture. In some embodiments, two or more layers are produced from membrane solutions having different components, agents, and / or concentrations.

[0081] In some embodiments, the film is treated to remove pologens, thereby forming pores in the film. The sacrificial pologen material is removed passively or forcibly, in conjunction with dissolution, phase transition, reversal of thermal gelation, or other techniques known in the art. In some embodiments, the sacrificial material may have thermally reversible gelation properties or be dissolved in a nonpolar solvent.

[0082] In some embodiments, the pologen material is Pluronic F127, which is removed by treatment with a nonpolar solvent (e.g., isopropanol).

[0083] In some embodiments, the membrane solution contains 3 to 35% by weight of gelatin or a gelatin polymer composite. In some embodiments, the solution contains about 3% by weight, about 4% by weight, about 5% by weight, about 6% by weight, about 7% by weight, about 8% by weight, about 9% by weight, about 10% by weight, about 11% by weight, about 12% by weight, about 13% by weight, about 14% by weight, about 15% by weight, about 16% by weight, about 17% by weight, about 18% by weight, about 19% by weight, about 20% by weight, about 21% by weight, about 22% by weight, about 23% by weight, about 24% by weight, about 25% by weight, about 26% by weight, about 27% by weight, about 28% by weight, about 29% by weight, about 30% by weight, about 31% by weight, about 32% by weight, about 33% by weight, about 34% by weight, or about 35% by weight of gelatin or a gelatin-polymer composite.

[0084] In some embodiments, the thin film layer may be dried, gelled, crosslinked, or otherwise solidified and removed from the substrate. In some embodiments, the thin film layer is crosslinked using a solution containing glutaraldehyde, transglutaminase, or other crosslinking enzymes or molecules. In some embodiments, the crosslinking agent is added to the film solution, for example, immediately before the solution is applied to the thin film on the substrate. In some embodiments, the crosslinking agent is brought into contact with the thin film layer after contact with the substrate. In some embodiments, the concentration of the crosslinking agent is about 0.01 to 5 g per 10 g of gelatin.

[0085] In some embodiments, the solution containing the scaffolding material also contains an extracellular matrix material. In some embodiments, the extracellular matrix material is gelatin.

[0086] In some embodiments, the scaffolding material is gelled by crosslinking with a solution containing glutaraldehyde, transglutaminase, or other crosslinking enzymes or molecules, and / or the scaffolding material is thermally crosslinked.

[0087] In some embodiments, the steps of immersing a plurality of membranes in a solution containing a scaffolding material and gelling the scaffolding material include: (a) providing a bottom mold (64) having a vascular channel system inflow conduit structure (63) and a vascular channel system outflow conduit structure (65) having an open top reservoir, each having an internal lumen filled with sacrificial material, wherein the reservoir is partially filled with gelled scaffolding material that partially embeds the vascular channel system inflow conduit structure and the vascular channel system outflow conduit structure; (b) providing a plurality of membranes in a frame; (c) filling the open top reservoir of the bottom mold (64) with a solution containing the scaffolding material; (d) positioning the frame on the top of the bottom mold such that the membranes in the frame are in contact with the solution; (e) gelling the solution and then removing the frame from the membranes; (f) positioning a spacer (62) having an internal volume around the top of the membranes; and (g) filling the internal volume of the spacer with the solution containing the scaffolding material. (h) filling the apparatus with (i) a spacer (57) on top of the final membrane which is configured to have a filtration channel system outflow conduit structure (56) having an internal lumen filled with sacrificial material (k) filling the internal volume of the spacer (57) with a solution containing scaffolding material and gelling the solution (l) adding a shaft filled with sacrificial material to the gelled solution which fluidly connects the first ends of the plurality of membranes to the vascular channel system inflow conduit structure (63), adding a shaft filled with sacrificial material to the gelled solution which fluidly connects the second ends of the plurality of membranes to the vascular channel system outflow conduit structure (65), adding a shaft filled with sacrificial material to the gelled solution which fluidly connects the third ends of the plurality of membranes to the filtration channel system outflow conduit structure (56), and (m) removing the sacrificial material from the structure. In some embodiments, the method for manufacturing the apparatus described herein includes the method described in "Manufacturing of Multilayer Devices" in the following embodiments.

[0088] In some embodiments, the method for manufacturing the device further includes adding cells to one or more segments of a vascular channel system and / or a filtration channel system. In some embodiments, the cells are added to each functional unit of the device. In some embodiments, the cells are added to each segment of each functional unit of the device (e.g., both or either of the vascular channel system and the filtration channel system located within each segment). In some embodiments, the cells are added to both the vascular channel system and the filtration channel system. The cells are not limited and may be any cells described herein.

[0089] In some embodiments, cells are added to the segment by (a) filling the vascular channel system and the filtration channel system with fluid; (b) placing the cells in a first volume of fluid approximately equal to the volume of fluid in the channel system of the target segment; (c) adding the first volume to the apparatus through a first fluid supply or fluid outlet that is in fluid communication with the target segment; and (d) adding a second volume of fluid approximately equal to the volume of fluid contained between the target segment and the first fluid supply or fluid outlet, and / or removing a third volume of fluid approximately equal to the volume of fluid contained between the target segment and a second fluid supply or fluid outlet that is in fluid communication with the first fluid supply or fluid outlet. In some embodiments, cells are added by methods described in the examples included herein (e.g., seeding).

[0090] A membrane containing pores and a method for manufacturing the same. Some aspects of this disclosure relate to membranes having pores having a diameter of about 1 μM to 15 μM, comprising biological or synthetic matrix materials. In some embodiments, the biological or synthetic matrix material comprises decellularized tissue, gelatin, gelatin complex, collagen, fibrin, hydrogel, hydrogel complex, chitosan, nitrocellulose, polylactic acid, or extracellular matrix. In some embodiments, the membrane may comprise any extracellular matrix material or scaffold material described herein. In some embodiments, the membrane has a thickness of about 0.1 μM to 100 μM. The membrane may have any thickness described herein and is not limited thereto.

[0091] Several aspects of this disclosure relate to methods for producing a membrane as described herein, comprising chemically or physically depositing, atomizing, spraying, electrospinning, immersion coating, or gelling a solution (i.e., membrane solution) containing liquefied or homogenized decellularized tissue, gelatin, gelatin complex, collagen, fibrin, hydrogel, hydrogel complex, chitosan, nitrocellulose, polylactic acid, or extracellular matrix in a thin film layer, and subsequently curing, crosslinking, polymerization, drying, or gelling the solution to form a membrane layer. The method for producing (i.e., manufacturing) the membrane is not limited and may be any method described herein or known in the art. In some embodiments, the membrane solution further comprises a pologen homogeneously mixed therein. The pologen is not limited and may be any pologen described herein. In some embodiments, the pologen 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 to 40% by weight in the membrane solution. The concentration of pologen in the membrane solution is not limited and may be any concentration disclosed herein.

[0092] In some embodiments, the membrane solution further comprises one or more agents that modify the mechanical or biological properties of the membrane. The one or more agents are not limited and may be any agents that modify the mechanical or biological properties of the membrane as described herein. In some embodiments, the one or more agents are selected from glycerin, sorbitol, propylene glycol, plasticizers, fibers or other longitudinal elements, and growth factors (e.g., encapsulated growth factors).

[0093] In some embodiments, the method for producing a membrane further includes adding one or more additional membrane layers to a first membrane layer by a method disclosed herein in order to produce a membrane of a mixed composition or architecture. In some embodiments, two or more layers are produced from membrane solutions having different components, agents, and / or concentrations.

[0094] In some embodiments, the film is treated to remove pologens, thereby forming pores in the film. The sacrificial pologen material is removed passively or forcibly, in conjunction with dissolution, phase transition, reversal of thermal gelation, or other techniques known in the art. In some embodiments, the sacrificial material may have thermally reversible gelation properties or be dissolved in a nonpolar solvent.

[0095] In some embodiments, the pologen material is Pluronic F127, which is removed by treatment with a nonpolar solvent (e.g., isopropanol).

[0096] In some embodiments, the membrane solution contains 3 to 35% by weight of gelatin or a gelatin polymer composite. In some embodiments, the solution contains about 3% by weight, about 4% by weight, about 5% by weight, about 6% by weight, about 7% by weight, about 8% by weight, about 9% by weight, about 10% by weight, about 11% by weight, about 12% by weight, about 13% by weight, about 14% by weight, about 15% by weight, about 16% by weight, about 17% by weight, about 18% by weight, about 19% by weight, about 20% by weight, about 21% by weight, about 22% by weight, about 23% by weight, about 24% by weight, about 25% by weight, about 26% by weight, about 27% by weight, about 28% by weight, about 29% by weight, about 30% by weight, about 31% by weight, about 32% by weight, about 33% by weight, about 34% by weight, or about 35% by weight of gelatin or a gelatin-polymer composite.

[0097] In some embodiments, the thin film layer may be dried, gelled, crosslinked, or otherwise solidified and removed from the substrate. In some embodiments, the thin film layer is crosslinked using a solution containing glutaraldehyde, transglutaminase, or other crosslinking enzymes or molecules. In some embodiments, the crosslinking agent is added to the film solution, for example, immediately before the solution is applied to the thin film on the substrate. In some embodiments, the crosslinking agent is brought into contact with the thin film layer after contact with the substrate. In some embodiments, the concentration of the crosslinking agent is about 0.01 to 5 g per 10 g of gelatin.

[0098] In some aspects of this disclosure, the membrane is used in a tissue or biological construct incorporating a membrane (e.g., a basement membrane) manufactured by the method described herein. In some embodiments, the tissue or biological construct comprising a membrane manufactured as described herein includes a hydrogel such as a complex of gelatin, collagen, PLA, chitosan, or other hydrogel materials and compounds. Without limiting this, 3 to 35% by weight of gelatin and gelatin-polymer complexes may be used using various thin-film deposition techniques. Sacrificial pologen materials of varying concentrations and compositions may enable substantial opportunities for tuning mechanical and biological properties, including, but not limited to, porosity, pore size, permeability, sieving, filtration, and other functions that can enhance and further specialize the tissue construct for a desired biological application.

[0099] In some embodiments, the membranes described herein are modified by techniques such as divalent metal ion removal or other techniques known in the art to obtain tunable mechanical and biological properties (see Qi et al., Scientific Reports 4:4706 (2013)).

[0100] In some embodiments of the methods for generating the film described herein, a second polymer or hydrogel material is generated that provides a support matrix for the film material. This second hydrogel or polymer may be constructed of a material similar to the film, or of a complementary hydrogel or polymer.

[0101] In some embodiments, the film is partially or completely constructed of gelatin, or other hydrogel materials modified to be photocurable using ultraviolet light of various wavelengths, such as gelatin methacrylic acid. Such materials, in various concentrations, can be prepared using published protocols or techniques known in the art.

[0102] Those skilled in the art will readily understand that the present invention is well adapted to perform its purpose and to obtain the objectives and benefits mentioned, as well as those inherent thereto. The details of the descriptions and examples herein are representative and illustrative of specific embodiments and are not intended to limit the scope of the invention. Modifications and other uses therein will be conceivable to those skilled in the art. These modifications are included within the spirit of the invention. It will be readily apparent to those skilled in the art that various substitutions and modifications can be made to the invention disclosed herein without departing from the scope and spirit of the invention.

[0103] The articles “a” and “an” as used herein in the specification and claims should be understood to include multiple references unless explicitly indicated otherwise. Any claim or description containing “or” between one or more members of a group shall be deemed satisfied if one, more than one, or all of the group members are present, used, or otherwise related to a given product or process, unless otherwise indicated or evident from the context. The present invention includes embodiments in which exactly one member of the group is present, used, or otherwise related to a given product or process. The present invention also includes embodiments in which more than one, or all of the group members are present, used, or otherwise related to a given product or process. Furthermore, it should be understood that the present invention provides all variations, combinations, and permutations in which one or more limitations, elements, clauses, descriptive terms, etc., from one or more of the enumerated claims are introduced into another claim (or any other claim, where applicable) dependent on the same basic claim, unless otherwise indicated, or unless it would be obvious to those skilled in the art that such a change would occur. All embodiments described herein are intended to be applicable to all different aspects of the present invention where appropriate. It is also intended that any embodiment or aspect may be freely combined with one or more other such embodiments or aspects as needed. Where elements are presented as a list, for example, in the form of a Markush group or similar, each subgroup of the elements is also disclosed, and it should be understood that any element(s) may be removed from a group. In general, where the present invention or an aspect of the present invention is referred to as including certain elements, features, etc., it should be understood that a particular embodiment or aspect of the present invention consists of or is essentially derived from such elements, features, etc. For the sake of simplification, these embodiments are not described in much detail in this specification in all cases.Regardless of whether specific exclusions are described herein, it should be understood that any embodiment or aspect of the present invention may be expressly excluded from the claims. For example, any one or more active agents, additives, components, optional agents, types of organisms, ailments, subjects, or combinations thereof may be excluded.

[0104] Where the claims or description relate to a composition of a substance, unless otherwise indicated, or unless it is obvious to a person skilled in the art that such a contradiction or inconsistency would arise, it should be understood that a method of preparing or using a composition of a substance by any method disclosed herein, and a method of using a composition of a substance for any purpose disclosed herein, constitutes an embodiment of the present invention. Where the claims or description relate to a method, for example, unless otherwise indicated, or unless it is obvious to a person skilled in the art that such a contradiction or inconsistency would arise, it should be understood that a method of preparing a composition useful for carrying out the method, and a product produced according to the method, constitutes an embodiment of the present invention.

[0105] Where a range is given herein, the present invention includes embodiments in which an endpoint is included, embodiments in which both endpoints are excluded, and embodiments in which one endpoint is included and the other is excluded. Unless otherwise indicated, it should be assumed that both endpoints are included. Furthermore, unless otherwise indicated, or unless it is evident from the context and the understanding of those skilled in the art, values ​​expressed as a range may take any specific value or subrange within the range described in different embodiments of the present invention, up to one-tenth of the lower limit of the range, unless the context otherwise explicitly indicates. Also, where a set of numerical values ​​is described herein, the present invention includes embodiments that similarly relate to any intermediate value or range defined by any two of the values ​​in the set, with the smallest value being the minimum and the largest value being the maximum. Numerical values ​​used herein include values ​​expressed as percentages. For any embodiment of the present invention in which a numerical value is preceded by "about" or "approximately," the present invention includes embodiments in which the exact value is described. With respect to any embodiment of the present invention in which the numerical value is not preceded by "approximately" or "about," the present invention includes embodiments in which the value is preceded by "approximately" or "about."

[0106] As used herein, “A and / or B” generally means at least one of A, B, or both A and B, if A and B are terms in different claims. For example, one sequence that is complementary to and / or hybridizes to another sequence includes (i) one sequence that is complementary to the other sequence, even if the one sequence may not necessarily hybridize to the other sequence under all conditions; (ii) one 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 to and hybridizes to the other sequence.

[0107] "Approximately" or "about" generally means a number (greater than or less than the number) in any direction that falls within 1 percent, or in some embodiments, within 5 percent of the number, or in some embodiments, within 10 percent of the number, unless otherwise stated or evident from the context (unless such number exceeds 100% of the possible value to an unacceptable degree). Unless expressly indicated to the contrary, 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 described, but it should be understood that the present invention includes embodiments in which the order is thus limited. Unless otherwise stated or evident from the context, it should also be understood that any product or composition described herein may be considered "isolated".

[0108] As used herein, the terms “comprising” or “comprises” are used in reference to compositions, methods, and their respective components that are essential to the present invention, and still permit the inclusion of unspecified elements, whether essential or not.

[0109] As used herein, the term “essentially consisting of” refers to the elements required for a given embodiment. This term allows for the presence of additional elements that do not substantially affect the basic and novel or functional characteristics of that embodiment of the invention.

[0110] The term "consisting of" refers to the compositions, methods, and their respective components described herein, excluding any elements not described in the description of the embodiments. [Examples]

[0111] Example 1 porous membrane manufacturing In step 1, a solution of gelatin, collagen, fibrin, or other biological or synthetic matrix material is prepared.

[0112] In step 2, the solution containing the sacrificial pologen material is combined with the matrix solution prepared in step 1 and thoroughly mixed.

[0113] In step 3, the matrix-pologen solution is deposited onto the substrate by spin coating, immersion coating, or other such thin-film deposition techniques, and then dried, gelled, or otherwise solidified. This deposition technique allows for precise control over the thickness of the thin film.

[0114] In step 4, an additional layer of similar or different composition is optionally deposited on the first layer to create a composite or layered thin film. Alternatively, the additional layer is deposited in a manner that allows for patterning or other spatial organization within the film.

[0115] In step 5, the sacrificial pologen material is removed by dissolution, decomposition, or other destructive techniques, leaving empty spaces in the thin film that act as pores.

[0116] In one example of the present invention, the sacrificial pologen material consists of a self-assembling triblock copolymer (Pluronic), such as F127 or other poloxamer formulations that form micelles above a certain concentration. These micelles are incorporated into a matrix solution by mixing, sonication, or other methods to produce sacrificial pologen homogeneously dispersed in a bulk matrix solution. Pore size can be controlled by using various polymers, but can also be controlled by varying the concentration, solution properties, and processing techniques to control micelle size and aggregation, as shown in Figure 1.

[0117] Subsequently, this matrix-pologen solution is deposited onto a substrate through spin coating or other thin-film deposition techniques, and depending on the technique and parameters, the thickness of the thin film may be in the range of 0.1 to 100 μm. This thin film can be dried, gelled, crosslinked, or otherwise solidified and removed from the substrate. Once removed from the substrate, the sacrificial pologen material is passively or forcibly removed, in conjunction with dissolution, phase transition, reversal of thermal gelation, or other techniques known in the art, to create an open-pore structure in the thin film. Figure 2 shows one such example of this type of thin film.

[0118] Specific examples of the application of the present invention include tissues or biological constructs incorporating basement membranes manufactured by the method described. Further examples include tissues or biological constructs comprising membranes manufactured as described using hydrogels such as gelatin, collagen, PLA, chitosan, or hydrogel composites, or other hydrogel materials and compounds. Without limitation, 3 to 35% by weight of gelatin and gelatin-polymer composites may be used using various thin-film deposition techniques. Various concentrations and compositions of sacrificial pologen materials may enable substantial opportunities for tuning mechanical and biological properties, including, but not limited to, porosity, pore size, permeability, sieving, filtration, and other functions that can enhance and further specialize tissue constructs for desired biological applications.

[0119] Further examples of the present invention include the membranes described in the previous example, which include composites of hydrogels, polymers, and materials modified by techniques such as divalent metal ion removal or other techniques known in the art to provide tunable mechanical and biological properties (see Qi et al., Scientific Reports 4:4706 (2013)).

[0120] Further examples of the present invention include the membranes described in the preceding examples, which include hydrogels, polymers, and composites of materials modified by the addition of enhancers or compounds to provide tunable mechanical and biological properties. Examples of techniques include, but are not limited to, the addition of glycerin, sorbitol, propylene glycol, or other plasticizers to gelatin or gelatin-composite hydrogels (see FMVanina et al., Food Hydrocolloids 19, 899-907 (2005)).

[0121] A further example of the present invention is the membrane described in the previous example, further comprising a second polymer or hydrogel material that provides a support matrix for the basement membrane material. This second hydrogel or polymer may be constructed from the same material as the basement membrane, or from a complementary hydrogel or polymer.

[0122] Further examples of the present invention include the films described in the preceding examples, in which the thin film is partially or completely composed of gelatin or other hydrogel materials that have been modified to be photocurable using ultraviolet light of various wavelengths, such as gelatin methacrylic acid. Such materials of various concentrations can be prepared using published protocols or techniques known in the art.

[0123] Further examples of the present invention include the films described in the previous example, which are manufactured in a multi-step process to produce films of mixed compositions or architectures.

[0124] Further examples of the present invention include the membranes described in the preceding examples, wherein the application of a curing solution or compound that acts to polymerize, gel, cure, or otherwise solidify a polymer or hydrogel material is introduced before, during, or after the formation of the membrane. For example, the membrane is prepared and then subjected to a crosslinking solution that may contain glutaraldehyde, transglutaminase, or other crosslinking enzymes or molecules at a concentration of 0.01 to 5 g per 10 g of gelatin, but is not limited to these. Alternatively, the crosslinking agent may be incorporated into the solution before the formation of the membrane.

[0125] Further examples of the present invention include membranes described in the previous example, comprising hydrogels and polymers that encapsulate or contain biological factors for promoting cell and tissue growth.

[0126] Example 2 Implantable IABBP device An implantable IABBP device for kidney replacement comprises one or more functional units. Within each of these functional units are two isolated channel systems separated by extracellular matrix material and lined with cells (Figures 4 and 6). One channel system (hereinafter referred to as the vascular channel or vascular channel system) is lined with endothelial cells and perfused with blood, which is gradually purified as it passes through the device. A second channel system (hereinafter referred to as the filtrate channel or filtrate channel system) is lined with epithelial cells and perfused with filtrate, which is gradually processed. Blood flows from the artery into the vascular channel system and returns to the vein via a vascular conduit. Filtrate is generated by the device and flows through the filtrate channel. Once completely processed, the filtrate is drained into an extracorporeal collection system via a conduit and a surgically fabricated fistula, or into the patient's bladder via a conduit and a surgical anastomosis. Within the IABBP device, the channel network consists of three segments that provide different functions. These segments are arranged in series based on the blood flowing through the device, so that the blood is processed sequentially by each segment before returning to the cardiovascular system.

[0127] In the first segment (referred to herein as the filtration segment), the IABBP device generates a primary ultrafiltrate via cell-enhanced ultrafiltration. A fraction of blood (the filtration fraction) is filtered from the vascular channel to the filtrate channel. Blood cells and larger molecules are retained in the vascular channel via cell- and matrix-mediated sieving, while water, glucose, uremic toxins such as blood urea nitrogen (BUN), and other solutes are freely filtered out.

[0128] The blood flow passing through the filtration segment is Q bf Defined as, arterial inflow Q A and filtration fraction FF f Depends on (Q bf =Q A -(Q A *FF f )) The filtrate flow from the filtration segment is the primary filtrate flow (Q fI ) is defined as blood flow Q bf and filtration fraction FF f Depends on (Q ff =Q bf *FF f ). The blood pressure during the filtration segment is P bf Defined as, blood flow Q bf , channel architecture, inflow blood pressure P bA , and back pressure P from the downstream vascular network bt These are dynamic parameters determined by [the specified factor]. (Figure 5)

[0129] Similar to blood pressure, filtrate pressure in a filtration segment is a dynamic parameter determined by the channel architecture, filtrate flow, and back pressure from the downstream filtrate channel system. (Figure 5)

[0130] In the second segment (referred to herein as the tubular segment), the primary ultrafiltrate is then subjected to further modification via cell-enhancing solute secretion (active and passive transport into the filtrate via cell and extracellular matrix material) and absorption (active and passive removal from the filtrate via cell and extracellular matrix material) to produce a secondary filtrate. In this segment, cells also contribute to active metabolic regulation through bicarbonate synthesis.

[0131] Blood flow through the tubular segment is Q bt This is defined as the inflow blood flow Q from the filtration segment. bf It is equal to the result of subtracting the relative filtration fraction of the tubular segment from (Q bt =Q bf -(FF t *Q bf )) The filtrate flow from the tubular segment is the secondary filtrate flow Q fII This is defined as the primary filtrate flow, the tubular filtration fraction FF t , and tubular blood flow Q bt Depends on (Q fII =Q fI +(Q bt *FF t )) The blood pressure in the tubular segment is the inflow blood flow Q bf , channel architecture, inflow blood pressure P from filtration segment bf , and back pressure P from the downstream vascular network bd These are dynamic parameters determined by [the specified factor]. (Figure 5)

[0132] Similar to blood pressure, the filtrate pressure in a tubular segment is P ft Defined as, and the inflow filtrate flow Q fI , channel architecture, and back pressure P from the downstream filtrate channel system fd These are dynamic parameters determined by [the specified factor]. (Figure 5)

[0133] In the third segment (referred to herein as a conduit segment or conduit segment system), the secondary filtrate is then concentrated (removal of water from the filtrate via the cellular and extracellular matrix) to produce a tertiary filtrate. The tertiary filtrate is then discharged from the device as described above.

[0134] Blood flow through the conduit-like segment is Q bd This is defined as the inflow blood flow Q through the tubular segment. bt Absorption fraction AF of the conduit-like segment d It is equal to the sum of (Q bd =Q bt +(AF d *Q fII Since the conduit-like segment is the final segment, the blood flow Q through the conduit-like segment bd is venous blood outflow Q bv Equal to (Q) bd =Q bv ). The filtrate flow in the conduit-like segment is the tertiary filtrate flow Q fIII This is defined as the secondary filtrate flow Q fII and tubular absorption fraction AF d Depends on (Q fII =Q fI +(Q bt *FF t )) The blood pressure in the conduit-like segment is the inflow blood flow Q bt , channel architecture, inflow blood pressure P from tubular segments bt , and venous back pressure P bV These are dynamic parameters determined by [the specified factor]. (Figure 5)

[0135] Similar to blood pressure, the filtrate pressure in a conduit-like segment is P fd Defined as the channel architecture, filtrate inflow from the tubular segment Q ft , and back pressure P from the filtrate discharge system fb These are dynamic parameters determined by [the specified factor]. (Figure 5)

[0136] Within each segment, cells are backed by various channels to support their respective functions. (Figure 6)

[0137] In the filtration segment, endothelial cells (e.g., primary human glomerular endothelial cells, induced pluripotent stem cell (iPSC)-derived endothelial cells, and / or human umbilical cord endothelial cells) line the vascular channel system. These endothelial cells can form a fenestrated lining, enabling filtration and sieving functions. The filtration channel system in the filtration segment may be lined with epithelial cells (e.g., primary human podocytes, human iPSC-derived podocytes), which can further enhance filtration and sieving functions.

[0138] In tubular segments, endothelial cells (e.g., primary human paratubular capillary endothelial cells, iPSC-derived endothelial cells, human umbilical cord endothelial cells) line the vascular channel system. The filtration channel system in tubular segments is lined with epithelial cells, enabling the absorption and secretion of solutes and water (e.g., primary human tubular epithelial cells, and / or iPSC-derived tubular epithelial cells).

[0139] In the ductal segment, endothelial cells (e.g., primary human renal medullary endothelial cells, iPSC-derived endothelial cells, and / or human umbilical cord endothelial cells) line the vascular channel system. The filtration channel system in the ductal segment is lined with epithelial cells, enabling water reabsorption and concentration of the secondary filtrate to form a tertiary filtrate (e.g., primary human tubular epithelial cells, and / or iPSC-derived tubular epithelial cells).

[0140] In each segment, vascular channels and filtrate channels are separated by membranes that support the function of their respective segments (Figures 4 and 7). In the filtration segment, this membrane allows for the formation of filtrate from the vascular space to the filtration space. In the tubular segment, this membrane allows for the exchange of solute and water between the vascular channel system and the filtration channel system. In the conduit segment, this membrane allows for the transfer of water and solute from the filtration channel system to the vascular channel system. The membranes separating each channel system may be in the form of collagen membranes 0.1–10 (e.g., 0.3–10) micrometers thick, supporting cell adhesion on both sides and resistant to membrane fouling. The membranes may be porous to facilitate higher filtration or reabsorption rates or increased solute exchange. The membranes may be crosslinked to varying degrees to alter their physical and biological properties.

[0141] Each channel system architecture in each segment is tailored to provide specific resistances and thus control hydrostatic pressure in vascular and filtrate channels to enable their respective functions. The channel system architecture is designed to minimize turbulence to reduce the risk of clot formation, cell activation, or occlusion. The channel system may include one or more branching networks and subnetworks to increase its surface area. Channels may have varying diameters to provide uniform pressure across each segment and its membrane.

[0142] To increase functional capabilities and meet the diverse needs of patients, the functional units of the purification device can be stacked and operated in parallel. (Figure 8)

[0143] Table 1 lists examples of target function specifications for clinically usable IABBP devices. [Table 1]

[0144] In vitro IABBP device In one example, the IABBP is not implanted but maintained in a sterile, heated enclosure (bioreactor) and connected to the patient's blood circulation via an arteriovenous fistula or central venous cannula insertion. Blood can be delivered to the device with or without the assistance of a mechanical pump.

[0145] Example 3 - Film Manufacturing In one example, a gelatin matrix solution is prepared at a concentration of 5–30% by weight to produce a membrane for hemofiltration. This solution can be prepared by dissolving gelatin in water with PBS, cell culture medium, growth factors, or other enhancers. This solution is heated to 45°C to maintain the gelatin in a sol state.

[0146] To generate a porous membrane, a specific volume of a pologen solution containing 1-40 wt% self-assembling triblock copolymer (Pluronic F127) is added to a gelatin matrix solution. This combined matrix-pologen solution is then mixed via sonication to disperse the F127 in the gelatin solution to a final concentration above the critical micelle concentration (CMC) and critical micelle temperature (CMT), causing the F127 to form micelles and micelle aggregates that can act as sacrificial pologens. This combined matrix-pologen solution is then deposited onto a substrate via spin coating or other thin-film deposition techniques, with film thicknesses ranging from 0.1 to 10 μm depending on the technique and parameters. The precise concentration of F127 in the final membrane is used to control the membrane pore size and bulk porosity, which directly correlate to the diffusion and filtration capacity of the cell-free membrane. This, in turn, influences the membrane's functional capacity when cellularized. For example, to prepare a membrane that provides a functional filtration rate (0.2–2 ml per minute) in the context of the first segment (filtration segment) of IABBP, a membrane is prepared using 25 ml of 30% gelatin mixed with 3.15 ml of 35% F127 stock solution, which provides an F127 concentration of approximately 4.00% in the working solution. This solution is then thoroughly mixed, degassed, deposited on the substrate during spin coating, and dried before removal and subsequent crosslinking. This membrane provides a range of filtration rates suitable for filtration function in the context of IABBP (Figure 12), and the precise rate and overall porosity can be increased or decreased to achieve the desired function. Pore size and distribution can be analyzed in a more quantitative way using experimental methods such as fluorescence bead analysis using the membrane testing system described below, or using pore image analysis which can provide distributions and other data not available through experimental methods (Figure 13).

[0147] Next, the thin film is dried and removed from the substrate. At this point, the thin film is dry and uncrosslinked, and can be incorporated into a scaffold or other such biological structure and crosslinked. This can be done by mounting the thin film into a frame, which allows for 3D printing or other deposition of sacrificial material on either side of the film to create a network of opposing channels. This thin film and channel construct can then be embedded in the scaffold and crosslinked. Once crosslinked, the sacrificial material can be removed and the channels perfused. It is possible to dissolve and remove Pluronic F127 micelles in the film using isopropanol or other nonpolar solvents that break down micelle cores, which can then be perfused through the scaffold to create pores in the film. Alternatively, this dried thin film may be crosslinked by immersion in a glutaraldehyde solution or other crosslinking agent (such as transglutaminase). The sacrificial pologen material can then be removed by further immersion of the thin film in the solvent. Once the pologen material is removed, the thin film can be rinsed with PBS or another solution to remove any remaining crosslinking agents or solvents, after which the cells can be rearranged as needed.

[0148] Cellularized or cell-free membranes can be tested in an in vitro isolation membrane apparatus (Figures 12A-12B). Briefly, the isolated membrane is placed between two highly porous and low-resistance support pieces or mesh screens designed to expose substantially the entire membrane surface area and allow uninhibited filtration or flow. These support pieces or mesh screens are clamped in place between two halves of a chamber containing ports that allow perfusion with fluid, gas, or a combination of both, to simulate in vivo membrane function. This type of chamber and test system allows for short-term or long-term testing of cellularized or cell-free membranes at high throughput. The data generated by this system can be correlated with known membrane surface area to provide surface area-normalized functional data, which can provide information for device design.

[0149] Example 4 - Fabrication of various channel patterns on thin films The channel architecture can be precisely controlled using extrusion-based 3D printing technology, where channels are printed from sacrificial material on either side of the membrane. The entire device is then embedded in an extracellular matrix material (e.g., a scaffold), the sacrificial material is removed, and one or two channel networks are left on either side of the membrane. The architecture and dimensions of the channels constituting these networks are designed using computer-aided design (CAD) software, then converted into G-code to control the 3D printing process. The channel diameter can be adjusted by varying the 3D printer motor speed, the amount of pressure-driven extrusion, and the extruder head temperature, all controlled through G-code and associated 3D printer electronics. For example, to create tapered channels with increasing diameter, the motor speed driving the extruder print head can be controlled to gradually slow down along the channel length, while keeping all other parameters constant. Alternatively, the pressure-driven extrusion can be increased along the channel length, while keeping all other parameters constant, to increase the rate of print material extrusion. The interaction of these varying parameters can be calibrated and used to print the desired channel configuration. By precisely controlling the channel arrangement and architecture, it becomes possible to control the hydrostatic pressure and flow patterns across the entire channel network and the thin film.

[0150] Example 5 - Manufacturing of a multilayer device Multiple layers of the channel network can be stacked to increase the functional surface area of ​​the IABBP device (Figure 9). The device support scaffold is first assembled by pouring 20 mL of 20% gelatin and 10% transglutaminase (20 U / mL) into the bottom mold (i.e., the extracellular matrix material). The prepared inflow and outflow conduits are then filled with Pluronic F127 and placed in the grooves of the bottom mold (Figure 10). The conduits are half-immersed in the gelatin. The gelatin in the mold is thermally and enzymatically crosslinked. Gelatin can be crosslinked at lower temperatures, but this crosslinking can be reversed by heating the gelatin. Crosslinking using enzymes such as transglutaminase or glutaraldehyde is permanent. The thin film is sandwiched between two frames. The framed film is printed on one side with the desired pattern using Pluronic F127 with air from a pressure-controlled extrusion assembly. Once dry, the framed membrane is lifted, inverted, and printed on the opposite side with Pluronic F127 in a mirror image pattern, thereby creating a channel network with opposing channels across the membrane. The printed membrane is then air-dried. Once the printed membrane is dry, another 10 mL of 20% gelatin and 10% transglutaminase is poured into the mold. This gelatin layer should completely cover the conduits. The membrane is placed on the gelatin without incorporating air, for example by mechanical means, aligning the channel network with the conduits to create a continuous network. As the membrane settles into place, it binds to the gelatin. After the gelatin has solidified, the excess membrane is cut from the frame and the frame is removed. Another membrane is framed, printed, and dried, and a spacer is placed on top of the first layer. At this point, shafts or pillars of F127 can be added to the channel pattern on the lower membrane, aligning with the upper membrane channel network and connecting to it, thereby creating a continuous multilayer network. If necessary, holes can be punched, dissolved, or otherwise removed from the membrane to enable interlayer connections. This next spacer is filled with 10 mL of 20% gelatin and 10% transglutaminase, and the next membrane is lowered into place. The membrane is lowered over the next layer so that the network on each layer is aligned.This process can be repeated for as many layers as needed. During the assembly as described above, or after the final membrane is in place, each layer may be connected via small shafts which may be filled with Pluronic F127 or other fluids or gels that can be removed. To connect the channel network of the various layers, these connections are made along the height of the graft. Care is taken to connect homogeneous to homogeneous (e.g., blood vessels to blood vessels, and filtrates to filtrates). Prepared conduits filled with Pluronic F127 are placed on the apical membrane to contact the vascular channel system or filtration channel system and to provide a position for anastomosis of the graft into the vascular structure or for cannula insertion. A higher spacer with notches for the conduits is then placed on the apical membrane, and the mold is filled with 20 mL of 20% gelatin and 10% transglutaminase to seal within the channel network and complete the multilayer graft. Alternatively, once the scaffold is fully assembled by removing the gelatin using a punch or other instrument and creating hollow shafts or other connections between the layers, internal connections between membrane layers may occur. The space may be filled with Pluronic F127, and then sealed, filled, or otherwise closed with gelatin or other material that can bond, crosslink, glue, or otherwise adhere to the space in place to close any remaining holes, thereby maintaining the integrity of the channel network.

[0151] Example 6 - Isolation of primary cells from discarded kidneys Primary cells are isolated from human kidneys deemed unsuitable for transplantation. The renal cortex is manually separated from the medulla and shredded into pieces less than 2 mm in diameter. The shredded tissue is digested in a 200 U / mL type IV collagenase solution at 37°C for 1 hour with constant stirring (150 RPM). After digestion, the collagenase in the tissue slurry is neutralized by adding 10% final concentration fetal bovine serum, and the digest is successively filtered through sieves with pore sizes of 250 μm and 125 μm. Glomeruli are collected from the top of the 125 μm sieve, and tubules are collected through the flow-through. Both parts are attached to gelatin-coated plastic. Culture medium formulations are designed to promote the maintenance of epithelial and endothelial cells and are specified in Table # (co-culture medium). After 1 week in the culture, cells are harvested by EDTA treatment to obtain single-cell suspensions, which are then separated into specific cell types. Glomerular endothelial cells and podocytes are obtained from the glomerular region by immunoisolysis using CD31 and nephrine antibodies, respectively. Contaminating fibroblasts are depleted by Thy1 immunoisolysis. Paratubular endothelial cells are separated from cortical epithelial cells (proximal and distal tubules) from the flow-through by CD31 and CK18 immunoisolysis, respectively. Contaminating fibroblasts are also depleted from these regions by Thy1 immunoisolysis. The medullary tissue is processed in the same manner, and ductal cells are collected by isolation using L1CAM immunoisolysis and fibroblast depletion using Thy1 immunoisolysis. Culture medium formulations for specific cell types are described in Tables 2-4. [Table 2] [Table 3]

[0152] Example 7 - Cell seeding on an IABBP scaffold Vascular and filtration channels are lined with a confluent monolayer of endothelial or epithelial cells, respectively, to provide their respective functions. Inlet and outlet conduits to both networks are cannula-inserted to seed cells into the channels, and a suspension of the appropriate cell type is injected into the inlet conduit for either the vascular or filtration channel. Fluid is simultaneously withdrawn from each outlet conduit at the same rate as the cell suspension injection until the entire network is filled with the cell suspension. The cells are allowed to adhere to the channel walls under static culture in a 37°C incubator for at least 1 hour, then the entire device is rotated 180°, and the same respective cell suspensions are seeded again in both the vascular and filtration networks to ensure complete seeding of the channel lumen. After this time, the network is perfused with culture medium and left for at least 24 hours to achieve full confluence. A direct flow of cell medium, blood, or serum may be introduced into each channel network to supply nutrients and oxygen to the cells and enhance the cellular function of both endothelial and epithelial cells. The flow can be controlled using either a pump or gravity-driven flow.

[0153] To seed multiple cell types within a single channel network, each type must be seeded sequentially based on its position within the scaffold. It is important to note that the scaffold channel network can be perfused in either direction, which enables this sequential seeding of types. To seed podocytes and other glomerular epithelial cell types in segment 1, followed by tubular cells in segment 2, and distal and collecting duct cells in segment 3, each cell type or mixture of cell types must be suspended in a volume proportional to the volume of the segment they occupy.

[0154] Once the cells are placed in a high-density suspension, the cells intended for segment 1 can be injected through a cannula adjacent to segment 1, resulting in segment 1 being entirely filled with the cell suspension and segment 2 being filled with the cell-free fluid previously located in segment 1. These cells are allowed to adhere for 30 minutes, up to 3 hours, or longer, and then a volume of fluid equal to the suspension volume is injected through a cannula adjacent to segment 3 to flush the remaining cell suspension medium and any cells that did not adhere in segment 1. The scaffold is then inverted, and the procedure is repeated to provide complete coverage of segment 1 with the desired cell type.

[0155] Next, a second cell suspension containing cells for segment 2, in a volume equal to the volume of the segment 2 channel, is injected into the scaffold through a cannula adjacent to segment 1. Then, a volume of cell-free solution equal to the volume of the segment 1 channel is injected through a cannula adjacent to segment 1, so that the cell suspension for segment 2 is pushed out through the channel and fills segment 2, but does not enter segment 3. The cells are allowed to adhere for 30 minutes, up to 3 hours. Then, a volume of cell-free solution equal to or greater than the combined volume of segments 1 and 2 is injected through a cannula adjacent to segment 3 to flush the remaining cell suspension medium and any cells that did not adhere. The scaffold is then inverted, and the procedure is repeated to provide complete coverage of segment 2 with the desired cell type.

[0156] Next, a third cell suspension containing cells for segment 3, in a volume equal to the volume of the segment 3 channel, is injected into the scaffold through a cannula adjacent to segment 1. Then, a cell-free solution, in a volume equal to the combined volume of the segment 1 and segment 2 channels, is injected into a cannula adjacent to segment 1 so that the cell suspension for segment 3 is pushed through the channel and fills segment 3. The cells are allowed to adhere for 30 minutes, up to 3 hours. Then, a cell-free solution, in a volume equal to or greater than the combined volume of segments 1, 2, and 3, is injected through a cannula adjacent to segment 3 to flush the remaining cell suspension medium and any cells that did not adhere. The scaffold is then inverted, and the procedure is repeated to provide complete coverage of segment 3 with the desired cell type.

[0157] At this point, the scaffold channel network is fully lined with the desired cell types positioned within the desired segments to produce coordinated function throughout the device. This process is repeatable and can be expanded if additional segments are to be included. The order in which the segments are filled with cells can also be rearranged as needed. Furthermore, the order of scaffold inversion can also be changed as needed. For example, all cells in each segment can be sequentially adhered to a membrane or matrix (e.g., a gelatin matrix), followed by a single inversion of the membrane to adhere the cells to each of the other segments. This type of sequential cell seeding can be achieved in other ways using known volumes of channels, and does not necessarily have to be done in the order or method described herein, and cells can be added in a sequential order via unidirectional perfusion without back perfusion to backflush the cell suspension and any cells that did not adhere.

[0158] Example 8 - In silico modeling of IABBP device and its function, and in vitro model of IABBP film A virtual model is generated using SolidWorks (SW). A 3D point cloud is transported from a 3D printer to Geomagic, and then to SW. The model in SolidWorks allows for the simulation of the device under various clinical conditions, enabling a better understanding of its performance. For example, applying 65 mmHg to the inflow conduit allows for the calculation of the ultrafiltration rate of the export-side device. Membranes are inserted into the device and given performance characteristics indicated by an in vitro membrane testing apparatus, enabling benchtop testing of various cell-free and cell membranes (Figures 12A-12B). These key characteristics are then used to operate and verify the output numbers obtained from the model. This makes it possible to adjust two key parameters of the device in parallel blood purification: surface area and the number of repeating layers.

[0159] Example 9 - Fiber film production In step 1, a solution of gelatin, collagen, fibrin, or other biological or synthetic matrix material is prepared. In step 2, dried silk nanoscale and microscale fibers are added to the matrix solution and mixed by stirring. The size of the fibers depends on the preparation, and the size composition of the fiber components can be adjusted to yield the desired mechanical properties. These fiber components may be crosslinked by themselves or bonded to matrix components in a later step to create an interpenetrating network of matrix and fibers. In step 3, a solution containing sacrificial pologen material is combined with the matrix and fiber solution prepared in step 1 and thoroughly mixed. In step 4, the matrix-pologen solution is deposited onto a substrate by spin coating, dipping coating, or other such thin-film deposition techniques, and then dried, gelled, or otherwise solidified. This deposition technique allows for precise control over the thickness of the thin film. In step 5, additional layers of similar or different compositions are optionally deposited on the first layer to produce a composite or layered thin film. Alternatively, an additional layer is deposited in a manner that allows for patterning or other spatial organization within the film. In step 6, the sacrificial pologen material is removed by dissolution, decomposition, or other destructive techniques, leaving empty spaces in the thin film that act as pores.

Claims

1. A device for restoring or supplementing the function of one or more glands of the endocrine system, The device comprises a functional unit, The aforementioned functional unit is A film comprising a first surface and a second surface, A first channel system comprising a first lumen space, wherein the first lumen space is adhered to the first surface of the membrane and is in fluid communication with the first surface of the membrane, and the first channel system comprises a first end configured to be connected in fluid communication with a fluid supply section, and a second end configured to be connected in fluid communication with a first fluid outlet, A second channel system comprising a second lumen space, wherein the second lumen space is adhered to the second surface of the membrane and is in fluid communication with the second surface of the membrane, and the second channel system comprises a third end configured to be in fluid communication with a second fluid outlet. Equipped with, The first channel system and the second channel system are in fluid communication with each other across the membrane. Each of the first channel system and the second channel system comprises a first channel system wall and a second channel system wall, The apparatus comprising a second channel system, wherein at least one cell type selected from the group consisting of adrenal cells, thyroid cells, ovarian epithelial cells, parathyroid cells, pituitary cells, thymocytes, and testicular epithelial cells is implemented to generate functional endocrine tissue adhered to the wall of the second channel system.

2. The apparatus according to claim 1, wherein the membrane comprises a biocompatible extracellular matrix membrane that separates the first channel system from the second channel system, and the biocompatible extracellular matrix membrane is embedded in a matrix material.

3. The apparatus according to claim 2, wherein the biocompatible extracellular matrix membrane includes a collagen membrane, and the collagen membrane supports cell adhesion on both the first surface and the second surface of the collagen membrane.

4. The apparatus according to claim 3, wherein the film has a thickness of 0.1 micrometers to 20 micrometers (0.1 μm to 20 μm).

5. The apparatus according to claim 1, wherein the membrane is configured to have a minimum shear stress of 0.5 Pascals and / or a minimum intermembrane pressure difference of 1333.22 Pascals.

6. The apparatus according to claim 1, wherein the functional unit comprises at least one biofluid inflow conduit that is in fluid communication with the first end and the first lumen space of the first channel system, and at least one biofluid outflow conduit that is in fluid communication with the second end and the first lumen space of the first channel system, wherein the at least one biofluid inflow conduit is in fluid communication with an arterial conduit, and the at least one biofluid outflow conduit is in fluid communication with a vascular conduit.

7. The apparatus according to claim 1, wherein the first lumen space and the second lumen space are embedded in the scaffold.

8. The apparatus according to claim 1, wherein the first channel system wall is lined with endothelial cells selected from induced pluripotent stem cell (iPSC)-derived endothelial cells and / or human umbilical cord endothelial cells.

9. The apparatus according to claim 8, wherein the endothelial cells are homogeneous or autologous to the patient using the apparatus.

10. The apparatus according to claim 1, wherein at least one cell type selected from the group consisting of adrenal cells, thyroid cells, ovarian epithelial cells, parathyroid cells, pituitary cells, thymocytes, and testicular epithelial cells is homogeneous or autologous to the patient using the apparatus.

11. The apparatus comprises a plurality of functional units, the plurality of functional units including the functional unit and additional functional units having the same configuration. Each of the aforementioned multiple functional units is: The first end and first lumen space of the first channel system, which is in fluid communication with the at least one biofluid inflow conduit, The second end and first lumen space of the first channel system, which is in fluid communication with the at least one biofluid outflow conduit, The third end of the second channel system and the second lumen space, which are in fluid communication with the second outflow conduit. It has, Each first end of the plurality of functional units is individually connected in parallel to one of the plurality of manifold ports of the at least one biofluid inflow conduit. Each second end of the plurality of functional units is individually connected in parallel to one of the plurality of manifold ports of the at least one biofluid outflow conduit. The apparatus according to claim 6, wherein each third end of the plurality of functional units is individually connected in parallel to one of the plurality of manifold ports of the second outflow conduit.

12. The apparatus according to claim 11, comprising the plurality of functional units, wherein the plurality of functional units are stacked in a parallel layer of functional units, and further comprising the functional units and additional functional units having the same configuration.

13. The apparatus according to claim 1, wherein the apparatus is configured for extracorporeal operation in a sterile heated enclosure.

14. The apparatus according to claim 1, wherein blood is delivered to the apparatus using a mechanical pump.

15. The apparatus according to claim 1, wherein the apparatus is located within a capsule, and the apparatus is sized and configured for placement in the human body to replace or enhance the function of one or more glands of the endocrine system.

16. The apparatus according to claim 1, wherein the membrane includes a porous membrane having pores arranged to interconnect the first surface and the second surface.

17. The apparatus according to claim 16, wherein the pores have a diameter of 1 μm to 15 μm.

18. The apparatus according to claim 8, wherein the apparatus is fluidly connected to the patient's circulatory system.

19. A method for manufacturing the apparatus described in Claim 1, The aforementioned method, The present invention provides a film comprising a sacrificial material in the form of a first channel system on a first surface and a sacrificial material in the form of a second channel system on a second surface. The film is immersed in a solution containing scaffolding material, The scaffolding material is gelled, By removing the sacrificial material, the lumen space and walls of the first channel system and the second channel system are formed. The second channel system is provided with at least one cell type selected from the group consisting of adrenal cells, thyroid cells, ovarian epithelial cells, parathyroid cells, pituitary cells, thymocytes, and testicular epithelial cells. Methods that include...

20. The method according to claim 19, further comprising mounting endothelial cells in the first channel system of the membrane.

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