Superporous gel matrices for cell encapsulation

Abstract

A biocompatible gel matrix is ​​provided that is produced from an emulsion that includes a water-soluble material capable of forming a gel and a biocompatible hydrophobic material. In certain aspects, the biocompatible gel matrix of the present disclosure may include a plurality of microchannels and a plurality of nanochannels, where the plurality of microchannels and the plurality of nanochannels are not patterned microchannels and nanochannels, and a plurality of cells, where the cells are adjacent to the plurality of microchannels and a majority of the plurality of cells are within a distance of 50 microns or less from at least one of the plurality of microchannels, where the plurality of microchannels have a width of 5 to 500 microns and the plurality of nanochannels have a width of 1 nm to 500 nm. Methods of using the matrix and methods of making the matrix are also provided.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 194,000, filed May 27, 2021, which is incorporated by reference in its entirety.

[0002] Introduction Type 1 diabetes (T1D) results from the autoimmune destruction of insulin-producing beta cells within the islets of Langerhans. Islet transplantation by direct injection of cadaveric islets into the portal vein of the recipient's liver provides a non-invasive cure for patients with type 1 diabetes (T1D mellitus 1). However, donor availability, poor engraftment, and side effects from general immunosuppression remain obstacles for a more widespread application of this approach. Furthermore, up to 60% of injected islets become non-viable within days after surgical delivery, and long-term insulin independence is often lost by 5 years of transplantation. Activation of innate and adaptive immunity is one of the main causes of islet graft failure. The idea of ​​encapsulating islets has attracted a great deal of interest. However, improved devices and methods are needed to provide encapsulated islets that maintain function and are protected from the patient's immune system. Summary of the Invention

[0003] A biocompatible gel matrix is ​​provided that is produced from an emulsion that includes a water-soluble material capable of forming a gel and a biocompatible hydrophobic material. The use of the biocompatible hydrophobic material allows for the production of a biocompatible gel matrix that is non-toxic to cells and includes microchannels that can support the flow of nutrients to the cells. The matrix also includes nanochannels that support the diffusion of nutrients to the cells.

[0004] In certain aspects, the biocompatible gel matrix of the present disclosure may include a plurality of microchannels and a plurality of nanochannels, where the plurality of microchannels and the plurality of nanochannels are not patterned microchannels and nanochannels, and a plurality of cells, where the cells are adjacent to the plurality of microchannels and a majority of the plurality of cells are within a distance of 50 microns or less from at least one of the plurality of microchannels, where the plurality of microchannels have a width of 5-500 microns, e.g., 5-100 microns or 5-50 microns, and the plurality of nanochannels have a width of 1 nm to 500 nm.

[0005] In certain aspects, an emulsion for producing a biocompatible gel matrix is ​​provided. The emulsion may be an oil-in-water emulsion comprising a water-soluble material capable of forming a gel, a biocompatible hydrophobic material, and optionally a surfactant. The emulsion may further comprise living cells. Upon cooling the emulsion, a matrix is ​​formed, which comprises microchannels and nanochannels as described herein.

[0006] In certain embodiments, the gel matrix is ​​made of a water-soluble material agarose, such as very low gelling agarose. Examples of low gelling agarose include type IX agarose, such as type IX-A agarose. In certain embodiments, the gel matrix is ​​made of a water-soluble material such as collagen, gelatin, polyethylene glycol, alginate, cellulose, PCL, or dextran.

[0007] In certain embodiments, the gel matrix is ​​in the form of a planar scaffold, a cylinder, a sphere, or a fiber. In certain embodiments, the gel matrix is ​​at least 1 cm 3 ~Approx. 10,000cm 3 In certain embodiments, the gel matrix comprises a volume of 1 cm 2 ~1000cm 2 Or 15cm 2 ~30cm2 It includes a surface area in the range of

[0008] In certain embodiments, the gel matrix comprises at least 100 cells. In certain embodiments, the cells may be uniformly dispersed in the matrix. In certain embodiments, the cells may be single cells or clusters of cells. In certain embodiments, the cells are insulin producing cells. In certain embodiments, the insulin producing cells are derived from the differentiation of stem cells. In certain embodiments, the insulin producing cells are pancreatic cells isolated from pancreatic islets. In certain embodiments, the insulin producing cells are in pancreatic islets isolated from the pancreas, and the islets are encapsulated in the matrix. In certain embodiments, the pancreatic islets each comprise about 1000 cells. In certain embodiments, each pancreatic islet has a diameter of about 100 microns. In certain embodiments, the insulin producing cells are in enriched beta clusters (eBCs) derived from stem cells. In certain embodiments, each eBC comprises about 1000 cells. In certain embodiments, each eBC has a diameter of about 100 microns.

[0009] In certain embodiments, the microchannels allow for the flow of nutrients to the plurality of cells, and at least 80% of the plurality of cells encapsulated in the matrix are viable for at least one day.

[0010] In certain embodiments, the microchannels allow for the flow of nutrients to the plurality of cells, and at least 80% of the plurality of cells encapsulated in the matrix are viable for up to one month.

[0011] In certain embodiments, the microchannels allow for the flow of nutrients to the plurality of cells, and at least 80% of the plurality of cells encapsulated in the matrix are viable and functional for at least one day.

[0012] In certain embodiments, the microchannels allow for the flow of nutrients to the plurality of cells, and at least 80% of the plurality of cells encapsulated in the matrix are viable and functional for up to one month.

[0013] In certain embodiments, the cells are insulin producing cells and the function of the cells is assessed by exposing the cells to glucose and measuring insulin production, hi certain embodiments, the cells are exposed to insulin by flowing blood through the matrix.

[0014] In certain aspects, a bioartificial ultrafiltration device is provided that includes a planar scaffold comprising a biocompatible gel matrix as disclosed herein. The device may include a first semi-permeable ultrafiltration membrane disposed on a first surface of the planar scaffold, a first compartment adjacent to the first surface of the planar scaffold and in fluid communication with the planar scaffold via the first semi-permeable ultrafiltration membrane and including an inlet and an outlet, and a second compartment adjacent to a second surface of the planar scaffold and including an outlet, the first semi-permeable ultrafiltration membrane including a plurality of pores having a width in the range of 5 nm to 5 microns, the first semi-permeable ultrafiltration membrane allows transport of ultrafiltrate from the first compartment to the matrix, and the ultrafiltrate crosses through the matrix to the second compartment.

[0015] In certain aspects, the device further comprises a second semi-permeable ultrafiltration membrane disposed on a second surface of the planar scaffold, and the ultrafiltrate traverses from the plurality of microchannels across the second semi-permeable ultrafiltration membrane to a second compartment.

[0016] In certain embodiments, the second semi-permeable ultrafiltration membrane comprises a plurality of pores having a width in the range of 5 nm to 5 microns. In certain embodiments, the first and second semi-permeable ultrafiltration membranes comprise a plurality of pores having a width in the range of 0.1 microns to 2 microns, 0.2 microns to 0.5 microns, 20 nm to 2 microns, or 20 nm to 50 nm.

[0017] In certain embodiments, the second semipermeable ultrafiltration membrane comprises a plurality of pores having a width greater than the width of the plurality of pores in the first semipermeable ultrafiltration membrane.

[0018] In certain embodiments, the inlet of the first compartment is attachable to a tube for connecting to a blood vessel of the subject, optionally the blood vessel is an artery of the subject. In certain embodiments, the outlet of the first compartment is attachable to a tube for connecting to a blood vessel of the subject, optionally the blood vessel is a vein of the subject or an artery of the subject. In certain embodiments, the artery connected to the outlet is the same artery as the artery connected to the inlet. In certain embodiments, the outlet of the second compartment is (i) attachable to a tube for connecting to a blood vessel of the subject, optionally providing ultrafiltrate to one or more blood vessels of the subject, (ii) one or more veins of the subject, (iii) one or more arteries of the subject, and / or (iv) an analyte analysis device.

[0019] In certain embodiments, the thickness of the first semi-permeable ultrafiltration membrane ranges from 0.1 microns to 100 microns or from 0.5 microns to 10 microns.

[0020] In certain embodiments, the surface of the first and / or second surface of the planar scaffold is less than 1 cm 2 ~1000cm 2 , 1cm 2 ~100cm 2 , 10cm 2 ~100cm 2 , or 15cm 2 ~30cm 2 In certain embodiments, the surface area of ​​the first semi-permeable ultrafiltration membrane is in the range of 1 cm 2 ~100cm 2 Or 15cm 2 ~30cm 2 The range is.

[0021] In certain embodiments, the pores are circular in shape, and the width refers to the diameter of the pores. In certain embodiments, the pores are slit-shaped. In certain embodiments, the pores are slit-shaped, and the pore width is 5 nm to 500 nm, for example, 5 nm to 300 nm, 5 nm to 200 nm, or 5 nm to 100 nm. In certain embodiments, the pores are slit-shaped, and the pore length is in the range of 0.1 microns to 5 microns. In certain embodiments, the pores are slit-shaped, and the pore length is in the range of 1 μm to 3 μm, and the pore width is 5 nm to 100 nm. In certain embodiments, the cells in the device are autologous to the subject, xenogeneic to the subject, or allogeneic to the subject.

[0022] In certain aspects, a bioartificial ultrafiltration device is provided that includes a planar scaffold comprising a matrix as disclosed herein, the device comprising a first semi-permeable ultrafiltration membrane as disclosed herein disposed on a first surface of the planar scaffold, a second semi-permeable ultrafiltration membrane as disclosed herein disposed on a second surface of the planar scaffold, a first compartment comprising a first inlet and a first outlet, the first compartment adjacent to the first surface of the planar scaffold, and a second compartment comprising a second inlet and a second outlet, the second compartment adjacent to the second surface of the planar scaffold. and a scaffold, the first inlet configured to connect to an artery of the subject, the first outlet connected to a second inlet of the second compartment, the second outlet of the second compartment configured to connect to a vein of the subject, the semi-permeable ultrafiltration membrane comprising a plurality of pores having a width in a range of 5 nm to 5 microns, the first semi-permeable ultrafiltration membrane enabling transport of ultrafiltrate from the first compartment to the scaffold, and the second semi-permeable ultrafiltration membrane enabling transport of ultrafiltrate from a plurality of microchannels in the scaffold to the second compartment. In certain aspects, the cells in the device are autologous to the subject, xenogeneic to the subject, or allogeneic to the subject. In certain aspects, the plurality of pores in the second semi-permeable ultrafiltration membrane have a width greater than the width of the plurality of pores in the first semi-permeable ultrafiltration membrane, or the plurality of pores in the second semi-permeable ultrafiltration membrane have a width less than the width of the plurality of pores in the first semi-permeable ultrafiltration membrane.

[0023] Disclosed is a method for providing a bioartificial ultrafiltration device that includes cells to a subject that requires the provision of the bioartificial ultrafiltration device that includes cells.The method can include connecting the bioartificial ultrafiltration device as disclosed herein to the subject, and connecting includes connecting the inlet of the first compartment to the artery of the subject, connecting the outlet of the first compartment to the blood vessel of the subject, connecting the outlet of the second compartment to the blood vessel or body cavity of the subject, or connecting the outlet of the second compartment to an analyte analysis device.

[0024] Disclosed is a method for providing a bioartificial ultrafiltration device that includes cells to a subject that requires the provision of the bioartificial ultrafiltration device that includes cells.The method can include connecting the bioartificial ultrafiltration device as disclosed herein to the subject, and connecting includes connecting a first inlet to the artery of the subject and connecting a second outlet to the vein of the subject.

[0025] In certain embodiments, the method includes providing insulin to a subject, and the cells include insulin-producing cells. In certain embodiments, connecting the bioartificial device to a subject requiring connection of the bioartificial device results in increased viability of the cells within the scaffold. In certain embodiments, the ultrafiltrate includes one or more of glucose and oxygen. In certain embodiments, the ultrafiltrate includes one or more of glucose and oxygen, and the insulin-producing cells excrete insulin in response to the presence of glucose in the ultrafiltrate, and the plurality of microchannels transport the insulin to the second compartment.

[0026] In certain aspects, the exported insulin is transported to a plurality of microchannels within the scaffold. In certain aspects, the semi-permeable ultrafiltration membrane prevents the passage of immune system components to the scaffold. In certain aspects, the semi-permeable ultrafiltration membrane prevents the passage of antibodies to the scaffold. In certain aspects, the semi-permeable ultrafiltration membrane prevents the passage of cytokines to the scaffold. In certain aspects, the semi-permeable ultrafiltration membrane prevents the passage of TNF-α, IFN-γ, and / or IL-1β to the scaffold.

[0027] In certain aspects, methods of making the biocompatible gel matrix disclosed herein are provided, the biocompatible gel matrix may be made from agarose, gelatin, polyethylene glycol, polycaprolactone (PCL), collagen, alginate, dextran, or cellulose, and comprises a plurality of microchannels and a plurality of nanochannels, the plurality of microchannels and the plurality of nanochannels being not patterned microchannels and nanochannels, the plurality of microchannels having a width of 5-500 microns, 5-100 microns, or 5-50 microns, and the plurality of nanochannels having a width of 1 nm to 500 nm. In certain aspects, the method includes dissolving agarose, gelatin, polyethylene glycol, PCL, collagen, alginate, dextran, or cellulose in an aqueous solution, adding a biocompatible hydrophobic material and a surfactant to the aqueous solution, mixing the aqueous solution under conditions sufficient to produce an emulsion comprising the dissolved agarose, gelatin, polyethylene glycol, PCL, collagen, alginate, dextran, or cellulose, the biocompatible hydrophobic material, and the surfactant, and generating the matrix by subjecting the emulsion to a temperature sufficient to permit gelation of the agarose, gelatin, polyethylene glycol, PCL, collagen, alginate, dextran, or cellulose, thereby creating the matrix. In certain aspects, the method may further include adding cells to the emulsion prior to the step of generating the matrix. In certain aspects, generating the matrix includes casting the emulsion into a mold comprising a flat surface, thereby creating a planar scaffold. In certain embodiments, the method further comprises disposing a first semi-permeable ultrafiltration membrane on a first surface of the planar scaffold. In certain embodiments, the method further comprises disposing a second semi-permeable ultrafiltration membrane on a second surface of the planar scaffold. In certain embodiments, the agarose is ultra-low gelling agarose. In certain embodiments, the agarose is present in an aqueous solution at a concentration of 1%-10% w / v, 2%-10% w / v, 2%-8% w / v, or 3%-6% w / v.In certain embodiments, dissolving the agarose comprises heating the aqueous solution to a temperature of about 37° C. and stirring the solution at about 300 revolutions per minute (RPM). In certain embodiments, generating the matrix comprises cooling the emulsion to a temperature at which the agarose, collagen, alginate, dextran, or cellulose forms a gel. In certain embodiments, the water-immiscible reagent is perfluorodecalin (PFD). [Brief description of the drawings]

[0028] [Figure 1A] SPA scaffold and endovascular bioartificial pancreas (iBAP) device assembly are shown. Figure 1A presents a schematic cross-sectional view of the SPA scaffold showing the convection and diffusion holes in the agarose. Figure 1B presents the SPA scaffold within the islet chamber housing. Figure 1C shows an exploded view of the components of the iBAP: the flow channel, silicon nanopore membrane (SNM), cell scaffold within the cell chamber, and polycarbonate (PC) backside and ultrafiltrate outlet. Figure 1D shows the assembled iBAP used for in vitro testing. [Figure 1B] SPA scaffold and endovascular bioartificial pancreas (iBAP) device assembly are shown. Figure 1A presents a schematic cross-sectional view of the SPA scaffold showing the convection and diffusion holes in the agarose. Figure 1B presents the SPA scaffold within the islet chamber housing. Figure 1C shows an exploded view of the components of the iBAP: the flow channel, silicon nanopore membrane (SNM), cell scaffold within the cell chamber, and polycarbonate (PC) backside and ultrafiltrate outlet. Figure 1D shows the assembled iBAP used for in vitro testing. [Figure 1C]Shows a SPA scaffold and a vascularized artificial pancreas (iBAP) device assembly. Figure 1A presents a schematic cross-sectional view of the SPA scaffold showing convective and diffusive pores in agarose. Figure 1B presents the SPA scaffold within the islet chamber housing. Figure 1C shows an exploded view of the components of the iBAP, namely the flow channel, the silicon nanopore membrane (SNM), the cell scaffold within the cell chamber, and the polycarbonate (PC) back surface and ultrafiltrate outlet. Figure 1D shows the assembled iBAP used in in vitro tests. [Figure 1D] Shows a SPA scaffold and a vascularized artificial pancreas (iBAP) device assembly. Figure 1A presents a schematic cross-sectional view of the SPA scaffold showing convective and diffusive pores in agarose. Figure 1B presents the SPA scaffold within the islet chamber housing. Figure 1C shows an exploded view of the components of the iBAP, namely the flow channel, the silicon nanopore membrane (SNM), the cell scaffold within the cell chamber, and the polycarbonate (PC) back surface and ultrafiltrate outlet. Figure 1D shows the assembled iBAP used in in vitro tests. [Diagram 2] Shows a water permeability test device consisting of an iBAP, a peristaltic pump, and a pressure gauge. Ultrafiltration measurements were determined using a graduated syringe and a timer. [Diagram 3] The water permeability values (mean ± SD, ** represents p < 0.001) of scaffolds made of SPA and non-emulsified agarose were shown to be statistically significant, with F(1,12) = 6986. [Figure 4A] Shows PFD droplet size analysis. Figure 4A shows a representative DIC image of the 3% SPA scaffold used for droplet analysis. Figure 4B shows a histogram representing the size distribution of PFD droplets represented as mean ± SD for each scaffold. Figure 4C shows the relative droplet area of the scaffold calculated for each DIC image and then averaged. The line represents the mean ± SD for each scaffold, and * represents p < 0.05. [Figure 4B]PFD droplet size analysis. Figure 4A shows a representative DIC image of the 3% SPA scaffold used for droplet analysis. Figure 4B shows a histogram showing the size distribution of PFD droplets representative of the PFD droplets expressed as the mean ± SD for each scaffold. Figure 4C shows the relative droplet area of ​​the scaffolds calculated for each DIC image and then averaged. Lines represent the mean ± SD for each scaffold, * represents p<0.05. [Figure 4C] PFD droplet size analysis. Figure 4A shows a representative DIC image of the 3% SPA scaffold used for droplet analysis. Figure 4B shows a histogram showing the size distribution of PFD droplets representative of the PFD droplets expressed as the mean ± SD for each scaffold. Figure 4C shows the relative droplet area of ​​the scaffolds calculated for each DIC image and then averaged. Lines represent the mean ± SD for each scaffold, * represents p<0.05. [Figure 5A] Degradation of 3% SPA scaffolds. Figure 5A shows that the change in body weight was measured in a 28-day degradation study and no significant differences were found at different time points. Data are shown as mean ± SD. Figure 5B shows representative images of scaffolds at 0, 7, 21, and 28 days in culture. [Figure 5B] Degradation of 3% SPA scaffolds. Figure 5A shows that the change in body weight was measured in a 28-day degradation study and no significant differences were found at different time points. Data are shown as mean ± SD. Figure 5B shows representative images of scaffolds at 0, 7, 21, and 28 days in culture. [Figure 6A] Histological assessment and viability of human islets and eBCs on 3% conventional agarose and SPA scaffolds are presented. Figure 6A shows representative images from H&E and viability staining of human islets and eBCs on conventional agarose and SPA scaffolds. Figure 6B presents viability results reported as mean + SD. [Figure 6B]Histological assessment and viability of human islets and eBCs on 3% conventional agarose and SPA scaffolds are presented. Figure 6A shows representative images from H&E and viability staining of human islets and eBCs on conventional agarose and SPA scaffolds. Figure 6B presents viability results reported as mean + SD. [Figure 7A] Figure 7 shows in vitro evaluation of human islet and eBC insulin production in optimized SPA formulations. Figure 7A shows insulin production in SPA scaffolds during GSIS exposure to 5 mM glucose (G5), 28 mM glucose (G28), followed by a second G5 stage, for both human islets (n=4) and eBCs (n=8) in Figure 7B. Data represent mean ± SD. [Figure 7B] Figure 7 shows in vitro evaluation of human islet and eBC insulin production in optimized SPA formulations. Figure 7A shows insulin production in SPA scaffolds during GSIS exposure to 5 mM glucose (G5), 28 mM glucose (G28), followed by a second G5 stage, for both human islets (n=4) and eBCs (n=8) in Figure 7B. Data represent mean ± SD.

[0029] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present teachings, some exemplary methods and materials are described below.

[0030] As used herein, the term "filtration" refers to the process of separating particulate matter from a fluid, such as air or liquid, by passing the fluid carrier through a medium that does not allow the passage of particulates (e.g., a semi-permeable membrane).

[0031] As used herein, the term "ultrafiltration" refers to subjecting a fluid to filtration, where the material being filtered is very small, typically the fluid contains colloidal, dissolved solutes, or very fine solid materials, and the filter is microporous or nanoporous. The filter may be a membrane, such as a semipermeable membrane. The fluid being filtered is referred to as the "feed fluid." In certain embodiments, the feed fluid may be arterial blood. During ultrafiltration, the feed fluid is separated into a "permeate" or "filtrate" or "ultrafiltrate" that has been filtered through the filter, and a "retentate," which is the portion of the feed fluid that has not been filtered through the membrane.

[0032] As used herein, the term "subject" or "patient" refers to a mammal, such as a primate (e.g., human or non-human primate), cow, horse, pig, dog, cat, or rodent. In certain embodiments, the subject or patient may be a human. In certain embodiments, the subject or patient may be pre-diabetic or have diabetes, such as type 1 diabetes (T1D) or type 2 diabetes. The terms "subject" and "patient" are used interchangeably herein.

[0033] As used herein, the terms "treat", "treatment", and "treating" refer to obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic, in that it completely or partially prevents the disease or its symptoms, and / or it may be therapeutic, in that it partially or completely cures the disease and / or the adverse effects caused by the disease. As used herein, "treatment" encompasses any treatment of a disease in a subject, particularly a human, and includes (a) preventing the disease from occurring in a subject who may be predisposed to the disease but has not yet been diagnosed as having the disease, (b) inhibiting the disease, i.e., arresting its development, and (c) alleviating the disease, e.g., causing regression of the disease, e.g., completely or partially eliminating the symptoms of the disease.

[0034] As used herein, the terms "layer," "film," or "membrane," and their plurals, used in the context of the devices of the present disclosure include layers, films, and / or layers of any material, including but not limited to silicon films, silicon nitride, silica, atomically thin films, such as graphene, silicon, silicene, molybdenum disulfide (MoS 2 ), or combinations thereof, or individual layers of a device that may be formed from a polymer. The "layers," "films," or "membranes" used to fabricate the porous layers of the present disclosure are typically porous and may be nanoporous or microporous. The terms "nanoporous layer," "nanopore layer," "nanoporous membrane," "nanopore membrane," "nanoporous film," and "nanopore film" are used interchangeably and all refer to a polymer layer in which nanopores have been fabricated. The nanoporous layer may include a frame to support the layer. The terms "microporous layer," "micropore layer," "microporous membrane," "micropore membrane," "microporous film," and "micropore film" are used interchangeably and all refer to a polymer layer in which micropores have been fabricated. The microporous layer may include a frame to support the layer.

[0035] As used herein, the term "encapsulated" in the context of cells disposed in a matrix as described herein refers to cells that are surrounded by the matrix rather than residing within microchannels in the matrix. The encapsulated cells are immobilized in the matrix so that they do not significantly move within the matrix. The encapsulated cells receive nutrients via the flow of solution within the microchannels in the matrix that surround the cells. The encapsulated cells receive nutrients via the diffusion of nutrients within the nanochannels in the matrix that surround the cells.

[0036] As used herein, the term "biocompatible" refers to a material, matrix, or device that does not exhibit significant toxicity to cells, eg, mammalian cells.

[0037] Before the present invention is further described, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0038] Where a range of values ​​is provided, it is understood that each intermediate value between the upper and lower limit of that range, to the tenth of the unit of the lower limit, and any other stated or intermediate value within this stated range, is encompassed within the invention, unless the context clearly dictates otherwise. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and may also be encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0039] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are described below. All publications mentioned herein are incorporated by reference to disclose and describe the methods and / or materials in connection with which the publications are cited.

[0040] It should be noted that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, a reference to a "channel" includes a plurality of such channels, a reference to an "agarose cell region" includes a reference to one or more agarose cell regions and equivalents thereof known to those skilled in the art, and so forth. It should be further noted that the claims may be drafted to exclude any optional element. Thus, this statement is intended to serve as a predicate for the use of exclusive terminology such as "solely" and "only" in connection with the recitation of claim elements, or for the use of a "negative" limitation.

[0041] It is understood that certain features of the invention that are described for clarity in the context of separate embodiments may also be provided in combination in a single embodiment. Conversely, for brevity, various features of the invention that are described in the context of a single embodiment may also be provided separately or in any suitable subcombination. All combinations of the embodiments according to the invention are specifically embraced by the present invention and are disclosed herein as if each and every combination were individually and expressly disclosed herein. In addition, all subcombinations listed in the embodiments describing such variables are also specifically embraced by the present invention and are disclosed herein as if each and every such subcombination were individually and expressly disclosed herein.

[0042] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such publications by virtue of prior invention. Further, the publication dates provided may be different from the actual publication dates, which may need to be independently confirmed. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0043] As summarized above, a biocompatible gel matrix is ​​provided that is produced from an emulsion that includes a water-soluble material capable of forming a gel and a biocompatible hydrophobic material. The use of the biocompatible hydrophobic material allows for the production of a biocompatible gel matrix that includes microchannels that are non-toxic to cells and can support the flow of nutrients to the cells. The matrix also includes nanochannels that support the diffusion of nutrients to the cells. In the absence of the use of a hydrophobic material to produce the emulsion, the biocompatible gel matrix formed from the water-soluble material capable of forming a gel does not include microchannels. The absence of microchannels significantly reduces the water permeability of the matrix, making it unsuitable for use to support living cells or as a device to provide cells for various applications. The use of a biocompatible hydrophobic material allows for the encapsulation of living cells in the matrix.

[0044] In certain aspects, the biocompatible gel matrix of the present disclosure may comprise a plurality of microchannels and a plurality of nanochannels, where the plurality of microchannels and the plurality of nanochannels are not patterned microchannels and nanochannels, and a plurality of cells, where the cells are adjacent to the plurality of microchannels and a majority of the plurality of cells are within a distance of 50 microns or less from at least one of the plurality of microchannels, where the plurality of microchannels have a width of 5-500 microns, e.g., 5-100 microns or 5-50 microns, and where the plurality of nanochannels have a width of 1 nm to 500 nm. As a result of the process of forming the microchannels, the microchannels are not straight channels and / or uniformly arranged channels such as those obtained from patterning.

[0045] In certain aspects, the biocompatible gel matrices of the present disclosure do not include laser cut voids or voids introduced by solidifying the matrix around hollow tubes to include through channels in the matrix.

[0046] In certain aspects, a majority of the plurality of cells is within a distance of 40 microns or less, 30 microns or less, 20 microns or less, 10 microns or less, 5 microns or less, 1 micron or less, or directly adjacent to at least one of the plurality of microchannels. In certain aspects, a majority of the plurality of cells is 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, or 85% or more.

[0047] In certain embodiments, the plurality of microchannels has a width of 5-500 microns, 5-100 microns, or 5-50 microns. In certain embodiments, the plurality of microchannels is circular, and the width refers to the average diameter of the microchannel. Thus, for example, the diameter of the microchannel may range from 5 to 100 microns. In certain embodiments, the plurality of microchannels has a width of 5 to 30 microns, 5 to 20 microns, 10 to 30 microns, or 20 to 30 microns.

[0048] In certain embodiments, the nanochannels have a width of 1 nm to 500 nm. In certain embodiments, the nanochannels are circular, and the width refers to the average diameter of the nanochannel. Thus, for example, the diameter of the nanochannel may be in the range of 1 nm to 500 nm, 1 nm to 250 nm, 1 nm to 200 nm, 1 nm to 100 nm, 10 nm to 100 nm, or 1 nm to 50 nm.

[0049] The micro- and nano-channels are not patterned or fabricated by using patterning techniques such as those used to produce channels from PCL or silicon carbide, or the like.

[0050] In certain aspects, an emulsion for producing a biocompatible gel matrix is ​​provided. The emulsion may be an oil-in-water emulsion comprising a water-soluble material capable of forming a gel, a biocompatible hydrophobic material, and optionally a surfactant. The emulsion may further comprise living cells. Upon cooling the emulsion, a matrix is ​​formed, which comprises microchannels and nanochannels as described herein.

[0051] In certain embodiments, the gel matrix is ​​composed of a water-soluble material, such as agarose, e.g., ultra-low gelling agarose. In certain embodiments, the gel matrix is ​​composed of a water-soluble material, such as collagen, gelatin, polyethylene glycol, PCL, alginate, cellulose, or dextran.

[0052] In certain aspects, the gel matrix is ​​in the shape of a planar scaffold, a cylinder, a sphere, or a fiber. For example, the emulsion can be transferred to a mold of any desired shape, cooled, and removed from the mold. In certain aspects, the mold can be formed from a semi-permeable ultrafiltration membrane. In certain aspects, a planar scaffold refers to a shape having a rectangular parallelepiped shape.

[0053] In certain embodiments, the gel matrix is ​​at least 1 cm 3 ~Approx. 10,000cm 3 For example, a gel matrix contains a volume of 1 cm 3 ~1000cm 3 , 10cm 3 ~Approx. 10,000cm 3 , or 10 cm 3 ~About 1000cm 3 In certain embodiments, the gel matrix may have a volume of 1 cm 2 ~1000cm 2 , e.g., 1 cm 2 ~50cm 2 , 10cm 2 ~100cm 2 , 10cm 2 ~50cm 2 , or 15cm2 ~30cm 2 It includes a surface area in the range of

[0054] In certain embodiments, the gel matrix can support at least 100 cells, e.g., 1000 cells, 10,000 cells, 100,000 cells, 10 6 Cells, 10 8 Cells, 10 10 Cells, 10 12 Cells, 10 14 In certain embodiments, the matrix may comprise 1000 cells, 500-5000 cells, or more. In certain embodiments, the cells may be uniformly dispersed in the matrix. In certain embodiments, the cells may be single cells or clusters of cells. In certain embodiments, the cells are insulin-producing cells. In certain embodiments, the insulin-producing cells are derived from the differentiation of stem cells. In certain embodiments, the insulin-producing cells are pancreatic cells isolated from pancreatic islets. In certain embodiments, the insulin-producing cells are in pancreatic islets isolated from the pancreas, and the pancreatic islets are encapsulated in the matrix. In certain embodiments, the pancreatic islets each comprise about 1000 cells, e.g., 500-5000 cells or 800-1500 cells. In certain embodiments, each pancreatic islet has a diameter of about 100 microns, e.g., 50-200 mm, 50-150 mm, 80-200 mm, 80-150 mm, or 90-125 mm. In certain embodiments, the insulin-producing cells are in enriched beta clusters (eBCs) derived from stem cells. In certain embodiments, each eBC comprises about 1000 cells, e.g., 500-5000 cells, or 800-1500 cells. In certain embodiments, each eBC has a diameter of about 100 microns, e.g., 50-200 mm, 50-150 mm, 80-200 mm, 80-150 mm, or 90-125 mm.

[0055] In certain aspects, the microchannels allow for the flow of nutrients to the plurality of cells, and at least 80%, at least 85%, or at least 90% of the plurality of cells encapsulated in the matrix are viable for at least 1 day, at least 10 days, at least 30 days, at least 3 months, at least 6 months, or more.

[0056] In certain aspects, the microchannels allow for the flow of nutrients to the plurality of cells, and at least 80%, at least 85%, or at least 90% of the plurality of cells encapsulated in the matrix are viable for up to 1 month, up to 2 months, up to 3 months, or up to 6 months.

[0057] In certain aspects, the microchannels allow for the flow of nutrients to the plurality of cells, and at least 80%, at least 85%, or at least 90% of the plurality of cells encapsulated in the matrix are viable and functional for at least 1 day, at least 10 days, at least 30 days, at least 3 months, at least 6 months, or more.

[0058] In certain aspects, the microchannels allow for the flow of nutrients to the plurality of cells, and at least 80%, at least 85%, or at least 90% of the plurality of cells encapsulated in the matrix are viable and functional for up to 1 month, up to 2 months, up to 3 months, or up to 6 months.

[0059] In certain embodiments, the cells are insulin producing cells and the function of the cells is assessed by exposing the cells to glucose and measuring insulin production, hi certain embodiments, the cells are exposed to insulin by flowing blood through the matrix.

[0060] In certain aspects, the biocompatible gel matrix has a higher water permeability than a biocompatible gel matrix formed without first forming an emulsion because the biocompatible gel matrix formed without first forming an emulsion does not contain a significant number of microchannels. As described in the Examples section, the microchannels are formed from gas bubbles formed by the use of a hydrophobic material, which gas bubbles coalesce during cooling of the emulsion during formation of the matrix.

[0061] In certain embodiments, the gel matrix comprises a water-soluble material capable of forming a gel upon cooling. Examples of such materials include agarose and collagen. In certain embodiments, the gel matrix does not comprise alginate, alginate derivatives, gelatin, collagen, fibrin, hyaluronic acid, matrigel, natural polysaccharides, synthetic polysaccharides, polyamino acids, polyesters, polyanhydrides, polyphosphazines, poly(vinyl alcohol), poly(alkylene oxide), modified styrene polymers, pluronic polyols, polyoxamers, poly(uronic acid), or poly(vinylpyrrolidone) polymers, polylactic acid, polyglycolic acid, PLGA polymers, polyesters, poly(allylamine) (PAM), poly(acrylates), polyethylene glycol, fibrin, PCL, and poly(methyl methacrylate), and copolymers or graft copolymers of any of the above.

[0062] As used herein, the term gel refers to a matrix that includes water and water-soluble materials that, when dissolved in an aqueous solution, form a gel or matrix when cooled below a certain temperature. The water-soluble materials are hydrophilic and swellable, and form crosslinks to create the gel. After formation, the gel can be dissolved by exposing the gel to a temperature above the temperature at which the water-soluble materials set to form a gel.

[0063] Cells that may be included in the matrices, scaffolds, and devices described herein include, but are not limited to, bone marrow cells; mesenchymal stem cells, stromal cells, pluripotent stem cells (e.g., induced pluripotent stem cells or embryonic stem cells), vascular cells, progenitor cells derived from adipose tissue, progenitor cells derived from bone marrow, intestinal cells, pancreatic islets, Sertoli cells, beta cells, progenitor cells of pancreatic islets, progenitor cells of beta cells, peripheral blood progenitor cells, stem cells isolated from adult tissues, retinal progenitor cells, cardiac progenitor cells, osteoprogenitor cells, neural progenitor cells, and genetically transformed cells, or combinations thereof. The population of cells may be from the subject (autologous cells), from another donor (allogeneic cells), or from another species (xenogeneic cells). The cells may be introduced into the matrix, and the matrix may be immediately (within one day) implanted into the subject, or the cells may be cultured for a longer period of time, e.g., more than one day, to allow cell proliferation prior to implantation.

[0064] In certain embodiments, the population of cells in the matrix are stem cells. In certain embodiments, the population of cells in the matrix are pancreatic progenitor cells. In certain embodiments, the population of cells in the matrix are pancreatic cells isolated from pancreatic islets. In certain embodiments, the population of cells in the matrix are pancreatic islets isolated from the pancreas. In certain embodiments, the population of cells in the matrix may be in the form of tissue fragments, such as islets of Langerhans, which may be isolated from the subject receiving the device or from another subject.

[0065] In certain embodiments, the devices disclosed herein may be used to treat people with diabetes, such as type 1 diabetes. The devices may include pancreatic islet cells or may include stem cells that can differentiate into insulin-producing pancreatic cells. In certain embodiments, pluripotent stem cells (PSCs) may be differentiated into insulin-producing pancreatic cells within the device, and then a bioartificial device containing the differentiated insulin-producing pancreatic cells is implanted in the subject (e.g., in the peritoneum, adjacent to the pancreas or liver, adjacent to the kidney, lung, or heart, or subcutaneously, e.g., in the arm or abdomen). In some cases, the device may include PSCs, and the device may be implanted adjacent to the subject's pancreas or liver.

[0066] In certain aspects, a bioartificial ultrafiltration device is provided that includes a planar scaffold comprising a biocompatible gel matrix as disclosed herein. The device includes a first semi-permeable ultrafiltration membrane disposed on a first surface of the planar scaffold, a first compartment adjacent to the first surface of the planar scaffold and in fluid communication with the planar scaffold via the first semi-permeable ultrafiltration membrane and including an inlet and an outlet, and a second compartment adjacent to a second surface of the planar scaffold and including an outlet, the first semi-permeable ultrafiltration membrane includes a plurality of pores having widths in the range of 5 nm to 5 microns, the first semi-permeable ultrafiltration membrane allows transport of ultrafiltrate from the first compartment to the matrix, and the ultrafiltrate crosses through the matrix to the second compartment.

[0067] In certain aspects, the device further comprises a second semi-permeable ultrafiltration membrane disposed on a second surface of the planar scaffold, and the ultrafiltrate traverses from the plurality of microchannels across the second semi-permeable ultrafiltration membrane to a second compartment.

[0068] In certain embodiments, the second semi-permeable ultrafiltration membrane comprises a plurality of pores having a width in the range of 5 nm to 5 microns. In certain embodiments, the first and second semi-permeable ultrafiltration membranes comprise a plurality of pores having a width in the range of 0.1 microns to 2 microns, 0.2 microns to 0.5 microns, 20 nm to 2 microns, or 20 nm to 50 nm.

[0069] In certain embodiments, the second semipermeable ultrafiltration membrane comprises a plurality of pores having a width greater than the width of the plurality of pores in the first semipermeable ultrafiltration membrane.

[0070] In certain embodiments, the inlet of the first compartment is optionally attachable to a tube for connecting to a blood vessel of the subject, the blood vessel being an artery of the subject. In certain embodiments, the outlet of the first compartment is optionally attachable to a tube for connecting to a blood vessel of the subject, the blood vessel being a vein of the subject or an artery of the subject. In certain embodiments, the artery connected to the outlet is the same artery as the artery connected to the inlet. In certain embodiments, the outlet of the second compartment is (i) attachable to a tube for connecting to a blood vessel of the subject, and optionally provides ultrafiltrate to one or more blood vessels of the subject, (ii) one or more veins of the subject, (iii) one or more arteries of the subject, and / or (iv) an analyte analysis device.

[0071] In certain embodiments, the thickness of the first semi-permeable ultrafiltration membrane ranges from 0.1 microns to 100 microns or from 0.5 microns to 10 microns.

[0072] In certain embodiments, the surface of the first and / or second surface of the planar scaffold is less than 1 cm 2 ~100cm 2 Or 15cm 2 ~30cm 2 In certain embodiments, the surface area of ​​the first semi-permeable ultrafiltration membrane is in the range of 1 cm 2 ~100cm 2 Or 15cm 2 ~30cm 2 The range is.

[0073] In certain embodiments, the plurality of pores are circular in shape, where the width refers to the diameter of the pores. In certain embodiments, the plurality of pores are slit-shaped. In certain embodiments, the plurality of pores are slit-shaped, and the width of the pores is 5 nm to 500 nm, 5 nm to 400 nm, 5 nm to 300 nm, 5 nm to 200 nm, 5 nm to 100 nm, or 5 nm to 50 nm. In certain embodiments, the plurality of pores are slit-shaped, and the length of the pores is in the range of 0.1 microns to 5 microns. In certain embodiments, the plurality of pores are slit-shaped, and the length of the pores is in the range of 1 μm to 3 μm, and the width of the pores is 5 nm to 100 nm. In certain embodiments, the cells in the device are autologous to the subject, xenogeneic to the subject, or allogeneic to the subject.

[0074] In certain aspects, a bioartificial ultrafiltration device is provided that includes a planar scaffold comprising a matrix as disclosed herein, the device comprising a first semi-permeable ultrafiltration membrane as disclosed herein disposed on a first surface of the planar scaffold, a second semi-permeable ultrafiltration membrane as disclosed herein disposed on a second surface of the planar scaffold, a first compartment comprising a first inlet and a first outlet, the first compartment adjacent to the first surface of the planar scaffold, and a second compartment comprising a second inlet and a second outlet, the second compartment adjacent to the second surface of the planar scaffold. and a scaffold, wherein the first inlet is configured to connect to an artery of the subject, the first outlet is connected to a second inlet of the second compartment, and the second outlet of the second compartment is configured to connect to a vein of the subject, the semi-permeable ultrafiltration membrane comprises a plurality of pores having a width in a range of 5 nm to 5 microns, the first semi-permeable ultrafiltration membrane allows transport of ultrafiltrate from the first compartment to the scaffold, and the second semi-permeable ultrafiltration membrane allows transport of ultrafiltrate from a plurality of microchannels in the scaffold to the second compartment. In certain aspects, the cells in the device are autologous to the subject, xenogeneic to the subject, or allogeneic to the subject. In certain aspects, the plurality of pores in the second semi-permeable ultrafiltration membrane have a width greater than the width of the plurality of pores in the first semi-permeable ultrafiltration membrane, or the plurality of pores in the second semi-permeable ultrafiltration membrane have a width less than the width of the plurality of pores in the first semi-permeable ultrafiltration membrane.

[0075] In some cases, the first compartment, where blood is introduced into the device, may have dimensions suitable to facilitate ultrafiltration of the blood. For example, the first compartment may have a height of 100 microns to 6 mm, e.g., 500 microns to 4 mm, 1 mm to 3 mm, or 2 mm to 3 mm.

[0076] In certain embodiments, the bioprosthetic device is sized to fit into a subject's body cavity. The device may be rectangular or cylindrical in shape. In certain cases, the device is sized to fit into a body cavity of a subject. 2 For example, 10 to 30 cm 2 , 10~25cm 2 , 15~25cm 2 , 20~25cm 2 , 15~30cm 2 In certain cases, the device may be rectangular and have a length of 3 cm to 10 cm, a width of 1 cm to 6 cm, and a height of 0.3 cm to 2 cm, for example, dimensions (length x width x height) of 3 cm x 1 cm x 0.5 cm to 6 cm x 4 cm x 1 cm, for example, dimensions of 3 cm x 1 cm x 0.5 cm, 5 cm x 2 cm x 1 cm, or 6 cm x 4 cm x 1 cm.

[0077] As described herein, the devices disclosed herein may maintain implanted cells in a functional and viable state for at least 1 month and up to at least 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 3 years, 5 years, 10 years, or up to 50 years or more, such as 1 month to 50 years, 1 year to 25 years, 5 years to 50 years, 5 years to 25 years, 10 years to 50 years, or 15 years to 25 years.

[0078] In certain embodiments, the devices disclosed herein can be encapsulated in a housing made from an inert material that will not decompose or decay when placed in a subject. Any material approved for use as a medical device placed in a subject can be utilized, including, but not limited to, medical grade plastics, inert metals such as titanium, stainless steel, etc.

[0079] In certain embodiments, the bioartificial device includes two or more semi-permeable ultrafiltration membranes. In certain embodiments, the semi-permeable ultrafiltration membranes are disposed on a first surface and a second surface of the planar scaffold. The semi-permeable ultrafiltration membrane disposed on the first surface of the scaffold may be the same as or different from the semi-permeable ultrafiltration membrane disposed on the second surface of the scaffold. For example, the semi-permeable ultrafiltration membrane adjacent to the compartment containing arterial blood may have smaller pores than the semi-permeable ultrafiltration membrane adjacent to the compartment containing ultrafiltrate flowing through the channels in the matrix. In some cases, the semi-permeable ultrafiltration membrane adjacent to the compartment containing arterial blood may have larger pores than the semi-permeable ultrafiltration membrane adjacent to the compartment containing ultrafiltrate flowing through the channels in the matrix. In certain embodiments, the semi-permeable membrane allows for filtration of ultrafiltrate from the compartment containing arterial blood, and the ultrafiltrate is transported to multiple microchannels in the scaffold. The microchannels are adjacent to the cells, providing efficient exchange of molecules in the ultrafiltrate in the microchannels with molecules released by the cells. These molecules diffuse in a concentration-dependent manner between the lumen of the microchannels and the matrix surrounding the cells. For example, molecules such as oxygen, glucose, lipids, vitamins, and minerals diffuse from the lumen of the channels into the matrix, and molecules secreted by the cells, such as urea, carbon dioxide, and insulin, are transported into the lumen of the microchannels. It is understood that in some embodiments, the diffusion and exchange of molecules in the ultrafiltrate can occur outside the microchannels, for example, the ultrafiltrate does not enter the microchannels but permeates the matrix, for example, via nanochannels.

[0080] In certain embodiments, the semi-permeable ultrafiltration membrane is configured for filtration of biological fluids. In certain embodiments, the membrane comprises a plurality of nanopores, and the shape and size of the pores are controlled. In certain embodiments, the membrane comprises a plurality of pores. In certain embodiments, the plurality of pores may be micropores and may have a width in the range of 0.1 μm to 5 μm, e.g., 0.1 μm to 3 μm, 0.1 μm to 0.5 μm, 0.5 μm to 1 μm, 1 μm to 1.5 μm, 1.5 μm to 2 μm, 0.1 μm to 1 μm, 0.1 μm to 0.8 μm, 0.2 μm to 0.7 μm, 0.2 μm to 0.6 μm, 0.2 μm to 0.5 μm. In certain embodiments, the pores may be nanopores and may have a width of 1 nm to 500 nm, e.g., 1 nm to 90 nm, 2 nm to 50 nm, 3 nm to 40 nm, 4 nm to 50 nm, 4 nm to 40 nm, 5 nm to 50 nm, 5 nm to 20 nm, 4 nm to 20 nm, 7 nm to 100 nm, 12 nm to 20 nm, or 5 nm to 10 nm. In certain embodiments, the pores are slit-shaped and have a width as enumerated herein and a length in the range of 1 μm to 10 μm, e.g., 2 μm to 3 μm, 3 μm to 4 μm, 4 μm to 5 μm, 5 μm to 6 μm, 6 μm to 7 μm, 7 μm to 8 μm, 8 μm to 9 μm, or 9 μm to 10 μm. In certain cases, the rectangular pores have a depth of 100-1000 nm, a width of 3 nm-50 nm, and a length of 1 micron-5 microns, for example a width x length x depth of 5 nm-50 nm x 1 micron-2 microns x 200 nm-500 nm.

[0081] In certain embodiments, the device of the present disclosure comprises a semipermeable ultrafiltration membrane having dimensions (length x width) of 6 mm x 6 mm, 5 mm x 5 mm, 7 mm x 7 mm, 8 mm x 8 mm, 9 mm x 9 mm, 10 mm x 10 mm, 10 cm x 10 cm, for example, 10 mm x 10 mm to 10 cm x 10 cm. In some embodiments, the semipermeable ultrafiltration membrane can be rectangular. In certain embodiments, the semipermeable ultrafiltration membrane can be rectangular in shape. In certain embodiments, the semipermeable ultrafiltration membrane can be rectangular in shape. 2 , for example, 30 to 100 cm 2 , 10~30cm 2 , 15~30cm 2 , 15~20cm 2 , 20~25cm2 , 25~30cm 2 , 0.5~10cm 2 , 0.75~5cm 2 , 0.75~3cm 2 , or 0.75 to 2 cm 2 The surface area is in the range of

[0082] In certain embodiments, the devices disclosed herein may be substantially planar and have a surface area of ​​20-100 cm 2 The devices disclosed herein may be dimensioned to have a surface area in the range of 1 cm to 3 cm (for each planar side) and a thickness of up to 500 cm. In certain embodiments, the devices disclosed herein may be dimensioned to have a surface area in the range of 1 cm to 3 cm. 3 , for example 50~500cm 3 , 100~500cm 3 , 100~300cm 3 , 100~150cm 3 In certain cases, the device may have a volume of 5 to 75 cm 2 , for example, 5 to 50 cm 2 , 10~30cm 2 , or 15~30cm 2 The size of the pores in the membrane may be between 10 nm and 100 nm, for example between 10 nm and 20 nm in width.

[0083] The semipermeable ultrafiltration membranes of the present disclosure include any membrane material suitable for use in filtration of biological fluids, which membrane is structurally capable of supporting the formation of pores. Examples of suitable membrane materials are known in the art and described herein.

[0084] In certain embodiments, the membrane material is synthetic, biological, and / or biocompatible (e.g., for use outside or inside the body). Materials include, but are not limited to, biocompatible silicon, coated silicon materials, polysilicon, silicon carbide, ultra-nanocrystalline diamond, diamond-like carbon (DLC), silicon dioxide, PMMA, SU-8, and PTFE. Other possible materials include metals (e.g., titanium), ceramics (e.g., silica or silicon nitride), and polymers (e.g., polytetrafluoroethylene, polymethylmethacrylate, polystyrene, and silicone). Materials for membranes can be found, for example, in U.S. Patent Application Publication No. 2009 / 0131858, which is incorporated herein by reference in its entirety.

[0085] The semipermeable ultrafiltration membrane of the present disclosure comprises a plurality of pores, the shape of the pores includes linear, square, rectangular (slit-shaped), circular, oval, elliptical, or other shapes. As used herein, the width of a pore refers to the diameter when the pore is circular, oval, or elliptical. In certain embodiments, the membrane comprises pores comprising a single shape or any combination of shapes. In certain embodiments, the size of the pores is highly uniform. In certain embodiments, the pores are microfabricated such that there is less than 20% size variation, less than 10% size variation, or less than 5% size variation between the dimensions of the slit-shaped pores. In certain embodiments, factors that determine the appropriate pore size and shape include a balance between water permeability and solute permselectivity. In certain embodiments, the plurality of pores are slit-shaped pores that provide optimal flux efficiency to enable efficient transport of molecules across the membrane. In certain embodiments, the membrane comprises slit-shaped nanopores. In certain embodiments, the semipermeable ultrafiltration membrane comprises a pore having a diameter of, for example, 1 cm. 2 , 0.5cm 2 , or 0.4 cm 2 On the membrane surface area of ​​about 10 3 ~10 8 10 rectangular slit-shaped nanopores (e.g., 10 4 ~10 8 Pieces or 10 5 ~107 In certain embodiments, the number of slit-shaped nanopores on the semipermeable ultrafiltration membrane is sufficient to allow the membrane to generate a physiologically sufficient ultrafiltration volume at capillary perfusion pressure. In certain embodiments, the porosity of the semipermeable ultrafiltration membrane is about 1% to 50%, such as, for example, 10% to 50%, 20% to 50%, or 20% to 75%.

[0086] How to Use Disclosed is a method for providing a bioartificial ultrafiltration device that includes cells to a subject that requires the provision of the bioartificial ultrafiltration device that includes cells.The method can include connecting the bioartificial ultrafiltration device as disclosed herein to the subject, and connecting includes connecting the inlet of the first compartment to the artery of the subject, connecting the outlet of the first compartment to the blood vessel of the subject, connecting the outlet of the second compartment to the blood vessel or body cavity of the subject, or connecting the outlet of the second compartment to an analyte analysis device.

[0087] Disclosed is a method for providing a bioartificial ultrafiltration device that includes cells to a subject that requires the provision of the bioartificial ultrafiltration device that includes cells.The method can include connecting the bioartificial ultrafiltration device as disclosed herein to the subject, and connecting includes connecting a first inlet to the artery of the subject and connecting a second outlet to the vein of the subject.

[0088] In certain embodiments, the method includes providing insulin to a subject, and the cells include insulin-producing cells. In certain embodiments, connecting the bioartificial device to a subject requiring connection of the bioartificial device results in increased viability of the cells within the scaffold. In certain embodiments, the ultrafiltrate includes one or more of glucose and oxygen. In certain embodiments, the ultrafiltrate includes one or more of glucose and oxygen, and the insulin-producing cells excrete insulin in response to the presence of glucose in the ultrafiltrate, and the plurality of microchannels transport the insulin to the second compartment.

[0089] In certain aspects, the excreted insulin is transported to a plurality of microchannels within the scaffold. In certain aspects, the semi-permeable ultrafiltration membrane prevents the passage of immune system components into the scaffold. In certain aspects, the semi-permeable ultrafiltration membrane prevents the passage of antibodies into the scaffold. In certain aspects, the semi-permeable ultrafiltration membrane prevents the passage of cytokines into the scaffold. In certain aspects, the semi-permeable ultrafiltration membrane prevents the passage of TNF-α, IFN-γ, and / or IL-1β into the scaffold. In certain embodiments, the bioartificial device of the present disclosure can reduce the passage of TNF-α, IFN-γ, and / or IL-1β while allowing the transport of nutrients from the subject's blood to cells within the device. In certain embodiments, the bioartificial device of the present disclosure can reduce the passage of immune system components (e.g., immune cells, antibodies, cytokines, e.g., TNF-α, IFN-γ, and / or IL-1β) by at least 50% (e.g., 60%-80%). In certain embodiments, the bioartificial device of the present disclosure has a semi-permeable ultrafiltration membrane with nanopores (e.g., a membrane that is sized to accommodate an effective number of cells within the bioartificial device for treatment of a subject in need of treatment. For example, the subject may suffer from a condition caused by a lack of functional cells, e.g., a molecule normally secreted by functional cells is not secreted or is secreted at a level that results in symptoms. Providing functional cells within the bioartificial device of the present disclosure can alleviate the symptoms. Exemplary conditions include type 1 diabetes, Parkinson's disease, muscular dystrophy, and the like.

[0090] The device may be implanted subcutaneously, intraperitoneally, or in any suitable location within the body, such as the brain, spinal cord, pancreas, liver, uterus, skin, bladder, kidney, muscle, etc. The implantation site may be selected based on the diseased / damaged tissue requiring treatment. For the treatment of diseases such as diabetes mellitus (DM), the device may be placed in a clinically convenient site, such as the subcutaneous cavity or peritoneum. The device may be connected to the subject's vasculature as described herein. In some cases, the device may be connected in-line to a vascular graft. In some cases, the device may be connected to a subject to deliver ultrafiltrate to an artery, vein, body cavity (e.g., peritoneal cavity), or combination thereof, of the subject. In some cases, the device may be connected to a catheter to deliver ultrafiltrate to a vein to which the catheter is connected.

[0091] The methods and devices disclosed herein can be used for both human clinical and veterinary applications. Thus, the subject or patient to whom the bioartificial device is administered may be a human, or in the case of veterinary applications, may be a laboratory animal, agricultural animal, livestock animal, or wild animal. The subject devices and methods can be applied to animals including, but not limited to, humans, laboratory animals such as monkeys and chimpanzees, livestock animals such as dogs and cats, agricultural animals such as cows, horses, pigs, sheep, goats, and captive wild animals such as bears, pandas, lions, tigers, leopards, elephants, zebras, giraffes, gorillas, dolphins, and whales.

[0092] During operation, blood is pumped from the patient's vascular system (i.e., arteries) to the inlet of the first compartment of the bioartificial device. The blood flows through the first compartment of the bioartificial device, and nutrients and small molecules from the blood pass through the semi-permeable ultrafiltration membrane, while larger molecules in the blood, such as immunoglobulins and cytokines, are prevented from contacting the cells in the device. The nutrients and small molecules include, but are not limited to, glucose, oxygen, and insulin. The small molecules and nutrients that pass through the semi-permeable ultrafiltration membrane are filtered to form an ultrafiltrate that contacts the matrix of the device containing the population of cells. In certain embodiments, the cell population releases insulin into the ultrafiltrate. The ultrafiltrate then passes through the ultrafiltration channels of the matrix, which then passes through a second semi-permeable ultrafiltration membrane. Optionally, the outlet of the second compartment can be configured to connect to a catheter. In certain embodiments, the catheter connects to a second vein.

[0093] The devices of the present disclosure provide high ultrafiltration rates producing ultrafiltrate at rates of 1-15 ml / min at physiological rates of blood flow.

[0094] Method of preparation In certain aspects, methods of making the biocompatible gel matrix disclosed herein are provided, the biocompatible gel matrix may be produced from a water-soluble gel-forming material such as agarose, collagen, alginate, dextran, or cellulose, and includes a plurality of microchannels and a plurality of nanochannels, the plurality of microchannels and the plurality of nanochannels being not patterned microchannels and nanochannels, the plurality of microchannels having a width of 5-100 microns, and the plurality of nanochannels having a width of 1 nm to 500 nm.

[0095] In certain aspects, the method includes dissolving agarose, gelatin, polyethylene glycol, PCL, collagen, alginate, dextran, or cellulose in an aqueous solution, adding a biocompatible hydrophobic material and a surfactant to the aqueous solution, mixing the aqueous solution under conditions sufficient to produce an emulsion comprising the dissolved agarose, gelatin, polyethylene glycol, collagen, alginate, dextran, or cellulose, the biocompatible hydrophobic material, and the surfactant, and generating the matrix by subjecting the emulsion to a temperature sufficient to permit gelation of the agarose, gelatin, polyethylene glycol, PCL, collagen, alginate, dextran, or cellulose, thereby creating the matrix. In certain aspects, the method may further include adding cells to the emulsion prior to the step of generating the matrix.

[0096] In certain aspects, dissolving the water-soluble gel-forming material in the aqueous solution may include mixing, e.g., stirring, the solution of the water-soluble gel-forming material and the buffer or balanced salt solution, hi certain aspects, dissolving may also include applying heat to the solution before, during, and / or after the mixing step.

[0097] In certain aspects, adding and mixing the biocompatible hydrophobic material and surfactant to the aqueous solution may include mixing a solution of the water-soluble gel-forming material with a solution of the biocompatible hydrophobic material, heating the solution to a temperature of 37° C., stirring at about 300 RPM (e.g., 100-500 RPM), adding a surfactant (e.g., Tween 80 or Tween 20), and stirring for a period of time. The solution may be stirred at a higher speed, e.g., greater than 500 RPM (e.g., greater than 600-1000 RPM), to create an emulsion.

[0098] The step of generating the matrix by placing the emulsion at a temperature sufficient to permit gelation of the agarose, gelatin, polyethylene glycol, PCL, collagen, alginate, dextran, or cellulose may include placing the emulsion at room temperature (e.g., 25° C.) and / or 4° C. for a period of time sufficient for gelation of the water-soluble gel-forming material.

[0099] In certain embodiments, generating the matrix includes casting the emulsion into a mold that includes a flat surface, thereby creating a planar scaffold. In certain embodiments, the method further includes disposing a first semi-permeable ultrafiltration membrane on a first surface of the planar scaffold. In certain embodiments, the method further includes disposing a second semi-permeable ultrafiltration membrane on a second surface of the planar scaffold.

[0100] In certain embodiments, the agarose is ultra-low gelling agarose. In certain embodiments, the agarose is present in the aqueous solution at a concentration of 1%-10% w / v, 2%-10% w / v, 2%-8% w / v, or 3%-6% w / v. In certain embodiments, dissolving the agarose comprises heating the aqueous solution to a temperature of about 37° C. and stirring the solution at about 300 revolutions per minute (RPM). In certain embodiments, creating the emulsion comprises stirring the solution at about 500-1000 RPM. In certain embodiments, generating the matrix comprises cooling the emulsion to a temperature at which the agarose, gelatin, polyethylene glycol, PCL, collagen, alginate, dextran, or cellulose forms a gel.

[0101] In certain embodiments, the biocompatible hydrophobic agent is a water-immiscible agent such as perfluorodecalin (PFD). In certain embodiments, the agarose solution, PFD solution, and detergent solution are combined at about 63%-65% (v / v) agarose solution, about 32%-33% (v / v) PFD, and about 5%-2% (v / v) detergent.

[0102] In certain aspects, the matrix may be washed with a solvent that dissolves and removes the water-immiscible reagents. In certain aspects, the matrix may be washed with an aqueous solution to remove the surfactant. In certain aspects, the matrix may be washed with a mixture of a hydrophobic material and an aqueous solution to remove the water-immiscible reagents and the surfactant.

[0103] experiment As can be understood from the disclosure provided above, the present disclosure has a wide variety of applications. Thus, the following examples are presented to provide those skilled in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as the invention, nor are they intended to represent that the following experiments are all or the only experiments performed. Those skilled in the art will readily recognize a variety of non-critical parameters that can be changed or modified to obtain essentially similar results. Thus, the following examples are presented to provide those skilled in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as the invention, nor are they intended to represent that the following experiments are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, dimensions, etc.), but some experimental error and deviation should be accounted for.

[0104] Materials and Methods Fabrication of SPA scaffolds Superporous agarose (SPA) scaffolds were constructed by dissolving ultra-low gelling agarose (Sigma-Aldrich: A2576) in 5 mL of Hank's Balanced Salt Solution (HBSS) (UCSF Cell Culture Facility: CCFAJ002) to make a 3% or 6% w / v solution in a beaker. The solution was further dissolved by heating in a microwave for five short cycles with gentle mixing between each cycle. The agarose solution was then heated to 37°C on a hotplate and stirred at 300 revolutions per minute (RPM). Perfluorodecalin (PFD) (Sigma-Aldrich: P9900) and Tween™ 80 (Sigma-Aldrich: P4780) were then added to the agarose solution to create an emulsion consisting of agarose (64%, v / v), PFD (33%, v / v), and Tween™ 80 (3%, v / v). PFD was selected instead of cyclohexane as the water-immiscible solvent due to its biocompatibility and similar cyclic structure [1]. The beaker was then sealed with parafilm (Bemis Co., Inc) and stirred at 750 rpm for approximately 10 min to thoroughly mix and emulsify as indicated by its cloudy color.

[0105] The thoroughly mixed superporous agarose emulsion was then cast into a 316L stainless steel cell scaffold housing. If the experiment included cells, 36 uL of emulsion was mixed with the desired number of cells before casting into the cell scaffold housing. To achieve gelation, the scaffold was left at room temperature (25 °C) for 20 min, followed by cooling at 4 °C for 10 min. The gelled scaffold is composed of convective pores created by the PFD droplets and diffusion pores already present within the native microstructure of the agarose (Figure 1A). The scaffold (Figure 1B) was then inserted into an intravascular bioartificial pancreas (iBAP) device (Figure 1C). For all in vitro tests, we used an iBAP device prototype for islet encapsulation under convective mass transport previously developed by our group (Figure 1D) [2].

[0106] Figures 1A-1D show the SPA scaffold and endovascular bioartificial pancreas (iBAP) device assembly. Figure 1A presents a schematic cross-sectional view of the SPA scaffold showing the convection and diffusion holes in the agarose. Figure 1B presents the SPA scaffold within the islet chamber housing. Figure 1C shows an exploded view of the components of the iBAP: the flow channel, silicon nanopore membrane (SNM), cell scaffold within the cell chamber, and polycarbonate (PC) backside and ultrafiltrate outlet. Figure 1D shows the assembled iBAP used for in vitro testing.

[0107] Water permeability measurement To test only the hydraulic permeability of the SPA cell scaffold, an iBAP prototype was assembled without an SNM and connected to a custom flow circuit (Figure 2). Masterflex L / S25 tubing (Cole-Parmer) was connected to both the inlet and outlet of the device. A peristaltic pump (Cole-Parmer) controlled the cross-flow rate within the device. A pressure gauge (General Electric) was placed in series with the tubing to assess the transmembrane pressure (TMP) across the scaffold. The ultrafiltrate outlet was connected to Masterflex L / S14 tubing (Cole-Parmer). The ultrafiltration rate was determined by the time required to produce 1 mL of ultrafiltrate, measured in triplicate per scaffold. The TMP, ultrafiltration rate (UF), and surface area (SA) were used to calculate hydraulic permeability (Lp) using an equation derived from Darcy's law (Equation 1):

number

[0108] Figure 2 shows the permeability test setup consisting of the iBAP, peristaltic pump, and pressure gauge. Ultrafiltration measurements were determined using a calibrated syringe and timer.

[0109] Droplet image acquisition and analysis SPA scaffolds used for imaging were prepared with 3% (w / v) agarose solution in the same manner as described above. After gelation, (n=3) scaffolds were fixed in 10% formalin to prevent bacterial growth and placed in glass-bottom microwell dishes (MatTek:P35G-1.5-14-C). Scaffolds were imaged with a differential interference contrast (DIC) 20x lens using a time-lapse wide-field microscope (Nikon Instruments). Z-stack images were taken at three different positions on the scaffold and analyzed using FIJI (ImageJ) software. The Hough Circle Transform plugin (UCB Vision Sciences) was used on three slices per stack (n=9 images per scaffold) to determine the PFD droplet size within the scaffold. The droplet size results were then used to determine the area of ​​the droplet and then calculate the relative droplet area by dividing by the total area of ​​the image. To visualize the variability between SPA batches, the droplet size and relative droplet area (%) results per scaffold were averaged.

[0110] Decomposition test Degradation studies were performed under static conditions for 28 days using SPA (n=15) scaffolds made with 3% (w / v) agarose solution. Scaffolds were placed in 12-well Transwell dishes (Millipore Sigma: PIXP01250) containing 1 mL of FBS (Corning: 35011CV) supplemented with 10% penicillin-streptomycin (UCSF Cell Culture Facility: CCFGK004). Scaffolds were maintained at 5% CO2 and 37°C throughout the experimental period, and FBS was replaced in the wells every 3-4 days. Every 7 days, three scaffolds were removed from the culture dish and weighed in a hydrated state.

[0111] Cell procurement, culture and encapsulation Adult human islets were obtained from both the UCSF Islet and Cell Production Facility (Mission Center Building, San Francisco, CA) and Prodo Laboratories, Inc. (Aliso Viejo, CA). Islets isolated from UCSF were shipped in CMRL1066 medium (Corning: 15110CV) within 24 hours of isolation. Islets from Prodo Labs were washed and incubated in PIM medium (Prodo Laboratories, Inc). Enriched β clusters (eBCs) were produced as previously described [3]. Briefly, MEL-1 INSGFP / w hPSCs were cultured with differentiation inducers in a manner that stepwise recapitulates pancreatic development, consisting of six stages over a 20-day period. Cells were then sorted by fluorescence-activated cell sorting (FACS), and immature β-like cells were recombined and further cultured to form eBCs. eBCs were ready for functional and physiological studies by day 28. eBCs were shipped in GN9 medium (CMRL medium supplemented with 1% penicillin-streptomycin, 10% FBS, 1:100 glutamax, 1:100 NEAA, 10 μM Alki II, 0.5 mM VitC, 1 μM T3, 1 mM cysteine, 10 μM zinc, and 10 μg / ml heparin). Cells from all sources were maintained overnight in non-treated T75 flasks at 5% CO2 and 37°C prior to encapsulation. Either 500 IEQ or 500 eBCs (approximately 1000 cells per cluster with an average diameter of 100 μm) in 36 μL of SPA were added to the 3% SPA emulsion after a mixing step and gelled as described above.

[0112] Viability and Histology Cell viability was assessed immediately after encapsulation. After gelation, scaffolds were placed into glass-bottom microwell dishes (MatTek: P35G-1.5-14-C) for confocal imaging. Scaffolds were incubated with live / dead cell stain (Invitrogen: L3224) for 15 min at room temperature and washed three times with PBS before imaging with a fluorescent spinning disk confocal microscope (Nikon Instruments). Viability was quantified by subtracting the area of ​​dead cells from the total cell area and then dividing by the total cell area using FIJI (ImageJ) software [4]. Viability was averaged for human islets (n=11) and eBCs (n=7) in SPA scaffolds, where n refers to the number of clusters analyzed. Both human islets (n=9) and eBCs (n=5) were compared using non-emulsified 3% agarose.

[0113] Scaffolds were fixed in 4% paraformaldehyde and processed at Gladstone Laboratories (San Francisco, CA). Scaffolds were embedded in paraffin before sectioning. Five micron thick sections were stained with hematoxylin and eosin (H&E). Images were taken with a light microscope (Leica) at 20x magnification. Images of islets and scaffold material were enhanced by increasing gamma radiation to 1.3 and contouring the pore area in FIJI.

[0114] In vitro glucose-stimulated insulin secretion assay Dynamic glucose-stimulated insulin secretion (GSIS) assays were performed at 37°C and 5% CO. 2A closed mock loop circuit was used containing either CMRL medium for human islets (supplemented with 10% fetal bovine serum (FBS) v / v) or GN9 medium for eBCs in a humidified incubator. Masterflex L / S14 and 25 tubing (Cole-Parmer) were connected to the ultrafiltrate outlet and both the inlet and outlet of the iBAP device, respectively. A peristaltic pump (Cole-Parmer) maintained constant flow through the scaffold in the iBAP device, resulting in an ultrafiltration rate of 50 μL / min. Cells were allowed to stabilize in low glucose (5 mM) medium for 120 min before performing GSIS testing. Ultrafiltrate samples were collected over 16 min during the first low glucose (5 mM) step. Glucose concentration was then increased (28 mM) by adding glucose to the medium, and ultrafiltrate collection was continued for 30 min. The choice of 28 mM for glucose stimulation was based on a protocol (SOP document: 3104, A03) from the National Institute of Allergy and Infectious Diseases (NIAID) [5]. Glucose concentrations were then lowered to basal concentrations and samples were collected for 44 min. Ultrafiltrate samples were kept at -20°C until they could be analyzed for insulin concentration. Secreted insulin concentrations in ultrafiltrate samples from GSIS experiments were determined using an enzyme-linked immunosorbent assay (ELISA) kit (Mercodia: 10-1113-01). The product of insulin concentration (pg / L) and ultrafiltration rate (mL / min), normalized by the number of islets or eBCs, was used to calculate insulin production (pg / min / IEQ). The stimulation index (SI) was calculated by dividing first-phase insulin production by averaged basal insulin production.

[0115] statistical methods Data are presented as mean ± SD for permeability, relative drop area, degradation and viability experiments. Statistical evaluation of mean values ​​in different tests was assessed using GraphPad Prism version 8.2. Permeability and viability data were analyzed by two-way analysis of variance (ANOVA), and relative drop area and degradation were assessed by one-way ANOVA. Post-hoc comparisons were performed using Tukey's multiple comparison test. A p-value <0.05 was accepted as statistically significant for all analyses.

[0116] result water permeability In optimization experiments, the water permeability (Lp) of SPA scaffolds made with agarose concentrations of 3% (w / v) and 6% (w / v) and non-emulsified agarose was compared (Figure 3). Two-way ANOVA found a significant effect of agarose concentration (3% and 6%) and a significant interaction on water permeability between whether the scaffolds were made with SPA or non-emulsified agarose (F(1,12)=81.6, p<0.001). SPA had higher water permeability than non-emulsified agarose (F(1,12)=6986, p<0.001) for both agarose concentrations. Furthermore, 3% SPA and 3% agarose had higher water permeability compared to their 6% counterparts (F(1,12)=86.1, p<0.001). Multiple comparison tests were performed using 3% and 6% non-emulsified agarose (0.0025 and 0.0008 mL / min / cm, respectively). 2 All groups showed significant differences (p<0.001) except for the scaffolds fabricated with 0.01% SPA (n=4) which had the highest permeability, followed by the 6% SPA (n=4) (0.66 mL / min / cm, respectively). 2 / mmHg, 0.53mL / min / cm 2 / mmHg) (p<0.001).

[0117] Figure 3 shows that the water permeability values ​​(mean ± SD, ** denotes p<0.001) of scaffolds made with SPA and non-emulsified agarose were statistically significant, F(1,12)=6986. There was also a significant effect of agarose concentration (3% vs. 6%), F(1,12)=86.14. Post-hoc tests showed significant differences between all groups except for scaffolds made with 3% and 6% non-emulsified agarose (p=0.998). Water permeability was significantly different for scaffolds made with 3% (w / v) SPA from the other three scaffolds.

[0118] PFD droplet analysis Three 3% SPA scaffolds were analyzed for PFD droplet size to compare variability between SPA batches (FIG. 4A). For each scaffold, z-stack images were acquired at three locations and three different heights, resulting in a total of nine DIC images per scaffold (n=9). The PFD droplets were well-dispersed and showed no visible trend in placement within the scaffold. The distribution of PFD droplet sizes for each scaffold was significantly skewed toward smaller diameters (FIG. 4B). The median diameters of the PFD droplets were 18.2, 13, and 14.3 μm for the three scaffolds, respectively. The relative droplet area of ​​the scaffold was calculated for each DIC image and then averaged (FIG. 4C). A one-way ANOVA was performed to compare the mean droplet area in the different scaffold batches (17.6 ± 5.0, 19.7 ± 6.9, and 13.2 ± 2.7%, respectively) and found significant differences between the means (F(2,24) = 3.8, p = 0.037). Multiple comparison tests found significant differences only between scaffolds 2 and 3 (p < 0.05), indicating some variability between the scaffolds.

[0119] Figures 4A-4B show the PFD droplet size analysis. Figure 4A shows a representative DIC image of the 3% SPA scaffold used for the droplet analysis. Figure 4B shows a histogram showing the size distribution of the PFD droplets, which represents the PFD droplets expressed as the mean ± SD for each scaffold. Figure 4C shows the relative droplet area of ​​the scaffolds, which was calculated for each DIC image and then averaged. Lines represent the mean ± SD for each scaffold, and * denotes p<0.05.

[0120] Scaffold Degradation Studies Changes in hydrated scaffold weight were assessed over 28 days in static culture conditions. The average weight of the scaffolds on day 0 was 45.8 ± 9.78 mg, while the average weight of the scaffolds on day 28 was 42.7 ± 4.81 mg (Figure 5A). One-way ANOVA determined that there was no significant change in the average weight of the scaffolds over time (F(4,10) = 0.048, p = 0.75). However, the appearance of the scaffolds changed throughout the 28-day study. After 21 days, the scaffolds were no longer translucent and appeared almost white on day 28 (Figure 5B), which may be due to protein deposition from the FBS in the medium.

[0121] Figures 5A-5B show the degradation of 3% SPA scaffolds. Figure 5A shows that the weight change was measured over a 28-day degradation study and no significant differences were found at different time points. Data are presented as mean ± SD. Figure 5B shows representative images of scaffolds at 0, 7, 21, and 28 days in culture.

[0122] Histology and cell viability assessment Histological sections were stained with H&E to visualize both cell and scaffold morphology (Figure 6A, top). Human islets and eBCs in both SPA and conventional agarose show similar morphology. SPA sections display open areas (pores) within the scaffold, indicated by dotted outlines. Pores appear distributed throughout the scaffold and close to the encapsulated cells (<10 μm, Figure 6A, top).

[0123] Adult human islet and eBC viability were measured immediately after encapsulation in both the optimized SPA formulation and conventional non-emulsified agarose (Figure 6A, bottom). Multiple cell clusters were imaged for both human islets and eBCs (Figure 6B). In SPA scaffolds, human islets were 95.8±3% (n=11) viable and eBCs were 95.8±2% (n=7) viable. In non-emulsified 3% agarose scaffolds, human islets and eBCs were 96.1±3% (n=9) and 96.6±3% (n=5) viable. Most of the dead cells were observed at the periphery of the islets. For eBCs, dead cells were mainly seen at the periphery, but a few were observed in the center of the clusters in the SPA scaffolds. Two-way ANOVA determined that there were no significant differences in mean viability between the different scaffolds (F(1,28)=0.2109, p=0.65) or between the different cell types (F(1,128)=0.050, p=0.83). Overall, the high viability demonstrated that the encapsulation process did not damage the cells, allowing for further dynamic glucose-stimulated insulin secretion assessment.

[0124] Figures 6A-6B present histological assessment and viability of human islets and eBCs in 3% conventional agarose and SPA scaffolds. Figure 6A shows representative images from H&E and viability staining of human islets and eBCs in conventional agarose and SPA scaffolds. H&E staining shows nuclei and cytoplasm, and dotted lines show pores (arrows) surrounding encapsulated cell clusters within the SPA. Scale bars represent 50 μm in length. Viability staining shows dead and live cells imaged by fluorescent microscopy. Figure 6B presents viability results reported as mean + SD. Human islets were 95.8 ± 3% viable in SPA scaffolds and 96.6 ± 3% viable in conventional agarose scaffolds. eBCs were 95.8 ± 2% viable in SPA scaffolds and 96.6 ± 3% viable in conventional agarose scaffolds. Two-way analysis of variance determined that there were no significant differences between the mean survival rates of the groups.

[0125] Assessment of insulin production SPA scaffolds containing either human islets (n=4) or eBCs (n=8) with 500 IEQs or eBCs in 36 μL of SPA were tested in iBAP using an in vitro GSIS assay. The mean insulin production during the first low glucose exposure was 6.8±2.7 pg / min / IEQ for human islets (FIG. 7A) and 2.1±0.9 pg / min / eBC for eBCs (FIG. 7B). When exposed to higher levels of glucose (28 mM), both adult human islets and eBCs increased insulin production and showed a biphasic insulin response. The first phase insulin production after exposure to a glucose concentration of 28 mM was 83.4±20.5 pg / min / IEQ for human islets and 8.0±2.7 pg / min / eBC for eBCs. During the second phase, insulin production gradually decreased from the peak of the first phase, with mean insulin production in human islets and eBCs of 27.8±14.0 pg / min / IEQ and 3.3±2.1 pg / min / eBC, respectively. All scaffolds maintained insulin production above baseline insulin levels during the second phase and showed a small spike in insulin production after the second low glucose exposure. During the second low glucose exposure, basal insulin production returned to the levels before high glucose stimulation (6.4±3.3 pg / min / IEQ and 2.0±1.6 pg / min / eBC). The stimulation index (SI), i.e., the ratio of first phase insulin production during high glucose exposure to basal insulin production, was 13.6±6.1 for human islets and 4.9±3.4 for eBCs.

[0126] Figures 7A-7B show in vitro evaluation of human islet and eBC insulin production in optimized SPA formulations. Insulin production in SPA scaffolds is shown for both human islets (n=4) in Figure 7A and eBCs (n=8) in Figure 7B during GSIS exposure to 5 mM glucose (G5), 28 mM glucose (G28), followed by a second G5 step. Data represent the mean ± SD.

[0127] Consideration We fabricated a novel superporous agarose-based cell scaffold that supports eBC and islet viability and insulin production in iBAPs. This scaffold aims to optimize mass transfer of key solutes to and from islets in two ways. First, the highly porous (superporous) structure allows ultrafiltrate permeation throughout the scaffold, as shown by permeability and porosity experiments, thus minimizing the diffusion distance (<10 μm) from ultrafiltrate to islets or eBCs. Second, the high permeability of the scaffold does not restrict the flow of SNM-generated ultrafiltrate through the cell scaffold. Most polymeric membranes used for ultrafiltration do not exhibit insufficient permeability due to the membrane thickness (30–100 μm) required to maintain islet viability and insulin production [6]. Unlike traditional polymer membranes, SNMs are less than 1 μm thick and have a very uniform pore size distribution, allowing for a higher amount of ultrafiltration and improving the functionality of islets [6,7,2]. For the iBAP design to deliver sufficient amounts of ultrafiltrate to the encapsulated islets, the water permeability of the cell scaffold must be greater than that of the SNM. High agarose concentration (6% w / v) is widely used and preferred in bioseparation and chromatography processes due to its small pore size [8]. It is known that the small pore size at high concentrations of agarose creates a rigid microenvironment and initiates mechanotransduction in encapsulated islets [9]. Also, the small pore size at high concentrations of agarose may limit water permeability

[10] , which is consistent with our finding that the 3% (w / v) SPA scaffold had significantly higher water permeability than the 6% (w / v) SPA scaffold. SPA scaffolds made with 3% (w / v) agarose have 70-fold higher water permeability than SNM (0.66 and 0.010 mL / min / cm, respectively). 2 / mmHg) [7]. Permeability is inversely proportional to the resistance to fluid flow, therefore, a high permeability scaffold will provide less resistance to fluid flow than an SNM. To maximize the ultrafiltration rate of the iBAP, the cell scaffold (L p ) and SNM(LSNM ) to obtain the total permeability (L o ) can be calculated (Equation 2)

[11] .

number

[0128] Human islets showed dynamic insulin production in response to glucose stimulation in SPA scaffolds. The baseline insulin production rate during the initial low glucose exposure was 6–7 pg / min / IEQ, consistent with other in vitro human islet studies at glucose concentrations of approximately 5 mM

[12] . Studies using perfused human islets showed insulin production peaks from 12–40 pg / min / IEQ when exposed to 16.7 mM glucose [12–14], whereas SPA scaffolds showed higher production (60–99 pg / min / IEQ) at 28 mM glucose. We chose the concentration of 28 mM as the glucose stimulation concentration because it is traditionally used in static GSIS assays for clinical transplant evaluation [5]. The distinct biphasic curves in response to glucose stimulation show promise for insulin therapy, but long-term evaluation of insulin response and diabetic pig studies are needed for confirmation. These studies demonstrated initial feasibility and a biphasic response to high glucose stimulation with a clear first phase and cessation of insulin production after return to low glucose levels, indicating physiological islet function in SPA scaffolds in vitro.

[0129] Recently, there has been progress in forming β-cells from hPSCs to improve insulin secretion and display responses similar to mature β-cells [3, 15, 16], but the incorporation of these cells into implantable devices is still in its infancy. Several groups, including Semma Therapeutics (a subsidiary of Vertex Pharmaceuticals, Boston, MA) and ViaCyte (San Diego, CA), have conducted preclinical studies and clinical trials to investigate BAP devices using previous generation hPSC-derived cells [17-20]. Human islets and eBCs were found to display similar insulin response profiles to glucose stimulation in SPA scaffolds. This study is believed to be the first to demonstrate dynamic insulin secretion kinetics with stem cell-derived β-cells in a macroencapsulated device in vitro. Phase 1 began for both cell types at the first time point of high glucose challenge showing no delay in glucose-insulin kinetics

[21] . The magnitude of insulin secretion was considerably smaller in eBCs than in human islets, a finding consistent with many other hPSC-derived β-cell studies [22–24], in addition to the fact that the MEL-1 INSGFP / w strain used in this study only has one functional insulin allele

[23] . The other insulin allele in eBCs was replaced by GFP for sorting purposes [3], and restoring the second insulin allele could theoretically double insulin production. Furthermore, in both eBCs and human islets, a second insulin production peak was observed after exposure to a second low glucose concentration medium. Henquin et al. made a similar observation, termed an off-response, in perfused infant and adult human islets where insulin peaks before returning to basal levels

[25] . Other reports have shown that hPSC-derived β-cells fail to terminate their insulin response to reduced glucose concentrations, a hallmark of immature β-cells [19, 26, 27]. Appropriate cessation of elevated insulin production in eBCs indicates that they function similarly to adult human islets and are able to prevent hypoglycemia [15, 28].Although overall insulin production was lower in eBCs, optimal mass transport in the SPA scaffold showed promise for supporting greater cell density while maintaining a normoxic environment in iBAPs. In summary, our findings demonstrate for the first time that eBCs have a similar insulin response profile to glucose stimulation as adult human islets in SPA scaffolds.

[0130] The SPA macroporous scaffold in the iBAP appeared to provide sufficient mass transfer for the macroencapsulated insulin-producing cells by minimizing the diffusion distance between the ultrafiltrate and the islets. Previous reports of SNM-based iBAPs have shown that ultrafiltrate-mediated convection-based mass transfer supports islet function across the SNM and through the agarose cell scaffold [2]. Traditional agarose, although widely used for islet encapsulation [29–31], does not allow for high flux of solutes. Further characterization of the SPA scaffold showed higher porosity based on relative PFD droplet area (>13.2 ± 2.7%) and minimal degradation over 28 days. It is currently unclear how much of the PFD remains as droplets and how much of the PFD is removed to form open pores. The apparent color change in the SPA scaffold could be due to either protein or albumin deposition from the FBS. Another hypothesis involves emulsion instability over time. Ongoing fluorine nuclear magnetic resonance (fNMR) spectroscopy studies will help determine PFD levels within the SPA scaffolds over time. Histological evaluation of the SPA scaffolds revealed pores (<10 μm from pore to islet surface) surrounding the outer surface of the islets, allowing ultrafiltrate to flow almost directly across the islet surface. Thus, the diffusion distance from ultrafiltrate to the center of the islet is defined by the diameter of the islet. Furthermore, changing the scaffold from agarose to SPA increases the permeability or fluid velocity, thus increasing the Reynolds number and improving overall mass transfer within the scaffold [11, 32]. The shorter diffusion distance and increased permeability significantly increases the transport of oxygen, glucose, and insulin within the cell scaffold. The SPA cell scaffold for islet encapsulation is ideal to maintain normoxic conditions and provide fast glucose-insulin kinetics to both human islets and stem cell-derived eBCs within a convection-based device, demonstrating its potential application in an endovascular bioartificial pancreas.

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Cross-flow Ultrafiltration through a Symmetric Microporous Membrane.Separation Science and Technology 1992;27:2121-2142.

Claims

1. A biocompatible gel matrix, comprising: A plurality of microchannels and a plurality of nanochannels, wherein the plurality of microchannels and the plurality of nanochannels are not patterned microchannels and nanochannels, A plurality of cells, wherein the cells are adjacent to the plurality of microchannels, and most of the plurality of cells are within a distance of 50 microns or less from at least one of the plurality of microchannels, The plurality of microchannels have a width of 5 to 500 microns, and the plurality of nanochannels have a width of 1 nm to 500 nm. A biocompatible gel matrix.

2. The gel matrix according to claim 1, wherein the gel matrix is composed of agarose.

3. The gel matrix according to claim 1, wherein the gel matrix is composed of collagen, alginate, cellulose, polyethylene glycol, polycaprolactone (PCL), gelatin, or dextran.

4. The gel matrix according to any one of claims 1 to 3, wherein the matrix is in the form of a planar scaffold, cylinder, sphere, or fiber.

5. The gel matrix according to claim 1, wherein the microchannels enable the flow of nutrients to the plurality of cells, and at least 80% of the plurality of cells encapsulated in the matrix are viable for at least one day.

6. The gel matrix according to claim 1, wherein the microchannels enable the flow of nutrients to the plurality of cells, and at least 80% of the plurality of cells encapsulated in the matrix are viable for up to one month.

7. The gel matrix according to claim 1, wherein the microchannels enable the flow of nutrients to the plurality of cells, and at least 80% of the plurality of cells encapsulated in the matrix are viable and functional for at least one day.

8. The gel matrix according to claim 1, wherein the microchannels enable the flow of nutrients to the plurality of cells, and at least 80% of the plurality of cells encapsulated in the matrix are viable and functional for up to one month.

9. The gel matrix according to claim 1, wherein the plurality of cells are insulin-producing cells.

10. The gel matrix according to claim 9, wherein the insulin-producing cells are derived from the differentiation of stem cells.

11. The gel matrix according to claim 9, wherein the insulin-producing cells are pancreatic cells isolated from islets of Langerhans.

12. The gel matrix according to claim 9, wherein the insulin-producing cells are within islets of Langerhans isolated from the pancreas, and the islets of Langerhans are encapsulated in the matrix.

13. The gel matrix according to claim 12, wherein each of the islets of Langerhans contains about 1000 cells.

14. The gel matrix according to claim 12 or 13, wherein each islet of Langerhans has a diameter of about 100 microns.

15. The gel matrix according to claim 9, wherein the insulin-producing cells are within a stem cell-derived enriched β-cluster (eBC).

16. The gel matrix according to claim 15, wherein each eBC contains about 1000 cells.

17. The gel matrix according to claim 15 or 16, wherein each eBC has a diameter of about 100 microns.

18. A bioartificial ultrafiltration device, comprising: a planar scaffold comprising the matrix according to claim 1; a first semipermeable ultrafiltration membrane disposed on a first surface of the planar scaffold; a first compartment adjacent to the first surface of the planar scaffold, in fluid communication with the planar scaffold via the first semipermeable ultrafiltration membrane, and including an inlet and an outlet; a second compartment adjacent to a second surface of the planar scaffold and including an outlet, wherein the first semipermeable ultrafiltration membrane includes a plurality of pores having a width in the range of 5 nm to 5 microns; the first semipermeable ultrafiltration membrane enables the transport of an ultrafiltered fluid from the first compartment to the matrix, and the ultrafiltered fluid traverses through the matrix to the second compartment, the bioartificial ultrafiltration device.

19. The device according to claim 18, further comprising a second semipermeable ultrafiltration membrane disposed on the second surface of the planar scaffold, wherein the ultrafiltered fluid traverses from the plurality of microchannels across the second semipermeable ultrafiltration membrane to the second compartment.

20. The device according to claim 19, wherein the second semipermeable ultrafiltration membrane comprises a plurality of pores having a width in the range of 5 nm to 5 microns.

21. The device according to claim 19 or 20, wherein the first and second semipermeable ultrafiltration membranes each comprise a plurality of pores having a width in the range of 0.1 micron to 2 microns.

22. The device according to claim 19, wherein the second semipermeable ultrafiltration membrane comprises a plurality of pores having a width greater than the width of the plurality of pores in the first semipermeable ultrafiltration membrane.

23. The device according to claim 18, wherein the inlet of the first compartment is attachable to a tube for connection to a blood vessel of a subject, and optionally, the blood vessel is an artery of the subject.

24. The device according to claim 18, wherein the outlet of the first compartment is attachable to a tube for connection to a blood vessel of a subject, and optionally, the blood vessel is a vein or an artery of the subject.

25. The device according to claim 18, wherein the outlet of the second compartment is (i) attachable to a tube for connection to a blood vessel of a subject, and optionally, the ultrafiltered fluid is provided to one or more blood vessels of the subject, (ii) one or more veins of the subject, (iii) one or more arteries of the subject, and / or (iv) an analyte analysis device.

26. The device according to claim 18, wherein the plurality of pores in the first semipermeable membrane have a width in the range of 0.2 μm to 0.5 μm, 20 nm to 2 microns, or 20 nm to 50 nm.

27. The device according to claim 19, wherein the plurality of pores in the second semipermeable membrane have a width in the range of 0.2 μm to 0.5 μm, 20 nm to 2 microns, or 20 nm to 50 nm.

28. The device according to claim 18, wherein the thickness of the first semipermeable ultrafiltration membrane is in the range of 0.1 micron to 100 microns, 0.5 μm to 10 μm.

29. The device according to claim 19, wherein the thickness of the second semipermeable ultrafiltration membrane is in the range of 0.1 micron to 100 microns or 0.5 μm to 10 μm.

30. The surface of the first and / or second surface of the planar scaffold is 1 cm ２ to 100 cm ２ or 15 cm ２ to 30 cm ２ within the range of, the device according to claim 18.

31. The surface area of the first semipermeable ultrafiltration membrane is 1 cm ２ to 1000 cm ２ or 15 cm ２ to 30 cm ２ The device according to claim 18, wherein the device is within the range of.

32. The surface area of the second semipermeable ultrafiltration membrane is 1 cm ２ to 1000 cm ２ or 15 cm ２ to 30 cm ２ within the range of, the device according to claim 19.

33. The device according to claim 18, wherein the plurality of pores are circular in shape and the width refers to the diameter of the pores.

34. The device according to claim 18, wherein the plurality of pores are in a slit shape.

35. The device according to claim 18, wherein the plurality of pores are in a slit shape and the width of the pores is 5 nm to 100 nm.

36. The device according to claim 18, wherein the plurality of pores are in a slit shape and the length of the pores ranges from 0.1 micron to 5 microns.

37. The device according to claim 18, wherein the plurality of pores are in a slit shape and the length of the pores is in the range of 1 μm to 3 μm.

38. The device according to claim 18, wherein the cells are autologous to the subject comprising the device.

39. The device according to claim 18, wherein the cells are heterologous to the subject comprising the device.

40. The device according to claim 18, wherein the cells are allogeneic to the subject comprising the device.

41. A bioartificial ultrafiltration device, a planar scaffold comprising the matrix according to claim 1, a first semipermeable ultrafiltration membrane according to claim 18 disposed on a first surface of the planar scaffold and a second semipermeable ultrafiltration membrane according to claim 19 disposed on a second surface, a first compartment including a first inlet and a first outlet, the first compartment being adjacent to the first surface of the planar scaffold, a second compartment including a second inlet and a second outlet, the second compartment being adjacent to the second surface of the planar scaffold, the first inlet being configured to connect to an artery of a subject, the first outlet being connected to the second inlet of the second compartment, the second outlet of the second compartment being configured to connect to a vein of the subject, the semipermeable ultrafiltration membrane including a plurality of pores having a width in the range of 5 nm to 5 microns, the first semipermeable ultrafiltration membrane enabling transport of an ultrafiltered fluid from the first compartment to the scaffold, and the second semipermeable ultrafiltration membrane enabling transport of the ultrafiltered fluid from the plurality of microchannels in the scaffold to the second compartment.

42. The device according to claim 41, wherein the cells are autologous to the subject.

43. The device according to claim 41, wherein the cells are heterologous to the subject.

44. The device according to claim 41, wherein the cells are allogeneic to the subject.

45. The device according to any one of claims 41 to 44, wherein the plurality of pores of the second semipermeable ultrafiltration membrane have a width greater than the width of the plurality of pores of the first semipermeable ultrafiltration membrane, or the plurality of pores of the second semipermeable ultrafiltration membrane have a width smaller than the width of the plurality of pores of the first semipermeable ultrafiltration membrane.

46. A method of providing a bioartificial ultrafiltration device containing cells to a subject in need thereof, the method comprising: connecting the bioartificial ultrafiltration device according to claim 18 to the subject, wherein the connecting comprises: connecting the inlet of the first compartment to an artery of the subject and connecting the outlet of the first compartment to a blood vessel of the subject; and connecting the outlet of the second compartment to a blood vessel or body cavity of the subject, or connecting the outlet of the second compartment to an analyte analysis device.

47. A method of providing a bioartificial ultrafiltration device containing cells to a subject in need thereof, the method comprising: connecting the bioartificial ultrafiltration device according to claim 41 to the subject, wherein the connecting comprises: connecting the first inlet to an artery of the subject; and connecting the second outlet to a vein of the subject.

48. The method according to claim 46, wherein the method comprises providing insulin to the subject, and the cells comprise insulin-producing cells.

49. Connecting the bioartificial device to the subject in need thereof results in an increase in the viability of the cells within the scaffold, according to the method of claim 46.

50. The method according to claim 46, wherein the ultrafiltered fluid contains one or more of glucose and oxygen.

51. The ultrafiltration liquid contains one or more of glucose and oxygen, the insulin-producing cells discharge insulin in response to the presence of glucose in the ultrafiltration liquid, and the plurality of microchannels transport the insulin to the second compartment. The method according to claim 46.

52. The method according to claim 51, wherein the discharged insulin is transported to the plurality of microchannels within the scaffold.

53. The method according to claim 46, wherein the semipermeable ultrafiltration membrane prevents the passage of immune system components into the scaffold.

54. The method according to claim 46, wherein the semipermeable ultrafiltration membrane prevents the passage of antibodies into the scaffold.

55. The method according to claim 46, wherein the semipermeable ultrafiltration membrane prevents the passage of cytokines into the scaffold.

56. The method according to claim 46, wherein the semipermeable ultrafiltration membrane prevents the passage of TNF-α, IFN-γ, and / or IL-1β into the scaffold.

57. A method of making a matrix comprising agarose, collagen, gelatin, polyethylene glycol, PCL, alginate, dextran, or cellulose, comprising a plurality of microchannels and a plurality of nanochannels, wherein the plurality of microchannels and the plurality of nanochannels are not patterned microchannels and nanochannels, the plurality of microchannels have a width of 5 to 500 microns, the plurality of nanochannels have a width of 1 nm to 500 nm, and the method comprises: Generating an aqueous solution containing dissolved agarose, gelatin, polyethylene glycol, PCL, collagen, alginate, dextran, or cellulose; Adding a water-immiscible reagent and a surfactant to the aqueous solution; Mixing the aqueous solution under conditions sufficient to produce an emulsion containing the dissolved agarose, gelatin, polyethylene glycol, PCL, collagen, alginate, dextran, or cellulose, the water-immiscible reagent, and the surfactant. A method comprising: generating the matrix by placing the emulsion at a temperature sufficient to enable gelation of agarose, gelatin, polyethylene glycol, PCL, collagen, alginate, dextran, or cellulose, thereby producing a matrix.

58. The method according to claim 57, further comprising adding cells to the emulsion prior to the step of generating the matrix.

59. The method according to claim 57 or 58, wherein generating the matrix comprises casting the emulsion into a mold comprising a plane, thereby producing a planar scaffold.

60. The method according to claim 59, wherein the method comprises placing a first semipermeable ultrafiltration membrane on a first surface of the planar scaffold.

61. The method according to claim 57, wherein the method comprises placing a second semipermeable ultrafiltration membrane on a second surface of the planar scaffold.

62. The method according to claim 57, wherein the agarose is low-gelling agarose.

63. The method according to claim 62, wherein the agarose is present in an aqueous solution at a concentration of 1% to 10% w / v, 2% to 10% w / v, 2% to 8% w / v, or 3% to 6% w / v.

64. The method according to claim 57, wherein dissolving the agarose comprises heating an aqueous solution to a temperature of about 37 °C and stirring the solution at about 300 revolutions per minute (RPM).

65. The method according to claim 57, wherein the water-immiscible reagent is perfluorodecalin (PFD).

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