Patent Application
20190216998
Type 1 diabetes (T1D) results from autoimmune destruction of the insulin-producing β-cells within the pancreatic islets of Langerhans. Islet transplantation by direct infusion of cadaveric islets into the portal vein of the recipient's liver offers a non-invasive cure for patients with T1D mellitus 1. However, donor availability, poor engraftment, and side effects from global immunosuppression remain as obstacles for wider application of this approach. Moreover, up to 60% of the infused islets become nonviable within a few days after surgical delivery and the long-term insulin independence is frequently lost by 5 years of transplantation. The activation of innate and the adaptive immune responses are among the main causes of islet graft failure. The idea of encapsulating islets has generated tremendous interest. However, there is a need for improved devices and methods for providing encapsulated islets that maintain function and are protected from the patient's immune system.
Bioartificial ultrafiltration devices for transplantation of cells in a subject are disclosed. These devices include a scaffold that encapsulates a population of cells while providing a plurality of channels adjacent the population of cells. In certain embodiments, a planar scaffold for facilitating exchange of molecules between a plurality of channels and cells adjacent the plurality of channels is disclosed. The planar scaffold may include a solid planar substrate comprising a first surface and a second surface; a void in the solid planar substrate, wherein the void extends from the first surface to the second surface; a matrix disposed in the void and extending from the first surface to the second surface, the matrix comprising: a plurality of channels extending from the first surface to the second surface, and a population of cells adjacent the plurality of channels.
In certain embodiments, the solid substrate has a thickness of 0.1 mm-10 mm, such as, 0.5 mm-5 mm, or 0.5 mm-3 mm and the first and second surfaces each have a surface area of 1 cm2-100 cm2. The matrix may have a surface area of 1 mm2-10,000 mm2, e.g., 1 mm2-5000 mm2, 1 mm2-1000 mm2, 1 mm2-100 mm2. In certain embodiments, the matrix comprises up to 25,000 channels. In certain embodiments, the channels comprise a width of 5 micron-1000 micron, e.g., a width of 10 micron-200 micron. In certain embodiments, the channels are circular and the width refers to diameter of the channels. In certain embodiments, the channels are rectangular. In certain embodiments, the length of the channels ranges from 100 micron to 1000 micron. In certain embodiments, a population of cells is separated from an adjacent channel by a distance of up to 500 microns or less, such as, 400 microns or less, or 300 microns or less. The population of cells may be insulin secreting cells. The population of cells may be pancreatic cells isolated from pancreatic islets, pancreatic islet cells, pancreatic beta cells, pancreatic islet cells, or cells in a pancreatic islet. Insulin secreting cells may be generated by differentiation of stem cells, such as, induced pluripotent stem cells (iPSCs).
The scaffold may include a semipermeable ultrafiltration membrane disposed on a first surface of the scaffold and covering the matrix on the first surface. In certain embodiments, a semipermeable ultrafiltration membrane may be disposed on a second surface of the scaffold and may be covering the matrix on the second surface. The semipermeable ultrafiltration membrane may be sized to immunoisolate the cells encapsulated in the matrix.
The planar scaffold may be used for culturing the population of cells, for example, for maintaining viability of the cells. The planar scaffold may be used for manufacturing a bioartificial ultrafiltration device as described herein.
In certain embodiments, the bioartificial ultrafiltration device may include a planar scaffold comprising a matrix comprising a population of cells and a plurality of channels adjacent to the population of cells, wherein the channels extend from a first surface to a second surface of the planar scaffold; a first semipermeable ultrafiltration membrane disposed on the first surface of the planar scaffold; a first compartment adjacent to the first surface of the planar scaffold and in fluidic communication with the planar scaffold via the first semipermeable ultrafiltration membrane and comprising an inlet and an outlet; a second compartment adjacent to the second surface of the planar scaffold and comprising an outlet, wherein the first semipermeable ultrafiltration membrane comprises a plurality of pores having a width in the range of 5 nm-5 micron, wherein the first semipermeable ultrafiltration membrane allows transport of ultrafiltrate from the first compartment to the plurality of channels and wherein the ultrafiltrate traverses from the plurality of channels into the second compartment.
In certain embodiments, the device may further include a second semipermeable ultrafiltration membrane disposed on the second surface of the planar scaffold and wherein the ultrafiltrate traverses from the plurality of channels across the second semipermeable ultrafiltration membrane into the second compartment. The second semipermeable ultrafiltration membrane may include a plurality of pores having a width in the range of 5 nm-5 micron. In certain embodiments, the first and second semipermeable ultrafiltration membranes may include a plurality of pores having a width in the range the range of 0.1 microns-2 microns.
In certain embodiments, the second semipermeable ultrafiltration membrane comprises a plurality of pores having a width larger than the width of the plurality of pores in the first semipermeable ultrafiltration membrane. In certain embodiments, the second semipermeable ultrafiltration membrane comprises a plurality of pores having a width smaller than the width of the plurality of pores in the first semipermeable ultrafiltration membrane.
In certain embodiments, the inlet of the first compartment is attachable to a conduit for connection to a blood vessel of a subject. In certain examples, this blood vessel may be an artery of the subject in whom the device is transplanted.
In certain embodiments, the outlet of the first compartment is attachable to a conduit for connection to a blood vessel of a subject. In certain examples, this blood vessel may be a vein or an artery of the subject. In certain cases, the outlet may be connect to an artery which may be the same artery as or different artery than the artery to which the inlet is connected.
In certain examples, the first compartment comprises a plurality of outlets that are each attachable to conduits for connection to (i) a plurality of different blood vessels of a subject or (ii) a plurality of connection sites on a single blood vessel.
In certain examples, the outlet of the second compartment is attachable to a conduit for connection to a blood vessel or a body cavity of a subject. In certain cases, the outlet of the second compartment provides the ultrafiltrate to one or more blood vessels or body cavity of the subject. In certain cases, the outlet of the second compartment is attachable to a tubing for connection to one or more veins of the subject. In other cases, the outlet of the second compartment is attachable to a tubing for connection to one or more arteries of the subject. In some cases, the outlet of the second compartment is attachable to an analyte analysis device. In yet other embodiments, the second compartment comprises a plurality of outlets for providing the ultrafiltrate to at least one blood vessel or a body cavity of the subject. In some cases, the second compartment comprises a plurality of outlets for providing the ultrafiltrate to an analyte analysis device.
In another embodiment, a bioartificial ultrafiltration device may include a first planar scaffold and a second planar scaffold each comprising a matrix comprising a population of cells and a plurality of channels adjacent the population of cells, wherein the channels extend from a first surface to a second surface of each of the planar scaffolds; a first semipermeable ultrafiltration membrane disposed on the first surface of the first and second planar scaffolds; a first compartment adjacent to and sandwiched between the first surface of the first and second planar scaffolds and comprising an inlet and an outlet, wherein the first semipermeable ultrafiltration membrane allows transport of ultrafiltrate from the first compartment to the scaffolds; a second compartment adjacent to the second surface of the first planar scaffold and comprising an outlet; a third compartment adjacent to the second surface of the second planar scaffold and comprising an outlet, wherein the first semipermeable ultrafiltration membrane comprises a plurality of pores having a width in the range of 5 nm-5 micron, and wherein the ultrafiltrate traverses from the plurality of channels in the scaffolds into the second compartment and the third compartment. In certain embodiments, the second semipermeable ultrafiltration membrane may include a plurality of pores having a width in the range of 5 nm-5 micron and wherein the second semipermeable ultrafiltration membrane is disposed on the second surface of the first and second planar scaffolds and wherein the ultrafiltrate traverses from the plurality of channels in the scaffolds into the second compartment and the third compartment via the second semipermeable ultrafiltration membrane.
In certain embodiments, the first semipermeable ultrafiltration membrane comprises a plurality of pores having a width in the range the range of 0.1 microns-2 microns. In certain embodiments, the second semipermeable ultrafiltration membrane comprises a plurality of pores having a width in the range the range of 0.1 microns-2 microns. In certain embodiments, the second semipermeable ultrafiltration membrane comprises a plurality of pores having a width larger than the width of the plurality of pores in the first semipermeable ultrafiltration membrane. In certain embodiments, the second semipermeable ultrafiltration membrane comprises a plurality of pores having a width smaller than the width of the plurality of pores in the first semipermeable ultrafiltration membrane.
In certain embodiments, the inlet of the first compartment is connectable to a conduit for connection to an artery of a subject. In certain embodiments, the outlet of the first compartment is connectable to a conduit for connection to an artery or a vein of a subject. In certain embodiments, the outlet of the second compartment is connectable to a conduit (e.g., a tubing) for connection to at least one blood vessel of a subject and/or to an analyte analysis device. In certain embodiments, the outlet of the third compartment is connectable to a tubing for connection to at least a blood vessel of a subject and/or to an analyte analysis device. In certain embodiments, the outlet of the third compartment is connectable to a tubing for connection to at least a vein of a subject and/or to an analyte analysis device. In certain embodiments, the outlet of the second compartment and the outlet of the third compartment are connected to a single tubing for connection to at least a blood vessel of a subject and/or to an analyte analysis device.
In certain embodiments, the plurality of pores in the first semipermeable membrane have a width in the range of 0.2 μm-0.5 μm and the plurality of pores in the second semipermeable membrane have a width in the range of 0.2 μm-0.5 μm. In certain embodiments, the thickness of the first semipermeable ultrafiltration membrane is in the range of 0.1 micron-1000 micron and the thickness of the second semipermeable ultrafiltration membrane is in the range of 0.1 micron-1000 micron.
In certain embodiments, the surface area of the first and second semipermeable ultrafiltration membrane is in the range of 1 cm2-100 cm2. In certain embodiments, the surface area of the first and second semipermeable ultrafiltration membrane is in the range 15 cm2-30 cm2.
As noted herein, the plurality of pores may be circular in shape and wherein the width refers to diameter of the pores. In some embodiments, the plurality of pores are slit-shaped and where the length of the pores is in the range of 0.1 micron-5 micron, e.g., 1 μm-3 μm.
The cells in the device may be insulin producing cells. For example, the insulin producing cells are derived from differentiation of stem cells or are pancreatic cells isolated from pancreatic islets. The cells may be autologous, allogenic, or xenogenic to the subject in whom the device will be transplanted.
A further embodiment of an bioartificial ultrafiltration device disclosed herein includes a planar scaffold comprising a matrix comprising a population of cells and a plurality of channels adjacent the population of cells, wherein the channels extend from a first surface to a second surface of the planar scaffold; a first semipermeable ultrafiltration membrane disposed on the first surface and a second semipermeable ultrafiltration membrane disposed on the second surface of the planar scaffold; a first compartment comprising a first inlet and a first outlet, wherein the first compartment is adjacent to the first surface of the planar scaffold; a second compartment comprising a second inlet and a second outlet, wherein the second compartment is adjacent to the second surface of the planar scaffold, wherein the first inlet is configured for connection to an artery of a subject and the first outlet is connected to the second inlet of the second compartment, wherein the second outlet of the second compartment is configured for connection to a vein of the subject, wherein the semipermeable ultrafiltration membranes comprise a plurality of pores having a width in the range of 5 nm-5 micron, wherein the first semipermeable ultrafiltration membrane allows transport of ultrafiltrate from the first compartment to the scaffold and the second semipermeable ultrafiltration membrane allows transport of the ultrafiltrate from the plurality of channels in the scaffold into the second compartment. The cells present in the device may insulin producing cells as described herein. In certain cases, the plurality of pores have a width in the range of 0.2 μm-0.5 μm.
In some embodiments, the plurality of pores in the second semipermeable ultrafiltration membrane have a width larger than the width of the plurality of pores in the first semipermeable ultrafiltration membrane. In some embodiments, the plurality of pores in the second semipermeable ultrafiltration membrane have a width smaller than the width of the plurality of pores in the first semipermeable ultrafiltration membrane.
In some embodiments, the thickness of the semipermeable ultrafiltration membranes is in the range of 0.1 micron-1000 micron, e.g., 200 μm-1000 μm. The surface area of the semipermeable ultrafiltration membrane is in the range of 1 cm2-100 cm2, e.g., 15 cm2-30 cm2.
As noted herein, the plurality of pores may be circular in shape and wherein the width refers to diameter of the pores.
In some embodiments, the plurality of pores are slit-shaped and wherein length of the pores is in the range of 1 micron-5 micron, e.g., 1 μm-3 μm.
Also disclosed herein are methods for transplanting the devices provided in the subject application into a patient. In certain cases, a method for providing a bioartificial ultrafiltration device comprising cells to a subject in need thereof includes connecting the bioartificial ultrafiltration device comprising a planar scaffold comprising a matrix comprising a population of cells and a plurality of channels adjacent to the population of cells, wherein the channels extend from a first surface to a second surface of the planar scaffold; a first semipermeable ultrafiltration membrane disposed on the first surface of the planar scaffold; a first compartment adjacent to the first surface of the planar scaffold and in fluidic communication with the planar scaffold via the first semipermeable ultrafiltration membrane and comprising an inlet and an outlet; a second compartment adjacent to the second surface of the planar scaffold and comprising an outlet, wherein the first semipermeable ultrafiltration membrane comprises a plurality of pores having a width in the range of 5 nm-5 micron, wherein the first semipermeable ultrafiltration membrane allows transport of ultrafiltrate from the first compartment to the plurality of channels and wherein the ultrafiltrate traverses from the plurality of channels into the second compartment, 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 a body cavity of the subject; or connecting the outlet of the second compartment to an analyte analysis device.
In certain embodiments, a method for providing a bioartificial ultrafiltration device comprising cells to a subject in need thereof includes connecting the bioartificial ultrafiltration device comprising a first planar scaffold and a second planar scaffold each comprising a matrix comprising a population of cells and a plurality of channels adjacent the population of cells, wherein the channels extend from a first surface to a second surface of each of the planar scaffolds; a first semipermeable ultrafiltration membrane disposed on the first surface of the first and second planar scaffolds; a first compartment adjacent to and sandwiched between the first surface of the first and second planar scaffolds and comprising an inlet and an outlet, wherein the first semipermeable ultrafiltration membrane allows transport of ultrafiltrate from the first compartment to the scaffolds; a second compartment adjacent to the second surface of the first planar scaffold and comprising an outlet; a third compartment adjacent to the second surface of the second planar scaffold and comprising an outlet, wherein the first semipermeable ultrafiltration membrane comprises a plurality of pores having a width in the range of 5 nm-5 micron, and wherein the ultrafiltrate traverses from the plurality of channels in the scaffolds into the second compartment and the third compartment, 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 outlets of the second and third compartments to a blood vessel or body cavity of the subject; or connecting the outlets of the second and third compartments to an analyte analysis device; or connecting the outlet of the second compartment to a blood vessel or a body cavity of the subject and connecting the outlet of the third compartment to an analyte analysis device; or connecting the outlet of the second compartment to an analyte analysis device and connecting the outlet of the third compartment to a second vein of the subject.
In another embodiment, a method for providing a bioartificial ultrafiltration device comprising cells to a subject in need thereof includes connecting the bioartificial ultrafiltration device comprising a planar scaffold comprising a matrix comprising a population of cells and a plurality of channels adjacent the population of cells, wherein the channels extend from a first surface to a second surface of the planar scaffold; a first semipermeable ultrafiltration membrane disposed on the first surface and a second semipermeable ultrafiltration membrane disposed on the second surface of the planar scaffold; a first compartment comprising a first inlet and a first outlet, wherein the first compartment is adjacent to the first surface of the planar scaffold; a second compartment comprising a second inlet and a second outlet, wherein the second compartment is adjacent to the second surface of the planar scaffold, wherein the first inlet is configured for connection to an artery of a subject and the first outlet is connected to the second inlet of the second compartment, wherein the second outlet of the second compartment is configured for connection to a vein of the subject, wherein the semipermeable ultrafiltration membranes comprise a plurality of pores having a width in the range of 5 nm-5 micron, wherein the first semipermeable ultrafiltration membrane allows transport of ultrafiltrate from the first compartment to the scaffold and the second semipermeable ultrafiltration membrane allows transport of the ultrafiltrate from the plurality of channels in the scaffold into the second compartment, to the subject, wherein the connecting comprises connecting the first inlet to an artery of a subject; and connecting the second outlet to a vein of the subject.
In some embodiments, the method comprises providing insulin to the subject and wherein the cells comprise insulin producing cells. In some cases, the insulin producing cells are derived from differentiation of stem cells or are pancreatic cells such as, pancreatic beta cells isolated from pancreatic islets. In some examples, the cells are islet cells, for example, the cells may be present in islets isolated from pancreas. The cells may be autologous, allogenic, or xenogenic to the subject. In certain embodiments, connecting the bioartificial device to the subject in need thereof results in increased viability of the cells in the scaffold. In certain embodiments, the ultrafiltrate comprises one or more of glucose and oxygen. In certain embodiments, the ultrafiltrate comprises one or more of glucose and oxygen and wherein the insulin producing cells excrete insulin in response to presence of glucose in the ultrafiltrate and wherein the plurality of channels transport the insulin to the second compartment and/or to the third compartment. In certain embodiments, the excreted insulin is transported to the plurality of channels in the scaffold.
In certain embodiments, the semipermeable ultrafiltration membranes reduce or prevent the passage of immune system components into the scaffold, e.g., passage of antibodies into the scaffold, passage of cytokines (e.g., TNF-α, IFN-γ, and/or IL-1β) into the scaffold.
SNM-encapsulated islets under convective conditions.
Unless defined otherwise, 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 now described.
As used herein, the term “filtration” refers to a process of separating particulate matter from a fluid, such as air or a liquid, by passing the fluid carrier through a medium (e.g., a semipermeable membrane) that will not pass the particulates.
As used herein, the term “ultrafiltration” refers to subjecting a fluid to filtration, where the filtered material is very small; typically, the fluid comprises colloidal, dissolved solutes or very fine solid materials, and the filter is a microporous or nanoporous. The filter may be a membrane, such as, a semi-permeable membrane. The fluid to be 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 “ultra- filtrate,” which has been filtered through the filter, and a “retentate,” which is that part of the feed fluid which did not get filtered through the membrane.
As used herein the terms “subject” or “patient” refers to a mammal, such as, a primate (e.g., humans or non-human primates), a bovine, an equine, a porcine, a canine, a feline, or a rodent. In certain embodiments, the subject or patient may be a human. In certain embodiments, the subject or patient may be pre-diabetic or may have diabetes, such as, type 1 diabetes (T1D) or type 2 diabetes. The terms “subject” and “patient” are used interchangeably herein.
As used herein, the terms “treat,” “treatment,” “treating,” and the like, refer to obtaining a desired pharmacologic and/or physiologic effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of a partial or complete cure for a disease and/or adverse effect attributable to the disease. “Treatment,” as used herein, covers any treatment of a disease in a subject, particularly in a human, and includes: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, e.g., causing regression of the disease, e.g., to completely or partially remove symptoms of the disease.
As used herein, the terms “layer”, “film”, or “membrane” and plurals thereof as used in the context of a device of the present disclosure refer to an individual layer of the device that may be formed from a silicon membrane, silicon nitride, silica, atomically thin membrane such as graphene, silicon, silicene, molybdenum disulfide (MoS2), etc., or a combination thereof or a polymer. The “layer”, “film”, or “membrane” used to manufacture a porous layer of the present disclosure is typically porous and can be nanoporous or microporous. The phrases “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 created. A nanoporous layer may include a frame for supporting the layer. The phrases “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 created. A microporous layer may include a frame for supporting the layer.
As used herein, the term “encapsulated” as used in the context of cells disposed in a matrix of a scaffold as described herein. The scaffold may be included into the devices provided herein. The cells may be encapsulated in a matrix that includes a plurality of channels adjacent the encapsulated cells. The cells may be encapsulated in a matrix that includes a biocompatible polymerizable polymer.
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.
Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also 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.
Unless defined otherwise, 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 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 now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited.
It must 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, reference to “channels” includes a plurality of such channels and reference to “the agarose-cell region” includes reference to one or more agarose-cell regions and equivalents thereof known to those skilled in the art, and so forth. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation.
It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of the embodiments pertaining to the invention are specifically embraced by the present invention and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all sub-combinations of the various embodiments and elements thereof are also specifically embraced by the present invention and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein.
The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.
As summarized above, bioartificial ultrafiltration devices comprising a population of cells is disclosed. Also described herein are methods for making the devices and methods of using the devices. The bioartificial devices provided herein may be used for providing an ultrafiltrate produced by filtration of blood across a semipermeable porous membrane. The ultrafiltrate may be provided to the population of cells and may provide nutrients, such as, oxygen and glucose to the cells. The ultrafiltrate may take up molecules secreted by the cells such as insulin and other metabolites and the ultrafiltrate may be returned to the blood. The ultrafiltrate may be enclosed in channels which are surrounded by the cells thereby increasing the rate of exchange of molecules. The devices of the present disclosure can have a variety of configurations and uses. The following sections provide a detailed description of various embodiments and uses of the device disclosed herein.
Provided herein are scaffolds that support a population of cells encapsulated in a matrix. In certain embodiments, the planar scaffold is three-dimensional and includes a first surface opposite a second surface. The planar scaffold may be include a sheet of a solid substrate in which a void has been created which void contains a matrix that supports the population of cells and a plurality of channels.
The solid substrate may be composed of any suitable material, such as, a polymer, such as a biocompatible polymer. Non-limiting examples of materials for the solid substrate region of the scaffold can be found in U.S. Pat. No. 9,132,210, which is herein incorporated by reference in its entirety. Non-limiting examples of materials for the solid substrate region of the scaffold: polylactic acid, polyglycolic acid, PLGA polymers, polyesters, poly(allylamines)(PAM), poly(acrylates), modified styrene polymers, pluronic polyols, polyoxamers, poly(uronic acids), poly(vinylpyrrolidone), alginate, polyethylene glycol, fibrin, and poly (methyl methacrylate) and copolymers or graft copolymers of any of the above. In certain embodiments, the solid substrate of the scaffold is composed of acrylate. In certain embodiments, the acrylate is in the form of an acrylate sheet.
In certain embodiments, the solid substrate may be laser-cut to create a void. In certain embodiments, a sheet of the solid substrate is laser-cut to create a void region. In certain embodiments, a sheet of the solid substrate is laser-cut to create two or more void regions which may be cut into individual pieces. The void or cut-out may have any shape, such as, square, or rectangular with edges that are substantially straight or undulating, or circular with substantially smooth periphery. The void may be of a size sufficient to contain a plurality of channels and a population of cells adjacent the plurality of channels. The size of the void may be proportional to the size of the device. In certain cases, the solid surface may be have a thickness of 0.1 mm-10 mm, such as, 0.5 mm-5 mm, or 0.5 mm-3 mm and may be in shape of a cube or a cuboid and the surface area of the first surface and the second surface may 1 cm2-200 cm2, e.g., 1 cm2-100 cm2, 1 cm2-50 cm2, 1 cm2-25 cm2, or 5 cm2-50 cm2. The matrix may have a surface area of 1 mm2-10,000 mm2, e.g., 1 mm2-5000 mm2, 1 mm2-1000 mm2, 1 mm2-100 mm2. In certain cases the void may have a surface area of 100 cm2 or less such as, 15 cm2 or less, 10 cm2 or less, 5 cm2 or less, 1 cm2 or less, 0.5 cm2 or less, for example, 20 cm2-0.5 cm2, 15 cm2-0.3 cm2, 10 cm2-0.5 cm2, 5 cm2-0.5 cm2, or 1 cm2-0.5 cm2. The depth of the void and the matrix disposed therein is determined by the thickness of the solid substrate used to form the scaffold and may be in the range of 200 micron to 1000 micron, such as, 200 micron-900 micron, 200 micron-800 micron, 300 micron-700 micron, 200 micron-700 micron, or 300 micron-600 micron. In certain embodiments, the void may have a dimension of about 1-5 mm (length)×1-5 mm (width)×0.5-1 mm (depth), e.g., 1 mm×3 mm×1 mm, 2 mm×3 mm×1 mm, 3 mm×3 mm×1 mm, or 4 mm×4 mm×1 mm.
In certain embodiments, the planar scaffold comprises a matrix. In certain embodiments, the matrix comprises a plurality of channels and a population of cells. In certain embodiments, the matrix is formed by disposing a plurality of elongate posts into the void where the elongate posts are oriented in a direction perpendicular to the first and second surfaces of the solid substrate in which the void was created. In certain embodiments, the elongate posts may be tubes or wires, such as, polytetrafluorethlene (PFTE) coated wires. In certain embodiments, the elongate posts may be substantially cylindrical in shape. The diameter of the elongate posts may range from 25 micron-500 micron, such as, 50 micron-500 micron, 50 micron-300 micron, 50 micron-200 micron, 75 micron-200 micron, 75 micron-150 micron, e.g., 100 μm in diameter. In other cases, the elongate posts may have a rectangular shape or an irregular shape. The matrix may be formed from a polymerizable biocompatible polymer that support viability of the encapsulated cells. For example, the matrix may enable transport of molecules (e.g., glucose, oxygen, insulin) to and from the cells. In certain embodiments, the matrix comprises a polylactic acid, polyglycolic acid, polyethylene glycol (PEG), poly(lactic-co-glycolic acid) (PLGA) polymer, alginate, alginate derivative, gelatin, collagen, fibrin, agarose, hyaluronic acid, hydrogel, matrigel, natural polysaccharide, synthetic polysaccharide, polyamino acid, polyester, polyanhydride, polyphosphazine, poly(vinyl alcohol), poly(alkylene oxide), modified styrene polymer, pluronic polyol, polyoxamer, poly(uronic acid), or poly(vinylpyrrolidone) polymer. In certain embodiments, the matrix is includes an agarose polymer, an extracellular matrix, hydrogel, matrigel, or a mixture thereof. In certain embodiments, the matrix further includes a population of cells. In certain embodiments, the matrix is formed by disposing a composition comprising an unpolymerized polymer and a population of cells as disclosed herein into the void region of the solid substrate of the scaffold, which void regions include the elongate posts. The composition is then polymerized and the elongate posts removed to provide a matrix that includes the population of cells and a plurality of channels created by removal of the wires. In certain embodiments, the plurality channels are cylindrical shaped channels. In certain embodiments, the channels are rectangular, cylindrical, or square shaped. In certain embodiments, the channels are at least 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm, 110 μm, 120 μm, 130 μm, 140 μm, or 150 μm in diameter. In certain embodiments, the channels 30-200 μm or 50-150 μm in diameter. In certain embodiments, the channels extend from a first surface to a second surface of the planar scaffold. The matrix may include two or more channels, such as, 3-25,000, 5-10,000, 10-10,000, 30-10,000, 50-10,000, 100-10,000, 1000-10,000, 100-3000, 3-100, 3-70, 3-50, 3-25, 3-20, 5-100, 6-75, 7-50, or 8-20 channels where the channels are adjacent a population of cells. The matrix is configured such that a channel is separated by a distance less than 500 μm from a cell in order to facilitate efficient exchange of molecules between the ultrafiltrate in the channel and the cell.
In certain embodiments, the scaffold includes a plurality of channels where at least one of the channels is surrounded by a hexagonal arrangement of the cells. In certain embodiments, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen, at least fifteen, at least sixteen, at least seventeen, at least eighteen, at least nineteen, or at least twenty channels are surrounded by a hexagonal arrangement of the cells. In certain embodiments, the cells are adjacent at least one of the plurality of channels such that the cells are separated from the channel by a distance less than 500 micron, such as, 400 micron, 300 micron, or 200 micron. The presence of cells adjacent to the channels facilitates diffusion of molecules 500 micron from at least one channel. In certain embodiments, the matrix includes a configuration of a channel surrounded by a hexagonal cluster of cells. In certain embodiments, the scaffold may include a cell density of at least 5% by volume, 10% (5,700 cell equivalents/cm2), 20% (11,400 cell equivalents/cm2) or more. The number of cells in the matrix of the bioartificial device may vary and may be determined empirically. In some cases, the scaffold may include at least 103 cells, 105 cells, 106 cells, 1010 cells, such as 103-1010 cells, 105-108 cells, 103-106 cells, or 105-106 cells. In certain embodiments, the matrix includes a plurality of channels surrounded by a population of cells, where the diameter of the channel-cell region is 100 μm, 200 μm, 300 μm, 400 μm, 500 μm, 600 μm, 700 μm, 800 μm, 900 μm, or 1000 μm, e.g., in the range of 800 μm-1000 μm in diameter. In certain embodiments, the scaffold includes eight 800 μm channel-cell regions with eight 100 μm diameter cylindrical channels. The scaffold thus includes a matrix in which the cells are encapsulated following polymerization of the composition containing the mixture of unpolymerized polymer and cells.
In certain cases, the matrix supported in a planar scaffold may be configured as depicted in
In certain cases, the matrix of insulin secreting cells (referred to as islet cells) may be configured according to the arrangement depicted in
Populations of cells that can be included in the 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), blood vessel cells, precursor cells derived from adipose tissue, bone marrow derived progenitor cells, intestinal cells, islets, Sertoli cells, beta cells, progenitors of islets, progenitors of beta cells, peripheral blood progenitor cells, stem cells isolated from adult tissue, retinal progenitor cells, cardiac progenitor cells, osteoprogenitor cells, neuronal progenitor cells, and genetically transformed cells, or a combination thereof. The population of cells may be from the subject (autologous cells), from another donor (allogeneic cells) or from other species (xenogeneic cells).The cells can be introduced into the scaffold and the scaffold may be immediately (within a day) implanted into a subject or the cells may cultured for longer period, e.g. greater than one day, to allow for cell proliferation prior to implantation.
In certain embodiments, the populations 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 islets of pancreas. In certain embodiments, the population of cells in the matrix are islets isolated from pancreas. In certain embodiments, the population of cells in the matrix may be in the form of a piece of tissue, such as, islet of Langerhans, which may have been isolated from the subject receiving the device or from another subject.
In certain embodiments, the devices disclosed herein may be used to treat a person having diabetes, such as, type 1 diabetes. The device may include pancreatic islet cells or may include stem cells that are capable of differentiating into insulin producing pancreatic cells. In certain embodiments, pluripotent stem cells (PSCs) may be differentiated into insulin producing pancreatic cells inside the device and then the bioartificial device containing the differentiated insulin producing pancreatic cells is placed in the subject (e.g., in the omentum, adjacent to pancreas or liver, adjacent to kidney, lung, or heart, or subdermally, e.g., in arm or abdomen). In some case, the device may include PSCs and the device may be implanted adjacent the pancreas or liver of the subj ect.
In certain embodiments, the bioartificial device may include a planar scaffold described herein, e.g., a planar scaffold that includes a matrix comprising a population of cells and a plurality of channels adjacent to the population of cells, wherein the channels extend from a first surface to a second surface of the planar scaffold; a first semipermeable ultrafiltration membrane disposed on the first surface of the planar scaffold; a first compartment adjacent to the first surface of the planar scaffold and in fluidic communication with the planar scaffold only via the first semipermeable ultrafiltration membrane and comprising an inlet and an outlet; a second compartment adjacent to the second surface of the planar scaffold and comprising an outlet, wherein the first semipermeable ultrafiltration membrane comprises a plurality of pores having a width in the range of 5 nm-5 micron, wherein the first semipermeable ultrafiltration membrane allows transport of ultrafiltrate from the first compartment to the plurality of channels and wherein the ultrafiltrate traverses from the plurality of channels into the second compartment.
In certain embodiments, the bioartificial device may include two planar scaffolds which sandwich a compartment containing arterial blood. For example, the device may include a first planar scaffold and a second planar scaffold each comprising a matrix comprising a population of cells and a plurality of channels adjacent the population of cells, wherein the channels extend from a first surface to a second surface of each of the planar scaffolds; a first semipermeable ultrafiltration membrane disposed on the first surface of the first and second planar scaffolds; a first compartment adjacent to and sandwiched between the first surface of the first and second planar scaffolds and comprising an inlet and an outlet, wherein the first semipermeable ultrafiltration membrane allows transport of ultrafiltrate from the first compartment to the scaffolds; a second compartment adjacent to the second surface of the first planar scaffold and comprising an outlet; a third compartment adjacent to the second surface of the second planar scaffold and comprising an outlet, wherein the first semipermeable ultrafiltration membrane comprises a plurality of pores having a width in the range of 5 nm-5 micron, and wherein the ultrafiltrate traverses from the plurality of channels in the scaffolds into the second compartment and the third compartment..
Aspects of the present disclosure include a bioartificial device that includes a planar scaffold comprising a matrix comprising a population of cells and a plurality of channels adjacent the population of cells, where the channels extend from a first surface to a second surface of the planar scaffold; a first semipermeable ultrafiltration membrane disposed on the first surface and a second semipermeable ultrafiltration membrane disposed on the second surface of the planar scaffold; a first compartment comprising a first inlet and a first outlet, wherein the first compartment is adjacent to the first surface of the planar scaffold; a second compartment comprising a second inlet and a second outlet, wherein the second compartment is adjacent to the second surface of the planar scaffold, wherein the first inlet is configured for connection to an artery of a subject and the first outlet is connected to the second inlet of the second compartment, where the second outlet of the second compartment is configured for connection to a vein of the subject, where the semipermeable ultrafiltration membranes comprise a plurality of pores having a width in the range of 5 nm-5 micron, where the semipermeable ultrafiltration membrane allows transport of ultrafiltrate filtered from the arterial blood in the first compartment to the scaffold and transport of the ultrafiltrate from the plurality of channels in the scaffold into the second compartment. In certain cases, the first outlet may include a means for reducing the rate of flow of blood to the second compartment. In some case, the means may include a pressure reduction manifold. In some cases, the means for reducing the rate of flow of blood to the second compartment may include a pressure reduction channel. In some cases, the rate of flow of blood through the second compartment may be controlled by directing the blood through a channel having a reduced width compared to the width of the first compartment. In some case, the rate of flow of blood through the second compartment may be controlled by sizing the second compartment to have a reduced width compared to the width of the first compartment.
In some cases, the first compartment into which the blood is introduced into the device may have a dimension suitable for facilitating ultrafiltration of the blood. For example, the first compartment may have a height of 100 micron-6 mm, e.g., 500 micron- 4 mm, 1 mm-3 mm, or 2 mm-3 mm.
In certain embodiments, the bioartificial device is dimensioned to fit in a body cavity of a subject. The device may be rectangular or cylindrical in shape. In certain case, the device may have a surface area of 50 cm2 or less, such as 10-30 cm2, 10-25 cm2, 15-25 cm2, 20-25 cm2, 15-30 cm2. In certain cases, the device may be rectangular and have a length of 3 cm-10 cm, a width of 1 cm-6 cm, and a height of 0.3 cm-2 cm, such as dimension (length x width x height) of 3 cm x 1 cm×0.5 cm to 6 cm×4 cm×1 cm, e.g., 3 cm×1 cm×0.5 cm, 5 cm×2 cm×1 cm, or 6 cm×4 cm×1 cm.
As noted herein, the devices disclosed herein may maintain the transplanted cells in a functional and viable state for at least 1 month and up to a period of 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 longer, such as, 1 month-50 years, 1 year-25 years, 5 years-50 years, 5 years-25 years, 10 years-50 years, or 15 years-25 years.
In certain embodiments, the devices disclosed herein may be enclosed in a housing made from an inert material that does not degrade or foul when placed in a subject. Any material approved for medical devices placed in a subject may be utilized including but not limited to medical grade plastic, inert metals, such as, titatnium, stainless steel, etc.
In certain embodiments, the bioartificial device comprises more than one semipermeable ultrafiltration membrane. In certain embodiments, the semipermeable ultrafiltration membrane is disposed on the first surface and the second surface of the planar scaffold. The semipermeable ultrafiltration membrane disposed on a first surface of the scaffold may be the same as the semipermeable ultrafiltration membrane disposed on the second surface of the scaffold or may be different. For example, the semipermeable ultrafiltration membrane adjacent to a compartment containing arterial blood may have smaller pores than the semipermeable ultrafiltration membrane adjacent a compartment containing the ultrafiltrate flowing through the channels in the matrix. In some cases, the semipermeable ultrafiltration membrane adjacent to a compartment containing arterial blood may have larger pores than the semipermeable ultrafiltration membrane adjacent a compartment containing the ultrafiltrate flowing through the channels in the matrix. In certain embodiments, the semipermeable membrane allows for filtration of an ultrafiltrate from the compartment containing arterial blood which ultrafiltrate is transported into the plurality of channels in the scaffold. The plurality of channels are adjacent the cells which provides for efficient exchange of molecules in the ultrafiltrate in the channels with the molecules released by the cells. These molecules diffuse in a concentration dependent manner between the lumen of the channels 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 to the cells and molecules secreted by the cells such as urea, carbon dioxide, insulin are transported into the lumen of the channels. It is understood that in some embodiments, the some diffusion and exchange of molecules in the ultrafiltrate may occur outside of the channels, such as, with the ultrafiltrate that does not enter the channels and permeates through the matrix.
In certain embodiments, the semipermeable ultrafiltration membrane comprises a plurality of pores having a width in the range of 5 nm-5 micron. In some embodiments, the present disclosure provides a membrane comprising fabricated pores of defined dimensions and structure, and density. In certain embodiments, one or more surface of the membrane may be treated to limit protein adsorption. Such a treatment may include treatments that alter or confer surface charge, surface free energy, or treatments that promote adhesion of specific cell types. In certain embodiments, at least one pore of the membrane comprises any combination of a surface treatment. Surface treatments function to effect restriction of size and electrostatic charge of solutes that may be passed through such pores. Examples of surface treatments can be found, for example, in U.S. Patent Application Publication No. 20090131858, which is hereby incorporated by reference in its entirety.
In certain embodiments, the semipermeable ultrafiltration membrane is configured for filtration of biological fluids. In certain embodiments, the membrane comprises a plurality of nanopores, where the shapes and sizes 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 have a width in the range of 0.1 μm-5 μm, e.g., 0.1 μm-3 μm, 0.1 μm-0.5 μm, 0.5 μm-1 μm, 1 μm-1.5 μm, 1.5 μm-2 μm, 0.1 μm-1 μm, 0.1 μm-0.8 μm, 0.2 μm-0.7 μm, 0.2 μm-0.6 μm, 0.2 μm-0.5 μm. In certain embodiments, the plurality of pores may be nanopores and may have a width of 1 nm-500 nm, e.g., 1 nm-90 nm, 2 nm-50 nm, 3 nm-40 nm, 4 nm-50 nm, 4 nm-40 nm, 5 nm-50 nm, 5 nm-20 nm, 4 nm-20 nm, 7 nm-100 nm, 12 nm-20 nm, or 5 nm-10 nm. In certain embodiments, the plurality of pores are slit shaped and have a width as listed herein and have a length in the range of 1 μm-10 μm, e.g., 2 μm-3 μm, 3 μm-4 μm, 4 μm-5 μm, 5 μm-6 μm, 6 μm-7 μm, 7 μm-8 μm, 8 μm-9 μm, or 9 μm-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 micron, e.g., a width×length×depth of 5 nm-50 nm×1 micron-2 micron×200 nm-500 nm.
In certain embodiments, the devices of the present disclosure include semipermeable ultrafiltration membranes having a dimension (length×width) of 6 mm×6 mm, 5 mm×5 mm, 7 mm×7 mm, 8 mm×8 mm, 9 mm×9 mm, 10 mm×10 mm, 10 cm x 10 cm, e.g., 10 mm×10 mm to 10 cm×10 cm. In some embodiments, the semipermeable ultrafiltration membrane may be rectangular.
In certain embodiments, the semipermeable ultrafiltration membrane has a surface area in the range of 0.5-100 cm2, e.g., 30-100 cm2, 10-30 cm2, 15-30 cm2, 15-20 cm2, 20-25 cm2, 25-30 cm2, 0.5-10 cm2, 0.75-5 cm2, 0.75-3 cm2, or 0.75-2 cm2.
In certain embodiments, the devices disclosed herein may be substantially planar and may be dimensioned to have a surface area ranging from 20-100 cm2 (on each planar side) and a thickness of 1 cm-3 cm. In certain embodiments, the devices disclosed herein may have a volume of up to 500 cm3, such as, 50-500 cm3, 100-500 cm3, 100-300 cm3, 100-150 cm3. In certain cases, the device may include a semipermeable membrane having a surface of 5-75 cm2, e.g., 5-50 cm2, 10-30 cm2, or 15-30 cm2. The size of pores in the membrane may be 10 nm-100 nm in width, such as, 10 nm-20 nm.
The semipermeable ultrafiltration membranes of the present disclosure include any membrane material suitable for use in filtering biological fluids, wherein the membranes are structurally capable of supporting formation of pores. Examples of suitable membrane materials are known in the art and are described herein.
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, silicon, which is biocompatible, coated silicon materials, polysilicon, silicon carbide, ultrananocrystalline diamond, diamond-like carbond (DLC), silicon dioxide, PMMA, SU-8, and PTFE. Other possible materials include metals (for example, titanium), ceramics (for example, silica or silicon nitride), and polymers (such as polytetrafluorethylene, polymethylmethacrylate, polystyrenes and silicones). Materials for membranes can be found in, for example U.S. Patent Application Publication No. 20090131858, which is hereby incorporated by reference in its entirety.
A semipermeable ultrafiltration membrane of the present disclosure comprises a plurality of pores, where pore shapes include linear, square, rectangular (slit-shaped), circular, ovoid, elliptical, or other shapes. As used herein, width of a pore refers to the diameter where the pore is circular, ovoid or elliptical. In certain embodiments, the membrane comprises pores comprising a single shape or any combination of shapes. In certain embodiments, the sizes of pores are highly uniform. In certain embodiments, the pores are micromachined such that there is less than 20% size variability, less than 10% size variability, or less than 5% size variability between the dimensions of the slit-shaped pores. In certain embodiments, factors that determine appropriate pore size and shape include a balance between hydraulic permeability and solute permselectivity. In certain embodiments, the plurality of pores are slit-shaped pores which provide for optimum flux efficiency enabling efficient transport of molecules across the membrane. In certain embodiments, the membrane comprises slit-shaped nanopores. In certain embodiments, the semipermeable ultrafiltration membrane has approximately 103-108 rectangular slit-shaped nanopores (e.g., 104-108, or 105-107) for example on a membrane surface area of 1 cm2, 0.5 cm2, or 0.4 cm2. In certain embodiments, the number of slit-shaped nanopores on the semipermeable ultrafiltration membrane is sufficient to allow the membrane to generate physiologically sufficient ultrafiltration volume at capillary perfusion pressure. In certain embodiments, the porosity of the semipermeable ultrafiltration membrane is approximately 1% -50%, e.g., 10%-50%, 20%-50%, or 20%-75%, etc.).
In certain embodiments, the present disclosure provides a series of membranes comprising sparse arrays of monodisperse slit-shaped pores, manufactured, for example, using silicon bulk and surface micromachining techniques (see e.g., Fissell W H, etal., JAm Soc Nephro12002; 13:602 A; incorporated herein by reference in its entirety). In certain embodiments, the semipermeable ultrafiltration membrane is prototyped using microelectromechanical systems (MEMS) technology. In certain embodiments, the process uses the growth of a thin SiO2 (oxide) layer on 400 μm-thick double side polished (DSP) silicon wafers followed by a low pressure chemical vapor deposition (LPCVD) of polysilicon (˜500 nm). In certain embodiments the wafers are then specifically patterned, dry oxidized, wet etched, deposited with a second polysilicon layer, and finally blanket-etched until 400 nm of polysilicon remains and the underlying vertical oxide layer is exposed. In certain embodiments, the vertical sacrificial oxide layer defines the critical nanoscale pore size of the membranes. In certain embodiments, the low temperature oxide (LTO) (˜1 μm) is deposited onto polysilicon of the wafers to serve as the hard mask for membrane protection. In certain embodiments, deep reactive ion etching (DRIE) removes the backside of each window until membranes were disclosed. In certain embodiments, the sacrificial oxide is etched away in 49% hydrofluoric acid (HF) during the final step of the fabrication process to leave behind open nanoscale slit pores. In certain embodiments, the wafers are subsequently cut into 1 cm×1 cm chips with an effective area of 6 mm2×6 mm2 containing 1500 windows each, with a total of 106 pores per membrane. In certain embodiments, each rectangular pore is 7 nm in width, 300 nm in depth, and 2 μm in length. In certain embodiments, silicon micropore membrane (SμM) is fabricated to produce wafer-scale arrays of 500 nm deep by 4 μm long rectangular slit pores with 1000 nm-wide slit width using similar process. In certain embodiments, the wafers are diced to form 1 cm×1 cm chips with an effective area of 6×6 mm2 containing 1500 windows each, with a total of 3.12×106 pores per membrane. In certain embodiments, all membranes may be cleaned using a conventional “piranha” clean procedure, which involve a 20 min-immersion in 3:1 sulfuric acid (H2SO4)/hydrogen peroxide (H2O2) mixture, followed by thorough rinses in deionized (DI) water.
In certain embodiments, the semipermeable ultrafiltration membrane is modified with PEG. Techniques for modification with PEG is well-known in the art, for example, in Papra et al. 2001 (Papra, A, et al., Langmuir 2001, 17 (5), 1457-1460.) In certain embodiments, the semipermeable ultrafiltration membrane is covalently modified with PEG. In certain embodiments, the surface modification with PEG prevents or minimizes protein fouling on the membrane surface. In certain embodiments, the technique used for PEG attachment involves a single reaction step which covalently couples silicon surface silanol group (Si—OH) to a chain of PEG polymer through a trimethoxysilane group forming a Si—O—Si—PEG sequence. In such embodiments, semipermeable ultrafiltration membranes were immersed in a solution of 3 mM 2-[methoxy(polyethyleneoxy)propyl]trimethoxysilane (PEG-silane) (Gelest: SIM6492.7) in toluene for 2 hrs at 70° C. In certain embodiments, a series of extensive washing steps involving toluene, ethanol, and DI water were used to rinse away unbounded PEG residue.
In certain embodiments, the compartments of the disclosed devices are of any appropriate shape and configuration to be compatible with the semipermeable ultrafiltration membranes and scaffold(s) included in the devices.
In certain embodiments, a device of the present disclosure may be as depicted in
In certain embodiments, the device may include two scaffolds to provide an increased amount of ultrafiltrate. For example, such a device may be configured as depicted in
Another exemplary device is depicted in
In certain aspects, the scaffold may also be referred to as an islet chamber (IC) or a cell scaffold and may be formed as depicted in
In certain embodiments, the devices provided herein provide formation of ultrafiltrate by convection rather than predominantly by diffusion thereby increasing the efficiency of ultrafiltrate formation and/or flow of the ultrafiltrate back to the subject.
The bioartificial devices of the present disclosure may be used for transporting nutrients to a planar scaffold comprising the population of cells in the device and release ultrafiltrate fluid containing molecules secreted by the cells to a subject. In certain embodiments, the cells are progenitor cells. In certain embodiments, the cells are pancreatic cells, such as, insulin producing cells isolated from pancreatic islets or derived by differentiation of stem cells (e.g., induced pluripotent stem cells). In certain embodiments, the cells excrete insulin. In certain embodiments, the cells release insulin into the ultrafiltrate fluid.
In certain embodiments, the bioartificial device of the present disclosure having semipermeable ultrafiltration membranes with pores are used for protecting cells encapsulated in the scaffolds described herein from an attack by the immune system of the subject. In certain embodiments, the bioartificial device of the present disclosure can reduce passage of immune system components such as cells, immune factors, such as, antibodies and cytokines. In certain embodiments, the bioartificial device of the present disclosure can reduce passage of immune factors. In certain embodiments, the bioartificial device of the present disclosure can reduce passage of cytokines. In certain embodiments, the bioartificial device of the present disclosure can reduce passage of TNF-α, IFN-γ, and/or IL-1β while permitting transport of nutrients from the blood of the subject to the cells in the device. In certain embodiments, the bioartificial device of the present disclosure can reduce passage of components of the immune system (e.g., immune cells, antibodies, cytokines, such as, 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 having semipermeable ultrafiltration membranes with nanopores (e.g. slit-shaped nanopore membranes with a PEG surface coating) are used for exchange of nutrients and small molecules to the population of cells. In certain embodiments, the bioartificial device may limit the diffusion of cytokines and immunoglobulins through the semipermeable ultrafiltration membrane.
In certain embodiments, the bioartificial device of the present disclosure comprising a population of pancreatic islet cells in the matrix increase pancreatic islet cell viability within the matrix.
The bioartificial device of the present disclosure are sized to house an effective number for cells within the bioartificial device for treatment of a subject in need thereof. For example, the subject may be suffering from a condition caused by lack of functional cells, e.g., wherein molecules typically secreted by functional cells are not secreted or are secreted at a level resulting in the condition. Providing functional cells within the bioartificial device of the present disclosure could alleviate the condition. Exemplary conditions include type 1 diabetes, Parkinson's disease, muscular dystrophy and the like.
The device may be transplanted into any suitable location in the body, such as, subcutaneously, intraperitoneally, or in the brain, spinal cord, pancreas, liver, uterus, skin, bladder, kidney, muscle and the like. The site of implantation may be selected based on the diseased/injured tissue that requires treatment. For treatment of a disease such as diabetes mellitus (DM), the device may be placed in a clinically convenient site such as the subcutaneous space or the omentum. The device may be connected to the vascular system of the subject as described herein. In some case, the device may be connected inline to a vascular graft. In some cases, the device may be connected to the subject to supply the ultrafiltrate to an artery, a vein, a body cavity (e.g., peritoneal cavity), or a combination thereof, of the subject. In some cases, the device may be connected to a catheter to supply the ultrafiltrate to a vein to which the catheter is connected.
In certain cases, the device may be connected to an artery of the subject and may supply the ultrafiltrate back to the same artery (e.g., at a connection downstream to the site at which the artery was connected to the device). In certain cases, the device may be connected to an artery of the subject and may supply the ultrafiltrate to a vein of the subject. In certain cases, the device may be connected to an artery of the subject and may supply the ultrafiltrate to a body cavity of the subject. In some embodiments, the device may include a sampling port for sampling the ultrafiltrate, for example, using an analyte analysis device for measuring concentration of insulin and/or glucose in the ultrafiltrate.
In certain embodiments, the bioartificial device disclosed herein may be used to treat a person having diabetes, such as, type 1 diabetes. The device may include pancreatic islet cells or may include stem cells that are capable of differentiating into pancreatic islet cells. In certain embodiments, pluripotent stem cells (PSCs) may be differentiated into pancreatic islet cells inside the device. In some case, the device may include PSCs and the device may be implanted adjacent the pancreas or liver of the subject.
As noted herein, the devices disclosed herein may maintain the transplanted cells in a functional and viable state for at least 1 month and up to a period of at least 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or up to a year or longer. In some embodiments, the present device provide immunoisolation while supporting supply of essential components to enable islets, beta cells, and other insulin producing cells to remain viable and functional for treatment of diabetes.
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 can be a human or, in the case of veterinary applications, can be a laboratory, agricultural, domestic, 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, domestic animals such as dogs and cats, agricultural animals such as cows, horses, pigs, sheep, goats, and wild animals in captivity such as bears, pandas, lions, tigers, leopards, elephants, zebras, giraffes, gorillas, dolphins, and whales.
In operation, blood is directed from a patient's vasculature (i.e. artery) into the inlet of the first compartment of the bioartificial device. Blood flows through the first compartment of the bioartificial device, and nutrients and small molecules from the blood are passed through the semipermeable ultrafiltration membrane, while large molecules, such as immunoglobulins and cytokines within the blood are prevented from coming in contact with the cells in the device. Nutrients and small molecules include, but are not limited to glucose, oxygen, and insulin. The small molecules and nutrients that pass through the semipermeable ultrafiltration membrane are filtered to form an ultrafiltrate which contacts the matrix of the device comprising the population of cells. In certain embodiments, the population of cells release insulin into the ultrafiltrate. The ultrafiltrate then passes through the ultrafiltrate channels of the matrix, which then passes through a second semipermeable ultrafiltration membrane. Optionally, the outlet of the second compartment can be configured to connect to a catheter. In certain embodiments, the catheter connects to the second vein.
The disclosed devices provide a high rate of ultrafiltration creating ultrafiltrate at the rate of 1-15 ml/min at physiological rate of blood flow.
The following examples are put forth so as to provide those of ordinary skill 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 their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric. Standard abbreviations may be used, e.g., room temperature (RT); base pairs (bp); kilobases (kb); picoliters (pl); seconds (s or sec); minutes (m or min); hours (h or hr); days (d); weeks (wk or wks); nanoliters (nl); microliters (ul); milliliters (ml); liters (L); nanograms (ng); micrograms (ug); milligrams (mg); grams ((g), in the context of mass); kilograms (kg); equivalents of the force of gravity ((g), in the context of centrifugation); nanomolar (nM); micromolar (uM), millimolar (mM); molar (M); amino acids (aa); kilobases (kb); base pairs (bp); nucleotides (nt); intramuscular (i.m.); intraperitoneal (i.p.); subcutaneous (s.c.); and the like.
The development and characterization of a new generation of semipermeable ultrafiltration membrane, the silicon nanopore membrane (SNM), designed with approximately 7 nm-wide slit-pores to provide middle molecule selectivity by limiting passage of proinflammatory cytokines is shown. The use of convective transport with a pressure differential across the SNM overcomes the mass transfer limitations associated with diffusion through nanometer-scale pores. The SNM exhibited a hydraulic permeability of 130 ml/hr/m2/mmHg. Analysis of sieving coefficients revealed 80% reduction in cytokines passage through SNM under convective transport. SNM protected encapsulated islets from infiltrating cytokines and retained islet viability over 6 hours and remained responsive to changes in glucose levels unlike non-encapsulated controls. The concept involves using the pressure difference between the artery and vein to generate ultrafiltrate and drive transport of glucose, insulin, and other small molecules through the SNM to support function of encased islets while preventing passage of immune components. SNM design and fabrication, followed by characterization of its immunobarrier properties under cytokine challenge with convective transport, and assessment of SNM encapsulated islet viability and glucose-insulin response were developed. Specifically, hydraulic permeability measurement and solute selectivity for SNM were determined. Mouse islets were encapsulated between SNM in a closed mock-loop fluid circuit (
Experimental overview
SNM were fabricated to produce an active membrane area (6×6 mm) consisting of ˜106 rectangular slit pores with ˜7 nm in width, 300 nm in depth, and 2 μm in length (
Silicon nanopore membranes (SNM) have been prototyped from silicon substrates by MEMS technology as previously reported with some modifications (
1.2.2 Surface Modification of SNM with poly(ethylene glycol) (PEG)
SNM were covalently modified with PEG using a previously reported protocol with some modifications to prevent protein fouling on the membrane surface. The technique used for PEG attachment involved a single reaction step which covalently couples silicon surface silanol group (Si—OH) to a chain of PEG polymer through a trimethoxysilane group forming a Si—O—Si—PEG sequence. Briefly, SNM were immersed in a solution of 3 mM2[methoxy(polyethyleneoxy)propyl] trimethoxysilane (PEGsilane) (Gelest: SIM6492.7) in toluene for 2 hr at 70° C. A series of extensive washing steps involving toluene, ethanol, and DI water were used to rinse away unbounded PEG residue.
An automated mass and pressure measurement system was utilized for characterizing liquid flow through the SNM under a tangential-flow filtration operation. The pore size of the SNM can be related to filtration flow parameters using
where h is pore width, H is the viscosity, 1 is the membrane thickness, Q is the volumetric flow rate, n is the number of pores per membrane, w is the pore length, and ΔP is the transmembrane pressure. To assemble the overall system for SNM pore size characterization (
Using the specifications for pore length, membrane thickness, and total number of pores provided based on individual wafer designs, the average pore size of SNM was calculated using Equation 1. All SNM membranes in this study were surface-modified with PEG and exhibited an average pore size of ˜7 nm.
1.3 Assessment of SNM Immunoisolation in vitro
Fluid was circulated by a peristaltic pump through a circuit that consisted of a differential pressure transducer, a polycarbonate flow cell with enclosed SNM, a three-way valve, and a fluid reservoir (
where S is the sieving coefficient, Cf is the concentration of the solute in the filtrate, and Cb is the molecule concentration in the bulk retentate solution.
A mock-loop circuit assembly with three flow cell components without cells to mimic the architecture of the final bioartificial pancreas device. Briefly, two SNM with customized silicone gasket frames were sandwiched in between three flow cell components. The middle flow cell was the encapsulation chamber comprised of a cylindrical chamber separating the two membranes. A peristaltic pump drove the fluid through the top of the flow cell mimicking the “artery”, then over the bottom of the flow cell resembling “vein”, and finally back to the original reservoir. For convective experiments, a three-way valve was used to create flow resistance for a physiological pressure difference psi between the top and the bottom compartments of the flow cell. Ultrafiltration occurred in the middle encapsulation chamber at this pressure difference. To study the transport of cytokines through the three-layered bioartificial pancreas device, solution consisting of mouse cytokines TNF-α (1000 U/ml), IFN-γ (1000 U/ml), and IL-1β (50 U/ml), glucose (400 mg/dL), insulin (150 mU/L) in 3% BSA was circulated through the circuit at a flow rate of 5 ml/min. Silicon membranes with 1000 nm-wide slit pores (SμM) were used as the control. Solutions were collected and analyzed with ELISA at the end of 6-hr experiments for the top, middle, and bottom chambers.
All procedures described involving isolation of mouse islets were performed in accordance with protocols approved by the Institutional Animal Care and Use Committee (IACUC) at the University of California, San Francisco (UCSF). Mouse islets were isolated from 8 to 10-week-old male B6 mice (Jackson Laboratories) based on previously described protocols 40. Harvested islets were maintained in suspension culture with RPMI 1640 with L-glutamine and 11.1 mM glucose (Gibco: 11875-093), 10% fetal bovine serum (FBS) (Gibco: 16000), and 1% penicillin-streptomycin (P/S) (UCSF Cell Culture Facility: CCFGK003). A group of 500 mouse islets were introduced into the middle encapsulation chamber of the mock-loop device (
Islet viability was assessed by double staining with fluorescein diacetate (FDA) (Sigma: F7378) and propidium iodide (PI) (Sigma: 287075) as described by protocol (SOP Document: 3104, A02) from National Institute of Allergy and Infectious Diseases (MAID). Briefly, mouse islets were incubated in phosphate buffered saline (PBS) containing 0.067 HM FDA and 4.0 HM PI for 30 min and extensively washed in PBS to remove excess staining. Images of mouse islets were obtained using laser scanning Nikon Spectral Clsi confocal microscope (Nikon Instruments). Viability of islets was calculated based on the ratio between the number of live cells in the islet and the area of that islet.
Mouse islets retrieved from the middle chamber of the mock-loop circuit were rested in RPMI 1640 containing 30 mg/dL glucose (Gibco: 11879) for 15 minutes before exposed to medium containing 300 mg/dL glucose for 15 minutes. After glucose stimulation, the islets were then returned to medium containing 3o mg/dL glucose. Supernatant was collected every 5 minutes during the series of incubations and insulin content was measured with mouse insulin ELISA kits (Mercodia:10-1247-01) and normalized by extracted total protein concentration (Thermo: 78505; 23225).
Sample pairs were analyzed using Student's t-test. Multiple samples were evaluated with oneway or two-way analysis of variance (ANOVA) followed by Bonferroni and multiple comparison using Graphpad Prism software (San Diego, Calif.). A p value of <0.05 was accepted as statistically significant for all analyses.
MEMS fabrication technologies offers unprecedented potential in reproducibility and precision to engineer controlled pore dimensions that can selectively block the passage of immune components while allowing transport of small molecules (e.g. glucose and insulin) to sustain the viability of the encased cells. In the present study, the permeability and selectivity of the SNM were characterized to prevent cytokine infiltration and assessed the functional performance of SNM-encapsulated mouse islets in a mock-loop device under convective transport.
A new generation of semipermeable membranes, SNM, with slit-pore designs initially investigated by Desai et al. 26, 27. The SNM exhibit a pore size distribution of ˜1%, and a consistent pore size control in the range of 5-15 nm (
The utilization of a sacrificial layer to define the nanopores resulted in a membrane with a straight slit pore path that presents a shorter distance for molecules to travel. The permeability-selectivity analysis for ultrafiltration demonstrated that membranes with slit-shaped pores showed higher performance and greater selectivity at a given value of permeability, than membranes with cylindrical pores for pore size below 100 nm. To circumvent the slow concentration-dependent diffusion occurred in size-restricted nanoporous membranes, the concept of using convection-dominated transport is advantageous in terms of creating faster solvent movement under transmembrane pressure gradient, which efficiently drags small molecules such as glucose and insulin across membranes to the encapsulated cells.
Permeability and selectivity of the SNM were characterized with the hydraulic permeability testing setup (
ΦKC=1−3.02 λ2+5.776λ3−12.3675 λ4+18.9775 λ5−15.2185 λ6+4.8525 λ7 (Equation 3),
where λ is the relative solute size indicating the ratio between the diameter of the molecule and the width of slit-pore channel. Based on the observed sieving coefficients of cytokines (
In summary, the SNM enables higher levels of ultrafiltrate production and demonstrate selective rejection against middle molecules like cytokines. Therefore, by encapsulating islets in SNM, it was postulated that the increased convective mass transport of nutrients and glucose can support islet viability and insulin production, while the selective rejection of immune components enables immunoisolation.
The feasibility of developing an implantable SNM-encapsulated bioartificial pancreas device using convective transport was demonstrated using a mock-loop setup. The middle cell chamber is sandwiched between two membranes to closely mimic the in vivo conditions where SNM-encapsulated islets will be mounted as an arterio-venous (AV) graft (
Additionally, the static culture without cytokines, mock-loop device without cytokines, and mock loop flow cell device with cytokines demonstrated a 3.0-fold, 2.6-fold, and 4.1-fold changes, respectively, in the amount of insulin secreted during high glucose challenge compared with those secreted during low glucose challenge, respectively (
An improved silicon nanopore membrane, SNM, for the encapsulation of pancreatic islets under convective flow was developed and characterized. The SNM structure was specifically designed to obtain a well-defined slit pore in the nanometer range with a remarkably high hydraulic permeability. Furthermore, SNM achieved high molecule selectivity against middle molecules such as cytokines under convective transport and provided adequate immuneprotection to the encapsulated islets while generating sufficient filtrate to support viability and functionality of the encapsulated islets.
Semipermeable membrane capsules can immunoprotect transplanted islets by blocking passage of the host's immune components while providing exchange of glucose, insulin and other small molecules. However, capsules-based diffusive transport often exacerbates ischemic injury to islets by reducing the rate of oxygen and nutrient transport. The efficacy of a newly-developed semipermeable ultrafiltration membrane, the silicon nanopore membrane (SNM) under convective-driven transport, limits the passage of pro-inflammatory cytokines while overcoming the mass transfer limitations associated with diffusion through nanometer-scale pores. SNM-encapsulated mouse islets perfused in culture solution under convection outperformed those under diffusive conditions in terms of magnitude (1.49-fold increase in stimulation index and 3.86-fold decrease in shut-down index) and rate of insulin secretion (1.19-fold increase and 6.45-fold decrease during high and low glucose challenges) respectively. SNM-encapsulated mouse islets under convection demonstrated rapid glucose-insulin sensing within a physiologically relevant time-scale while retaining healthy islet viability even under cytokine exposure. The encapsulation of islets with SNM under convection improves graft function and survival.
This study showed that SNM and silicon micropore membrane (SμM) with 7 nm and 1000 nm-wide slit-shaped pores respectively, were used to encapsulate mouse islets under diffusive and convective conditions with and without cytokine exposure. The islets were then exposed to varying concentration of glucose inside the reservoir culture medium, and glucose-stimulated insulin responses and islet viability were evaluated. In addition, to determine the immunoprotective effect of the membranes, a highly concentrated cocktail of pro-inflammatory cytokines was added to the circulating system to challenge the encapsulated islets.
SNM were designed to have an active membrane area (6×6 mm) consisting of ˜106 rectangular slit pores with an average pore size of 7 nm in width, 2 μm in length, and 300 nm in depth (
1.1.1 Silicon Nanopore Membranes (SNM) and Silicon Micropore Membrane (SμM): architecture and Fabrication
Silicon nanopore membranes (SNM) have been prototyped from silicon substrates by
MEMS technology as previously reported19, 36-37 with some modifications (
1.1.2 Surface Modification of SNM with poly(ethylene glycol) (PEG)
SNM were covalently modified with PEG using a previously reported protocol38 with some modifications to prevent protein fouling on the membrane surface. The technique used for PEG attachment involved a single reaction step which covalently couples silicon surface silanol group (Si—OH) to a chain of PEG polymer through a trimethoxysilane group forming a Si—O—Si—PEG sequence. Briefly, SNM were immersed in a solution of 3 mM 2-[methoxy(polyethyleneoxy)propyl]trimethoxysilane (PEG-silane) (Gelest: SIM6492.7) in toluene for 2 hr at 70 oC. A series of extensive washing steps involving toluene, ethanol, and DI water were used to rinse away unbounded PEG residue.
An automated mass and pressure measurement system was utilized for characterizing liquid flow through the SNM under a tangential-flow filtration operation.9 The pore size of the SNM can be related to filtration flow parameters using (Equation 1), where h is pore width, μ is the viscosity, 1 is the membrane thickness, Q is the volumetric flow rate, n is the number of pores per membrane, w is the pore length, and ΔP is the transmembrane pressure. To assemble the overall system for SNM pore size characterization (
All procedures described involving isolation of mouse islets were performed in accordance with protocols approved by the Institutional Animal Care and Use Committee (IACUC) at the University of California, San Francisco (UCSF). Mouse islets were isolated from 8 to 10-week-old male B6 mice (Jackson Laboratories) based on previously described protocols. Harvested islets were maintained in suspension culture with RPMI 1640 with L-glutamine and 11.1 mM glucose (Gibco: 11875-093), 10% fetal bovine serum (FBS) (Gibco: 16000), and 1% penicillin-streptomycin (P/S) (UCSF Cell Culture Facility: CCFGK003).
A mock-loop circuit was assembled with two flow cell components. Briefly, one SNM with customized silicone gasket frames were sandwiched in between two flow cell components. A group of 40-50 mouse islets were introduced into the bottom chamber separated by the SNM from the circulating fluid (5 ml/min) in the top chamber. A peristaltic pump drove the fluid through the top of the flow cell component, and finally back to the original medium reservoir. For convective experiments, a three-way valve was used to create flow resistance for a physiological pressure difference ˜2 psi between the top and the bottom compartments of the flow cell. The membrane Peclet number (Pe) for the pressure-driven ultrafiltration system was significantly greater than 1, suggesting that convective transport dominates. For diffusive experiments, no transmembrane pressure was induced and fluid still circulated throughout the system. To study the effects of cytokines on SNM-encapsulated islets, solution consisting of mouse cytokines TNF-α (2,000 U/ml), IFN-γ (1,000 U/ml), and IL-1β (10,000 U/ml) was added to the original reservoir. Silicon membranes with 1 μm-wide slit pores (SμM) were used as the control with adjusted pressure (˜0.127 psi) and flow rate (˜20 μl/min) to produce similar amount of ultrafiltrate as the SNM in this mock-loop system. Naked mouse islets cultured under static conditions were also used as controls.
The membrane-encapsulated mouse islets in the mock-loop systems were exposed to a series of low (1.6 mM), high (16.6 mM), and low (1.6 mM) glucose (Gibco: 11879) stimulation for 30 min each. Supernatant was sampled every 10 min from the bottom islet chamber during this series of glucose challenge. For convective experiments, an ultrafiltrate rate of ˜3.5 ul/min was observed for the SNM with ˜7 nm pore size and the same ultrafiltrate rate was obtained for the SμM with lowered transmembrane membrane pressure and system flow rate. For diffusive experiments, islet chambers were re-filled after individual sampling to ensure that the volume of islet chamber was kept constant at all time. This step minimized the any bubbles that might potentially be formed during the process which could hinder mass transfer within the system. Insulin content was measured with mouse insulin enzyme-linked immunosorbent assay (ELISA) kits (Mercodia: 10-1247-01) with accounted dilutions and normalized by extracted total protein concentration (Thermo: 78505; 23225). Naked mouse islets were also challenged under static culture condition as controls. About 7-10 μl chamber fluid per islet were used in all cases.
A stimulation index was calculated as the ratio of stimulated to basal insulin secretion. In our study, the stimulation index (SI) was the ratio of (1) the first insulin collection point in the high glucose phase to the last insulin collection point of the previous low glucose phase (Immediate Stimulation), and (2) the highest insulin secretion in the high glucose phase to the last insulin collection point of the previous low glucose phase (Maximum Stimulation). The shut-down index (SDI) was calculated as the ratio of (1) the first insulin collection point in the subsequent low glucose phase to the last insulin collection point in the high glucose phase (Immediate Shutdown), and (2) the lowest insulin secretion in the subsequent low glucose phase to the last insulin collection point in the high glucose phase (Maximum Shutdown). The stimulation index indicates the magnitude of insulin released as stimulated by a higher concentration of glucose, whereas the shut-down index reflects the magnitude of cessation in insulin production once glucose concentration returns to normal.
The rate of change in insulin secretion was calculated for the stimulation and shut-down phases. For the stimulation phase, a curve was fitted on the glucose-insulin kinetic graph with the last point of insulin produced during low glucose exposure to the highest point of insulin produced during high glucose exposure. For the shut-down phase, a curve was fitted on the glucose-insulin kinetic graph with the last point of insulin produced during high glucose exposure to the first point of insulin produced during low glucose exposure. The rate of change was obtained by taking derivatives of those curves to study the quickness of insulin being secreted during changes in glucose concentration.
Islet viability was assessed by double staining with live green and dead red solutions (Invitrogen: R37601). Briefly, mouse islets were incubated in live green and dead red solutions for 15 min at room temperature followed by extensive washes in PBS to remove excess staining. Images of mouse islets were obtained using laser scanning Nikon Spectral C1si confocal microscope (Nikon Instruments). Viability of islets was calculated based on the percentage of live cells in the islets as described by protocol on assessment of islet viability by fluorescent dyes from Department of Surgery Division of Transplantation at University of Wisconsin-Madison.
Sample pairs were analyzed using Student's t-test. Multiple samples were evaluated with one-way or two-way analysis of variance (ANOVA) followed by Bonferroni and multiple comparison using Graphpad Prism software (San Diego, Calif.). A p value of <0.05 was accepted as statistically significant for all analyses.
A construction of a benchtop flow loop circuit consisting of a single membrane that separated islets from the circulating fluid (
The slit pore microarchitecture of SNM is produced by dry oxidation of polysilicon for the growth of silicon dioxide (SiO2) followed by backside patterning with deep ion-reactive etching (DRIE) that produces vertical walls in each membrane window (
A benchtop flow loop circuit incorporating membrane-encapsulated islets under applied physiological transmembrane pressure (
Once the circuit was switched back to low glucose concentration (1.6 mM) from 60 to 90 minutes, the SNM-encapsulated islets under convection exhibited a rapid shutdown in insulin production whereas a gradual decrease in insulin production occurred for the capsule under the diffusive mode. The shut-down index (SDI), calculated as the ratio of the first insulin collection in the subsequent low glucose phase to the last insulin collection in the previous high glucose phase (Immediate Shutdown), showed that the amount of insulin that was secreted significantly decreased for SNM-encapsulated islets under convection (0.20±0.03) compared with the naked islets under static culture (0.59±0.17) and SNM-encapsulated islets under diffusion (0.93±0.19) (
As illustrated in Table 1 of
Further comparison with the silicon micropore membrane (SμM)-encapsulated islets under convection showed that pressure-driven convection yields faster mass transport as the pore size becomes larger (1 μm). The naked islets under static culture, SNM-encapsulated islets under convection, and SμM-encapsulated islets under convection all quickly released more insulin during high glucose exposure from 40 to 60 minutes (
In addition, the SμM-encapsulated islets under convection showed the fastest rate of response when switching from low to high glucose condition (3.15 normalized insulin content min−1 (×10−2)) to the high to low glucose situation (−3.36 normalized insulin content min−1 (×10−2)) (
A highly concentrated solution of pro-inflammatory cytokines consisting of TNF-α, IFN-γ, and IL-1β was used to investigate how the glucose-insulin kinetics of SNM-encapsulated islets are influenced by cytokine exposure. When challenged with high glucose concentration, SNM-encapsulated islets under convection immediately secreted insulin to the maximum level within first 10 minutes followed by a slight decrease in insulin secretion in the next 20 minutes (
Unlike the SNM-encapsulated islets under convection in which the maximum level of insulin secreted within 10 minutes of high glucose challenge, SμM-encapsulation under convection showed a continuous rise in insulin secretion and reached the highest peak within 30 minutes of high glucose exposure (
Comparing previous conditions that were not subjected to cytokines, it was observed that conditions with cytokine exposure had a slight decrease in SI values (including
In addition to the glucose-insulin kinetics of SNM- and SμM-encapsulation described above, the islet viability was investigated to understand if cytokines caused excessive islet dysfunction (
In this study, the glucose-insulin kinetics of an improved silicon nanopore membrane was characterized, SNM, for the encapsulation of pancreatic islets under convective flow. The glucose-insulin responsiveness of membrane-encapsulated islets was analyzed under a series of low, high, and low glucose challenge by: (1) SI and SDI values, which show the magnitude of insulin secreted when transitioning from low to high glucose condition or vice versa; and (2) rate of change in insulin secretion, which indicates how quickly the system responds from low to high glucose condition or vice versa. Based on these parameters, it was found that convective mode performed better than diffusive mode in both SNM and SμM encapsulations. In addition, once exposed under cytokines, convective transport with SNM encapsulation demonstrated superior performance over SμM encapsulation in terms of the magnitude of insulin produced and ceased during high and low glucose phases with healthy islet viability, while the rate of changes in insulin secretion was on the same scale as that for the SμM encapsulation. In summary, SNM encapsulation under convective transport enables rapid glucose-insulin sensing to activate and cease insulin production based on the surrounding glucose concentration while retaining healthy islet viability even under cytokine exposure. Our data demonstrates the importance of using convective transport to obtain faster insulin activation and shut-down, which is a critical issue to address in many islet-encapsulating devices5, 35 with undesired delay in glucose-insulin response. Successful islet encapsulation with selective SNM under convective transport could potentially lower the immunosuppressive drugs and their side effects resulted from current therapies, lead to the possibility of using xenogeneic or stem-cell derived cell sources to overcome donor shortage, and reduce dangerous episodes of hypoglycemia for T1D patients in the future.
A peristaltic pump circulated liquid through a flow cell that connected to a 3-way valve to establish transmembrane pressure. The permeated ultrafiltrate through the membrane was collected at various time for up to 6 hrs.
based on the sieving coefficients for cytokines at 6 hrs. Theoretical values were determined using the Stokes-Einstein's equation 14.
Diffusion-based bioartificial pancreas (BAP) devices are limited by poor islet viability and functionality due to inadequate mass transfer resulting in islet hypoxia and delayed glucose-insulin kinetics. While intravascular ultrafiltration-based BAP devices possess enhanced glucose-insulin kinetics, the polymer membranes used in these devices provide inadequate ultrafiltrate flow rates and result in excessive thrombosis. Here, the silicon nanopore membrane (SNM) exhibits a greater hydraulic permeability and a superior pore size selectivity compared to polymer membranes for use in BAP applications. Specifically, the SNM-based intravascular BAP with ˜10 and ˜40 nm pore sized membranes support high islet viability (>60%) and functionality (<15 minute insulin response to glucose stimulation) at clinically relevant islet densities (5,700 and 11,400 IE/cm2) under convection in vitro. In vivo studies with ˜10 nm pore sized SNM in a porcine model showed high islet viability (>85%) at clinically relevant islet density (5,700 IE/cm2), c-peptide concentration of 144 pM in the outflow ultrafiltrate, and hemocompatibility under convection.
Silicon nanopore membranes (SNM) have been prototyped from silicon substrates by MEMS technology as previously reported26. Briefly, the process used the growth of a thin SiO2 (oxide) layer on 400 μm-thick double side polished (DSP) silicon wafers followed by a low pressure chemical vapor deposition (LPCVD) of polysilicon (˜500 nm). The wafers were then specifically patterned, dry oxidized, wet etched, deposited with a second polysilicon layer, and finally blanket-etched until 400 nm of polysilicon remained and the underlying vertical oxide layer was exposed. The vertical sacrificial oxide layer defined the critical nanoscale pore size of the membranes. The low temperature oxide (LTO) (˜1 μm) was deposited onto polysilicon of the wafers to serve as the hard mask for membrane protection. Deep reactive ion etching (DRIE) removed the backside of each window until membranes were disclosed. Eventually, the sacrificial oxide was etched away in 49% hydrofluoric acid (HF) during the final step of the fabrication process to leave behind open nanoscale slit pores. The wafers were subsequently cut into 1×1 cm chips with an effective area of 6×6 mm2 containing 1500 windows each, with a total of 106 pores per membrane. Each rectangular pore was 300 nm in depth and 2 μm in length. The SNM with an average pore size width of ˜10 nm and ˜40 nm were used in this study. All membranes were cleaned using a conventional “piranha” clean procedure, which involved a 20 min-immersion in 3:1 sulfuric acid (H2SO4)/hydrogen peroxide (H2O2) mixture, followed by thorough rinses in deionized (DI) water. Images of SNM were obtained using scanning electron microscope (SEM) (Leo 1550) (
Surface Modification of SNM with poly(ethylene glycol) (PEG)
SNM were covalently modified with PEG using a previously reported protocol to prevent protein fouling on the membrane surface26. The technique used for PEG attachment involved a single reaction step which covalently couples silicon surface silanol group (Si—OH) to a chain of PEG polymer through a trimethoxysilane group forming a Si—O—Si—PEG sequence. Briefly, SNM were immersed in a solution of 3 mM 2-[methoxy(polyethyleneoxy)propyl]trimethoxysilane (PEG-silane) (Gelest: SIM6492.7) in toluene for 2 hr at 70° C. A series of extensive washing steps involving toluene, ethanol, and DI water was used to remove unbounded PEG residue.
An automated mass and pressure measurement system was utilized for characterizing liquid flow through the SNM under a tangential-flow filtration operation. The pore size of the SNM can be related to filtration flow parameters using
where h is pore width, μ is the viscosity, l is the membrane thickness, Q is the volumetric flow rate, n is the number of pores per membrane, w is the pore length, and ΔP is the transmembrane pressure. To assemble the overall system for SNM pore size characterization, air was applied through a syringe pump (Sigma: Z675709) into a water reservoir. Water was circulated by a peristaltic pump (Masterflex: 07551-00) through a differential pressure transducer (Omega: PX429 015GI), a flow cell with enclosed membrane, and returned to the original water reservoir. The flow cell was assembled with the SNM submerged under water to remove air bubbles from all compartments. Specifically, a membrane was positioned with the polysilicon interface facing down with a customized silicone gasket positioned on top of the membrane, followed by the final placement of a filtrate chamber on top of the gasket. All sections were fastened together and secured to the base with hand-tightened hex bolts until the gasket was visibly compressed. The ultrafiltrate permeated through the membrane and was routed to a liquid collection container that rested on a precision mass balance (Mettler Toledo: XS205). Measurements from the differential pressure transducer and the mass balance were automatically collected with a data acquisition laptop. A typical membrane hydraulic permeability test consisted of 5 ml/min flow rate and 4 pressure cycles (5, 1, 5, and 1 psi) for durations of 150 s each. Using the specifications for pore length, membrane thickness, and total number of pores provided based on individual wafer designs, the average pore size of SNM was calculated using Equation 1. All SNM membranes in this study were surface-modified with PEG and exhibited an average pore size of ˜10 nm and ˜40 nm.
In this study, IC possessed a thickness of 1000 μm and high islet densities of 10% (5,700 IE/cm2) and 20% (11,400 IE/cm2). Islet density by percentage was calculated as the ratio of total islet volume expressed in islet equivalents and the IC volume. Islet density by surface area was calculated by dividing the total number of islet equivalents (IE) by the device membrane surface area. A biocompatible acrylic sheet (McMaster: 8589K11) was first laser-cut to create ˜2.4 mm x ˜2.4 mmט1 mm thick void region which was inserted with eight 100 μm diameter polytetrafluoroethlene (PTFE) coated wires (McMaster: 1749T11). A 2% agarose-islet mixture was then poured into this void region of acrylic sheets. After the agarose-islet mixture was cured, all wires were removed (
Assembly of an Intravascular Bioartificial Pancreas Device (iBAP) for Islet Encapsulation
The intravascular bioartificial pancreas device (iBAP) is shown in an exploded view in
Testing of the Intravascular Bioartificial Pancreas Device (iBAP) in vitro
All procedures involving isolation of mouse islets were performed in accordance with protocols approved by the Institutional Animal Care and Use Committee (IACUC) at the University of California, San Francisco (UCSF). Mouse islets were isolated from 8 to 10-week-old male B6 mice (Jackson Laboratories) based on previously described protocols. Harvested islets were maintained in suspension culture with RPMI 1640 with L-glutamine and 11.1 mM glucose (Gibco: 11875-093), 10% fetal bovine serum (FBS) (Gibco: 16000), and 1% penicillin-streptomycin (P/S) (UCSF Cell Culture Facility: CCFGK003). A 2% agarose gel mixed with appropriate amount of mouse islets was dispensed into the previously described IC to create high islet densities of 10% or 20% by volume, respectively. The SNM with ˜10 nm and ˜40 nm pore sizes were chosen to encapsulate the IC with 10% and 20% islet density. For in vitro viability tests, a mock-loop circuit was set up with a peristaltic pump flowing culture medium through the iBAP at TMP of ˜80 mmHg to generate ultrafiltrate for convective condition (
Implantation of the Intravascular Bioartificial Pancreas Device (iBAP) in Pigs
A preliminary proof-of-concept study was designed with a swine model because of the comparably sized vasculature and hematologic similarities with humans. The study was approved by the IACUC review committee at PMI Preclinical CRO, San Carlos, Calif.
For pig #1, the device was assembled as previously described with each chamber containing a 5% islet equivalents (IE) density by volume of mouse islets suspended in agarose gel. Oral aspirin (81 mg) and clopidogrel (75 mg) were given to a 75 kg female Yorkshire pig for 3 days preoperatively and then daily thereafter. After general anesthesia was induced, a vertical incision was made to the left of midline to expose the left external jugular vein. A 15Fr double-lumen tunneled catheter (NextStep , Teleflex, Morrisville, N.C.) was placed in the left external jugular vein for blood sampling. The right carotid artery and right external jugular vein were then exposed via a similar vertical incision on the right side of the neck. Once the vessels were exposed a subcutaneous pocket was created caudally for eventual device placement. Heparin was given intraoperatively targeting an activated clotting time (ACT) of greater than 200 seconds. The 6 mm externally-supported polytetrafluoroethylene (PTFE) grafts were then anastomosed end-to-side to the internal carotid artery for inflow and the external jugular vein for outflow. The device was then placed in the subcutaneous pocket and anchored to surrounding soft tissue. The inflow and outflow grafts were then connected to the device and clamps were removed to allow blood flow through the device, which was visually confirmed (
For pig #2, the device was assembled as previously described with 10% density by volume for each chamber. One chamber had a channel in communication with the islets that was open to atmosphere, allowing for ultrafiltrate flow through the chamber (
Assessment of Islet Function in vivo
Islet function was assessed using standard intravenous glucose tolerance tests (IVGTT) with administration of glucose (0.5g/kg in 40% solution) via the tunneled venous catheter. Blood was drawn to measure serum glucose using a standard glucometer (Accu-Chek Compact Plus: 1002-5021) at time 0, 5, 10, 15, 30, 60 and 90 minutes. The IVGTT was administered on post-operative day (POD) 1 and 2 prior to the animals eating their morning meals. On POD 3 the test was performed intra-operatively prior to planned explant of the device and islet retrieval. For pig #1, blood sampling for the intra-operative IVGTT was performed via direct cannulation of the external jugular venous outflow tract immediately distal to the anastomosis. All samples from systematic circulation and directly collected from ultrafiltration port were stored on ice prior to testing for mouse insulin enzyme-linked immunosorbent assay (ELISA) (Mercodia: 10-1247-01) and c-peptide ELISA kits (EMD Millipore: EZRMCP2-21K).
Patency Assessment and Device Explant with Islet Retrieval
On POD 3 both animals were taken back to the operating room for assessment of patency and device retrieval. Once the animal was intubated and sedated the incision was re-opened and the device was delivered into the superficial tissue for visual assessment and confirmation of maintained patency. A final IVGTT was administered. As mentioned, for pig #1, blood was sampled directly from the outflow vein via direct cannulation of the external jugular vein distal to the anastomosis. For pig #2, blood was sampled from the tunneled catheter. Once the IVGTT was completed, the carotid was cannulated proximal to the anastomosis with a 5Fr catheter. Radiopaque contrast media was then injected (VisipaqueTM, GE Healthcare, Little Chalfont, United Kingdom) to fluoroscopically confirm flow through the device (
Islet viability was assessed by double staining with the Live/Dead Cell Imaging Kit (488/570) (Life Technology: R37601). Live cells are distinguished by the presence of intensely fluorescent calcein (green) which is well-retained within live cells, whereas dead cells are stained with red. Briefly, agarose-encapsulated mouse islets were incubated in the mixture of live (green) and dead (red) kit components for 15 min and extensively washed in phosphate buffered saline (PBS) to remove excess staining. Images of mouse islets were obtained using laser scanning Nikon Spectral Clsi confocal microscope (Nikon Instruments). The percentage of viability was calculated based on the ratio of non-dead or the green area over the entire area of that islet.
For observation, SNM were fixed in a solution containing 3% glutaraldehyde (Sigma:
G7651), 1 M sodium cacodylate (Polysciences) and 0.1 M sucrose (Sigma). After 2 days, the substrates were washed with distilled water. Dehydration was achieved by placing these scaffolds in an increasing concentration of ethanol (50-100%). Dehydrated samples were then mounted on aluminum stubs, sputter-coated with gold-palladium, and examined with scanning electron microscopy (SEM) (Ultra 55, Carl Zeiss).
The SNM were fixed with 4% paraformaldehyde followed by PBS washes and incubated in blocking solution (PBS, 1% bovine serum albumin (BSA)) for 30 min. Samples were then incubated with CD41 antibody (Biorbyt: orb181793) for platelet adhesion (green) and CD62p antibody (Bioss: bs-0561R-Cy3) for platelet activation (red) at a dilution of 1:300 for 4 h and repeatedly washed with PBS to remove residues. Images were obtained using 6D High Throughput Perfect Focus System (Nikon Instruments).
Sample pairs were analyzed using Student's t-test. Multiple samples were evaluated with one-way or two-way analysis of variance (ANOVA) followed by Bonferroni and multiple comparison using Graphpad Prism software (San Diego, Calif.). A p value of <0.05 was accepted as statistically significant for all analyses.
iBAP Testing in vitro
The iBAP comprising a ˜10 nm pore sized SNM with 10% (5,700 IE/cm2) or 20% (11,400 IE/cm2) mouse islet densities was investigated for glucose-stimulated insulin response and viability of the encapsulated islets after three days. Under convection, the iBAP with 10% mouse islet density and ˜10 nm pore sized SNM showed an increase in insulin secretion within 10 minutes of high glucose exposure (
The viability study of ˜10 nm pore sized SNM in the iBAP demonstrated that 10% mouse islet density under convection (40±11%) showed a higher viability compared to that under diffusion (4.0±1.3%) (
To verify whether the pore size was the limiting factor in causing cell death at the higher density, the ˜40 nm pore sized SNM with 10% or 20% mouse islet densities was studied in the iBAP under diffusion and convection. The glucose-stimulated insulin study showed that both 10% (
Compared to the previous in vitro experiments using ˜10 nm pore sized SNM, the ˜40 nm pore sized SNM enhanced the viability at 10% and 20% mouse islet densities to 66±4.8% and 61 ±3.0% as compared to 40±11% and 17±11% for the ˜10 nm pore sized SNM under convection, respectively (
iBAP Implantation in Pigs
As a first step to study the device and membrane patency, the diffusion-based iBAP with ˜10 nm pore sized SNM and a 5% mouse islet density (2,850 IE/cm2) was intravascularly grafted in the porcine model for three days. The angiogram showed no thrombosis formation and obstruction in the blood flow path of the device during explant (
Next, the performance of the iBAP with SNM-encapsulated mouse islets under diffusion and convection at a higher islet density was evaluated to demonstrate the effectiveness of convective mass transfer for supporting islet viability and functionality. Specifically, the iBAP with ˜10 nm pore size SNM and a 10% mouse islet density (5,700 IE/cm2) with convective and diffusive mechanism was grafted to the carotid artery and vein of a pig for three days. A pro-inflammatory response was also observed for this pig immediately after the surgery (
The silicon nanopore membrane possesses ultra-high-hydraulic permeability. A range of different pore-size SNM (5-500 nm) has been tested to generate the appropriate ultrafiltrate rates to deliver the necessary convective mass transfer of nutrients and insulin, while still maintaining immunoisolation.
The silicon nanopore membrane possesses ultra-selective precise slit-shaped nanopores to achieve immunoisolation.
SNM with highly precise pore were fabricated using an innovative process and demonstrated excellent hydraulic permeability. Microelectromechanical systems (MEMS) fabrication technology produced the SNM. A thin sacrificial SiO2 layer is grown defining the submicron pore size of the membrane. Thermal oxidation of silicon substrates provides oxides down to 3 nm in thickness with <1% variation. The oxide is etched leaving behind open parallel-plate nanochannel pores. 0.25-1.00 μm-thick polycrystalline silicon membranes with pore sizes between 5-500 nm supported by a 400 μm-thick support structure were fabricated. The SNM hydraulic permeability was tested in a custom flow cell with cross-flow and a transmembrane (TMP) pressure. Hydraulic permeability experiments demonstrated greater hydraulic permeability of SNM than conventional polymer membranes.
Polymer coated SNM reduced protein adsorption and provided a hemocompatible surface. Polymer coatings were evaluated on silicon substrates to retard protein fouling. Polyethylene glycol (PEG), polysulfobetaine methyacrylate (pSBMA), and poly(N-vinyldextran aldonamide-co-N-vinylhexanamide) (PVAm-Dex/Hex) were chosen to reduce nonspecific protein adsorption. A Rapid In Vivo IntraVascular Evaluation (RIVIVE) protocol inserted PEG-coated and uncoated (bare) silicon darts in femoral veins of Wistar rats. After 30 days of implantation, the darts and vessels were explanted and evaluated by gross examination, histology, and scanning electron microscopy (SEM) (
SNM demonstrated successful immunoisolation. Islet immunoisolation was also studied in vitro using cell culture medium with or without cytokines (IL-1β, TNF-α, and IFN-γ). Islets were encapsulated in between two 7 nm pore SNM. After six hours, the islets were removed and stained for viability. Control static culture experiments were performed to determine the effect of SNM.
A small-scale cell scaffold has been developed and enables testing of enriched insulin producing cells under convection. In order to test ultrafiltrate formation under convective mass transport, a small-scale Cell Scaffold has been developed and tested with islets. The Cell Scaffold consists of a hexagonal arrangement of eight 100 μm diameter cylindrical ultrafiltrate channels (solid circles in
A small-scale iBAP prototype has been developed for vitro and in vivo Cell Scaffold testing and in vivo hemocompatibility testing.
In vitro small-scale Cell Scaffold testing demonstrated increased islet viability with convection versus diffusion and promising glucose-insulin kinetics. Freshly isolated mouse islets at either 10% or 20% islet density by volume (or 5,700 IEQ/cm2 or 11,400 IEQ/cm2 respectively) within the small-scale Cell Scaffold were loaded into an iBAP containing 40 nm SNM. The iBAP devices were connected to a mock circuit loop (
Human islet function was assessed in a 90-minute glucose-insulin kinetics study. Freshly isolated human islets at 10% islet density in the small-scale Cell Scaffold were loaded into an iBAP with a 40 nm SNM and then stabilized in low glucose cell culture medium for 1 hour in the mock circuit loop. At time zero, the glucose concentration was increased for 70 minutes and ultrafiltrate samples were collected from the Ultrafiltrate Outlet.
A prototype full-scale iBAP was designed, CFD modeled, and demonstrated blood flow path patency in an in vivo 7-day hemocompatibility study. Full-scale iBAP blood flow path designs were generated in SolidWorks and analyzed in ANSYS Fluent to determine the feasibility of extending the blood flow path from the small-scale iBAP prototype. This first generation full-scale iBAP was manufactured from medical grade polycarbonate and loaded with SNM. The full-scale iBAP was intravascularly implanted into a healthy pig (
The iBAP will be connected to arterio-venous grafts and a pressure drop between the artery and vein will produce ultrafiltrate flow through the SNM encapsulated Cell Scaffold seeded with insulin producing cells, carrying nutrients to the cells and insulin to the ultrafiltrate vein (
This application claims priority to U.S. Provisional Application No. 62/304,758 filed Mar. 7, 2016 and to U.S. Provisional Application No. 62/328,298 filed Apr. 27, 2016, which are both herein incorporated by reference in their entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2017/021196 | 3/7/2017 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62328298 | Apr 2016 | US | |
| 62304758 | Mar 2016 | US |
