Biocompatible membrane composite

Abstract

To provide a biocompatible membrane composite capable of providing an environment capable of mitigating or controlling a foreign body reaction.SOLUTION: The membrane composite includes a conformable layer and a vascularizing layer. A reinforcing component can optionally be included to provide support to the biocompatible membrane composite invivo and prevent distortion of the membrane composite. The relief layer may be coupled or attached to the implantable device and / or the cell system. The biocompatible membrane composite can be used as a surface layer for implantable devices or cell systems that require vascularization for function but require protection from a host immune response, such as the formation of foreign body giant cells. The biocompatible membrane composite can partially or completely cover the exterior of the implantable device or cell system. The relief layer is positioned between the implantable device or bioactive scaffold and the vascularizing layer.SELECTED DRAWING: Figure 7

Description

[Technical Field]

[0001] The present invention relates generally to implantable devices, and specifically to biocompatible membrane composites and uses thereof. [Background technology]

[0002] Biological therapy is an increasingly viable method of treating peripheral arterial disease, aneurysms, heart disease, Alzheimer's and Parkinson's disease, autism, blindness, diabetes, and other pathologies.

[0003] With regard to biological therapies in general, cells, viruses, viral vectors, bacteria, proteins, antibodies, and other biologically active moieties can be introduced into a patient by surgical or interventional methods that place the biologically active moiety within the patient's tissue bed. Often, the biologically active moiety is first placed into a device that is then inserted into the patient. Alternatively, the device may be inserted into the patient first, and the biologically active moiety may be added later.

[0004] Implantation of a foreign device (e.g., a cell encapsulation device, a sensor, and / or a monitor for measuring physical parameters and / or analytes within the body) elicits an immune response. The immune response results in the formation of foreign body giant cells that at least partially encapsulate the implanted device. The device can be formed from one or more biocompatible membranes or other biocompatible materials that allow nutrients or other therapeutically useful substances to pass through but prevent the passage of cells. The presence of foreign body giant cells at or near a cell-impermeable interface makes it difficult, if not impossible, for blood vessels to form adjacent to this surface, thereby limiting access to oxygen, nutrients, analytes, or other signaling across the device interface necessary for adequate device function.

[0005] Thus, there remains a need in the art for materials that can be utilized in or provide an environment in which the foreign body response can be mitigated or regulated so that sufficient vascularization occurs at or near the surface of the cell-impermeable interface, thereby allowing implanted encapsulated cells to survive and secrete therapeutically useful substances, while allowing the implanted device access to analytes and physical parameters for measurement. Summary of the Invention [Means for solving the problem]

[0006] According to one embodiment (“Embodiment 1”), a biocompatible membrane composite includes a first layer having first solid features with a first solid feature spacing, wherein a majority of the first solid feature spacing is less than about 50 microns; and a second layer having second solid features with a second solid feature spacing, wherein a majority of the second solid feature spacing is greater than about 50 microns.

[0007] According to another embodiment ("embodiment 2") in addition to embodiment 1, the first layer comprises a majority of a representative minor axis of about 3 microns to about 20 microns.

[0008] According to another embodiment ("embodiment 3") in addition to embodiment 1 or embodiment 2, the second layer has a first pore size greater than about 9 microns in effective diameter.

[0009] According to another embodiment ("embodiment 4") in addition to any one of embodiments 1 to 3, the first thickness of the first layer is less than about 200 microns.

[0010] According to another embodiment ("embodiment 5") in addition to any one of embodiments 1 to 4, the first layer has a second pore size with an effective diameter of about 1 micron to about 9 microns.

[0011] According to another aspect ("Aspect 6") in addition to Aspect 5, the solid features of at least one of the first layer and the second layer are connected by fibrils, and the fibrils are deformable.

[0012] According to another embodiment ("embodiment 7") in addition to any one of embodiments 1 to 5, the second thickness of the second layer is from about 30 microns to about 200 microns.

[0013] According to another aspect ("Aspect 8") of any one of Aspects 1 to 6, at least one of the first layer and the second layer comprises a polymer selected from an expanded polytetrafluoroethylene (ePTFE) membrane, a fluorinated ethylene propylene (FEP) membrane, and a modified expanded polytetrafluoroethylene (ePTFE) membrane.

[0014] According to another aspect ("Aspect 9") of any one of Aspects 1 to 8, the biocompatible membrane composite comprises a surface coating thereon, the surface coating comprising one or more members selected from an antimicrobial agent, an antibody, a pharmaceutical agent, and a biologically active molecule.

[0015] According to another aspect ("Aspect 10") in addition to any one of Aspects 1 to 9, at least one of the first layer and the second layer is an expanded polytetrafluoroethylene membrane.

[0016] According to another embodiment (“embodiment 11”) in addition to any one of embodiments 1 through 10, the second layer is a spunbond nonwoven polyester material.

[0017] According to another embodiment ("embodiment 12") in addition to any one of embodiments 1 through 10, a reinforcing component is included.

[0018] According to another embodiment ("embodiment 13") in addition to embodiment 12, the reinforcing component is a woven or nonwoven fabric.

[0019] According to another embodiment (“Embodiment 14”) in any one of Embodiments 1 to 13, the solid features of the first layer further comprise a representative minor axis, a representative major axis, and a solid feature depth, and a majority of at least two of the representative minor axis, the representative major axis, and the solid feature depth is greater than about 5 microns.

[0020] In another embodiment (“Embodiment 15”) of any one of Embodiments 1 through 14, the first layer includes a first layer having a first pore size of about 1 micron to about 9 microns effective diameter, a first thickness of less than about 200 microns, first solid features with a majority of the first solid feature spacing less than about 50 microns, and a majority of the first solid features having a first representative minor axis of about 3 microns to about 20 microns; and a second layer.

[0021] According to another embodiment (“embodiment 16”) in any one of embodiments 1 to 15, the second layer has a pore size that is greater than about 9 microns in effective diameter.

[0022] According to another embodiment (“Embodiment 17”) in any one of Embodiments 1 to 16, the second layer includes second solid features, the majority of the second solid feature spacing being greater than about 50 microns.

[0023] According to another embodiment ("embodiment 18") in addition to any one of embodiments 15 to 17, the second thickness of the second layer is from about 30 microns to about 200 microns.

[0024] According to another embodiment ("Embodiment 19") in any one of Embodiments 15 to 18, the first solid feature of the first layer comprises a first representative major axis and a first solid feature depth, and at least two of the first representative minor axis, the first representative major axis, and the first solid feature depth are greater than about 5 microns.

[0025] According to another embodiment ("embodiment 20") in addition to any one of embodiments 15 to 19, the solid features are connected by fibrils, and the fibrils are deformable.

[0026] According to another embodiment ("embodiment 21") in any one of embodiments 15 to 20, the second layer includes second solid features, and a majority of the second solid features have a second representative minor axis of less than about 40 microns.

[0027] According to another embodiment ("Embodiment 22") in any one of Embodiments 15 to 21, the second layer comprises a second representative major axis and a second solid feature depth, and a majority of at least two of the second representative minor axis, the second representative major axis, and the second solid feature depth is greater than about 5 microns.

[0028] According to another aspect ("Aspect 23") in any one of Aspects 15 to 22, at least one of the first layer and the second layer is a polymer selected from an expanded polytetrafluoroethylene (ePTFE) membrane, a fluorinated ethylene propylene (FEP) membrane, and a modified expanded polytetrafluoroethylene (ePTFE) membrane.

[0029] According to another embodiment (“embodiment 24”) in addition to any one of embodiments 15 to 23, the second layer is a spunbond nonwoven polyester material.

[0030] According to another embodiment (“Aspect 25”) in any one of Aspects 15 to 24, at least one of the first layer and the second layer comprises a polymer, a fluoropolymer membrane, a non-fluoropolymer membrane, a woven fabric, a nonwoven fabric, a woven or nonwoven collection of fibers or yarns, a fibrous matrix, and combinations thereof.

[0031] According to another aspect ("Aspect 26") in any one of Aspects 15 to 25, the first solid features of the first layer include a member selected from a thermoplastic polymer, a polyurethane, a silicone, a rubber, an epoxy, and combinations thereof.

[0032] According to another embodiment ("embodiment 27") in addition to any one of embodiments 15 to 26, a reinforcing component is included.

[0033] According to another embodiment ("embodiment 28") in addition to embodiment 27, the reinforcing component is a woven or nonwoven fabric.

[0034] According to another embodiment ("embodiment 29") of any one of embodiments 15 to 28, the biocompatible membrane composite comprises a surface coating thereon, and the surface coating comprises one or more members selected from an antimicrobial agent, an antibody, a pharmaceutical agent, and a biologically active molecule.

[0035] According to another embodiment (“embodiment 30”) in any one of embodiments 15 to 29, the biocompatible membrane composite includes a hydrophilic coating thereon.

[0036] According to another embodiment ("Embodiment 31") in any one of embodiments 15 to 30, the first layer includes bondable solid features, and the bondable solid features are bonded to an implantable device or an implantable cell system.

[0037] According to another embodiment ("embodiment 32") in addition to embodiment 31, the implantable device is a scaffold.

[0038] According to another embodiment ("embodiment 33") in addition to embodiment 32, the scaffold is a cell culture matrix.

[0039] According to another embodiment ("embodiment 34") in addition to embodiment 32, the scaffold is an explant.

[0040] According to another embodiment ("embodiment 35") in addition to embodiment 31, the first solid feature is at least partially attached to the cellular system.

[0041] According to another embodiment ("embodiment 36") in addition to embodiment 35, the cell system is a cell container.

[0042] According to another aspect ("Aspect 37") in addition to Aspect 31, the implantable device is a sensor.

[0043] According to another embodiment ("Embodiment 38") in addition to embodiment 31, the cell system is a bioactive scaffold.

[0044] According to another embodiment ("Embodiment 39") in any of the preceding embodiments, a method for lowering blood glucose levels in a mammal comprises implanting a cell encapsulation device comprising a biocompatible membrane composite of any of the preceding claims, wherein cells encapsulated within the cell encapsulation device comprise a population of PDX1-positive pancreatic endoderm cells, and wherein the pancreatic endoderm cells grow to become insulin-secreting cells, thereby lowering blood glucose levels.

[0045] According to another embodiment ("Embodiment 40") of any of the preceding embodiments, the PDX1-positive pancreatic endoderm cells comprise a mixture of cells further comprising endocrine and / or endocrine precursor cells, wherein the endocrine and / or endocrine precursor cells express chromogranin A (CHGA).

[0046] According to another embodiment ("Embodiment 41") in addition to any of the preceding embodiments, the method includes implanting the cell encapsulation device of claim 1, wherein the cells encapsulated within the cell encapsulation device comprise a population of PDX1-positive pancreatic endoderm cells, and the pancreatic endoderm cells grow to become insulin-secreting cells, thereby lowering blood glucose levels.

[0047] According to another embodiment ("Embodiment 42") of any of the preceding embodiments, the PDX1-positive pancreatic endoderm cells comprise a mixture of cells further comprising endocrine and / or endocrine precursor cells, wherein the endocrine and / or endocrine precursor cells express chromogranin A (CHGA).

[0048] According to another embodiment (“Embodiment 43”) in addition to any of the preceding embodiments, a method of lowering blood glucose levels in a mammal comprises implanting a cell encapsulation device, the cell encapsulation device comprising at least one sensor and a biocompatible membrane composite, the biocompatible membrane composite at least partially covering the sensor, the biocompatible membrane composite comprising: a first layer having first solid features with a first solid feature spacing, a majority of the first solid feature spacing being less than about 50 microns; and a second layer having second solid features with a second solid feature spacing, a majority of the second solid feature spacing being greater than about 50 microns, the first layer being positioned between the sensor and the second layer, at least some of the bonded features being closely bonded to the first layer; and the cell encapsulation device comprising a cell population comprising PDX1-positive pancreatic endoderm cells, and the pancreatic endoderm cells growing to become insulin-secreting cells, thereby lowering blood glucose levels.

[0049] According to another embodiment ("Embodiment 44") of any of the preceding embodiments, the PDX1-positive pancreatic endoderm cells comprise a mixture of cells further comprising endocrine and / or endocrine precursor cells, wherein the endocrine and / or endocrine precursor cells express chromogranin A (CHGA).

[0050] According to another embodiment ("Embodiment 45") in any of the preceding embodiments, a method for lowering blood glucose levels in a mammal comprises implanting at least one sensor and a biocompatible membrane composite, wherein the biocompatible membrane composite at least partially covers the sensor, the biocompatible membrane composite comprising: a first layer having first solid features with a first solid feature spacing, wherein a majority of the first solid feature spacing is less than about 50 microns; and a second layer having second solid features with a second solid feature spacing, wherein a majority of the second solid feature spacing is greater than about 50 microns, the first layer being positioned between the sensor and the second layer, and at least some of the bonded features being closely bonded to the first layer; and the biocompatible membrane composite comprising a cell population comprising PDX1-positive pancreatic endoderm cells, and the pancreatic endoderm cells growing to become insulin-secreting cells, thereby lowering blood glucose levels.

[0051] According to another embodiment ("Embodiment 46") of any of the preceding embodiments, the PDX1-positive pancreatic endoderm cells comprise a mixture of cells further comprising endocrine and / or endocrine precursor cells, wherein the endocrine and / or endocrine precursor cells express chromogranin A (CHGA).

[0052] According to another embodiment ("Embodiment 47") of any of the preceding embodiments, the encapsulated in vitro PDX1-positive pancreatic endoderm cells comprise a mixture of cell subpopulations including at least a pancreatic progenitor population that co-expresses PDX-1 / NKX6.1.

[0053] According to another embodiment ("Embodiment 48") of any of the preceding embodiments, the encapsulated in vitro PDX1-positive pancreatic endoderm cells comprise a mixture of cell subpopulations including at least a pancreatic progenitor population that co-expresses PDX-1 / NKX6.1 and a pancreatic endocrine and / or endocrine precursor population that expresses PDX-1 / NKX6.1 and CHGA.

[0054] According to another embodiment ("Embodiment 49") of any of the preceding embodiments, at least 30% of the population comprises a pancreatic progenitor population that co-expresses PDX-1 / NKX6.1.

[0055] According to another embodiment ("Embodiment 50") of any of the preceding embodiments, at least 40% of the population comprises a pancreatic progenitor population that co-expresses PDX-1 / NKX6.1.

[0056] According to another embodiment ("Embodiment 51") of any of the preceding embodiments, at least 50% of the population comprises a pancreatic progenitor population that co-expresses PDX-1 / NKX6.1.

[0057] According to another embodiment ("Embodiment 52") of any of the preceding embodiments, at least 20% of the population is an endocrine and / or endocrine precursor population that expresses PDX-1 / NKX6.1 / CHGA.

[0058] According to another embodiment ("Embodiment 53") of any of the preceding embodiments, at least 30% of the population is an endocrine and / or endocrine precursor population that expresses PDX-1 / NKX6.1 / CHGA.

[0059] According to another embodiment ("Embodiment 54") of any of the preceding embodiments, at least 40% of the population is an endocrine and / or endocrine precursor population that expresses PDX-1 / NKX6.1 / CHGA.

[0060] According to another embodiment ("embodiment 55") of any of the preceding embodiments, the pancreatic progenitor cells and / or endocrine or endocrine precursor cells are capable of growing into insulin-secreting cells in vivo.

[0061] According to another embodiment ("Embodiment 56") in any of the preceding embodiments, a method for producing insulin in vivo comprises implanting a cell encapsulation device comprising a biocompatible membrane composite of any of the preceding claims and a population of PDX1-positive pancreatic endoderm cells that grow into insulin-secreting cells, wherein the insulin-secreting cells secrete insulin in response to glucose stimulation.

[0062] According to another embodiment ("Embodiment 57") of any of the preceding embodiments, the PDX1-positive pancreatic endoderm cells comprise a mixture of cells further comprising endocrine and / or endocrine precursor cells, wherein the endocrine and / or endocrine precursor cells express chromogranin A (CHGA).

[0063] According to another embodiment ("Embodiment 58") of any of the preceding embodiments, at least about 30% of the population is an endocrine and / or endocrine precursor population that expresses PDX-1 / NKX6.1 / CHGA.

[0064] According to another embodiment ("Embodiment 59") of any of the preceding embodiments, the in vitro human PDX1-positive pancreatic endoderm cell culture comprises a mixture of PDX1-positive pancreatic endoderm cells and at least a transforming growth factor beta (TGF-beta) receptor kinase inhibitor.

[0065] According to another embodiment ("embodiment 60") of any of the preceding embodiments, further comprising a bone morphogenetic protein (BMP) inhibitor.

[0066] According to another embodiment ("embodiment 61") of any of the preceding embodiments, the TGF-beta receptor kinase inhibitor is a TGF-beta receptor type 1 kinase inhibitor.

[0067] According to another embodiment ("embodiment 62") of any of the preceding embodiments, the TGF-beta receptor kinase inhibitor is ALK5i.

[0068] According to another embodiment ("embodiment 63") of any of the preceding embodiments, the BMP inhibitor is noggin. [Brief explanation of the drawings]

[0069] The accompanying drawings are included to provide a further understanding of the disclosure, and are incorporated in and constitute a part of this specification, illustrating embodiments and, together with the description, serving to explain the principles of the disclosure.

[0070] [Figure 1A] FIG. 1A is a schematic diagram illustrating the determination of solid feature spacing according to an embodiment described herein, where three adjacent solid features represent the corners of a triangle, the circumscribing circle of the triangle has an interior without additional solid features, and the solid feature spacing is the linear distance between two of the solid features that form the triangle.

[0071] [Figure 1B] FIG. 1B is a schematic diagram illustrating the determination of non-adjacent solid features according to an embodiment described herein, where the solid features represent corners of a triangle and the circumscribing circle of the triangle contains at least one additional solid feature.

[0072] [Figure 2] FIG. 2 is a scanning electron micrograph showing the spacing (white lines) between solid features (white shapes) within an ePTFE membrane according to an embodiment described herein.

[0073] [Figure 3A] FIG. 3A is a schematic diagram illustrating a method for determining the major and minor axes of a solid feature according to embodiments described herein.

[0074] [Figure 3B] FIG. 3B is a schematic diagram illustrating the depth of solid features according to embodiments described herein.

[0075] [Figure 4] FIG. 4 is a schematic diagram showing effective diameters of holes according to embodiments described herein.

[0076] [Figure 5] FIG. 5 is a scanning electron micrograph (SEM) showing pore sizes according to an embodiment described herein.

[0077] [Figure 6A] FIG. 6A is a cross-sectional view that schematically illustrates an implantable device that can be at least partially covered by a biocompatible membrane composite according to embodiments described herein.

[0078] [Figure 6B] FIG. 6B is a schematic diagram illustrating a bioactive scaffold that can be at least partially covered by a biocompatible membrane composite according to embodiments described herein.

[0079] [Figure 7] FIG. 7 is a schematic diagram illustrating a biocompatible membrane composite according to an embodiment described herein.

[0080] [Figure 8] FIG. 8 is a schematic diagram illustrating another biocompatible membrane composite according to embodiments described herein.

[0081] [Figure 9] FIG. 9 is a schematic diagram illustrating yet another biocompatible membrane composite according to embodiments described herein.

[0082] [Figure 10] FIG. 10 is a scanning electron micrograph (SEM) showing the top surface of the ePTFE relaxed layer of Example 1 according to an embodiment described herein.

[0083] [Figure 11] FIG. 11 is a scanning electron micrograph (SEM) showing the top surface of a vascularized layer formed from nonwoven polyester utilized in Example 1 according to an embodiment described herein.

[0084] [Figure 12] FIG. 12 is an exploded view showing the materials and fastener configuration utilized in Example 1 according to an embodiment described herein.

[0085] [Figure 13] FIG. 13 is a representative SEM image showing the second ePTFE layer of Structures A, B, and C of Example 2 with FEP according to an embodiment described herein.

[0086] [Figure 14] Figure 14 is a representative SEM image showing the node-fibril structure of the second ePTFE membrane in Structure A of Example 2, according to an embodiment described herein.

[0087] [Figure 15] Figure 15 is a representative SEM image showing the node-fibril structure of the second ePTFE membrane in Structure B of Example 2, based on an embodiment described herein.

[0088] [Figure 16] Figure 16 is a representative SEM image showing the node-fibril structure of the second ePTFE membrane in Structure C of Example 2, based on an embodiment described herein.

[0089] [Figure 17] FIG. 17 is an SEM image showing a cross section of a biocompatible membrane composite of Structure A of Example 2 according to an embodiment described herein.

[0090] [Figure 18]FIG. 18 is an SEM image showing a cross section of a biocompatible membrane composite of Structure B of Example 2 according to an embodiment described herein.

[0091] [Figure 19] FIG. 19 is an SEM image showing a cross section of a biocompatible membrane composite of Structure C of Example 2 according to an embodiment described herein. DETAILED DESCRIPTION OF THE INVENTION

[0092] It will be readily apparent to those skilled in the art that various aspects of the present disclosure may be implemented by any number of methods and apparatuses configured to perform their intended functions. Furthermore, for clarity, the accompanying drawings referred to herein are not necessarily drawn to scale and may be exaggerated to illustrate various aspects of the present disclosure, and the drawings should not be construed as limiting in this regard. Directional references, such as "up," "down," "top," "left," "right," "front," and "back," among others, are intended to refer to the orientation shown and described in the drawings in which the components and directions are referenced. It should be understood that the terms "biocompatible membrane composite" and "membrane composite" are used interchangeably herein. It should be noted that all ranges described herein are exemplary in nature and include any and all values ​​therebetween. Additionally, all references cited herein are incorporated by reference in their entirety.

[0093] The present disclosure relates to a biocompatible membrane composite capable of providing an environment capable of mitigating or modulating a foreign body response. The biocompatible membrane composite contains a first layer and a second layer. Each layer is distinguishable from the other and serves a unique function of helping to mitigate the formation of foreign body giant cells on a cell-impermeable layer of an implantable device or bioactive entity (e.g., a bioactive scaffold). In certain embodiments, the first layer functions as a relaxing layer, and the second layer functions as an angiogenic layer. For convenience, the term "first layer" is used interchangeably with "relaxing layer," and the term "second layer" is used interchangeably with "angiogenic layer" herein. The relaxing layer is positioned between the implantable device or bioactive entity and the angiogenic layer. In at least one embodiment, the relaxing layer includes solid features (e.g., nodes) inherently present within the membrane forming the relaxing layer. Reinforcement components can be optionally positioned on either side (i.e., outside) or within (i.e., inside) the biocompatible membrane composite to provide support and prevent distortion of the biocompatible membrane composite. The relaxed layer can be bonded (e.g., point bonded or welded) to the implantable device and / or bioactive entity. In some embodiments, the relaxed layer and the vascularized layer can be intimately bonded or connected to each other to form a composite layer having an open / open structure. As used herein, the terms "intimately bonded" and "intimately bonded" refer to a layer of a biocompatible composite that cannot be easily separated or detached at any point on its surface or a solid feature within the biocompatible composite. Of course, as used herein, the term "about" refers to ±10% of the specified unit of measurement.

[0094] In at least one embodiment, the relaxed layer and the vascularized layer are bonded together with one or more biocompatible adhesives to form a biocompatible membrane composite. The adhesives can be applied to the surfaces of one or both of the relaxed and vascularized layers to form discontinuous or intimate bonds between the layers. As used herein, the terms "discontinuous bonds" or "discontinuously bonded" are intended to include bonds or bonds that form an intentional pattern of dots and / or lines around the continuous perimeter of a defined area. Suitable biocompatible adhesives include, but are not limited to, fluorinated ethylene propylene (FEP), polycarbonate urethane, thermoplastic fluoropolymers consisting of TFE and PAVE, EFEP (ethylene fluorinated ethylene propylene), PEBAX (polyether amide), PVDF (polyvinylidene fluoride), Carbosil® (ab silicone polycarbonate urethane), Elasthane® (polyether urethane), PurSil® (silicone polyether urethane), polyethylene, high density polyethylene (HDPE), ethylene chlorotetrafluoroethylene (ECTFE), perfluoroalkoxy (PFA), polypropylene, polyethylene terephthalate (PET), and combinations thereof.

[0095] In some embodiments, the biocompatible membrane composites described herein can be utilized as a biological interface for implantable sensors used to detect molecules produced within the body (e.g., glucose or other bioactive molecules) or molecules produced outside the body (e.g., molecules from ingested food). In another embodiment, the biocompatible membrane composites can be used as a biocompatible cover for implantable devices, such as pacemakers, that provide or require molecules, signals, or activity within the body, thereby deriving these functions. The implantable devices can be used to measure physical parameters of the body, such as blood pressure. The term "implantable device" is used herein to encompass any implantable sensor or device. In other embodiments, the biocompatible membrane composites can be used as a surface layer or surrounding cover for implantable devices that require angiogenesis for function but also require protection from a host immune response, such as foreign body giant cell formation. The implantable device may contain a third layer (i.e., a cell-impermeable layer) thereon. The cell-impermeable layer serves as a microporous immunoisolation barrier, rejecting vascular ingrowth and preventing cellular contact from the host. In another embodiment, the biocompatible membrane composite may be used in conjunction with tissues, cell scaffolds, or cell encapsulation devices. Some examples include explants, two-dimensional (2D) and three-dimensional (3D) cell culture systems or cell containers. The generic term "cell system" is used herein to describe any biological entity that may be used in conjunction with the biocompatible membrane composite.

[0096] Some examples of implantable device elements that can benefit from the functionality of biocompatible membrane composites include switches, sensors, bolometers, biosensors, chemical sensors, inertial sensors, acoustic sensors, microphones, microspeakers, pressure sensors, resonators, ultrasonic resonators, temperature sensors, vibration sensors, microengines, actuators, thermal actuators, bimorph and unimorph actuators (e.g., piezoelectric and thermal), electrical rotating micromachines, microgears, micropumps, microtransmitters, microengines, optical microelectromechanical systems (MEMS), micromirrors, optical switches, and biological microelectromechanical systems (MEMS).

[0097] The interface between the biocompatible membrane composite and the implantable device is a relaxing layer. The relaxing layer is sufficiently porous to allow vascular tissue growth into the relaxing layer. Thus, in some cases, the relaxing layer acts as an early vascularization layer. The relaxing layer creates a favorable environment on or near the surface of the implantable device to minimize or prevent the formation of an adjacent foreign body giant cell layer, while allowing blood vessels to access the surface of the implantable device. A layer with openings large enough to allow vascular ingrowth is sometimes referred to herein as an "open" layer. Blood vessels, which are a source of oxygen and nutrients for the implantable device, need to be formed at a distance from the implantable device so that signals can be easily detected and transmitted. Non-limiting examples of signals include glucose, oxygen, growth factors, or any analyte requiring detection or monitoring.

[0098] The relaxed layer is characterized, at least in part, by including a plurality of solid features having a solid feature spacing. As used herein, "solid features" can be defined as three-dimensional elements within the relaxed layer that are generally immobile and resistant to deformation when exposed to environmental forces, such as cell movement (e.g., cell migration and ingrowth, host angiogenesis / endothelialization). The solid features within the relaxed layer may be formed from thermoplastic polymers, polyurethanes, silicones, rubbers, epoxies, and combinations thereof.

[0099] In embodiments in which the relaxation layer has a node-fibril microstructure (e.g., formed from a fibrillating polymer), the nodes are solid features and the fibrils are not solid features. Indeed, in some embodiments, the fibrils can be removed, leaving only the nodes in the relaxation layer. In embodiments in which the nodes within the relaxation layer are solid features, these nodes are intimately bonded to the device or sensor interface and are referred to herein as "bonded solid features." "Unbonded solid features" are solid features within the relaxation layer that are not bonded (intimately bonded or otherwise) to the device or sensor interface. In one embodiment, the relaxation layer is formed from an expanded polytetrafluoroethylene (ePTFE) membrane having a node-fibril microstructure.

[0100] The majority of solid feature spacing for adjacent solid features in an implantable device or cell system is less than about 50 microns, less than about 40 microns, less than about 30 microns, less than about 20 microns, or less than about 10 microns. As used herein, the term "majority" refers to more than half (i.e., greater than 50%) of the measured parameter. In some embodiments, the majority of solid feature spacing may be between about 5 microns and about 45 microns, between about 10 microns and about 40 microns, between about 10 microns and about 35 microns, or between about 15 microns and about 35 microns. The phrase "solid feature spacing" is defined herein as the linear distance between two adjacent solid features. In the present disclosure, solid features are considered adjacent if their centroids represent the corners of a triangle whose circumscribing circle has an empty interior. As pictorially shown in FIG. 1A, a triangle 100 is formed by connecting a designated solid feature (P) to an adjacent solid feature (N). In the triangle, the circumscribing circle 110 does not contain any solid features within it. Solid feature (X) designates a solid feature that is not an adjacent solid feature. Thus, in the example shown in FIG. 1A, solid feature spacing 130 is the linear distance between designated solid features (P) and (N). In contrast, circumscribed circle 150 shown in FIG. 1B, drawn from triangle 160, contains solid feature (N) and, as such, cannot be used to determine solid feature spacing within the relaxed layer (or vascularized layer). FIG. 2 is a scanning electron micrograph showing measured distances, e.g., white lines 200, between solid features 210 (white shapes) within a relaxed layer formed from an expanded polytetrafluoroethylene membrane.

[0101] A solid feature also includes a representative minor axis, a representative major axis, and a solid feature depth. The representative minor axis of a solid feature is defined herein as the length of the minor axis of an ellipse fitted to the solid feature. The ellipse has the same area, orientation, and centroid as the solid feature. The representative major axis of a solid feature is defined herein as the length of the major axis of an ellipse fitted to the solid feature. The ellipse has the same area, orientation, and centroid as the solid feature. The major axis is greater in length than or equal to the minor axis. The minor and major axes of an ellipse 320 for fitting solid feature 310 are shown pictorially in FIG. 3A. The representative minor axis of solid feature 310 is indicated by arrow 300, and the representative major axis of solid feature 310 is indicated by arrow 330. The majority of solid features have a minor axis ranging in size from about 3 microns to about 20 microns, from about 3 microns to about 15 microns, or from about 3 microns to about 10 microns. The solid feature depth is the projected length of the solid feature in an axis perpendicular to the surface of a layer (e.g., the relaxed layer or the vascularized layer). The solid feature depth of solid feature 310 is shown pictorially in FIG. 3B. The depth of solid feature 310 is indicated by line 340. In at least one embodiment, the solid feature depth is equal to or less than the thickness of the relaxed layer. In at least one embodiment, a majority of at least two of the representative minor axis, the representative major axis, and the solid feature depth within the layer are greater than 5 microns.

[0102] In embodiments where the solid features are interconnected by fibrils or fibers, the boundaries interconnecting the solid features form pores. These pores are sufficiently open to allow cell ingrowth and vascularization and not form a resistance to mass transport of oxygen and nutrients. Pore effective diameter is measured by quantitative image analysis (QIA) performed on scanning electron microscope (SEM) images. The term "effective diameter" of a pore is defined as the diameter of a circle having an area equal to the measured area of ​​the surface pores. This relationship is defined by the following equation:

number

[0103] Referring to Figure 4, the effective diameter is the diameter of circle 400, and surface pores are designated by the numeral 420. The total pore area of ​​a surface is the sum of the areas of all pores on that surface. The pore size of a layer is the effective pore diameter that defines the point where approximately half of the total pore area consists of pores with diameters smaller than the pore size, and half of the total pore area consists of pores with diameters greater than or equal to the pore size. Figure 5 shows pore size 500 (white), smaller sized pores 510 (light gray), and larger sized pores 520 (dark gray). Pores 530 that intersect the edge of the image are excluded from the analysis and are shown in black.

[0104] The pore size of the relaxation layer, as measured by quantitative image analysis (QIA) performed on scanning electron micrograph (SEM) images, can be from about 1 micron to about 9 microns in effective diameter, from about 3 microns to about 9 microns in effective diameter, or from about 4 microns to about 9 microns in effective diameter. The thickness of the relaxation layer is less than about 200 microns, less than about 290 microns, less than about 280 microns, less than about 270 microns, less than about 260 microns, less than about 200 microns, less than about 190 microns, less than about 180 microns, less than about 170 microns, less than about 160 microns, less than about 150 microns, less than about 140 microns, less than about 130 microns, less than about 120 microns, less than about 110 microns, less than about 100 microns, less than about 90 microns, less than about 80 microns, less than about 70 microns, or less than about 60 microns, less than about 50 microns, less than about 40 microns, less than about 30 microns, less than about 20 microns, or less than about 10 microns. The thickness of the relaxation layer may be about 60 microns to about 200 microns, about 60 microns to about 170 microns, about 60 microns to about 150 microns, about 60 microns to about 125 microns, about 60 microns to about 100 microns, about 3 microns to about 60 microns, about 10 microns to about 50 microns, about 10 microns to about 40 microns, or about 15 microns to about 35 microns. In some embodiments, the porosity of the relaxation layer is greater than about 60%. In other embodiments, the porosity of the relaxation layer is greater than about 70%, greater than about 80%, greater than about 90%, or greater than about 95%. In some embodiments, the porosity may be about 98% or about 99%. The porosity of the relaxation layer may be about 60% to about 98%, about 70% to about 98%, or about 80% to about 98%.

[0105] Anchoring of the implantable device and ingrowth of vascular tissue through the biocompatible membrane composite to the surface of the device are further facilitated by a second layer (i.e., the vascularization layer). The vascularization layer is an "open" layer. This open layer allows additional vascular penetration from the host and also allows rapid anchoring and attachment of the biocompatible membrane composite within the host's tissue. In addition, the vascularization layer provides a porous matrix to accommodate a sufficient amount of additional new blood vessel growth, for example, to the implantable device or cellular system. In embodiments in which the vascularization layer does not meet the same criteria as the relaxed layer, the relaxed layer and the vascularization layer are considered separate and distinct layers. The vascularization layer is configured with solid features to allow integration and attachment to the host. These solid features have increased spacing and pore size compared to the solid features of the relaxed layer, thereby facilitating more rapid tissue ingrowth into the layer.

[0106] In some embodiments, the majority of the solid features in the vascularized layer have a solid feature spacing greater than about 50 microns, greater than about 60 microns, greater than about 70 microns, or greater than about 80 microns. The majority of the solid features in the vascularized layer have a solid feature spacing of about 50 microns to about 90 microns, about 60 microns to about 90 microns, or about 70 microns to about 90 microns. The pore size and overall thickness of the vascularized layer are sufficient to provide space for the additional vascular volume necessary to provide nutrients and oxygen to the cells. The pore size of the vascularized layer, as measured by quantitative image analysis (QIA) performed on SEM images, may be greater than about 9 microns in effective diameter, greater than about 25 microns in effective diameter, greater than about 50 microns in effective diameter, greater than about 75 microns in effective diameter, greater than about 100 microns in effective diameter, greater than about 125 microns in effective diameter, greater than about 150 microns in effective diameter, greater than about 175 microns in effective diameter, or greater than about 200 microns in effective diameter. In some embodiments, the pore size of the vascularized layer may be from about 9 microns effective diameter to about 200 microns effective diameter, from about 9 microns effective diameter to about 50 microns effective diameter, from about 15 microns effective diameter to about 50 microns effective diameter, from about 25 microns effective diameter to about 50 microns effective diameter, from about 50 microns effective diameter to about 200 microns effective diameter, or from about 75 microns effective diameter to about 175 microns effective diameter, as measured by quantitative image analysis (QIA) performed on SEM images.

[0107] In addition, the thickness of the vascularized layer may be greater than about 30 microns, greater than about 50 microns, greater than about 75 microns, greater than about 100 microns, greater than about 125 microns, greater than about 150 microns, or greater than about 200 microns. In addition, the thickness of the vascularized layer may be about 30 microns to about 300 microns, about 30 microns to about 200 microns, about 30 microns to about 100 microns, about 100 microns to about 200 microns, or about 100 microns to about 150 microns. In addition, the representative minor axis of the majority of solid features within the vascularized layer is less than about 40 microns, less than about 30 microns, less than about 20 microns, less than about 10 microns, less than about 5 microns, or less than about 3 microns. In some embodiments, the representative minor axis may be about 3 microns to about 40 microns, about 3 microns to about 30 microns, about 3 microns to about 20 microns, about 3 microns to about 10 microns, or about 20 microns to about 40 microns. Solid features within the vascularized layer, such as continuous fibers of a nonwoven fabric, may also have a major axis that is greater in length than the minor axis and may be virtually unlimited in length. Solid features within the vascularized layer may have a depth that is less than or equal to the entire thickness of the vascularized layer.

[0108] Optional reinforcing components can be included to provide mechanical support to the biocompatible membrane composite to minimize in vivo distortion. Such additional optional reinforcing components provide the biocompatible membrane composite with greater stiffness than the biocompatible membrane composite itself. Such optional reinforcing components can be continuous in nature or present in discrete regions on the biocompatible membrane composite, e.g., patterned across the entire surface of the biocompatible membrane composite, or positioned at specific locations, e.g., around the periphery of the biocompatible membrane composite. Non-limiting patterns suitable for the surface of the membrane composite include dots, straight lines, angled lines, curved lines, dotted lines, grids, etc. Patterns forming reinforcing components can be used alone or in combination. Additionally, reinforcing components can be temporary in nature (e.g., formed from a bioabsorbable material) or permanent in nature (e.g., polyethylene terephthalate (PET) mesh or nitinol). The ultimate determination of a component's stiffness depends not only on the stiffness of a single reinforcing component, but also on the location and constraint of the reinforcing component in the final device configuration.

[0109] In at least one embodiment, a reinforcing component can be provided on the outer surface of the vascularized layer to strengthen the biocompatible membrane composite against environmental forces. In such an orientation, the reinforcing component has a pore size sufficient to allow vascular ingrowth and is therefore considered an "open" layer. Materials useful as the reinforcing component include materials that have significantly higher stiffness than the biocompatible membrane composite. Examples of such materials include open mesh biomaterial fabrics, woven fabrics, nonwoven fabrics (e.g., collections of fibers or threads), and fibrous matrices, alone or in combination.

[0110] In some embodiments, the relaxed layer and the vascularized layer are bonded together with one or more biocompatible adhesives to form a biocompatible membrane composite. The adhesive can be applied to the surface of one or both of the relaxed layer and the vascularized layer to form a discontinuous or intimate bond. Non-limiting examples of suitable biocompatible adhesives include fluorinated ethylene propylene (FEP), polycarbonate urethane, thermoplastic fluoropolymers consisting of TFE and PAVE, EFEP (ethylene fluorinated ethylene propylene), PEBAX (polyetheramide), PVDF (polyvinylidene fluoride), CarbOsil® (ab silicone polycarbonate urethane), Elasthane® (polyether urethane), PurSil® (silicone polyether urethane), polyethylene, high density polyethylene (HDPE), ethylene chlorotetrafluoroethylene (ECTFE), perfluoroalkoxy (PFA), polypropylene, polyethylene terephthalate (PET), and combinations thereof.

[0111] In some embodiments, at least one of the mitigating layer and the vascularization layer may be formed from a polymer membrane, a woven or nonwoven collection of fibers or threads, or a fibrous matrix, alone or in combination. Non-limiting examples of polymers that may be used include alginate, cellulose acetate, polyalkylene glycols such as polyethylene glycol and polypropylene glycol, panvinyl polymers such as polyvinyl alcohol, chitosan, polyacrylates such as polyhydroxyethyl methacrylate, agarose, hydrolyzed polyacrylonitrile, polyacrylonitrile copolymers, polyvinyl acrylates such as polyethylene-co-acrylic acid, polyalkylenes such as polypropylene, polyethylene, polyvinylidene fluoride, fluorinated ethylene propylene (FEP), perfluoroalkoxyalkanes (PFA), polyestersulfones (PES), polyurethanes, polyesters, and copolymers and combinations thereof. In some embodiments, the vascularization layer may be a spunbond nonwoven polyester or expanded polytetrafluoroethylene (ePTFE) membrane.

[0112] In some embodiments, at least one of the relief layer, vascularization layer, or reinforcement component is formed from a nonwoven fabric. There are many types of nonwoven fabrics, each of which can vary in weave tightness and sheet thickness. The filament cross-section can be trilobal. The nonwoven fabric can be a bonded fabric, a molded fabric, or an engineered fabric produced by a process other than weaving or knitting. In some embodiments, a nonwoven fabric is a porous textile-like material, usually in flat sheet form, composed primarily or entirely of fibers, such as staple fibers assembled into a web, sheet, or batt. The structure of a nonwoven fabric is typically based on a randomly arranged, e.g., staple fiber arrangement. Additionally, nonwoven fabrics can be formed by various techniques known in the textile industry. Various methods can form carded, wet-laid, meltblown, spunbonded, or air-laid nonwoven materials. Methods and substrates are described, for example, in U.S. Patent Application Publication No. 2010 / 0151575 to Colter et al. In one embodiment, the nonwoven fabric is polytetrafluoroethylene (PTFE). In another embodiment, the nonwoven fabric is spunbond polyester. The density of the nonwoven fabric may vary depending on processing conditions. In one embodiment, the nonwoven fabric has a basis weight of about 10 to about 20 g / m 2 The nonwoven fabric is a spunbond polyester having a nominal thickness of about 75 to about 150 microns and a fiber diameter of about 20 to about 40 microns. The filament cross section is trilobal. In some embodiments, the nonwoven fabric is bioabsorbable.

[0113] In some embodiments, the polymer forming the polymer membrane of the relaxation layer and / or the vascularization layer is a fibrillizable polymer. As defined herein, fibrillizable refers to the ability to introduce fibrils into the polymer membrane, e.g., the ability to convert solid features into fibrils. For example, fibrils are solid elements that span gaps between solid features. Fibrils generally do not resist deformation upon exposure to environmental forces and are therefore deformable. The diameter of the majority of the deformable fibrils in the relaxation layer and / or the vascularization layer may be less than about 2 microns, less than about 1 micron, less than about 0.75 microns, less than about 0.50 microns, or less than about 0.25 microns. In some embodiments, the diameter of the fibrils may be between about 0.25 microns and about 2 microns, between about 0.5 microns and about 2 microns, or between about 0.75 microns and about 2 microns.

[0114] In some embodiments, the solid features in one or both of the relaxed layer and the vascularized layer can be formed by microlithography, micromolding, machining, selective deposition, or printing (or laying down) a polymer (e.g., a thermoplastic) onto the relaxed layer or the vascularized layer to form at least a portion of the solid features. Any conventional printing technique, such as transfer coating, screen printing, gravure printing, inkjet printing, patterned imbibing, and knife coating, can be used to deposit the thermoplastic polymer onto the relaxed layer and / or the vascularized layer. Optionally, a pattern can be printed on a liner and then applied to the relaxed layer, the vascularized layer, or the implantable device.

[0115] Examples of materials used to form the solid features include thermoplastics, polyurethanes, polypropylenes, silicones, rubbers, epoxies, polyethylenes, polyetheramides, polyetheretherketones, polyphenylsulfones, polysulfones, silicone polycarbonate urethanes, polyether urethanes, polycarbonate urethanes, silicone polyether urethanes, polyesters, polyester terephthalates, melt-processable fluoropolymers such as fluorinated ethylene propylene (FEP), tetrafluoroethylene-(perfluoroalkyl)vinyl ethers (PFA), alternating copolymers of ethylene and tetrafluoroethylene (ETFE), terpolymers of tetrafluoroethylene (TFE), hexafluoropropylene (HFP), and vinylidene fluoride (THV), polyvinylidene fluoride (PVDF), and combinations thereof. In some embodiments, polytetrafluoroethylene can be used to form the pattern features. In further embodiments, the solid features can be formed separately and attached to the surface of the vascularization layer or implantable device (not shown).

[0116] Examples of fibrillizable polymers that can be used to form one or more of the relaxing layer, the vascularization layer, and the optional cell impermeable layer include tetrafluoroethylene (TFE) polymers, such as polytetrafluoroethylene (PTFE), expanded PTFE (ePTFE), modified PTFE, TFE copolymers, polyvinylidene fluoride (PVDF), poly(p-xylylene) (eP), as taught in U.S. Patent Application Publication No. 2016 / 0032069 to Sbriglia, and the like. PX), porous ultra-high molecular weight polyethylene (eUHMWPE) as taught in U.S. Pat. No. 9,926,416 to Sbriglia, porous ethylene tetrafluoroethylene (eETFE) as taught in U.S. Pat. No. 9,932,429 to Sbriglia, and porous vinylidene fluoride-co-tetrafluoroethylene or trifluoroethylene [VDF-co-(TFE or TrFE)] polymers as taught in U.S. Pat. No. 9,441,088 to Sbriglia.

[0117] In some embodiments, the fibrillizable polymer is a fluoropolymer membrane, such as an expanded polytetrafluoroethylene (ePTFE) membrane. Expanded polytetrafluoroethylene (ePTFE) (and other fibrillating polymers) have a node-fibril microstructure. In a node-fibril microstructure, nodes are interconnected by fibrils, and pores are spaces located between the nodes and fibrils throughout the membrane. As used herein, the term "node" refers to a solid feature composed mostly of polymeric material. When deformable fibrils are present, these nodes are located at the junctions of multiple fibrils. In some embodiments, the fibrils can be removed from the membrane, for example, by plasma etching. In at least one embodiment, an expanded polytetrafluoroethylene membrane is used in one or more of the relaxed layer, the vascularized layer, and the optional cell-impermeable layer. Expanded polytetrafluoroethylene membranes, such as those prepared according to the methods described in U.S. Pat. No. 3,953,566 to Gore, U.S. Pat. No. 7,306,729 to Bacino et al., U.S. Pat. No. 5,476,589 to Bacino, WO 94 / 13469 to Bacino, U.S. Pat. No. 5,814,405 to Branca et al., or U.S. Pat. No. 5,183,545 to Branca et al., may be used herein.

[0118] In some embodiments, one or more of the mitigating layer and the vascularization layer may be formed from a fluoropolymer membrane, such as an expanded polytetrafluoroethylene (ePTFE) membrane, a modified ePTFE membrane, a tetrafluoroethylene (TFE) copolymer membrane, a polyvinylidene fluoride (PVDF), or a fluorinated ethylene propylene (FEP) membrane. In further embodiments, the vascularization layer may comprise biocompatible fabrics, including woven and nonwoven fabrics (e.g., spunbond nonwovens, meltblown fibrous materials, electrospun nanofibers, etc.), non-fluoropolymer membranes, such as polyvinylidene fluoride (PVDF), nanofibers, polysulfone, polyethersulfone, polyarylsulfone, polyetheretherketone (PEEK), polyethylene, polypropylene, and polyimide. In some embodiments, the vascularization layer is a spunbond nonwoven polyester or an expanded polytetrafluoroethylene (ePTFE) membrane.

[0119] In some embodiments, it may be desirable for one or more of the vascularized layer and the reinforcement component to be impermeable (e.g., biodegradable). In such cases, biodegradable materials may be used to form the vascularized layer and / or the reinforcement component. Suitable examples of biodegradable materials include polyglycolide:trimethylene carbonate (PGA:TMC), polyalphahydroxy acids such as polylactic acid, polyglycolic acid, poly(glycolide), and poly(lactide-co-caprolactone), poly(caprolactone), poly(carbonate), poly(dioxanone), poly(hydroxybutyrate), poly(hydroxyvalerate), poly(hydroxybutyrate-co-valerate), expanded polyparaxylylene (ePLLA), as taught in U.S. Patent Application Publication No. 2016 / 0032069 to Sbriglia, and copolymers and blends thereof. Alternatively, the vascularized layer may be coated with a bioabsorbable material, or the bioabsorbable material may be incorporated in or on the vascularized layer in powder form. The coated material can promote reduced infection sites, vascularization, and favorable type 1 collagen deposition.

[0120] The biocompatible membrane composite may at least partially have a surface coating, such as a zwitterionic non-fouling coating, a hydrophilic coating, or a CBAS® / heparin coating (commercially available from W.L. Gore & Associates, Inc.). The surface coating may additionally or alternatively contain antimicrobial agents, antibodies (e.g., anti-CD47 antibodies (anti-fibrotic)), pharmaceutical agents, and bioactive molecules (e.g., stimulators of angiogenesis such as FGF, VEGF, endoglin, PDGF, angiopoietins, and integrins, anti-fibrotic agents such as TGFβ inhibitors, sirolimus, CSF1R inhibitors, anti-inflammatory / immunomodulatory agents such as CXCL12, and corticosteroids), and combinations thereof.

[0121] Referring to FIG. 6A, in at least one embodiment, a biocompatible membrane composite can be used in combination with an implantable device 600. Specifically, the biocompatible membrane composite (not shown) can partially or completely cover an enclosure 605. The enclosure 605 can be a pouch or container for carrying a sensor, pacemaker, or electrical lead component 610, or it can be the implantable device itself. In another embodiment, shown in FIG. 6B, the biocompatible membrane composite (not shown) can partially or completely cover the exterior of a cell system 620 and / or some or all of the structural elements 650. Section 630 is enlarged to show the individual structural elements 650 of the cell system and cells 640 growing with the cell system 620.

[0122] A biocompatible membrane composite 700 is shown in FIG. 7. As shown in FIG. 7, the biocompatible membrane composite 700 includes a relaxed layer (i.e., first layer) 720 and a vascularized layer (i.e., second layer) 730. The biocompatible membrane composite 700 can be utilized to at least partially cover, surround, or enclose an implantable device 710. In the illustrated embodiment, solid features 750 are attached to the surface of the implantable device 710 to form the relaxed layer 720. As used herein, "attached" is intended to include closely attached or discontinuously attached. In some embodiments, the solid features 750 do not penetrate into the vascularized layer 730. While the solid features 750 are shown in FIG. 7 as being essentially the same height and width and extending between the implantable device 710 and the vascularized layer 730, it will be appreciated that this is by way of example only, and the solid features 750 may vary in height and / or width. The distance between solid features 750 is solid feature spacing 760 .

[0123] Figure 8 illustrates a biocompatible composite 800. As shown in Figure 8, the biocompatible membrane composite 800 includes a relaxed layer 820 and a vascularized layer 830. In the illustrated embodiment, the solid features 850 are nodes of varying height and width and may or may not extend the distance between the implantable device 810 and the vascularized layer 830. The solid features 850 are connected by fibrils 870. In Figure 8, the majority of the solid feature depth is less than the total thickness of the relaxed layer 820. Bondable solid features 880 can be attached to the surface of the implantable device 810.

[0124] Referring to FIG. 9 , a biocompatible membrane composite 900 is shown. The biocompatible membrane composite 900 includes a cell-impermeable layer 920 and a vascularization layer 930. The biocompatible membrane composite 900 can at least partially cover or surround an implantable device 910. In this embodiment, the solid features within the relaxed layer 920 are nodes formed from an expanded polytetrafluoroethylene membrane. The nodes 950 are interconnected by fibrils 970. The nodes 950, 980 are positioned within the relaxed layer 920. However, the bondable solid features or nodes 980 are not only within the relaxed layer 920 but are also in contact with the implantable device 910 and can be tightly bonded to the implantable device 910.

[0125] Of course, in each of the embodiments illustrated in Figures 7-9, an implantable device may be used in place of the cell system, and such embodiments are considered to be within the scope of the present invention.

[0126] Test Method Porosity The porosity of a layer is defined herein as the fraction of the layer volume that consists of pore space compared to the total volume of the layer. Porosity is calculated by comparing the bulk density of the porous structure, consisting of the solid and void fractions, to the density of the solid fraction using the following equation:

number

[0127] mass / area The sample was cut (by hand, laser, or die) to a known geometry. The sample dimensions were measured or verified, and the area was calculated in m 2 The sample was then weighed in grams on a calibrated scale. The mass (grams) was then converted to the area (m 2 ) to get the mass per unit area in g / m 2 was calculated.

[0128] Thickness The layer thicknesses within the biocompatible membrane composite were measured by quantitative image analysis (QIA) of cross-sectional SEM images. Cross-sectional SEM images were generated by clamping the membrane to an adhesive, manually cutting the film using a liquid nitrogen-cooled razor blade, and then holding the adhesive-backed film upright so that the cross section was vertical. The samples were then sputter coated using an Emitech K550X sputter coater (commercially available from Quorum Technologies Ltd, UK) and a platinum target. The samples were then imaged using a Thermo Scientific FEI Quanta 400 scanning electron microscope.

[0129] The thickness of the layers within the cross-sectional SEM images was then measured using ImageJ 1.51h from the National Institutes of Health (NIH). The image scale was set relative to the scale provided by the SEM. The layer of interest was isolated and clipped using the freehand tool. At least 10 equally spaced lines were then drawn in the direction of the layer thickness. The length of all the lines was measured and averaged to define the layer thickness.

[0130] rigidity Stiffness testing was performed according to ASTM D790-17 standard test method for flexural properties of unreinforced and reinforced plastic and electrical insulating materials, and was used to determine the stiffness of the biocompatible membrane composite layer and / or the final device.

[0131] ASTM Method Procedure B was followed, which includes strains greater than 5% and a type 1 crosshead position for deflection. The fixture dimensions were adjusted to have a span of 16 mm and a support and nosebead radius of 1.6 mm. The test parameters used were a deflection of 3.14 mm and a test speed of 96.8 mm / min. If the test width was different from the standard 1 cm, the force was normalized to a 1 cm specimen width by a linear ratio.

[0132] The load was reported in N / cm at maximum deflection.

[0133] SEM sample preparation SEM samples were prepared by first fixing the membrane composite or membrane composite layer to an adhesive for handling, with the side opposite the side to be imaged facing the adhesive. The film was then cut to provide an approximately 3 mm x 3 mm area for imaging. The samples were then sputter coated using an Emitech K550X sputter coater and a platinum target. Images were taken using a Thermo Scientific FEI Quanta 400 scanning electron microscope at a magnification and resolution that allowed visualization of a sufficient number of features for robust analysis while ensuring that the smallest dimension of each feature was at least 5 pixels long.

[0134] Solid Feature Spacing Solid feature spacing was determined by analyzing SEM images in National Institutes of Health (NIH) ImageJ 1.51h. The image scale was set based on the scale provided by the SEM image. Features were identified and isolated by a combination of size-based thresholding / shading and / or manual identification. In cases where the structure consisted of a continuous structure, such as a nonwoven fabric or an etched surface, as opposed to a structure with discrete solid features, the solid feature was defined as the portion of the structure surrounding the void, and the corresponding spacing of the solid feature extended from one side of the void to the other. After feature isolation, Delaunay triangulation was performed to identify adjacent features. Triangulations with circumscribing circles extending beyond the edge of the image were ignored in the analysis. The spacing between adjacent features was defined by drawing a line between the nearest edges of the adjacent features and measuring its length (see, for example, Figure 1A).

[0135] The median of all measured solid feature spacing marks the value that is less than or equal to half the measured solid feature spacing and greater than or equal to half the measured solid feature spacing. Thus, if the measured median is above or below a certain value, the majority of the measurements will be above or below that value as well. As such, the median is used as a summary statistic to represent the majority of solid feature spacings.

[0136] Measurement of the representative minor axis and the representative major axis The representative minor axis was measured by analyzing membrane surface SEM images in NIH ImageJ 1.51h. The image scale was set based on the scale provided by the SEM image. Features were identified and isolated using a combination of size-based thresholding / shading and / or manual identification. After feature isolation, the built-in particle analysis capability was used to determine the major and minor axes of a representative ellipse. The minor axis of this ellipse is the representative minor axis of the measured feature. The major axis of this ellipse is the representative major axis of the measured feature. The median of all measured minor axes marks the value that is less than or equal to half the measured minor axis and greater than or equal to half the measured minor axis. Similarly, the median of all measured major axes marks the value that is less than or equal to half the measured major axis and greater than or equal to half the measured major axis. In both cases, if the measured median value is above or below a certain value, the majority of the measurements will also be above or below that value. As such, the median is used as a summary statistic to represent the majority of the representative minor and major axes.

[0137] Solid Feature Depth Solid feature depth was determined using quantitative image analysis (QIA) of SEM images of membrane cross sections. Cross-sectional SEM images were generated by clamping the film to an adhesive, manually cutting the film using a liquid nitrogen-cooled razor blade, and then holding the adhesive-backed film upright so that the cross section was vertical. The sample was then sputter coated using an Emitech K550X sputter coater (commercially available from Quorum Technologies Ltd, UK) and a platinum target. The sample was then imaged using a Thermo Scientific FEI Quanta 400 scanning electron microscope.

[0138] The depth of features within the cross-sectional SEM images was then measured using National Institutes of Health (NIH) ImageJ 1.51h. The image scale was set relative to the scale provided by the SEM. Features were identified and isolated by a combination of size-based thresholding / shading and / or manual identification. After feature isolation, the built-in particle analysis capability was utilized to calculate the ferret diameter and the angle formed by the ferret diameter axis of each solid feature and the axis defined by the horizontal plane. The ferret diameter is the longest distance between any two points on the boundary of the feature within the plane of the SEM image. The ferret diameter axis is the line defined by these two points. The projection of the ferret diameter of each solid feature in the direction of the layer thickness was calculated using the following equation:

number

[0139] The projection of the longest axis in the layer thickness direction is the solid feature depth of the measured feature. The median of all measured solid feature depths marks the value that is less than or equal to half the measured solid feature depth and greater than or equal to half the measured solid feature depth. Thus, if the measured median value is above or below some value, the majority of the measurements will be above or below that value as well. As such, the median value is used as a summary statistic to represent the majority of the solid feature spacing.

[0140] Hole Size Pore ​​size was determined by analyzing SEM images in NIH ImageJ 1.51h. The image scale was set based on the scale provided by the SEM image. Pores were identified and isolated by a combination of size-based thresholding / shading and / or manual identification. After pore isolation, the area of ​​each pore was determined using the built-in particle analysis capability. The measured pore area was converted to an "effective diameter" using the following equation:

number

[0141] The pore areas are summed to define the total area of ​​the surface defined by the pores. This is the total pore area of ​​the surface. The pore size of the layer is the effective diameter of the pores that defines the point where approximately half of the total pore area consists of pores with diameters smaller than the pore size and half of the total pore area consists of pores with diameters greater than or equal to the pore size.

[0142] In vitro generation of human PDX1-positive pancreatic endoderm and endocrine cells The differentiation methods herein directed to pluripotent stem cells, e.g., hES and iPS cells, can be described as having at least four, five, six, or seven stages, depending on the desired end-stage cell culture or cell population (e.g., a PDX1-positive pancreatic endoderm cell population (or PEC), or an endocrine precursor cell population, an endocrine cell population, or an immature beta cell population, or a mature endocrine cell population).

[0143] Stage 1 is the generation of definitive endoderm from pluripotent stem cells, which takes approximately 2 to 5 days, preferably 2 or 3 days. Growth, survival, proliferation, and / or cell-cell adhesion are promoted by suspending pluripotent stem cells in a medium containing RPMI, a TGFβ superfamily member growth factor such as activin A, activin B, GDF-8, or GDF-11 (100 ng / mL), a Wnt family member or Wnt pathway activator such as Wnt3a (25 ng / mL), or a rho-kinase or ROCK inhibitor such as Y-27632 (10 μM). After approximately 24 hours, the medium is replaced with medium containing serum, e.g., 0.2% FBS, and a TGFβ superfamily member growth factor, e.g., activin A, activin B, GDF-8, or GDF-11 (100 ng / mL), or a rho-kinase or ROCK inhibitor, for an additional 24 hours (day 1) to 48 hours (day 2). Alternatively, after culturing in medium containing activin / Wnt3a for approximately 24 hours, the cells are cultured in medium containing only activin (i.e., the medium does not contain Wnt3a) for the next 24 hours. Importantly, the generation of definitive endoderm requires cell culture conditions that are low in serum and, therefore, low in insulin or insulin-like growth factors. See McLean et al. (2007) Stem Cells 25: 29-38. McLean et al. also show that contacting hES cells with insulin at concentrations as low as 0.2 μg / mL at stage 1 can be detrimental to the generation of definitive endoderm.Still others skilled in the art will recognize the benefits of this method substantially as set forth herein and in D'Amour et al. (2005), and see, for example, at least, Agarwal et al., Efficient Differentiation of Functional Hepatocytes from Human Embryonic Stem Cells, Stem Cells (2008) 26:1117-1127; Borowiak et al., Small Molecules Efficiently Direct Endodermal Differentiation of Mouse and Human Embryonic Stem Cells, (2009) Cell Stem Cell 4:348-358; Brunner et al., Distinct DNA methylation patterns characterize differentiated human embryonic stem cells and developing human fetal liver.), (2009) Genome Res. 19:1044-1056, Rezania et al. Reversal of Diabetes with Insulin-producing Cells Derived In Vitro from Human Pluripotent Stem Cells (2014) Nat Biotech 32(11): 1121-1133 (GDF8 & GSK3beta inhibitor, e.g., CHIR99021); and Pagliuca et al. (2014) Generation of Functional Human Pancreatic B-cell In Vitro, Cell 159: 428-439 (Activin A & CHIR), altering the differentiation of stage 1 pluripotent stem cells into definitive endoderm. Proper differentiation, specification, characterization, and identification of definitive endoderm are required to derive other endoderm lineages. Definitive endoderm cells at this stage co-express SOX17 and HNF3β (FOXA2) and do not appreciably express at least HNF4alpha, HNF6, PDX1, SOX6, PROX1, PTF1A, CPA, cMYC, NKX6.1, NGN3, PAX3, ARX, NKX2.2, INS, GSC, GHRL, SST, or PP.The absence of HNF4alpha expression in definitive endoderm is supported and detailed at least in Duncan et al. (1994), “Expression of transcription factor HNF-4 in the extraembryonic endoderm, gut, and nephrogenic tissue of the developing mouse embryo: HNF-4 is a marker for primary endoderm in the implanting blastocyst,” Proc. Natl. Acad. Sci, 91:7598-7602, and Si-Tayeb et al. (2010), “Highly Efficient Generation of Human Hepatocyte-Like Cells from Induced Pluripotent Stem Cells,” Hepatology 51:297-305.

[0144] Stage 2 generated foregut endoderm or PDX1-negative foregut endoderm by incubating the cells in suspension for 24 hours (days 2-3) in a 1:1000 dilution of RPMI containing low serum levels in ITS, e.g., 0.2% FBS, 25 ng of KGF (or FGF7), or a ROCK inhibitor. After 24 hours (days 3-4), the medium was replaced with the same medium minus a TGFβ inhibitor or a ROCK inhibitor, promoting cell growth, survival, and proliferation for an additional 24 days (days 4-5) to 48 hours (day 6). A critical step for proper specification of foregut endoderm is the removal of TGFβ family growth factors. Thus, a TGFβ inhibitor, such as 2.5 μM TGFβ inhibitor No. 4, or 5 μM SB431542, a specific inhibitor of the TGFβ type I receptor, activin receptor-like kinase (ALK), can be added to cell cultures at stage 2. Foregut endoderm or PDX1-negative foregut endoderm cells generated from stage 2 express SOX17, HNF1β, and HNF4alpha, and do not appreciably co-express at least HNF3β (FOXA2), nor do they appreciably co-express HNF6, PDX1, SOX6, PROX1, PTF1A, CPA, cMYC, NKX6.1, NGN3, PAX3, ARX, NKX2.2, INS, GSC, GHRL, SST, or PP. These are characteristics of definitive endoderm, PDX1-positive pancreatic endoderm cells, or pancreatic progenitor cells, or endocrine progenitor / precursor cells, as well as typically polyhormonal cells.

[0145] Stage 3 (days 5-8) for PEC generation involves taking foregut definitive endoderm cell cultures and generating PDX1-positive foregut endoderm cells over approximately 24 hours (day 7) to 48 hours (day 8) with DMEM or RPMI in 1% B27, 0.25 μM KAAD cyclopamine, a retinoid such as 0.2 μM retinoic acid (RA) or a retinoic acid analog such as 3 nM TTNPB (or CTT3, which is a combination of KAAD cyclopamine and TTNPB), and 50 ng / mL Noggin. Specifically, applicant has been using DMEM high glucose since approximately 2003, and all patent and non-patent disclosures to that time have employed DMEM high glucose, even if they do not refer to it as "DMEM high glucose." This is in part because manufacturers such as Gibco did not designate these DMEMs as such, e.g., DMEM (Cat. No. 11960) and Knockout DMEM (Cat. No. 10829). It is worth noting that, as of the filing date of this application, Gibco, while offering many more DMEM products, has yet to designate some of these DMEM products containing high glucose, e.g., Knockout DMEM (Cat. No. 10829-018), with the "high glucose" designation. Thus, in each instance where DMEM is mentioned, this is intended to mean DMEM with high glucose. This has been clear to others conducting research and development in this field. Again, a ROCK inhibitor or rho-kinase inhibitor, e.g., Y-27632, can be used to promote growth, survival, proliferation, and / or promote cell-cell adhesion. Examples of additional substances and factors include, but are not limited to, ascorbic acid (e.g., vitamin C), a BMP inhibitor (e.g., noggin, LDN, chordin), an SHH inhibitor (e.g., SANT, cyclopamine, HIP1), and / or a PKC activator (e.g., PdBu, TBP, ILV), or any combination thereof. Alternatively, Stage 3 is performed without an SHH inhibitor, e.g., Stage 3 cyclopamine.PDX1-positive foregut endoderm cells generated from stage 3 co-express PDX1 and HNF6 as well as SOX and PROX and do not appreciably express the markers indicative of definitive endoderm or foregut endoderm (PDX1-negative foregut endoderm) cells described above in stages 1 and 2, or PDX1-positive foregut endoderm cells.

[0146] The Stage 3 method described above is one of four stages for generating PEC populations. In addition to Noggin, KAAD-cyclopamine and retinoids, activins, Wnt and heregulin, thyroid hormone, TGFβ receptor inhibitors, protein kinase C activators, vitamin C, and ROCK inhibitors are used alone and / or in combination to suppress early expression of NGN3 and expansion of CHGA-negative cells to generate endocrine progenitors / precursors and endocrine cells, as described in detail below.

[0147] Stage 4 (approximately days 8-14) PEC culture production involves taking the stage 3 medium and replacing it with medium containing DMEM in 1% vol / vol B27 supplement plus 50 ng / mL KGF and 50 ng / mL EGF, and sometimes also 50 ng / mL Noggin and a ROCK inhibitor; this medium also contains activin alone or in combination with hereglint. Alternatively, stage 3 cells can be further differentiated using KGF, RA, SANT, PKC activator, and / or vitamin C, or any combination thereof. These methods yield pancreatic progenitor cells that co-express at least PDX1 and NKX6.1, as well as PTF1A. These cells do not appreciably express markers indicative of definitive endoderm or foregut endoderm (PDX1-negative foregut endoderm) cells as described above for stages 1, 2, and 3.

[0148] Stage 5 production involves taking the Stage 4 PEC cell population described above and further differentiating them to generate endocrine progenitor / precursor or progenitor-type cells and / or monohormonal or polyhormonal pancreatic endocrine cells over a period of about 1-6 days (preferably about 2 days, i.e., days 13-15) in medium containing DMEM with 1% vol / vol B27 supplement, Noggin, KGF, EGF, RO (a gamma secretase inhibitor), nicotinamide and / or an ALK5 inhibitor, or any combination thereof, such as a combination of Noggin and an ALK5 inhibitor. Alternatively, stage 4 cells can be further differentiated using retinoic acid (e.g., RA or an analog thereof), thyroid hormone (e.g., T3, T4 or an analog thereof), TGFβ receptor inhibitors (ALK5 inhibitors), BMP inhibitors (e.g., noggin, chordin, LDN), or gamma secretase inhibitors (e.g., XXI, XX, DAPT, XVI, L685458), and / or betacellulin, or any combination thereof. Endocrine progenitors / precursors generated from stage 5 co-express at least PDX1 / NKX6.1 and also express CHGA, NGN3, and Nkx2.2, and do not appreciably express markers indicative of definitive endoderm or foregut endoderm (PDX1-negative foregut endoderm) as described above in stages 1, 2, 3, and 4 for PEC production.

[0149] Stage 6 and 7 can be further differentiated from stage 5 cell populations by adding any of a combination of substances or factors including, by way of example, PDGF plus SSH inhibitors (e.g., SANT, cyclopamine, HIP1), BMP inhibitors (e.g., Noggin, Chordin, LDN), nicotinamide, insulin-like growth factors (e.g., IGF1, IGF2), TTNBP, ROCK inhibitors (e.g., Y27632), TGFβ receptor inhibitors (e.g., ALK5i), thyroid hormones (e.g., T3, T4 and analogs thereof), and / or gamma secretase inhibitors (XXI, XX, DAPT, XVI, L685458), or combinations thereof, to achieve cell culture populations or appropriate ratios of endocrine cells, endocrine precursors, or immature beta cells.

[0150] Stage 7 or immature beta cells are considered endocrine cells, but may or may not be sufficiently mature to physiologically respond to glucose. Stage 7 immature beta cells may express MAFB, whereas cells that express MAFA and MAFB are fully mature cells that can physiologically respond to glucose.

[0151] Stages 1-7 cell populations are derived from human pluripotent stem cells (e.g., human embryonic stem cells, induced pluripotent stem cells, or stem cells that have been genetically engineered, e.g., using any of the currently available or later developed gene editing tools and applications) and may not have their exact naturally occurring counterparts, since they are derived from immortal human pluripotent stem cells developed in vitro (i.e., in artificial tissue culture), and not from the inner cell mass in vivo (i.e., in vivo human development has no human ES cell equivalent).

[0152] Pancreatic cell therapy alternatives contemplated herein can use any of the stage 4, 5, 6 or 7 cell populations, encapsulated within the devices described herein comprised of the membranes described herein, loaded into the macroencapsulation device, fully enclosed, and implanted into a patient, and the pancreatic endoderm-lineage cells mature into pancreatic hormone-secreting cells, or pancreatic islets, e.g., insulin-secreting beta cells in vivo (also referred to as "in vivo functional"), capable of responding normally to blood glucose.

[0153] Encapsulation of pancreatic endoderm-lineage cells and in vivo insulin production is described in detail in U.S. Application No. 12 / 618,659, filed November 13, 2009, entitled ENCAPSULATION OF PANCREATIC LINEAGE CELLS DERIVED FROM HUMAN PLURIPOTENT STEM CELLS (the '659 application). The '659 application claims priority to U.S. Provisional Patent Application No. 61 / 114,857, entitled ENCAPSULATION OF PANCREATIC PROGENITORS DERIVED FROM HES CELLS, filed November 14, 2008, and U.S. Provisional Patent Application No. 61 / 121,084, entitled ENCAPSULATION OF PANCREATIC ENDODERM CELLS, filed December 9, 2008, and now U.S. Patent Nos. 8,278,106 and 8,424,928. The methods, compositions, and devices described herein are presently representative of preferred embodiments and are exemplary and are not intended as limitations on the scope of the invention. Modifications and other uses will readily occur to those skilled in the art that are within the scope of the invention and defined by the scope of the disclosure. Accordingly, it will be apparent to those skilled in the art that various substitutions and modifications can be made in the invention disclosed herein without departing from the scope and spirit of the invention.

[0154] Additionally, the embodiments described herein are not limited to pluripotent stem cells, or any one type of human pluripotent stem cell, but include, by way of example, human embryonic stem (hES) cells and human induced pluripotent stem (iPS) cells, or other pluripotent stem cells later developed. At the time of filing this application, it is also well known to those skilled in the art that methods for forming human pluripotent stem cells can be performed without the destruction of human embryos, and such methods are anticipated for the generation of any human pluripotent stem cell.

[0155] Methods for generating pancreatic cell lineages from human pluripotent stem cells are substantially similar to those described, by way of example only, in at least the following publications: PCT / US2007 / 62755 (WO2007101130), PCT / US2008 / 80516 (WO2009052505), PCT / US2008 / 82356 (WO2010053472), PCT / US2005 / 28829 (WO2006020919), PCT / US20 14 / 34425(WO2015160348),PCT / US2014 / 60306(WO2016080943),PCT / US2016 / 61442(WO2018089011),PCT / US20 14 / 15156(WO2014124172),PCT / US2014 / 22109(WO2014138691),PCT / US2014 / 22065(WO2014138671),PCT / US200 5 / 14239(WO2005116073),PCT / US2004 / 43696(WO2005063971),PCT / US2005 / 24161(WO2006017134),PCT / US200 6 / 42413(WO2007051038),PCT / US2007 / 15536(WO2008013664),PCT / US2007 / 05541(WO2007103282),PCT / US2008 / 61053 (WO2009131568), PCT / US2008 / 65686 (WO2009154606), PCT / US2014 / 15156 (WO2014124172), PCT / US2018 / 41648 (WO2019014351), PCT / US2014 / 26529 (WO2014160413), PCT / US2009 / 64459 (WO2010057039), ViaCyte, Publications assigned to the Company, Inc., and d'Amour et al. 2005 Nature Biotechnology 23:1534-41; D'Amour et al. 2006 Nature Biotechnology 24(11):1392-401; McLean et al., 2007 Stem Cells 25:29-38, Kroon et al. 2008 Nature Biotechnology 26(4): 443-452, Kelly et al.2011 Nature Biotechnology 29(8): 750-756, Schulz et al., 2012 PLos One 7(5):e37004; and / or Agulnick et al. 2015 Stem Cells Transl. Med. 4(10):1214-22.

[0156] Methods for generating pancreatic cell lineages from human pluripotent stem cells are essentially those described, by way of example only, in at least the following: PCT / US2008 / 68782 (WO200906399), PCT / US2008 / 71775 (WO200948675), PCT / US2008 / 71782 (WO200918453), PCT / US2008 / 84705 (WO200970592), PCT / US2009 / 41348 (WO2009132063), PCT / US2009 / 41356 (WO2009132068), PCT / US2009 9 / 49183(WO2010002846),PCT / US2009 / 61635(WO2010051213),PCT / US2009 / 61774(WO2010051223),PCT / US2010 / 42390(WO2011011300),PCT / US2 010 / 42504(WO2011011349),PCT / US2010 / 42393(WO2011011302),PCT / US2010 / 60756(WO2011079017),PCT / US2011 / 26443(WO2011109279),PCT / US 2011 / 36043(WO2011143299),PCT / US2011 / 48127(WO2012030538),PCT / US2011 / 48129(WO2012030539),PCT / US2011 / 48131(WO2012030540),PCT / US2011 / 47410(WO2012021698),PCT / US2012 / 68439(WO2013095953),PCT / US2013 / 29360(WO2013134378),PCT / US2013 / 39940(WO2013169769),PCT / US2013 / 44472 (WO2013184888), PCT / US2013 / 78191 (WO2014106141), PCTU / S2014 / 38993 (WO2015065524), PCT / US2013 / 75939 (WO2014105543), CT / US2013 / 75959(WO2014105546),PCT / US2015 / 29636(WO2015175307),PCT / US2015 / 64713(WO2016100035),PCT / US2014 / 41988(WO2015002724),Publications assigned to Janssen, including PCT / US2017 / 25847 (WO2017180361), PCT / US2017 / 37373 (WO2017222879), PCT / US2017 / 37373 (WO2017222879); PCT / US2009 / 049049 (WO2010 / 002785), PCT / US2010 / 060770 (WO2011 / 079018), PCT / US2014 / 042796 (WO2015 / 065537), PCT / US2008 / 070418 (WO2009 / 012428), and Bruin et al. 2013 Diabetologia. 56(9): 1987-98, Fryer et al. 2013 Curr. Opin. Endocrinol. Diabetes Obes. 20(2): 112-7, Chetty et al. 2013 Nature Methods. 10(6):553-6, Rezania et al. 2014 Nature Biotechnologyy 32(11):1121-33, Bruin et al. 2014 Stem Cell Res.12(1): 194-208, Hrvatin 2014 Proc. Natl. Acad. Sci. US A. 111(8): 3038-43, Bruin et al. 2015 Stem Cell Reports. 5, 1081-1096, Bruin et al.2015 Science Transl. Med., 2015, 7, 316ps23, and / or performed as described in Bruin et al. 2015 Stem Cell Reports. 14;4(4):605-20.

[0157] In one embodiment, human pluripotent stem cells were differentiated into PDX1-positive pancreatic endoderm cells, which contain pancreatic progenitors and endocrine precursors, according to one of the following preferred conditions A and / or B: [Table 1]

[0158] Table 1 Legend: r0.2FBS: RPMI 1640 (Mediatech); 0.2% FBS (HyClone), 1x GlutaMAX-1 (Life Technologies), 1% v / v penicillin / streptomycin; db: DMEM Hi glucose (HyClone) supplemented with 0.5x B-27 supplement (Life Technologies); A100, A50, A5: 100 ng / mL recombinant human activin A (R&D Systems); A5i: 1 μM, 5 μM, 10 μM ALK5 inhibitor; TT3: 3 nM TTNPB (Sigma-Aldrich); E50: 50 ng / mL recombinant human EGF (R&D Systems); ITS: Insulin-Transferrin-Selenium (Life Technologies) diluted 1:5000 or 1:1000; IV: 2.5 mM TGF-β RI kinase inhibitor IV (EMD Bioscience); K50, K25: 50 ng / mL, 25 ng / mL recombinant human KGF (R&D Systems, or Peprotech); N50, N100: 50 ng / mL, or 100 ng / mL recombinant human Noggin (R&D Systems); W50: 50 ng / mL recombinant mouse Wnt3A (R&D Systems).

[0159] As will be apparent to those skilled in the art, there may also be other methods for generating PDX1-positive pancreatic endoderm cells or PDX1-positive pancreatic endoderm-lineage cells, including pancreatic progenitor or endocrine cells and endocrine precursor cells, and PDX1-positive pancreatic endoderm cells described in at least Kroon et al. 2008, Rezania et al. 2014 (see above), and Pagliuca et al. 2014 Cell 159(2):428-439 (see above).

[0160] As will be apparent to those skilled in the art, the embodiments described herein for generating PDX1-positive pancreatic endoderm cells consist of a mixture of mixed populations or subpopulations. Unlike mammalian in vivo development, which occurs along an anterior-posterior axis and in which cells and tissues are named accordingly, cell cultures in any culture vessel lack such directional patterning and are therefore specifically characterized based on the expression of these markers. Therefore, mixed cell subpopulations at any stage of differentiation do not occur in vivo. Thus, an example of a PDX1-positive pancreatic endoderm cell culture includes: i) endocrine precursors (e.g., as indicated by early endocrine markers, chromogranin A or CHGA); ii) monohormonal or polyhormonal cells expressing typical pancreatic hormones, such as insulin (INS), somatostatin (SST), pancreatic polypeptide (PP), glucagon (GCG), or gastrin, incretin, or cholecystokinin; iii) pre-pancreatic cells, such as cells expressing PDX-1 but not NKX6.1 or CHGA; iv) PDX-1 / NKX6.1 and CHGA (PDX-1 / NKX6.1 / CHGA), or non-endocrine cells, such as endocrine cells that co-express PDX-1 / NKX6.1 but do not express CHGA (PDX-1+ / NKX6.1+ / CHA-), and v) further cells that do not express PDX-1, NKX6.1, or CHGA (e.g., triple-negative cells).

[0161] This PDX1-positive pancreatic endoderm cell population, which contains mixed cell subpopulations, often contains at least a PDX-1-expressing subpopulation, specifically a PDX-1 / NKX6.1-expressing subpopulation. The PDX-1 / NKX6.1 subpopulation is also referred to as "pancreatic progenitor," "pancreatic epithelial," or "PEC" or versions of PEC, e.g., PEC-01. While Table 1 lists stage 4 cell populations, these various subpopulations are not limited to stage 4. Some of these subpopulations can be found as early as stage 3, and later, including stages 5, 6, and 7 (immature beta cells). The ratio of each subpopulation varies depending on the cell culture medium conditions employed. For example, in Agulnick et al. 2015 (see above), 73–80% PDX-1 / NKX6.1 cells were further differentiated into pancreatic islet-like cells (ICs), which contained approximately 74–89% endocrine cells, and 40–50% of these expressed insulin (INS). Therefore, different cell culture conditions can generate different ratios of cell subpopulations, which can affect in vivo function and, consequently, serum c-peptide levels. Whether modified methods for generating PDX1-positive pancreatic endoderm-lineage cell culture populations affect in vivo function can only be determined using the in vivo studies detailed below. Furthermore, just because a particular cell type has been generated and is well characterized, it cannot and should not be assumed that such methods will produce the same cellular intermediates unless they are also well characterized.

[0162] In one embodiment, there is provided a method for generating mature beta cells in vivo, comprising forming human definitive endoderm-lineage cells induced in vitro from human pluripotent stem cells using at least a TGFβ superfamily member and / or at least a TGFβ superfamily member and a Wnt family member, preferably a TGFβ superfamily member and a Wnt family member, preferably activin A, B, or GDF-8, GDF-11, or GDF-15 and Wnt3a, preferably activin A and Wnt3a, preferably GDF-8 and Wnt3a, and forming PDX1-positive pancreatic endoderm cells using at least KGF, a BMP inhibitor, and retinoic acid (RA) or an RA analog, and preferably KGF, noggin, and RA. This method can further include differentiating PDX1-positive pancreatic endoderm cells into immature beta cells or MAFA-expressing cells using thyroid hormone and / or a TGFb-RI inhibitor, a BMP inhibitor, KGF, EGF, thyroid hormone and / or a protein kinase C activator, preferably using noggin, KGF, and EGF, preferably in addition to T3 or T4 and an ALK5 inhibitor or T3, or T4 alone, or an ALK5 inhibitor alone, or T3 or T4, an ALK5 inhibitor, and a PKC activator, such as ILV, TPB, and PdBu. Alternatively, preferably using noggin and ALK5i, the PDX1-positive pancreatic endoderm cell or MAFA immature beta cell population can be transplanted into a mammalian host and matured in vivo to generate a cell population containing insulin-secreting cells capable of responding to blood glucose.

[0163] In one aspect, unipotent human immature beta cells or PDX1-positive pancreatic endoderm cells are provided that express INS and NKX6.1 and express little or no NGN3. In one embodiment, the unipotent human immature beta cells are capable of maturing into mature beta cells. In one embodiment, the unipotent human immature beta cells further express MAFB in vitro and in vivo. In one embodiment, the immature beta cells express INS, NKX6.1, and MAFA and express little or no NGN3.

[0164] In one embodiment, pancreatic endoderm-lineage cells that express at least CHGA (or CHGA+) refer to endocrine cells, and pancreatic endoderm cells that do not express CHGA (or CHGA-) refer to non-endocrine cells. In another embodiment, these endocrine and non-endocrine subpopulations can be pluripotent progenitor / precursor subpopulations, such as non-endocrine pluripotent pancreatic progenitor subpopulations or endocrine pluripotent pancreatic progenitor subpopulations, or they can be unipotent subpopulations, such as immature endocrine cells, preferably immature beta cells, immature glucagon cells, and the like.

[0165] In one embodiment, greater than 10%, preferably greater than 20%, 30%, 40%, and more preferably greater than 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 100% of the pancreatic endoderm cell population or PDX1-positive pancreatic endoderm cell population (stage 4) is a non-endocrine (CHGA-) pluripotent progenitor subpopulation, which gives rise to mature insulin-secreting cells and responds to glucose in vivo when implanted into a mammalian host.

[0166] One embodiment provides compositions and methods for differentiating pluripotent stem cells in vitro substantially into pancreatic endoderm cultures and further differentiating the pancreatic endoderm cultures into endocrine cells or endocrine precursor cells in vitro. In one embodiment, the endocrine cells or endocrine precursor cells express CHGA. In one embodiment, the endocrine cells are capable of producing insulin in vitro. In one embodiment, the in vitro endocrine insulin-secreting cells are capable of producing insulin in response to glucose stimulation. In one embodiment, more than 10%, preferably more than 20%, 30%, 40%, and more preferably more than 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 100% of the cells in a cell population are endocrine cells.

[0167] Embodiments described herein provide compositions and methods for differentiating pluripotent human stem cells into endocrine cells in vitro. In one embodiment, the endocrine cells express CHGA. In one embodiment, the endocrine cells are capable of producing insulin in vitro. In one embodiment, the endocrine cells are immature endocrine cells, e.g., immature beta cells. In one embodiment, the in vitro insulin-producing cells are capable of producing insulin in response to glucose stimulation.

[0168] One embodiment provides a method for producing insulin in vivo in a mammal, the method comprising: (a) loading a population of pancreatic endoderm cells, endocrine cells, or endocrine precursor cells into an implantable semipermeable device; (b) implanting the device containing the cell population into a mammalian host; and (c) maturing the cell population in the device in vivo, wherein at least some of the endocrine cells are insulin-secreting cells that produce insulin in response to glucose stimulation in vivo, thereby producing insulin in vivo in the mammal. In one embodiment, the endocrine cells are derived from a cell composition comprising PECs using a higher level of a non-endocrine pluripotent pancreatic progenitor subpopulation (CHGA-). In another embodiment, the endocrine cells are derived from a cell composition comprising PECs using a reduced endocrine subpopulation (CHGA+). In another embodiment, the endocrine cells are immature endocrine cells, preferably immature beta cells.

[0169] In one embodiment, endocrine cells formed in vitro from pluripotent stem cells express more PDX1 and NKX6.1 than PDX1-positive pancreatic endoderm populations or PDX1 / NKX6.1-positive non-endocrine (CHGA-) subpopulations. In one embodiment, endocrine cells formed in vitro from pluripotent stem cells express relatively more PDX1 and NKX6.1 than PEC non-endocrine multipotent pancreatic progenitor subpopulations (CHGA-). In one embodiment, endocrine cells were obtained by adding bone morphogenetic protein (BMP) and retinoic acid (RA) analogs, alone or in combination, to cell cultures. The endocrine cells exhibit increased expression of PDX1 and NKX6.1 compared to PEC non-endocrine multipotent pancreatic progenitor subpopulations (CHGA-). In one embodiment, the BMP is selected from the group including BMP2, BMP5, BMP6, BMP7, BMP8, and BMP4, more preferably BMP4. In one embodiment, the retinoic acid is selected from the group including all-trans retinoic acid and TTNPB (4-[(E)-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl]benzoic acid arotinoid acid), or 0.1 to 10 μM AM-580 (4-[(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)carboxamido]benzoic acid), more preferably TTNPB.

[0170] One embodiment provides a method for differentiating pluripotent stem cells in vitro into endocrine cells and immature endocrine cells, preferably immature beta cells, comprising dissociating and recombining aggregates. In one embodiment, the dissociation and recombination occurs at stage 1, stage 2, stage 3, stage 4, stage 5, stage 6, or stage 7, or a combination thereof. In one embodiment, definitive endoderm, PDX1-negative foregut endoderm, PDX1-positive foregut endoderm, PECs, and / or endocrine and endocrine progenitor / precursor cells are dissociated and recombined. In one embodiment, the dissociated and recombined cell aggregates at stage 7 comprise a reduced non-endocrine (CHGA-) subpopulation relative to the endocrine (CHGA+) subpopulation. In one embodiment, more than 10%, preferably more than 20%, 30%, 40%, and more preferably more than 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 100% of the cells in the cell population are endocrine (CHGA+) cells.

[0171] One embodiment provides a method for differentiating pluripotent stem cells in vitro into endocrine cells by removing endocrine cells formed during stage 4 PEC generation, thereby enriching for a subpopulation of non-endocrine pluripotent pancreatic progenitors (CHGA-) that are PDX1+ and NKX6.1+.

[0172] In one embodiment, PEC cultures enriched for non-endocrine pluripotent progenitor subpopulations (CHGA-) are formed by not adding a Noggin family member at stage 3 and / or stage 4. In one embodiment, PEC cultures relatively enriched for cells committed to the endocrine lineage (CHGA+) are formed by not adding a Noggin family member at stage 3 and / or stage 4. In one aspect, the Noggin family member is a compound selected from the group including Noggin, Chordin, Follistatin, Follistatin-like protein, Cerberus, Coco, Dan, Gremlin, Sclerostin, and PRDC (protein related to Dan and Cerberus).

[0173] One embodiment provides a method for maintaining endocrine cells in culture by culturing the cells in a medium containing a high level of exogenous glucose, wherein the exogenous glucose added is between about 1 mM and 25 mM, about 1 mM and 20 mM, about 5 mM and 15 mM, about 5 mM and 10 mM, or about 5 mM and 8 mM. In one aspect, the medium is a DMEM-, CMRL-, or RPMI-based medium.

[0174] One embodiment provides methods for differentiating pluripotent stem cells in vitro, with and without dissociation and reassociation of cell aggregates. In one aspect, undissociated or dissociated-reassociated cell aggregates are cryopreserved or frozen at stage 6 and / or stage 7 without affecting the in vivo function of the endocrine cells. In one aspect, cryopreserved endocrine cell cultures are thawed, cultured, and function in vivo upon transplantation.

[0175] Another embodiment provides a culture system for differentiating pluripotent stem cells, the culture system comprising at least a substance capable of suppressing or inhibiting endocrine gene expression during early differentiation stages and a substance capable of inducing endocrine gene expression during later differentiation stages. In one embodiment, the substance capable of suppressing or inhibiting endocrine gene expression is added to a culture system comprising pancreatic PDX1-negative foregut cells. In one embodiment, the substance capable of inducing endocrine gene expression is added to a culture system comprising PDX1-positive pancreatic endoderm progenitors or PECs. In one embodiment, the substance capable of suppressing or inhibiting endocrine gene expression is a substance that activates the TGF-beta receptor family, preferably activin, and preferably high levels of activin followed by low levels of activin. In one embodiment, the substance capable of inducing endocrine gene expression is N-[N-(3,5-difluorophenacetyl-L-alanyl)]-S-phenylglycine t-butyl ester (DAPT), RO44929097, DAPT (N-[N-(3,5-difluorophenacetyl-L-alanyl)]-S-phenylglycine t-butyl ester), 1-(S)-endo-N-(1,3,3)-trimethylbicyclo[2.2.1]hept-2-yl)-4-fluorophenylsulfonamide, WPE-III31C, S-3-[N′-(3,5-difluorophenyl-alpha-hydroxyacetyl)-L-alanyl]amino-2,3-dihydro-1-methyl and a gamma secretase inhibitor selected from the group consisting of (N)-[(S)-2-hydroxy-3-methyl-butyryl]-1-(L-alaninyl)-(S)-1-amino-3-methyl-4,5,6,7-tetrahydro-2H-3-benzazepin-2-one, (N)-[(S)-2-hydroxy-3-methyl-butyryl]-1-(L-alaninyl)-(S)-1-amino-3-methyl-4,5,6,7-tetrahydro-2H-3-benzazepin-2-one, BMS-708163 (avagacestat), BMS-708163, semagacestat (LY450139), semagacestat (LY450139), MK-0752, MK-0752, YO-01027, YO-01027 (dibenzazepine, DBZ), LY-411575, LY-411575, or LY2811376.In one embodiment, high levels of activin refer to levels greater than 40 ng / mL, 50 ng / mL, and 75 ng / mL. In one embodiment, high levels of activin are used during stage 3 or prior to the generation of pancreatic foregut endoderm cells. In one embodiment, low levels of activin refer to levels less than 30 ng / mL, 20 ng / mL, 10 ng / mL, and 5 ng / mL. In one embodiment, low levels of activin are used during stage 4 or for PEC generation. In one embodiment, the endocrine gene inhibited or induced is NGN3. In another embodiment, activin A and Wnt3A are used alone or in combination to inhibit endocrine expression, preferably inhibiting NGN3 expression prior to the generation of pancreatic foregut endoderm cells or preferably during stage 3. In one embodiment, a gamma secretase inhibitor, preferably RO44929097 or DAPT, is used in the culture system to induce endocrine gene expression after PEC generation, or preferably during stages 5, 6, and / or 7.

[0176] An in vitro cell culture comprising endocrine cells, wherein at least 5% of the human cells express any of the following genes: insulin (INS), NK6 homeobox 1 (NKX6.1), pancreatic and duodenal homeobox 1 (PDX1), transcription factor-associated locus 2 (NKX2.2), paired box 4 (PAX4), neurogenic differentiation 1 (NEUROD), forkhead box A1 (FOXA1), forkhead box A2 (FOXA2), snail family zinc finger 2 (SNAIL2), and musculoaponeurotic fibrosarcoma oncogene family A and B (MAFA and MAFB) genes. ), and are substantially free of a marker selected from the group consisting of neurogenin 3 (NGN3), islet 1 (ISL1), hepatocyte nuclear factor 6 (HNF6), GATA binding protein 4 (GATA4), GATA binding protein 6 (GATA6), pancreas-specific transcription factor 1a (PTF1A), and SRY (sex determining region Y)-9 (SOX9), wherein the endocrine cells are unipotent and capable of maturing into pancreatic beta cells. [Example]

[0177] Example 1 The two discontinuous layers were thermally bonded together to form a bilayer bonded composite.

[0178] The first layer of the two-layer biocompatible membrane composite was expanded polytetrafluoroethylene (ePTFE) (relaxed layer) prepared according to the teachings of U.S. Patent No. 5,814,405 to Branca et al. The scanning electron micrograph (SEM) image shown in Figure 10 is a representative image of the surface of the first layer (i.e., relaxed layer) ePTFE membrane. The properties of this ePTFE layer are listed in Table 1. The second layer was a commercially available spunbond polyester nonwoven material (vascularized layer). A representative surface structure of the third layer is shown in the SEM image in Figure 11. The properties of this layer are listed in Table 2. [Table 2]

[0179] The relaxed layer and vascularized layer were bonded together by stacking them adjacent to each other and restraining them within an aluminum clamping ring with an aluminum backing block. The vascularized layer (nonwoven fabric layer) was oriented so that it was in contact with the aluminum backing block. The ePTFE membrane faced outward within the clamping hoop. The material within the clamping ring with backing block was then sandwiched between two steel plates and placed in a carver press. Figure 12 is an exploded view showing the configuration of the materials used. Specifically, the materials included a carver press upper platen 1220, an upper steel plate 1240, a clamping ring with backing block 1260, a lower steel plate 1280, and a carver press lower platen 1225. The two-layer biocompatible membrane composite 1210 included a first layer (relaxed layer) 1230 and a second layer (relaxed layer) 1250.

[0180] The Carver press was set to a temperature of 235°C, and minimal pressure was applied so that the Carver press platens were in contact with the steel plates but no pressure was registered on the pressure gauge. After a 45-second dwell time, contact from the Carver press platens was removed. The relaxed and vascularized layers were removed from the clamping ring and bonded together as a biocompatible membrane composite.

[0181] Example 2 Three biocompatible membrane composites, each with two distinct layers, were similarly assembled. The three structures shared a similar first layer (relaxation layer) but had different second layers (vascularization layers). The three different biocompatible membrane composites are hereafter referred to as Structure A, Structure B, and Structure C.

[0182] A first expanded polytetrafluoroethylene (ePTFE) membrane was prepared according to the teachings of U.S. Patent No. 5,814,405 to Branca et al. The ePTFE tape precursor for the first ePTFE layer was processed by a below-the-melt machine direction (MD) expansion process according to the teachings of U.S. Patent No. 5,814,405 to Branca et al. During the below-the-melt MD expansion process of the first ePTFE tape precursor, an FEP film was applied according to the teachings of WO 94 / 13469 to Bacino. The ePTFE tape precursor for the second ePTFE membrane was processed by an amorphous lock process and above-the-melt MD expansion according to the teachings of U.S. Patent No. 5,814,405 to Branca et al. The properties of the tape precursor and the amount of MD expansion applied to the second layer varied among the three structures. During the first below-melt MD stretching step of the second ePTFE tape precursor, an FEP film was applied according to the teachings of Bacino, WO 94 / 13469. The expanded ePTFE tape precursor of the second ePTFE membrane was laminated to the expanded ePTFE tape precursor of the first ePTFE membrane, with the FEP side of the second ePTFE tape in contact with the PTFE side of the ePTFE tape precursor of the first ePTFE membrane.

[0183] The two-layer biocompatible membrane composite was then simultaneously stretched in the machine and transverse directions at a temperature above the melting point of PTFE. A representative surface microstructure of the first ePTFE layer with FEP1320 on top is shown in the scanning electron micrograph (SEM) image in Figure 13. The SEM images shown in Figures 14, 15, and 16 are representative images of the node-fibril structure of the second ePTFE membranes 1400, 1500, and 1600 (the vascularized layer), respectively. The SEM images shown in Figures 17, 18, and 19 are representative images of the cross-sectional structure of the two-layer biocompatible membrane composite including the first ePTFE membranes 1720, 1820, and 1920 (the relaxed layer), respectively, and the second ePTFE membranes 1740, 1840, and 1940 (the vascularized layer), respectively.

[0184] Characterization of biocompatible membrane composites Each individual layer of the biocompatible membrane composite was evaluated and characterized for relevant parameters of layer-by-layer function. The methods used to characterize the relevant parameters were performed according to the test methods described in the "Test Methods" section above. The results are summarized in Table 3. [Table 3]

[0185] The invention of this application has been described generally and with reference to specific embodiments. As will be apparent to those skilled in the art, various modifications and variations can be made in the embodiments without departing from the scope of the disclosure. Accordingly, the embodiments are intended to cover the modifications and variations of the present invention provided they come within the scope of the appended claims and their equivalents. The following are embodiments of the present invention: [Aspect 1] A biocompatible membrane composite, comprising: a first layer having first solid features with a first solid feature spacing, the majority of the first solid feature spacing of the first solid features being less than about 50 microns; a second layer having second solid features with a second solid feature spacing, the majority of the second solid feature spacing of the second solid features being greater than about 50 microns; A biocompatible membrane composite comprising: [Aspect 2] 2. The biocompatible membrane composite of embodiment 1, wherein the first layer comprises bondable solid features, and the bondable solid features are bonded to an implantable device or an implantable cell system. [Aspect 3] 2. The biocompatible membrane composite of embodiment 1, wherein the first layer comprises a representative minor axis of about 3 microns to about 20 microns. [Aspect 4] 2. The biocompatible membrane composite of embodiment 1, wherein the first layer has a first thickness of less than about 200 microns. [Aspect 5] 2. The biocompatible membrane composite of claim 1, wherein at least one of the first solid features of the first layer and the second solid features of the second layer are connected by fibrils, and the fibrils are deformable. [Aspect 6] 5. The biocompatible membrane composite of embodiment 4, wherein the second layer has a second thickness of about 30 microns to about 200 microns. [Aspect 7] 2. The biocompatible membrane composite of claim 1, wherein the biocompatible membrane composite comprises a surface coating thereon, the surface coating comprising one or more elements selected from an antimicrobial agent, an antibody, a pharmaceutical agent, and a biologically active molecule. [Aspect 8] 2. The biocompatible membrane composite of embodiment 1, wherein the biocompatible membrane composite comprises a hydrophilic coating thereon. [Aspect 9] 2. The biocompatible membrane composite of claim 1, wherein at least one of the first layer and the second layer is a fluoropolymer membrane. [Aspect 10] 2. The biocompatible membrane composite of embodiment 1, wherein the second layer is a spunbond nonwoven polyester material. [Aspect 11] 10. The biocompatible membrane composite of embodiment 1, comprising a reinforcing component. [Aspect 12] 12. The cell encapsulation device of embodiment 11, wherein the reinforcing component is a woven or nonwoven fabric. [Aspect 13] the first solid feature includes a representative minor axis, a representative major axis, and a solid feature depth; and a majority of the first solid features of the first layer have at least two of the representative minor axis, the representative major axis, and the solid feature depth greater than about 5 microns; 2. The biocompatible membrane composite of embodiment 1. [Aspect 14] the first layer having a first thickness of less than about 200 microns and first solid features, the first solid features having a majority of first solid feature spacing of less than about 50 microns; The second layer and wherein a majority of the first solid features have a first representative minor axis of between about 3 microns and about 20 microns. [Aspect 15] 15. The biocompatible membrane composite of embodiment 14, wherein the second layer comprises second solid features and second solid feature spacings, and a majority of the second solid feature spacings of the second solid features are greater than about 50 microns. [Aspect 16] 15. The biocompatible membrane composite of embodiment 14, wherein the second layer has a second thickness of about 30 microns to about 200 microns. [Aspect 17] the first solid feature includes a first representative minor axis, a first representative major axis, and a first solid feature depth; and a majority of the first solid features of the first layer having at least two of the first representative minor axis, the first representative major axis, and the first solid feature depth greater than about 5 microns; 15. The biocompatible membrane composite of embodiment 14. [Aspect 18] 15. The biocompatible membrane composite of embodiment 14, wherein the first solid features are connected by fibrils, and the fibrils are deformable. [Aspect 19] 15. The biocompatible membrane composite of embodiment 14, wherein the second layer comprises second solid features, and a majority of the second solid features have a second representative minor axis of less than about 40 microns. [Aspect 20] 15. The biocompatible membrane composite of embodiment 14, wherein the second layer is a spunbond nonwoven polyester material. [Aspect 21] 15. The biocompatible membrane composite of claim 14, wherein the first solid features of the first layer comprise a member selected from a thermoplastic polymer, a polyurethane, a silicone, a rubber, an epoxy, and combinations thereof. [Aspect 22] 15. The biocompatible membrane composite of embodiment 14, comprising a reinforcing component. [Aspect 23] 23. The cell encapsulation device of embodiment 22, wherein the reinforcing component is a woven or nonwoven fabric. [Aspect 24] 15. The biocompatible membrane composite of claim 14, wherein the biocompatible membrane composite comprises a surface coating thereon, and the surface coating comprises one or more elements selected from an antimicrobial agent, an antibody, a pharmaceutical agent, and a biologically active molecule. [Aspect 25] 15. The biocompatible membrane composite of embodiment 14, wherein the biocompatible membrane composite comprises a hydrophilic coating thereon. [Aspect 26] 15. The biocompatible membrane composite of embodiment 14, wherein the first layer comprises bondable solid features, and the bondable solid features are bonded to an implantable device or an implantable cell system. [Aspect 27] 27. The biocompatible membrane composite of embodiment 26, wherein the implantable device comprises a switch, a sensor, a bolometer, a biosensor, a chemical sensor, an inertial sensor, an acoustic sensor, a microphone, a microspeaker, a pressure sensor, a resonator, an ultrasonic resonator, a temperature sensor, a vibration sensor, a microengine, an actuator, a thermal actuator, a bimorph and unimorph actuator, an electric rotating micromachine, a microgear, a micropump, a microtransmitter, a microengine, an optical microelectromechanical system, a micromirror, an optical switch or a biological microelectromechanical system, or a combination thereof. [Aspect 28] 15. The biocompatible membrane composite of embodiment 14, wherein the biocompatible membrane composite is configured for use with a tissue, a scaffold, a two-dimensional cell culture system, a three-dimensional cell culture system, a cell container, a cell encapsulation device, a cell system, or a combination thereof. [Aspect 29] 15. The biocompatible membrane composite of claim 14, wherein at least one of the first layer and the second layer is configured as a biological interface for an implantable sensor used to detect molecules produced in the body or molecules produced outside the body. [Aspect 30] A biocompatible membrane composite as described in aspect 14, wherein at least one of the first layer and the second layer is configured as a biocompatible cover for an implantable device that provides or requires molecules, signals, or activity within the body and induces these functions. [Aspect 31] 15. The biocompatible membrane composite of embodiment 14, wherein the first solid feature is at least partially attached to the cell system or implantable device. [Aspect 32] 32. The biocompatible membrane composite of embodiment 31, wherein the cell system is a cell container or a bioactive scaffold. [Aspect 33] 10. A method of lowering blood glucose levels in a mammal, comprising implanting a cell encapsulation device comprising the biocompatible membrane composite of embodiment 1, wherein the cells encapsulated within the cell encapsulation device comprise a population of PDX1-positive pancreatic endoderm cells, and wherein the pancreatic endoderm cells grow to become insulin-secreting cells, thereby lowering blood glucose levels. [Aspect 34] 10. A method for producing insulin in vivo, comprising implanting a cell encapsulation device comprising the biocompatible membrane composite of embodiment 1, wherein the cell encapsulation device contains a population of PDX1-positive pancreatic endoderm cells that grow into insulin-secreting cells, and wherein the insulin-secreting cells secrete insulin in response to glucose stimulation.

Claims

1. A biocompatible membrane composite, comprising: a first layer having first solid features with a first solid feature spacing, a majority of the first solid feature spacing being less than about 50 microns; a second layer having second solid features with a second solid feature spacing, a majority of the second solid feature spacing being greater than about 50 microns; A biocompatible membrane composite comprising:

2. 10. The biocompatible membrane composite of claim 1, wherein the first layer comprises a typical minor axis of about 3 microns to about 20 microns.

3. 3. The biocompatible membrane composite of claim 1 or 2, wherein the second layer has a first pore size greater than about 9 microns effective diameter.

4. 4. The biocompatible membrane composite of claim 1, wherein the first layer has a first thickness of less than about 200 microns.

5. 5. The biocompatible membrane composite of claim 1, wherein the first layer has a second pore size with an effective diameter of about 1 micron to about 9 microns.

6. The biocompatible membrane composite of claim 5 , wherein the solid features of at least one of the first and second layers are connected by fibrils, and the fibrils are deformable.

7. 7. The biocompatible membrane composite of claim 1, wherein the second layer has a second thickness of about 30 microns to about 200 microns.

8. 8. The biocompatible membrane composite of claim 1, wherein at least one of the first and second layers comprises a polymer selected from expanded polytetrafluoroethylene (ePTFE) membranes, fluorinated ethylene propylene (FEP) membranes, and modified expanded polytetrafluoroethylene (ePTFE) membranes.

9. 9. The biocompatible membrane composite of claim 1, wherein the biocompatible membrane composite comprises a surface coating thereon, the surface coating comprising one or more members selected from antimicrobial agents, antibodies, pharmaceuticals, and biologically active molecules.

10. 10. The biocompatible membrane composite of claim 1, wherein at least one of the first and second layers is an expanded polytetrafluoroethylene membrane.

11. 11. The biocompatible membrane composite of claim 1, wherein the second layer is a spunbond nonwoven polyester material.

12. 12. The biocompatible membrane composite of claim 1, comprising a reinforcing component.

13. The cell encapsulation device of claim 12 , wherein the reinforcing component is a woven or nonwoven fabric.

14. the first layer further comprising a representative minor axis, a representative major axis, and a solid feature depth; and a majority of at least two of the typical minor axis, the typical major axis, and the solid feature depth is greater than about 5 microns; The biocompatible membrane composite of any one of claims 1 to 13.

15. a first layer having a first pore size having an effective diameter of about 1 micron to about 9 microns, a first thickness of less than about 200 microns, and first solid features having a majority of first solid feature spacings of less than about 50 microns, the majority of the first solid features having a first representative minor axis of about 3 microns to about 20 microns; A second layer; A biocompatible membrane composite comprising:

16. 16. The biocompatible membrane composite of claim 15, wherein the second layer has a pore size that is greater than about 9 microns in effective diameter.

17. 17. The biocompatible membrane composite of claim 15 or claim 16, wherein the second layer includes second solid features with a majority of the second solid feature spacing greater than about 50 microns.

18. 18. The biocompatible membrane composite of claim 15, wherein the second layer has a second thickness of about 30 microns to about 200 microns.

19. the first layer includes a first representative major axis and a first solid feature depth; and at least two of the first representative minor axis, the first representative major axis, and the first solid feature depth are greater than about 5 microns; 19. The biocompatible membrane composite of any one of claims 15 to 18.

20. 20. The biocompatible membrane composite of claim 15, wherein the solid features are connected by fibrils, and the fibrils are deformable.

21. 21. The biocompatible membrane composite of claim 15, wherein the second layer comprises second solid features, and a majority of the second solid features have a second representative minor axis of less than about 40 microns.

22. the second layer includes a second representative major axis and a second solid feature depth; and a majority of at least two of the second representative minor axis, the second representative major axis, and the second solid feature depth is greater than about 5 microns; 22. The biocompatible membrane composite of any one of claims 15 to 21.

23. 23. The biocompatible membrane composite of any one of claims 15 to 22, wherein at least one of the first and second layers is a polymer selected from expanded polytetrafluoroethylene (ePTFE) membranes, fluorinated ethylene propylene (FEP) membranes, and modified expanded polytetrafluoroethylene (ePTFE) membranes.

24. 24. The biocompatible membrane composite of any one of claims 15 to 23, wherein the second layer is a spunbond nonwoven polyester material.

25. 25. The biocompatible membrane composite of any one of claims 15 to 24, wherein at least one of the first and second layers comprises a polymer, a fluoropolymer membrane, a non-fluoropolymer membrane, a woven fabric, a non-woven fabric, a woven or non-woven collection of fibers or yarns, a fibrous matrix, and combinations thereof.

26. 26. The biocompatible membrane composite of claim 15, wherein the first solid features of the first layer comprise a member selected from a thermoplastic polymer, a polyurethane, a silicone, a rubber, an epoxy, and combinations thereof.

27. 27. The biocompatible membrane composite of any one of claims 15 to 26, further comprising a reinforcing component.

28. 28. The cell encapsulation device of claim 27, wherein the reinforcing component is a woven or nonwoven fabric.

29. 29. The biocompatible membrane composite of any one of claims 15 to 28, wherein the biocompatible membrane composite comprises a surface coating thereon, and the surface coating comprises one or more members selected from antimicrobial agents, antibodies, pharmaceuticals, and biologically active molecules.

30. 30. The biocompatible membrane composite of any one of claims 15 to 29, wherein the biocompatible membrane composite comprises a hydrophilic coating thereon.

31. 31. The biocompatible membrane composite of claim 15, wherein the first layer comprises bondable solid features, and the bondable solid features are bonded to an implantable device or an implantable cell system.

32. 32. The biocompatible membrane composite of claim 31, wherein the implantable device is a scaffold.

33. 33. The biocompatible membrane composite of claim 32, wherein the scaffold is a cell culture matrix.

34. 33. The biocompatible membrane composite of claim 32, wherein the scaffold is an explant.

35. 32. The biocompatible membrane composite of claim 31, wherein the first solid feature is at least partially bonded to the cellular system.

36. The biocompatible membrane composite of claim 35 , wherein the cell system is a cell container.

37. The biocompatible membrane composite of claim 31 , wherein the implantable device is a sensor.

38. 32. The biocompatible membrane composite of claim 31, wherein the cell system is a bioactive scaffold.

39. 10. A method for lowering blood glucose levels in a mammal, comprising implanting a cell encapsulation device comprising the biocompatible membrane composite of any one of the preceding claims, wherein the cells encapsulated within the cell encapsulation device comprise a population of PDX1-positive pancreatic endoderm cells, and wherein the pancreatic endoderm cells grow to become insulin-secreting cells, thereby lowering blood glucose levels.

40. 40. The method of claim 39, wherein the PDX1-positive pancreatic endoderm cells comprise a mixture of cells further comprising endocrine and / or endocrine precursor cells, wherein the endocrine and / or endocrine precursor cells express chromogranin A (CHGA).

41. 10. The method of lowering blood glucose levels in a mammal as described in claim 1, wherein the cells encapsulated in the cell encapsulation device comprise a population of PDX1-positive pancreatic endoderm cells, and wherein the pancreatic endoderm cells grow to become insulin-secreting cells, thereby lowering blood glucose levels.

42. 42. The method of claim 41, wherein the PDX1-positive pancreatic endoderm cells comprise a mixture of cells further comprising endocrine and / or endocrine precursor cells, wherein the endocrine and / or endocrine precursor cells express chromogranin A (CHGA).

43. 10. A method of lowering blood glucose levels in a mammal of any one of the preceding claims, comprising implanting a cell encapsulation device comprising: a first layer having first solid features with a first solid feature spacing, wherein a majority of the first solid feature spacing is less than about 50 microns; and a second layer having second solid features with a second solid feature spacing, wherein a majority of the second solid feature spacing is greater than about 50 microns, at least a portion of the bonded features being intimately attached to the first layer; and wherein the cell encapsulation device comprises a cell population comprising PDX1-positive pancreatic endoderm cells, and wherein the pancreatic endoderm cells grow to become insulin-secreting cells, thereby lowering blood glucose levels.

44. 44. The method of claim 43, wherein the PDX1-positive pancreatic endoderm cells comprise a mixture of cells further comprising endocrine and / or endocrine precursor cells, wherein the endocrine and / or endocrine precursor cells express chromogranin A (CHGA).

45. 10. A method of lowering blood glucose levels in a mammal according to any one of the preceding claims, comprising implanting a biocompatible membrane composite comprising: a first layer having first solid features with a first solid feature spacing, wherein a majority of the first solid feature spacing is less than about 50 microns; a second layer having second solid features with a second solid feature spacing, wherein a majority of the second solid feature spacing is greater than about 50 microns; and a cell population comprising PDX1-positive pancreatic endoderm cells, wherein the pancreatic endoderm cells grow into insulin-secreting cells, thereby lowering blood glucose levels.

46. 46. ​​The method of claim 45, wherein the PDX1-positive pancreatic endoderm cells comprise a mixture of cells further comprising endocrine and / or endocrine precursor cells, wherein the endocrine and / or endocrine precursor cells express chromogranin A (CHGA).

47. 47. The method of claim 45 or 46, wherein the encapsulated in vitro PDX1-positive pancreatic endoderm cells comprise a mixture of cell subpopulations including at least a pancreatic progenitor population that co-expresses PDX-1 / NKX6.

1.

48. The method of any one of claims 45 to 47, wherein the encapsulated in vitro PDX1-positive pancreatic endoderm cells comprise a mixture of cell subpopulations including at least a pancreatic progenitor population that co-expresses PDX-1 / NKX6.1 and a pancreatic endocrine and / or endocrine precursor population that expresses PDX-1 / NKX6.1 and CHGA.

49. 49. The method of any one of claims 45-48, wherein at least 30% of said population comprises a pancreatic progenitor population that co-expresses PDX-1 / NKX6.

1.

50. 50. The method of any one of claims 45-49, wherein at least 40% of said population comprises a pancreatic progenitor population that co-expresses PDX-1 / NKX6.

1.

51. 51. The method of any one of claims 45-50, wherein at least 50% of said population comprises a pancreatic progenitor population that co-expresses PDX-1 / NKX6.

1.

52. 52. The method of any one of claims 45 to 51, wherein at least 20% of said population is an endocrine and / or endocrine precursor population that expresses PDX-1 / NKX6.1 / CHGA.

53. 53. The method of any one of claims 45 to 52, wherein at least 30% of said population is an endocrine and / or endocrine precursor population that expresses PDX-1 / NKX6.1 / CHGA.

54. 54. The method of any one of claims 45 to 53, wherein at least 40% of said population is an endocrine and / or endocrine precursor population that expresses PDX-1 / NKX6.1 / CHGA.

55. 55. The method of any one of claims 45 to 54, wherein the pancreatic progenitor cells and / or endocrine or endocrine precursor cells are capable of growing into insulin-secreting cells in vivo.

56. 10. A method for producing insulin in vivo according to any one of the preceding claims, comprising implanting a cell encapsulation device comprising the biocompatible membrane composite of any one of the preceding claims and a population of PDX1-positive pancreatic endoderm cells that grow into insulin-secreting cells, wherein the insulin-secreting cells secrete insulin in response to glucose stimulation.

57. 57. The method of claim 56, wherein said PDX1-positive pancreatic endoderm cells comprise a mixture of cells further comprising endocrine and / or endocrine precursor cells, wherein said endocrine and / or endocrine precursor cells express chromogranin A (CHGA).

58. 58. The method of claim 56 or 57, wherein at least about 30% of said population is an endocrine and / or endocrine precursor population that expresses PDX-1 / NKX6.1 / CHGA.

59. 59. The method of any one of claims 56-58, wherein the in vitro human PDX1-positive pancreatic endoderm cell culture comprises a mixture of PDX1-positive pancreatic endoderm cells and at least a transforming growth factor beta (TGF-beta) receptor kinase inhibitor.

60. 60. The method of any one of claims 56 to 59, further comprising a bone morphogenetic protein (BMP) inhibitor.

61. 61. The method of any one of claims 56 to 60, wherein the TGF-beta receptor kinase inhibitor is a TGF-beta receptor type 1 kinase inhibitor.

62. 62. The method of any one of claims 56 to 61, wherein the TGF-beta receptor kinase inhibitor is an ALK5i.

63. 63. The method of any one of claims 56 to 62, wherein the BMP inhibitor is noggin.

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