PDX1 pancreatic endoderm cells in cell delivery devices and methods thereof

Abstract

To provide a device and method for grafting a cell such as a pancreatic endoderm cell to a host.SOLUTION: A cell delivery device includes a nonwoven fabric outside a cell excluding membrane, and can drill the nonwoven fabric and / or the cell excluding membrane. Treatment of a host by immune suppression reagent is required in order to suppress allograft rejection by drilling of the device, and does not impair maturation or function of a grafted pancreatic endoderm cell.SELECTED DRAWING: Figure 1A

Description

[Technical Field]

[0001] The present invention relates to the field of devices and uses for delivering cells, such as pancreatic endoderm cells.

[0002] Description of support This invention was funded in part by the California Institute for Regenerative Medicine. [Background technology]

[0003] Several methods for implanting living cells or tissues in a device have been attempted. For example, perforated cell delivery devices have previously been reported. See U.S. Patent Application No. 12 / 618,659 and International Publication No. WO 1993002635, the entire contents of which are incorporated herein by reference. In these disclosures, each layer of the cell delivery device is perforated, i.e., perforations traverse each wall of the device or the entire device (all layers). If each layer of the device were perforated, cells would escape from the device.

[0004] Valentini et al., U.S. Patent Application No. 4,877,029, the entire contents of which are incorporated herein by reference, describes a semipermeable nerve guidance channel or tube composed of a smooth, cell-impermeable inner membrane surface and an outer surface with pores of 1-20 microns in size that form a trabecular structure that does not contain holes (pores) that traverse the thickness of the tube, and therefore does not allow vascular growth into the interior compartment.

[0005] Nonwoven fabrics have been used within cell delivery devices. The nonwoven fabric provides an inert scaffold within the delivery device that provides a structure for adhering and distributing cells within the device. See U.S. Patent Application No. 5,853,717 (incorporated by reference in its entirety).

[0006] Nonwoven fabrics have also been used to surround glucose monitoring devices. U.S. Patent Application No. 8,527,026, the entire contents of which are incorporated herein by reference, describes a sensor surrounded by a vascularized layer of expanded polytetrafluoroethylene (ePTFE), onto which a NWF may be laminated. In the '026 patent, the sensor measures a patient's blood glucose level.

[0007] Many strategies have been developed to encapsulate cells and promote their survival in vivo, but only mixed results have been reported. Thus, there is a need for devices and methods for transplanting cells that promote the maturation or function of the transplanted cells. Summary of the Invention

[0008] Provided are medical devices for delivering cells and methods for promoting cell viability, differentiation, and maturation of transplanted cells. In particular, embodiments described herein improve the interface between the host tissue and the delivery device by providing a nonwoven fabric (NWF), e.g., a polyester nonwoven fabric (NWPF), on the exterior of a cell-exclusion membrane, thereby improving vascularization of the implant. Embodiments described herein include perforations in the NWF and cell-exclusion membrane to improve vascularization of the implant. Embodiments described herein control the host immune response, improving cell viability.

[0009] In some embodiments, a combination product is disclosed that includes (a) a cell delivery device that includes a nonwoven layer and (B) PDX1-positive pancreatic endoderm cells.

[0010] In another embodiment, a method for producing insulin in a mammal is disclosed. The method comprises: a) administering an immunosuppressant to a mammalian host; and b) administering to the mammal a perforated device containing pancreatic endoderm cells. and c) maturing a pancreatic endoderm cell population in the perforated apparatus of the mammalian host, with the progenitor cell population producing insulin-secreting cells. [Brief explanation of the drawings]

[0011] [Figure 1A] 1A-1D show a delivery device in accordance with the present disclosure. [Figure 1B] Same as above [Figure 1C] Same as above [Figure 1D] Same as above [Figure 2A] 2A-2D each illustrate one embodiment of a perforated cell delivery device. [Figure 2B] Same as above [Figure 2C] Same as above [Figure 2D] Same as above [Figure 3] Figure 3 is a graph showing the correlation between total C-peptide protein content and beta cell mass, designated as islet equivalents (IEQ). This graph can be used to determine the mass of beta cells produced after transplantation of pancreatic cells into a perforated cell encapsulation device, designated as islet equivalents (IEQ). [Figure 4] FIG. 4 is a cross-sectional view of a cell delivery device with an added nonwoven layer. [Figure 5] Figure 5 is a graph showing serum human C-peptide concentrations in rats transplanted with pancreatic precursors delivered with needle-perforated delivery devices (control, CON) and laser-perforated devices. Secreted C-peptide levels were analyzed 12 weeks post-transplantation in a fasting state and 60 minutes after intraperitoneal administration of glucose. Mean C-peptide concentrations (+ / - SEM). "M" refers to manual hole creation using a needle, approximately 2 mm apart. [Figure 6] Figure 6 is a graph showing serum human C-peptide concentrations in rats transplanted with pancreatic precursors delivered with a (manual) needle-perforated delivery device (control, CON) and a laser-perforated device. Secreted C-peptide levels were analyzed 34 weeks post-transplantation in a fasting state and 15 and 30 minutes after intraperitoneal administration of glucose. "M" refers to the creation of manual holes using a needle, spaced approximately 2 mm apart. [Figure 7]Figure 7 is a graph showing the total C-peptide protein content of grafts explanted from rats previously transplanted with pancreatic precursors delivered via a perforated device at 10, 16, 36, and 39 weeks after transplantation. The perforated device used to generate the 10- and 16-week data was similar to that shown in Figure 1D, except that the NWF was not layered on a cell-exclusion membrane and had holes spaced 2 mm apart. The NWF used had a basis weight of 1.00 oz / yd², a nominal thickness of 228 µm, and a fiber diameter of 26 µm. The perforated device used to generate the 36-week data was identical to the 16-week device except that it did not have an NWF. The perforated device used to generate the 39-week data was identical to the 16-week device except that the NWF used had a basis weight of 0.75 oz / yd² and holes spaced 2 mm apart. [Figure 8] Figure 8 is a graph showing that the beta cell mass achieved in individual grafts by implanting perforated device pancreatic endoderm cells into rats for approximately 9 months was increased approximately 5-fold compared to intact devices implanted into mice for a similar period. [Figure 9] Figure 9 shows cross-sections of intact and perforated cell delivery devices after C-peptide plateau. The perforated device is larger and contains more insulin-producing cells (beta cells) than the intact device. [Figure 10A] 10A and 10B show graphs of serum rat or human C-peptide concentrations in nude rats fed a normal diet (control) or a diet containing 250 mg / kg of cyclosporine A (CsA-250) transplanted with pancreatic endoderm delivered via a perforated delivery device. Figure 10A shows a complete lack of endogenous rat beta cell function in rats treated with cyclosporine A at 12 weeks. Figure 10B shows that human C-peptide levels in CsA-treated rats were slightly increased due to CsA toxicity in endogenous beta cells. [Figure 10B] Same as above [Figure 11]Figure 11 shows serum human C-peptide concentrations in nude rats (control) fed a normal diet, rats fed a diet containing 250 mg / kg cyclosporine A (CsA-250), rats fed a diet containing 250 mg / kg cyclosporine A and 500 mg / kg mycophenolate mofetil (CsA-250 + MMF500), and rats fed a diet containing 150 mg / kg tacrolimus and 500 mg / kg mycophenolate mofetil (TAC-150 + MMF500), each transplanted with perforated delivery device pancreatic endoderm. Blood glucose levels in CsA-treated rats were slightly increased due to endogenous beta cell ISD toxicity. [Figure 12] Figure 12 is a graph showing serum human C-peptide concentrations in nude rats fed a normal diet (control) or rats fed a diet containing CsA-MMF that were transplanted with pancreatic endoderm delivered using either perforated or non-perforated (intact) delivery devices at 11 weeks (◆), 14 weeks (■), and 18 weeks (●). DETAILED DESCRIPTION OF THE INVENTION

[0012] Reference will now be made in detail to the embodiments of the present disclosure, examples of which are illustrated in the accompanying drawings. Wherever possible, the same reference numbers will be used throughout the drawings to refer to the same or like parts.

[0013] Terminology Unless otherwise noted, technical terms will be used according to normal usage. Definitions of common terms in molecular biology can be found in Benjamin Lewin, Genes V, Oxford University Press, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a It can be found in Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8).

[0014] Therapeutic Agent: In some embodiments, the cell delivery device includes a therapeutic agent. The term "therapeutic agent" refers to a drug or pathogenic agent, such as a microorganism, that provides a therapeutic or preventative result for a given disease. Preferably, the therapeutic agent is a toxin directed against encapsulated cells, cell aggregates, organoids, clusters, masses, tissues, or cancer cells, such as doxorubicin, daunomycin, epirubicin, vinblastine, vincristine, mitoxantrone, bleomycin, mitomycin, mechlorethamine, etc. The therapeutic agent may be an antibody or fragment thereof, a DNA molecule, or an RNA molecule. Additionally, the therapeutic agent may further include an antiviral agent, an antibacterial agent, an antifungal agent, or other agent that further facilitates treatment or prevention of a cellular disease or disorder. In some embodiments, the therapeutic agent is a cell to be transplanted, as described below.

[0015] Transplanted Cells: In one embodiment, the implantation site or delivery device is loaded with "cells," also referred to as "implanted cells," "exogenous cells," "cells suitable for transplantation," "transplanted cells," "implanted cells," "therapeutic cells," "implanted encapsulated cells," "encapsulated cells," "therapeutic allogeneic cells," or "therapeutic autologous cells." A variety of cells can be used in the disclosed methods. Transplanted cells can be allogeneic or heterogeneous cell populations, or cells that produce one or more biologically active substances of interest. Transplanted cells, e.g., pancreatic progenitors or PDX1-positive pancreatic endoderm, may not be therapeutically active when initially transplanted, but once transplanted, they further develop, mature, and have a therapeutic effect. As used herein, "cells" refers to cells that are capable of being transplanted. "Cells to be transplanted" refers to cells or cell populations that are sufficiently viable and / or functional, or to be functional for the in vivo treatment of a metabolic disorder.

[0016] "Induced pluripotent stem cells," "iPS cells," or "iPS" refer to a type of pluripotent stem cell artificially prepared from non-pluripotent cells, such as fibroblasts, hematopoietic cells, muscle cells, neurons, and epidermal cells, generally from adult somatic cells or highly differentiated cells, by inserting specific genes or gene products. They are also called reprogrammed cells. Takahashi et al., Cell 131:861-872 (2007); Wernig et al., Nature 448:318-324 (2007); Park et al., Nature 451:141-146 (2008), and see also PCT Application WO 2010048567, PCT Application PCT / US2009 / 061935, PCT Application PCT / JP2009 / 063906, and U.S. Patent Application Nos. 2011 / 0039338US and 2011 / 0039338, which are incorporated herein by reference in their entireties. These and other methods for generating iPSCs are known, and the manner in which iPSCs are derived or generated is not a limitation of the disclosed methods. Human iPSCs provide a source of pluripotent stem cells without the use of embryos.

[0017] Transplanted cells include reprogrammed cells. As used herein, the terms "reprogramming," "reprogrammed," or equivalents thereof, refer to a process that endows cells with a measurably increased ability to form at least one new cell type of progeny, either in culture or in vivo, that they would have under the same conditions without reprogramming.

[0018] Transplant cells include differentiated, dedifferentiated, and transdifferentiated cells. Thus, as used herein, the phrase "differentiation" refers to the process by which an unspecialized cell becomes a more specialized cell type. Conversely, the phrase "dedifferentiation" refers to the cellular process by which a partially or highly differentiated cell reverts to an earlier developmental stage, such as a pluripotent or multipotent cell. In further contrast, the phrase "transdifferentiation" refers to the process of transforming one differentiated cell type into another differentiated cell type.

[0019] Transplant cells include single hormone cells or polyhormonal cells. As used herein, "single hormone" cells or their equivalents refer to cells that express only one hormone (e.g., immature beta cells and beta cells express only insulin protein and do not express glucagon or somatostatin proteins). As used herein, "polyhormonal" cells express one or more hormones (e.g., endocrine precursor or progenitor cells have subpopulations of cells that express two, three, or four or more hormones within the same cell).

[0020] The transplanted cells include mesendoderm cells. The term "mesendoderm cells" or its equivalents refers to pluripotent cells that have relatively high expression levels of brachyury, FGF4, SNAI1 MIXL1 and / or WNT3 marker genes compared to SOX17 low, CXCR4 low, FOXA2 low, SOX7 low and SOX1 low.

[0021] The transplanted cells include definitive endoderm cells. The terms "definitive endoderm," "DE," "definitive endoderm lineage," or equivalents thereof, refer to pluripotent endodermal cells that can differentiate into cells of the gut or gut-derived organs.

[0022] The transplanted cells include PDX1-negative foregut endoderm cells. "PDX1-negative foregut endoderm cells," "foregut endoderm cells," or equivalents thereof, are cells that express the markers SOX17, HNF1β (HNF1B), HNF4 alpha (HNF4A), and FOXA1, but do not substantially express PDX1, AFP, SOX7, or SOX1. PDX1-negative foregut endoderm cell population and methods for producing the same are described in U.S. patent application Ser. No. 11 / 588,693, filed Oct. 27, 2006, entitled "PDX1-expressing dorsal and ventral foregut endoderm," which is incorporated herein by reference in its entirety.

[0023] The transplanted cells include PDX1-positive, dorsally-biased, foregut endoderm cells. The term "PDX1-positive, dorsally-biased, foregut endoderm cells" (dorsal PDX1-positive foregut endoderm cells) refers to cells that express one or more markers selected from Table 1 of U.S. Patent Application No. 13 / 761,078, filed February 6, 2013, and U.S. Patent Application No. 9,109,245, both of which are incorporated herein by reference in their entireties.

[0024] Transplant cells include pancreatic endoderm cells. The terms "pancreatic endoderm," "pancreatic epithelial," "pancreatic epithelial" (which may be abbreviated as "PE"), "intrapancreatic precursor," "PDX1-positive pancreatic endoderm," or "PEC cell product," or equivalents thereof, e.g., "pancreatic endoderm cells" (PECs), are all precursor or progenitor pancreatic cells. PECs, as described herein, are a progenitor cell population after stage 4 differentiation (approximately days 12-14) and comprise at least two major distinct populations: i) pancreatic progenitor cells that express PDX1 and NKX6.1 but do not express CHGA (or CHGA negative, CHGA-), a "non-endocrine pluripotent progenitor subpopulation (CHGA-)," a "non-endocrine (CHGA-) subpopulation," "non-endocrine (CHGA-) cells," or equivalents; and ii) CHGA (CHGA positive CHGA+), an "endocrine pluripotent progenitor subpopulation (CHGA+)," or an "endocrine (CHGA+) subpopulation," "endocrine (CHGA+) cells," or equivalents. The PEC pancreatic progenitor subpopulation that expresses PDX1 and NKX6.1 but not CHGA is also referred to as the "non-endocrine multipotent pancreatic progenitor subpopulation (CHGA-)," "non-endocrine progenitor subpopulation," "non-endocrine (CHGA-) subpopulation," "non-endocrine (CHGA-) subpopulation," or "multipotent progenitor subpopulation." The PEC multihormonal endocrine cell subpopulation that expresses CHGA is also referred to as "endocrine-committed cells (CHGA+)" or "CHGA+ cells." Without being bound by theory, the cell population that expresses NKX6.1 but not CHGA is hypothesized to represent a more active or therapeutic component of PECs, and CHGA-positive multihormonal endocrine cells are hypothesized to further differentiate and mature into glucagon-expressing pancreatic islet cells in vivo.See Kelly et al. (2011) Cell-surface markers for the isolation of pancreatic cell types derived from human embryonic stem cells, Nat Biotechnol. 29(8):750-756, published online July 31, 2011, and Schulz et al. (2012), A Scalable System for Production of Functional Pancreatic Progenitors from Human Embryonic Stem Cells, PLosOne 7(5):1-17, e37004, incorporated herein by reference in their entireties.

[0025] Pancreatic endoderm is sometimes used without reference to the aforementioned PECs, but is generally used to refer to at least stage 3 and 4 type cells. Its use and meaning will be clear from the context. Pancreatic endoderm has high levels of expression of markers selected from PDX1, NKX6.1, PTF1A, CPA1, cMYC, NGN3, PAX4, ARX, and NKX2.2 markers, but does not substantially express genes characteristic of pancreatic endocrine cells, e.g., CHGA, INS, GCG, GHRL, SST, MAFA, PCSK1, and GLUT1. Furthermore, some "endocrine precursor cells" that express NGN3 can differentiate into other non-pancreatic structures (e.g., the duodenum). Pancreatic endoderm or endocrine precursor cell populations and methods thereof are described in U.S. patent application Ser. No. 11 / 773,944, filed July 5, 2007, entitled "Methods of Producing Pancreatic Hormone-Induced Endocrine Cells." nes,” and U.S. patent application Ser. No. 12 / 107,020, filed April 21, 2008, entitled “METHODS FOR PURIFYING ENDODERM AND PANCREATIC ENDODERM CELLS DERIVED FORM.” and in "HUMAN EMBRYONIC STEM CELLS," which is incorporated herein by reference in its entirety.

[0026] Transplant cells include endocrine precursor cells. "Endocrine precursor cells" or equivalents thereof, as used herein, refer to pluripotent cells of the definitive endoderm lineage that express at least one marker from the list consisting of neurogenin 3 (NEUROG3), PDX1, PTF1A, SOX9, NKX6.1, HNF1b, GATA4, HNF6, FOXA1, FOXA2, GATA6, MYT1, ISLET1, NEUROD, SNAIL2, MNX1, IA1, RFX6, PAX4, PAX6, NKX2.2, and MAFB, and can be further differentiated into cells of the endocrine lineage, including, but not limited to, pancreatic islet hormone-expressing cells. Endocrine precursor cells are described in detail at least in U.S. Patent Application No. 8,129,182, filed March 6, 2012, entitled "ENDOCRINE PRECURSOR CELLS, PANCREATIC HORMONE-EXPRESSING CELLS AND METHODS OF PRODUCTION," and U.S. Patent Application No. 8,859,286, filed October 14, 2014, entitled "IN VITRO DIFFERENTIATION OF PLURIPOTENT STEM CELLS TO PANCREATIC ENDODERM CELLS (PEC) AND ENDOCRINE CELLS," both of which are incorporated by reference in their entireties.

[0027] Transplant cells include endocrine cells. "Endocrine cells" or "pancreatic islet hormone-expressing cells," "pancreatic endocrine cells," "pancreatic islet cells," "pancreatic islets," "stem cell-derived beta cells," "beta cells" ("β cells"), "SC-beta cells," or equivalents thereof, are pancreatic endocrine cells that can express insulin but do not express glucagon, somatostatin, ghrelin, or pancreatic polypeptide. Pancreatic endocrine cells that express markers characteristic of beta cells can be characterized by expression of insulin and at least one of the following transcription factors: PDX1, NKX2.2, NKX6.1, NeuroD1, ISL1, HNF3β, HB9, MAFA, and PAX6.

[0028] The transplanted cells properly specify endocrine cells. As used herein, the phrases "properly specified endocrine cells" or "stage 7 culture," "immature endocrine cells," including "immature beta cells," or equivalents thereof, refer to an in vitro-generated endocrine cell population that, when transplanted, can function in vivo, e.g., immature beta cells, to secrete insulin in response to blood glucose. The properly specified endocrine cells or stage 7 culture may have additional characteristics, including the following: In one embodiment, the properly specified endocrine cells, upon transplantation, develop and mature into functional pancreatic islet cells. In one embodiment, the properly specified endocrine cell population is enriched for endocrine cells (or depleted for non-secreting cells). In one embodiment, greater than about 50% of the cells in the properly specified endocrine cell population are CHGA+. In one embodiment, greater than about 60%, about 70%, 80%, 90%, 95%, 98%, or 100% of the cells in the properly specified endocrine cell population are CHGA+. In one embodiment, less than about 50% of the cells in a properly specified endocrine cell population are CHGA-. In one embodiment, less than about 15% of the cells in a properly specified endocrine cell population are CHGA-. In one embodiment, less than about 10%, 5%, 3%, 2%, 1%, 0.5%, or 0% of the cells in a properly specified endocrine cell population are CHGA-. Furthermore, expression of certain markers may be present in properly specified endocrine cells, such as NGN3 expression during stage 3. In one embodiment, properly specified endocrine cells have increased expression of NGN3 at stage 5. In one embodiment, properly specified endocrine cells are hormone-single (e.g., INS only, GCG only, or SST only). In one embodiment, In some embodiments, properly specified endocrine cells co-express other immature endocrine cell markers, including NKX6.1 and PDX1. In one embodiment, properly specified endocrine cells may co-express both a single hormone and other immature endocrine cell markers, including NKX6.1 and PDX1. In one embodiment, properly specified endocrine cells may have a greater number of INS cells expressing a single hormone as a percentage of the total INS population. In one embodiment, properly specified endocrine cells have at least 50% INS cells expressing a single hormone as a percentage of the total INS population. In one embodiment, properly specified endocrine cells are CHGA+ / INS+ / NKX6.1+ (triple positive). In one embodiment, more than about 25% of the cells in the cell population are CHGA+ / INS+ / NKX6.1+ (triple positive). In one embodiment, greater than about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100% of the cells in the cell population are CHGA+ / INS+ / NKX6.1+ (triple positive).

[0029] Transplanted cells include immature endocrine cells. The term "immature endocrine cells," particularly "immature beta cells" or equivalents thereof, refers to cells derived from endocrine precursor cells, pancreatic endoderm cells (PE), pancreatic foregut cells, definitive endoderm cells, mesendoderm cells, or other endocrine cells, including cells of early origin as described below, and express at least one marker selected from the group consisting of INS, NKX6.1, PDX1, NEUROD, MNX1, NKX2.2, MAFA, PAX4, SNAIL2, FOXA1, or FOXA2. Preferably, the immature beta cells described herein express INS, NKX6.1, and PDX1, and more preferably, co-express INS and NKX6.1. The terms "immature endocrine cell," "immature pancreatic hormone-expressing cell," "immature pancreatic islet," or equivalents thereof, refer to at least one unipotent immature beta cell or pre-beta cell, e.g., as described in Figure 45 of U.S. Patent Application No. 8,859,286, which is incorporated herein by reference in its entirety, and do not include other immature cells, e.g., the terms do not include immature alpha (glucagon) cells, immature delta (somatostatin) cells, immature epsilon (ghrelin) cells, or immature pancreatic polypeptide (PP) cells. Methods for differentiating pluripotent cells into pancreatic endocrine precursor cells and pancreatic endocrine cells are further described in U.S. Patent Application Nos. 9,012,218, 9,181,528, 9,234,178, 9,150,833, 9,096,832, and 9,062,290, which are incorporated by reference in their entireties.

[0030] The transplanted cells include functional beta cells. The terms "functional beta cells," "mature beta cells," or equivalents thereof, refer to pancreatic endocrine cells that exhibit established processes to ensure rapid and controlled glucose-stimulated insulin secretion ("GSIS"), particularly increased mitochondrial respiration / activity followed by the first and second phases of insulin secretion ("biphasic GSIS"). Specifically, functional beta cells exhibit at least one of the following characteristics of biphasic GSIS: (i) coupling of mitochondrial respiration / activity with insulin secretion, (ii) rapid insulin secretion in response to high demand (herein, high glucose concentrations), (iii) the ability to rapidly cease insulin secretion after demand subsides, (iv) the ability to "on-off" insulin secretion multiple times, (v) the ability to secrete adequate amounts of insulin as required, and (vi) the ability to respond to multiple insulin secretagogues (e.g., exendin-4 or the amino acids L-glutamine and L-arginine). Functional beta cells may be characterized by the expression of insulin and at least one of the following transcription factors: PDX1, NKX2.2, NKX6.1, NeuroD1, ISL1, HNF3β, HB9, PAX6, MAFA, SLC2A1, UCN3, and GLP1R.

[0031] As used herein, the terms "developed from pluripotent cells," "differentiated from pluripotent cells," "mature from pluripotent cells," or "produced from pluripotent cells," "derived from pluripotent cells," "differentiated from pluripotent cells," and their equivalents refer to cells that are produced in vitro or in vitro from pluripotent cells. Refers to the production of differentiated cell types in vivo.

[0032] Transplanted cells include cell aggregates. Cell aggregates can be aggregates of the cell types listed above. Aggregates can be substantially of one cell type or a mixed cell population. As used herein, the terms "cluster," "clump," "aggregate," or equivalents thereof, can be used interchangeably and generally refer to a group of cells that have been dissociated into single cells and aggregated to form clusters or have close cell-to-cell contacts. The term "reaggregated," as used herein, refers to clusters, clumps, and / or aggregates that have been dissociated into smaller clusters, clumps, and / or aggregates or single cells and reaggregated into clusters, clumps, and / or aggregates, thereby forming new cell-to-cell contacts. This dissociation is typically manual in nature (using a Pasteur pipette), although other dissociation methods are contemplated. The aggregate suspension pluripotent cell cultures are substantially as described in International Publication Nos. PCT / US2007 / 062755, entitled "COMPOSITIONS AND METHODS FOR CULTURING DIFFERENTIAL CELLS," and PCT / US2008 / 082356, entitled "STEM CELL AGGREGATE SUSPENSION CELLS." COMPOSITIONS AND METHODS OF DIFFERENTIATION THEREOF.

[0033] Transplant cells include single cell suspensions. The term "single cell suspension" or its equivalents refers to a pluripotent or differentiated single cell suspension, or a single cell suspension derived from pluripotent cells by mechanical or chemical means. See U.S. Patent Application No. 7,964,402, filed June 21, 2011, entitled "Methods for culture and This is described in detail in "Production of Single Cell Populations of Human Embryonic Stem Cells."

[0034] "Cell" refers to an individual cell, a cell line, or a culture derived from such a cell. "Culture" refers to a composition containing isolated cells of the same or different types. "Culture," "population," or "cell population," as used herein, can be used interchangeably, and the meaning will be clear depending on the context. For example, the term "population" can be a cell culture of one or more cells with the same distinguishing characteristic, or a culture of one or more cell types with different distinguishing characteristics. The term "subpopulation," when used to describe a specific cell type within a cell culture or cell population, refers to a subset of the cell culture or population.

[0035] The term "cell lineage," as used herein, refers to all stages in which a cell type develops from the earliest progenitor cell to a fully mature (specialized) cell. For example, "definitive endoderm lineage cells," "PDX1-negative endoderm lineage cells," "PDX1-positive pancreatic endoderm lineage cells," "endocrine precursor lineage cells," "endocrine lineage cells," or "immature beta lineage cells" refer to cells derived or differentiated from definitive endoderm cells, PDX1-negative endoderm cells, PDX1-positive pancreatic endoderm cells, etc. Definitive endoderm cells are a lineage of mesendoderm cells, one of their precursors. PDX1-positive pancreatic endoderm cells are a lineage of definitive endoderm cells, one of their precursors. The endocrine precursors of the PDX1-positive pancreatic cell, definitive endoderm cell, and mesendoderm cell lineages are all precursors. The immature beta cells of the endocrine precursor cell, PDX1-positive pancreatic cell, definitive endoderm cell, and mesendoderm cell lineages are all precursors. Beta cells, for example, are the only lineage of immature beta cells. All endodermal cells described herein are hES lineage cells.

[0036] The terms "treatment," "amelioration," or "cure," or equivalents thereof, refer to a therapeutic intervention that improves signs or symptoms. "Amelioration" refers to a reduction in the number or severity of signs or symptoms.

[0037] The terms "patient," "host," "mammalian host," "subject," or equivalents thereof, refer to a living multi-cellular vertebrate, a category that includes both humans and non-human mammals. In some embodiments, the subject is a human subject. Preferred patients to be treated are human subjects. Patients implanted with a perforated combination product are "high-risk insulin-requiring patients," and example populations include hypoglycemic unaware, unstable (brittle) T1D, and transplant patients. The target patient population may vary over time with clinical use / experience, independent of the perforated combination product itself, but rather is related to the nature of the immunosuppressive regimen. For example, a perforated combination product may be used for the "all T1D population" using an ISD regimen that achieves operational tolerance.

[0038] The terms "effective amount," "therapeutically effective amount," and equivalents refer to an amount of drug sufficient to achieve a desired effect in a subject or cell being treated. For example, it may refer to the amount of cells required to suppress or measurably reduce blood glucose levels and ultimately achieve homeostatic glycemic control. It may also refer to an amount of drug effective to alter the function or structure of a cell or subject. An effective amount of drug can be administered in a single dose or multiple doses. However, the effective amount will depend on the particular drug being administered, the subject being treated, the severity and type of affliction, and the mode of administration.

[0039] The terms "biofouling," "device fouling," or equivalents thereof, as used herein, refer to a process at the interface of an implantable device with a biological environment (e.g., a host environment, including, but not limited to, a subcutaneous environment) that occurs in part through nonspecific absorption of proteins into the device material, which in turn promotes the attachment of host cells, such as macrophages and fibroblasts, to the device surface. This process is commonly referred to as a foreign body response. Accordingly, embodiments described herein are encapsulated devices that include biofouling-resistant surfaces. Such surfaces can be fabricated or used based on surface hydrophilicity and charge, biomolecule functionalization, and drug elution. Reduced device biofouling generally reduces the foreign body response and restores or maintains cell viability, development, maturation, and function. Embodiments described herein contemplate the use of nonwoven fabrics to inhibit or reduce biofouling on device surfaces and promote vascularization, thereby integrating the device and cells within it.

[0040] As used herein, "reduced hypoglycemia" refers to a reduction in the number of hypoglycemic episodes without worsening glycemic control, as defined by an increase in HbA1c of 0.2% or less.

[0041] As used herein, "reduced insulin dependence" refers to a reduction in the frequency and / or dosage of exogenous insulin infusions without worsening glycemic control, as defined by an increase in HbA1c of 0.2% or less.

[0042] As used herein, "maintenance" refers to the amount of PEC cells maintained within the perforated combination product. The perforated device maintains the cell product, shelf life, surgical engraftment, maturation, and function within the formulation.

[0043] As used herein, "tissue capsule" refers to a foreign body capsule that forms around the implant. It is intended that the perforated device and the majority of its cellular content remain within the capsule during implantation. The device maintains cellular product within the lumen during initial implantation, prior to capsule formation.

[0044] "Engraftment" refers to the differentiation of a precursor or immature cell population into a mature cell type. For example, transplantation of a PDX1-positive pancreatic endoderm cell population results in maturation into a pancreatic endocrine cell population.

[0045] "Graft" refers to a differentiated cell population encapsulated or delivered in a device described herein, such as a mature pancreatic endocrine cell transplant.

[0046] The terms "essentially" or "substantially" refer to only a minor or minor amount of components or cells present in a cell population or culture, e.g., immature beta cells are "immature beta cells that essentially or substantially express INS, NKX6.1, and PDX1, and essentially or substantially do not express NGN3." Other examples include, but are not limited to, "essentially or substantially hES cells," "essentially or substantially definitive endoderm cells," "essentially or substantially foregut endoderm cells," "essentially or substantially PDX1-negative foregut endoderm cells," "essentially or substantially PDX1-positive pancreatic endoderm cells," "essentially or substantially pancreatic endocrine precursor cells," "essentially or substantially pancreatic endocrine cells," and the like.

[0047] With respect to cells in a cell culture or cell population, the term "substantially free" means that the particular cell type that does not comprise the cell culture or cell population is present in an amount of at least less than about 10%, at least less than about 9%, at least less than about 8%, at least less than about 7%, at least less than about 6%, at least less than about 5%, at least less than about 4%, at least less than about 3%, at least less than about 2%, or at least less than about 1% of the total number of cells present in the cell culture or cell population.

[0048] The term "nonwoven" or its equivalents includes, but is not limited to, bonded, formed, or engineered fabrics made by processes other than weaving or knitting.

[0049] Unless otherwise noted, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The singular forms "a," "an," and "the" include plural unless expressly stated otherwise. Similarly, the term "or" is intended to include "and" unless expressly stated otherwise. It is further understood that nucleic acid or polypeptide base sizes or amino acid sizes, and total molecular weight or molecular mass values ​​are appropriate and are provided for descriptive purposes. Methods and materials similar or equivalent to those described herein can be used in practicing or testing the present disclosure; suitable methods and materials are described below. The term "comprises" means "includes." All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including explanations of terms, will control. Furthermore, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0050] As used in the following claims and in this disclosure, the phrase "consisting essentially of" is meant to include the elements listed after the phrase, and is limited to other elements that do not interfere with or contribute to the function or activity identified in the disclosure of the listed elements. Thus, the phrase "consisting essentially of" indicates that the listed elements are necessary or essential, but that other elements are optional and may or may not be present depending on whether they affect the function or activity of the listed elements.

[0051] cell delivery device Cell delivery devices, also known as cell encapsulation devices, cell maintenance devices, cell containment devices or bioartificial organs, are devices that provide a housing that fully or partially encapsulates the cells of a host, including but not limited to the host immune system.

[0052] The cells may be cells of interest. The cells may be homogeneous or heterogeneous cell populations, or cells that produce one or more biologically active substances of interest. Transplanted cells, e.g., pancreatic progenitors or PDX1-positive pancreatic endoderm, may not be therapeutically active when initially transplanted, but may be useful in treating various conditions. Once transplanted, they can further develop, mature, and have a therapeutic effect. The transplanted cells can be individual cells in suspension or cell aggregates.

[0053] For example, diabetes or one or more symptoms thereof may improve or decrease for a period of time after transplantation of cells suitable for transplant into a subject suffering from diabetes. In one embodiment, the cell delivery device is loaded with PDX1-positive pancreatic endoderm cells. In one embodiment, the cell delivery device is loaded with pancreatic progenitor cells. In one embodiment, the cell delivery device is loaded with pancreatic endocrine progenitor cells. In one embodiment, the cell delivery device is loaded with pancreatic endocrine cells. In one embodiment, the cell delivery device is loaded with mature beta cells.

[0054] In one embodiment, the transplanted cells comprise totipotent cells. In one embodiment, the transplanted cells comprise pluripotent cells. In one embodiment, the transplanted cells comprise multipotent cells. Pluripotent cells include endocrine precursor cells, PDX1-positive pancreatic endoderm cells, definitive endoderm cells, or mesendoderm cells, which can give rise to pancreatic alpha, beta, delta, and gamma islet cells. In one embodiment, the transplanted cells comprise unipotent cells. Unipotent cells include, for example, immature beta cells that have the ability to differentiate only into insulin beta cells, but not into glucagon (alpha) cells, somatostatin (delta) cells, and pancreatic polypeptide (gamma) cells. In one embodiment, the transplanted cells comprise highly differentiated cells.

[0055] In one embodiment, the transplant cells are well-known, publicly available immortalized cell lines. The invention described herein is useful with all commercially available hES cell lines, including at least hESCs and iPSCs, such as CyT25, CyT203, CyT212, BG01, BG02, and BG03, available on the WiCell website at wicell.org / home / stem-cell-lines / order-stem-cell-lines / obtain-stem-cell-lines.cmsx. Suitable cells for practicing the invention are known or subsequently generated pluripotent cells. WiCell lists hundreds of other commercially available hES stem cell lines. Those skilled in the art will know how to identify and purchase commercially available stem cells for use in the invention from literature and publicly available databases, such as the National Institutes of Health (NIH) Stem Cell Registry, the Human Embryonic Stem Cell Registry, and the International Stem Cell Registry at the University of Massachusetts Medical School (Worcester, Massachusetts, USA). These databases are updated regularly as cell lines become available and are registered. The International Stem Cell Registry lists at least 254 commercially available iPSC lines and 1211 commercially available hESC lines. In one embodiment, the pluripotent transplant cells are human embryonic stem (hES) cells, human embryonic germ (hEG) cells, induced pluripotent stem cells (i.e., "iPS cells" or "iPSC cells"), parthenogenetic cells, embryos generated by somatic cell transfer, etc. In one embodiment, the transplant cells are differentiated cells derived from pluripotent cells, such as human embryonic stem (hES) cells, human embryonic germ (hEG) cells, induced pluripotent stem cells (i.e., "iPS cells" or "iPSC cells"), parthenogenetic cells, embryos generated by somatic cell transfer, etc. Pluripotency can also be determined by characterizing the cells for surface markers, transcriptional markers, karyotype, and the ability to differentiate into cells of the three germ layers.These characteristics are well known to those skilled in the art. For example, human pluripotent stem cells express several transcription factors that form core regulatory complexes that repress genes that direct differentiation and maintenance of pluripotency, cell surface proteins including the transcription factors Oct-4, Nanog, and Sox-2, glycolipids SSEA3, SSEA4, keratan sulfate antigen, Tra-1-60, Tra-1-81, and alkaline phosphatase. It can be defined or characterized by the presence of surface antigens.

[0056] Those skilled in the art can practice the disclosed methods and use the disclosed devices without the need to use human embryos and without assuming the destructive use of human embryos at an earlier time point. Indeed, deriving hES cell lines from parthenogenetically activated oocytes (e.g., as described in International Publication No. WO 03 / 046141, incorporated herein by reference in its entirety) is one way of practicing the present invention. Other methods exist for deriving pluripotent stem cells, such as mammalian ES cells, without destroying the embryo. Briefly, Advanced Cell Technology (Worcester, Massachusetts, USA) published three scientific journal articles deriving mouse and human ES cells from a single blastomere without destroying the embryo, thus leaving it intact. In late 2005, Chung et al. first described a method for generating mouse ES cells from a single blastomere. See Chung et al. (2006) Nature 439:216-219, published online October 16, 2005. Chung et al. (2006) described the extraction of histological specimens from embryos using micromanipulation techniques similar to those used for preimplantation genetic diagnosis (PGD), see p. 217. At that time, Chung et al. (2006) co-cultured blastomere cell lines with other embryonic stem cells. Chung et al. (2008) Human Embryonic Stem Cell Lines Generated without Embryonic Stem Cells See "Human Embryonic Stem Cell Lines Generated without Embryonic Destruction," Cell Stem Cell 2:113-117. However, in late 2008, the aforementioned study by Chung et al. demonstrated that culturing isolated blastomeres in medium with laminin enhanced their ability to generate hESCs, so that hESC cell lines did not require co-culture with ES cells at all. See Chung et al. (2008), "Human Embryonic Stem Cell Lines Generated without Embryonic Destruction," Cell Stem Cell(2):113-117, p. 116, published online January 10, 2008. Furthermore, we demonstrated that the hES cells obtained by this method have the same characteristics as other human pluripotent stem cells, including hES cells, which can maintain an undifferentiated state for more than six months, exhibit a normal karyotype and express pluripotency markers including Oct-4, SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, Nanog, and alkaline phosphatase, and can differentiate and form derivatives of the three embryonic germ layers in vitro and even form teratomas in vivo.

[0057] The transplanted cells may include differentiated, dedifferentiated, and transdifferentiated cells. In additional embodiments, the transplanted cells may include a hormone or polyhormonal cell. In further embodiments, the transplanted cells include reprogrammed cells. In some embodiments, the transplanted cells include mesodermal cells.

[0058] In other embodiments, the transplanted cells comprise definitive endoderm cells. In certain embodiments, the definitive endoderm cells are mammalian cells, and in preferred embodiments, the definitive endoderm cells are human cells. In some embodiments, one or more markers selected from SOX17, CXCR4, MIXL1, GATA4, HNF3β, GSC, FGF17, VWF, CALCR, FOXQ1, CMKOR1, and CRIP1 are expressed in the definitive endoderm cells. In other embodiments, one or more markers selected from OCT4, alpha-fetoprotein (AFP), thrombomodulin (TM), SPARC, SOX7, and HNF4α are not expressed or are significantly expressed in the definitive endoderm cells. Definitive endoderm cell populations and methods for producing them are also described in U.S. Patent Application No. 11 / 021,618, filed December 23, 2004, entitled "DEFINITIVE ENDODERM," which is incorporated by reference in its entirety.

[0059] In some embodiments, the transplanted cells comprise endocrine precursor cells. Thus, the transplanted cells include functional beta cells. In further embodiments, the transplanted cells include, but are not limited to, human islets of Langerhans, porcine islets, and rat islets. The transplanted cells include highly differentiated alpha (α), beta (β), delta (δ), and / or pancreatic peptide (PP) cells. In one aspect, embodiments consistent with the present disclosure are used for transplantation of pancreatic islets of Langerhans cells. In some embodiments, the transplanted cells include stem cells, other cell types such as spleen, pancreas, gallbladder, kidney, and other tissues with exocrine function. In some embodiments, the transplanted cells include neuroendocrine cells, proliferating cells, and cell lines that secrete hormones, cytokines, lymphokines, cell growth regulators, or other cells with metabolic function. As will be apparent to one of skill in the art, these cells and tissues can be obtained from mammalian tissues, primary cultured cells, cultured cell lines that produce biological products, and genetically engineered cultured cell lines. The transplanted cells can also be genetically engineered to produce desired molecules, such as proteins, peptides, nucleic acids, or other bioactive agents. Examples include cells engineered to express an enzyme that is missing or defective in the recipient, or an enzyme that expresses a therapeutic agent, such as a toxin, against cancer cells.

[0060] In some embodiments, the transplanted cells comprise PDX1-negative foregut endoderm cells that express the markers SOX17, HNF1 beta (HNF1B), HNF4 alpha (HNF4A), and FOXA1, but do not substantially express PDX1, AFP, SOX7, or SOX1. In other embodiments, the transplanted cells comprise PDX1-positive, dorsally-biased, foregut endoderm cells.

[0061] In an embodiment, the transplanted cells are present in a medium that is free of animal-derived products. In another embodiment, the transplanted cells are present in a xeno-free medium. The transplanted cells can be present in a medium supplemented with growth factors. The term "supplementary growth factor" is used in the broadest sense to refer to substances that are effective in promoting the growth of pluripotent cells, maintaining cell survival, stimulating cell differentiation, and / or stimulating the reversal of cell differentiation. Such substances include, but are not limited to, cytokines, chemokines, small molecules, neutralizing antibodies, and proteins. Growth factors also include intracellular signaling polypeptides that control the development and maintenance of cells and tissue morphology and function. In preferred embodiments, the supplemental growth factor is selected from steel cell factor (SCF), oncostatin M (OSM), ciliary neurotrophic factor (CNTF), interleukin-6 (IL-6) in combination with soluble interleukin-6 receptor (IL-6R), fibroblast growth factor (FGF), bone morphogenetic protein (BMP), tumor necrosis factor (TNF), granulocyte-macrophage colony-stimulating factor (GM-CSF).

[0062] In one embodiment, the transplanted cells are prepared as described by D'Amour et al., "Production of Pancreatic Hormone-Expressing Endocrine The cells are substantially similar to those described in "Cells From Human Embryonic Stem Cells" (November 1, 2006) Nature Biotechnology 24, 1392-1401, which is incorporated herein by reference in its entirety. D'Amour et al. describe a five-step differentiation protocol: Stage 1 (mostly produces definitive endoderm), Stage 2 (mostly produces PDX1-negative foregut endoderm), Stage 3 (mostly regenerates PDX1-positive foregut endoderm), Stage 4 (mostly produces pancreatic endoderm, also called pluripotent pancreatic progenitor cells or pancreatic endocrine progenitor cells), and Stage 5 (mostly produces hormone-expressing endocrine cells).

[0063] In one embodiment, the transplanted cells are derived from the cells of Schulz et al., A Scalable System for Production of Functional Pancreatic Progenitors from Human Embryonic Stem Cells PLoS One 7:5 1-17(2012), in its entirety by reference. The cells are substantially similar to those described in [Schulz et al., 2004], which is incorporated herein by reference. describe a method for expanding, banking, and a suspension-based differentiation system for hESCs. Specifically, undifferentiated pluripotent cells were aggregated into clusters in dynamic rotating suspension cultures and then differentiated en masse for 2 weeks in a four-stage protocol. Briefly, hESC monolayers were dissociated from the hES cell aggregate suspension with Accutase (Innovative Cell Technologies) and cultured at 1 x 10 cells / ml in StemPro hESC SFM (Life Technologies; a combination of DMEM / F12 with Glutamax, StemPro hESC supplements, BSA, and 1% (v / v) penicillin / streptomycin; excluding FGF-2 and 2-mercaptoethanol). 6The cells were harvested and resuspended at 100 cells / mL. One cell suspension was dispensed into a non-TC-treated 6-well plate (5.5 mL / well) and spun at 95 rpm in an Innova 2000 rotator (New Brunswick Scientific), and one into a 500 mL Nalgene filter receiver reservoir (150 mL / vessel) and spun at 65 rpm in a Sartorius Certomat RM-50 rotator (configured with a 5 cm rotating shaft). The cells were rotated overnight in a 37°C / 8% CO2 incubator to form aggregates of approximately 100-200 μm diameter. For aggregate diameters of 100-200 μm, rotation speeds of 60-140 rpm for 6-well dishes and 5-20 rpm for 500 mL reservoirs can be used. The differentiation of suspension aggregates was performed using a method adapted from D'Amour, with some modifications. TGF-βRI kinase inhibitor IV was included during stage 2, and retinoic acid was replaced with a more stable retinoid analog, TTNPB (3 nM), during stage 3. Growth factors KGF (50 ng / mL) and EGF (50 ng / mL) were added in stage 4 to preserve cell mass. Noggin (50 ng / mL) was also added in stage 4.

[0064] In one embodiment, the transplanted cells are substantially similar to those described in Agulnick et al., "Insulin-Producing Endocrine Cells Differentiated In Vitro From Human Embryonic Stem Cells Function in Macroencapsulation Devices In Vivo Stem Cells Translational Medicine 4:1-9 (2015), incorporated herein by reference in its entirety. Agulnick et al. described a modified protocol for generating pancreatic progenitor cells such that 73%-80% of the cell population consisted of PDX1-positive (PDX1+) and NKX6.1+ pancreatic progenitors. The pancreatic progenitor cells reproducibly contained 73%-89% endocrine cells, of which approximately 40%-50% were further differentiated into insulin-expressing islet-like cells (ICs). A large fraction of these insulin-positive cells were hormone-positive and expressed the transcription factors PDX1 and NKX6.1. We modified our protocol to generate pancreatic progenitor cells, resulting in 73%–80% PDX1-positive (PDX1+) and NKX6.1+ PPs. PPs reproducibly contained 73%–89% endocrine cells, of which approximately 40%–50% expressed insulin. A large fraction of these insulin-positive cells were hormone-positive and expressed the transcription factors PDX1 and NKX6.1. Agulnick et al. (2012) modified the protocol by additionally treating the PPs with activin A, Wnt3A, and heregulin β1 at stage 3 (days 5–7) and activin A and heregulin β1 at stage 4 (days 7–13).

[0065] Various cell compositions derived from pluripotent stem cells are described herein and can be found in the following U.S. patent applications: U.S. Patent Application Nos. 10 / 486,408, entitled "METHODS FOR CULTURE OF HESC ON FEEDER CELLS," filed August 6, 2002; 11 / 021,618, entitled "DEFINITIVE ENDODERM," filed December 23, 2004; and 11 / 115,868, entitled "PDX1 EXPRESSING ENDODERM," filed April 26, 2005. Filed on June 23, 2005; No. 11 / 165,305, Specification title: ``METHODS FOR IDENTIFYING FACTORS FOR DIFFERENTIATING DEFINITIVE ENDODERM''; Filed on June 23, 2005; No. 11 / 573,662, Specification title: ``METHODS FOR INCREASING DEFINITIVE ENDODERM DIFFERENTIATION OF PLURIPOTENT HUMAN EMBRYONIC STEM CELLS WITH PI-3 KINASE INHIBITORS”, filed on August 15, 2005; No. 12 / 729, 084, specification name: “PDX1-EXPRESSING DORSAL AND VENTRAL FOREGUT ENDODERM”, filed on October 27, 2005; No. 12 / 093,590, specification name: “MARKERS OF DEFINITIVE ENDODERM”, filed on November 14, 2005; No. 11 / 993,399, specification title “EMBRYONIC STEM CELL CULTURE COMPOSITIONS AND "METHODS OF USE THEREOF" was vowed on June 20, 2006; No. 11 / 588,693, titled "PDX1 - EXPRESSING DORSAL AND VENTRAL FOREGUT ENDODERM", was vowed on October 27, 2006; No. 11 / 681,687, titled "ENDOCRINE PROGENITOR / PRECURSOR CELLS, PANCREATIC HORMONE - EXPRESSING CELLS AND METHODS OF PRODUCTION", was vowed on March 2, 2007; No. 11 / 807,223, titled "METHODS FOR "CULTURE AND PRODUCTION OF SINGLE CELL POPULATIONS OF HESC" was vowed on May 24, 2007; Vote No. 11 / 773,944, titled "METHODS OF PRODUCING PANCREATIC HORMONES", was vowed on July 5, 2007; Vote No. 11 / 860,494, titled "METHODS FOR INCREASING DEFINITIVE ENDODERM PRODUCTION", was vowed on September 24, 2007; Vote No. 12 / 099,759, titled "METHODS OF PRODUCING PANCREATIC HORMONES", was vowed on April 8, 2008; Vote No. 12 / 107,020, titled "METHODS FOR PURIFYING ENDODERM AND PANCREATIC ENDODERM CELLS DERIVED FORM HUMAN" EMBRYONIC STEM CELLS, submitted on April 21, 2008; No. 12 / 618,659, titled "ENCAPSULATION OF PANCREATIC LINEAGE CELLS DERIVED FROM HUMAN PLURIPOTENT STEM CELLS", submitted on November 13, 2009; No. 12 / 765,714 and No. 13 / 761,078, both submitted on April 22, 2010, and February 6, 2013; No. 11 / 838,054, titled "COMPOSITIONS AND METHODS USEFUL FOR CULTURING DIFFERENTIABLE". "CELLS", registered August 13, 2007; No. 12 / 264,760, titled "STEM CELL AGGREGATE SUSPENSION COMPOSITIONS AND METHODS OF DIFFERENTIATION THEREOF", registered November 4, 2008; No. 13 / 259,15, titled "SMALL MOLECULES SUPPORTING PLURIPOTENT CELL GROWTH", registered April 27, 2010; PCT / US11 / 25628, titled "LOADING SYSTEM". FOR AN ENCAPSULATION DEVICE", filed on February 21, 2011; No. 13 / 992,931, specification title: "AGENTS AND METHODS FOR INHIBITING PLURIPOTENT STEM CELLS, filed December 28, 2010; U.S. Design Patent Application No. 29 / 408,366, filed December 12, 2011; US ​​Design Patent Application No. 29 / 408,368, filed December 12, 2011; U.S. Patent Application No. 14 / 201,630, entitled "3-DIMENSIONAL LARGE CAPACITY CELL ENCAPSULATION DEVICE ASSEMBLY," filed March 7, 2014; and U.S. Patent Application No. 14 / 106,330, entitled "IN VITRO DIFFERENTIATION OF PLURIPOTENT STEM CELLS TO PANCREATIC ENDODERM CELLS (PEC) AND ENDOCRINE CELLS," filed December 13, 2013, each of which is incorporated by reference in its entirety.

[0066] Various cell compositions derived from pluripotent stem cells are described herein and can be found in the following exclusive patent applications: U.S. Patent Application No. 2009 / 0269845, entitled "Pluripotent cells," filed April 24, 2008; U.S. Patent Application No. 2011 / 0014703, entitled "Differentiation of Human Embryonic Stem Cells," filed July 20, 2010; U.S. Patent Application No. 2011 / 0014702, entitled "Differentiation of Human Embryonic Stem Cells," filed July 19, 2010; U.S. Patent Application No. 2011 / 0151561, entitled "Differentiation of Human Embryonic Stem Cells," filed December 16, 2010; and U.S. Patent Application No. 2010 / 0112692, entitled "Differentiation of Human Embryonic Stem Cells." U.S. Patent Application No. 2012 / 0052576, entitled "Differentiation of Pluripotent Stem Cells," filed October 22, 2009; U.S. Patent Application No. 2012 / 0052576, entitled "Differentiation of Pluripotent Stem Cells," filed August 17, 2011; U.S. Patent Application No. 2010 / 0112693, entitled "Differentiation of human pluripotent stem cells," filed October 23, 2009; U.S. Patent Application No. 2011 / 0151560, entitled "Differentiation of human embryonic stem cells," filed December 16, 2010; U.S. Patent Application No. 2010 / 0015100, entitled "Differentiation of U.S. Patent Application No. 2009 / 0170198, entitled "Differentiation of human embryonic stem cells," filed July 31, 2008; U.S. Patent Application No. 2009 / 0170198, entitled "Differentiation of human embryonic stem cells," filed November 25, 2008; U.S. Patent Application No. 2015 / 0329828, entitled "Use of Small Molecules to Enhance Mafa Expression in Pancreatic Endocrine Cells," filed May 7, 2015; U.S. Patent Application No. 2013 / 0330823, entitled "Differentiation of Human Embryonic Stem Cells into Pancreatic Endocrine Cells," filed June 6, 2013; International Patent Application No. WO 2013 / 192005, entitled "Differentiation of human embryonic stem cells into pancreatic endocrine cells." "Suspension cells" filed June 13, 2013; U.S. Patent Application No. 2014 / 0242693, specification title: and clustering of human pluripotent stem cells for differentiation into pancreatic endocrine cells,” filed December 30, 2013; U.S. Patent Application No. U.S. Patent Application No. 2014 / 0295552, entitled “Suspension and clustering of human pluripotent stem cells for differentiation into pancreatic endocrine cells,” filed June 17, 2014; International Patent Application No. WO 2015 / 065524, entitled “Suspension and clustering of human pluripotent stem cells for differentiation into pancreatic endocrine cells.” "Differentiation of Human Embryonic Stem Cells into Pancreatic Endocrine Cells," filed May 21, 2014; U.S. Patent Application No. 2013 / 0330823, entitled "Differentiation of Human Embryonic Stem Cells into Pancreatic Endocrine Cells," filed June 6, 2013; U.S. Patent Application No. 2014 / 0186953, entitled "Differentiation of Human Embryonic Stem Cells into Pancreatic Endocrine Cells," filed June 6, 2013; "Stem Cells Into Pancreatic Endocrine Cells Using HB9 Regulators," filed December 18, 2013; U.S. Patent Application No. 14 / 963,730, filed December 9, 2015; U.S. Patent Application No. 14 / 898,015, filed December 11, 2015, each of which is incorporated by reference in its entirety.

[0067] In one embodiment, the transplanted cells are encapsulated using biocompatible polyethylene glycol (PEG). PEG-based encapsulation is described in the following patent applications: U.S. Patent Application No. 7,427,415, entitled "IMPLANTATION OF CARDIAC IMPLANTATION SYSTEMS." "GELS FOR ENCAPSULATION OF BIOLOGICAL MATERIALS FOR TREATING DISEASES"; U.S. Patent Application No. 6,911,227, entitled "GELS FOR ENCAPSULATION OF BIOLOGICAL MATERIALS FOR TREATING DISEASES" Nos. 6,911,227, 5,529,914, 5,801,033, and 6,258,870, entitled "GELS FOR ENCAPSULATION OF BIOLOGICAL MATERIALS," which are incorporated herein by reference in their entireties.

[0068] In another embodiment, the delivery device is a TheraCyte (formerly Baxter) device (Irvine, Calif.). TheraCyte cell delivery devices are disclosed in U.S. Patent Application Nos. 6,773,458; 6,156,305; 6,060,640; 5,964,804; 5,964,261; 5,882,354; 5,807,406; 5,800,529; 5,782,912; 5,741,330; 5,733,336; 5,713,888; 5,653 ,756; 5,593,440; 5,569,462; 5,549,675; 5,545,223; 5,453,278; 5,421,923; 5,344,454; 5,314,471; 5,324,518; 5,219,361; 5,100,392; and 5,011,494, each of which is incorporated by reference in its entirety.

[0069] In another embodiment, the delivery device is a fusion-enhanced device, such as those described in U.S. Patent Application No. 8,278,106, U.S. Patent Application No. 14 / 201,630, filed March 7, 2014, U.S. Design Patent Application Nos. 29 / 447,944, 29 / 509,102, 29 / 484,363, 29 / 484,360, 29 / 484,359, 29 / 484,357, 29 / 484,356, 29 / 484,355, 29 / 484,362, 29 / 484,358, 29 / 484,3 66, 29 / 517,319, 29 / 408,368, 29 / 518,513, 29 / 518,516, 29 / 408,370, 29 / 517,144, 29 / 423,365, 29 / 530,325, which are incorporated herein by reference in their entireties.

[0070] The cell delivery device embodiments described herein are not intended to be limited to a particular size, shape, design, volume capacity, and / or material used to fabricate the cell delivery device, so long as the transplanted cells are capable of producing insulin in response to blood glucose.

[0071] The graft or cells in the core (also called the cell chamber or lumen) of the cell delivery device may be immobilized in an immobilization matrix, such as a hydrogel or extracellular matrix component. The core of the cell delivery device can include an insert to create a "cell-free" region in the center of the core, further reducing the likelihood of a necrotic core of cells developing in the center of the device.

[0072] The cell delivery device has a structure suitable for maintaining biological activity and providing access for delivering a product or function, including, for example, cylindrical, rectangular, disk-shaped, patch-shaped, oval, star-shaped, or spherical shapes. Furthermore, the cell delivery device can be wrapped, tubular, or encased in a mesh-like or nested structure. If the cell delivery device is to be retrieved at some point after implantation, structures that tend to cause the cell delivery device to migrate from the implantation site should be avoided (e.g., a spherical device would be too small to navigate the recipient's blood vessels). Embodiments of the present invention include shapes that provide high structural integrity and are easily retrieved from the host. Such shapes include rectangular patches, disks, cylinders, and flat sheets.

[0073] In other embodiments, the cell delivery device or bulk assembly is comprised of one or more seals that further divide the lumen of the cell delivery device, i.e., partition seals. See, e.g., U.S. Design Patent Application Nos. 29 / 408366, 29 / 408368, 29 / 408370, and 29 / 423,365.

[0074] The cell delivery device may be implanted subcutaneously, but may also be implanted in other locations, such as the peritoneal cavity or abdominal wall, an intramuscular site, an abdominal fat pad, or another suitable location. Alternatively, the disclosed cell delivery device may be partially implanted into a lumen of the host's body, including the omentum or other suitable site, and extend into the subcutaneous environment. In one embodiment, cells may be added to the portion of the device that extends into the subcutaneous environment, while the remainder of the device is in the intraperitoneal environment. In another embodiment, the cell delivery device may be implanted into the brain, spinal cord region, or other organ where a therapeutic effect from the transplanted cells is desired. In many instances, the host is a human, but may also be another mammal or a non-mammal.

[0075] Expanding Device: In one embodiment, a cell delivery device or expandable large volume assembly is provided.

[0076] Refilling Device: Replacing cells within an implanted cell delivery device can be accomplished by removing the cells from the cell delivery device and then injecting a therapeutic agent or cells directly into a reservoir, chamber, lumen, container, or compartment of the implanted cell delivery device, e.g., subcutaneously. Injection of cells / therapeutic agents can be accomplished using a syringe inserted into a port.

[0077] Alternatively, in another embodiment, the devices or assemblies provided herein do not include inlet or outlet ports, the devices are so-called portless, and the cells are loaded into the delivery device prior to implantation at the implantation site.

[0078] Multi-chamber modular equipment In one embodiment, the transplant cells are delivered into a macro cell encapsulation / delivery device, also referred to as a bulk assembly or bulk device. As used herein, "bulk assembly" or "bulk device" refers to a cell encapsulation device comprised of multiple or multiple cell chambers. In one embodiment, the bulk assembly is comprised of at least 1, 2, 4, 5, 6, 7, 8, 9, or 10 or more cell chambers. In another embodiment, the bulk assembly is fabricated such that the bulk assembly is comprised of several cell chambers (or modular units). For example, a modular unit may be comprised of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more cell chambers, depending on the number or dose of cells required to treat a disease as described in U.S. Patent Application No. 8,278,106, incorporated herein by reference in its entirety. The number of chambers in the delivery device may be determined based on the number of cells to be transplanted. This is determined based on volume and / or number.

[0079] Three-dimensional large-volume device: In one embodiment, a cell delivery device or large-volume assembly is provided that includes multiple or numerous cell chambers interconnected by cell-free regions, and that can be folded and bent, for example, as described in U.S. Patent Application No. 14 / 201,630, filed March 7, 2014. In one embodiment, the large-volume assembly includes at least two cell chambers and at least two folded and unfolded configurations, where the folded configuration has a smaller footprint than the unfolded configuration. Thus, as used herein, the term "cell encapsulation device" or "cell delivery device" can refer to a device consisting of one cell chamber, or a device consisting of multiple cell chambers, such as the large-volume device or multiple cell chambers in the three-dimensional device or device assembly described in U.S. Patent Application No. 8,278,106 and U.S. Patent Application No. 14 / 201,630, filed March 7, 2014, both of which are incorporated herein by reference in their entireties. Thus, cell delivery device, large-volume assembly, and three-dimensional device can be used interchangeably.

[0080] Various cell delivery device configurations The cell delivery device comprises various layers, each of which serves one or more functions, hi some embodiments, the cell delivery device comprises both a cell-exclud- ing membrane and a nonwoven fabric.

[0081] Cell-Exclusion Membrane: This layer inhibits cellular components of the immune system, such as T cells, from entering the device. This layer also prevents therapeutic cells from exiting the device. This layer allows the passage of the encapsulated bioactive agent of interest (insulin, glucagon, pancreatic polypeptide, etc.), making the active agent available to target cells outside the cell delivery device and within the patient's body. This layer ideally allows nutrients naturally present in the host to pass through the membrane, providing essential nutrients to the encapsulated cells. Cell-exclusion membranes are disclosed in U.S. Patent Application Nos. 6,773,458; 6,520,997; 6,156,305; 6,060,640; 5,964,804; 5,964,261; 5,882,354; 5,807,406; 5,800,529; 5,782,912; 5,741,330; 5,733,336; 5,713,888; 5,653,756; 5,593,440 and 5,011,494, all of which are incorporated herein by reference in their entireties. In some embodiments, the layer is perforated.

[0082] Membrane: In some embodiments, the cell delivery device includes a membrane layer, membrane ring, or membrane weld. The membrane is a bonding or adhesive layer present only at the weld that helps adhere or bond at least two or more layers together. In some embodiments, the membrane is present only on the inner surface (chamber face) of a nonwoven (see below), eliminating the smooth surface it would create if it were on the outer surface (host face) that inhibits fixation. In some embodiments, the membrane is present on the inner surface (chamber face) of a cell-exclusion membrane. The membrane is not part of the chamber, but is placed at the weld.

[0083] Mesh: The woven mesh provides structural rigidity to each device and protects the cell-exclusion membrane by serving as a protective exoskeleton. In some embodiments, a nonwoven mesh is not included in the device's construction. To address the lack of rigidity resulting from not including a woven mesh in the device design, a double layer of nonwoven fabric and / or nonwoven rings can be used on the outside of the device.

[0084] Nonwoven Fabrics: Providing cell encapsulation devices that successfully integrate into the host after implantation. To this end, reducing, inhibiting, or decreasing biofouling at the device-host interface is essential for device integration. In one embodiment, nonwoven fabrics are used to investigate whether they can provide structural integrity while also increasing vascularization and reducing or inhibiting biofouling. In one embodiment, nonwoven fabrics prevent direct contact of the cell-exclusion membrane with the woven mesh, providing additional material for anchoring the device to the host or for device integration. There are many types of nonwoven fabrics with varying weave tightness and sheet thickness. In one embodiment, the filament cross-section is trilobal. Nonwoven fabrics can be bonded, formed, or engineered fabrics and are manufactured by processes other than weaving or knitting. In some embodiments, they are porous textile-like materials, usually in flat sheet form, composed primarily or entirely of fibers, such as staple fibers, fabricated into woven fabrics, sheets, or batts. Nonwoven fabric structures, for example, are typically based on a more or less randomly arranged arrangement of staple fibers.

[0085] Nonwoven fabrics can be made by a variety of techniques well known in the textile industry. Various methods can be used to make carded, wet-laid, meltblown, spunbonded, or airlaid nonwoven fabrics. Exemplary methods and substrates are described in U.S. Patent Application No. 2010 / 0151575, which is incorporated herein by reference in its entirety. In one embodiment, the nonwoven fabric is polytetrafluoroethylene (PTFE). In one embodiment, the nonwoven fabric is spunbonded polyester.

[0086] The density of the nonwoven fabric can vary depending on processing conditions. In one embodiment, the nonwoven fabric has a basis weight of about 0.40 to about 1.00 oz / yd 2 ), a spunbond polyester having a nominal thickness of about 127 to about 228 μm and a fiber diameter of about 0.5 to about 26 μm. In one embodiment, the filament cross section is trilobal. In some embodiments, the nonwoven fabric is biocompatible and / or bioabsorbable.

[0087] Historically, cell delivery devices have had simple configurations including a cell exclusion membrane for containment of therapeutic cells, a membrane for welding, and / or a woven mesh (EN-A configuration).

[0088] Figures 1A-D are exploded views of specific embodiments of cell delivery devices. Illustrated in the figures is EN20, a simplified representation of a drug delivery device that, in a non-porous form, is capable of supporting approximately 20 μl of transplanted cells at maturity. As shown in Figures 1A-D, the device can have various additional layers of mesh, film, membrane, and nonwoven fabric. Each structure is fabricated as a stack of materials, fabricated like a sealed "sandwich."

[0089] Table 1 below describes the construction of a cell delivery device. Each wall of the device can be composed of the same number and type of layers and materials, or different numbers and types of layers, depending on the function required and imparted by the layers. Chambers or housings of the device are created by welding or bonding around the perimeter, and the addition of chambers is accomplished with port tubing. The first and bottom rows of Table 1 are the layers that will be exposed to or in contact with the host upon implantation.

[0090] [Table 1]

[0091] For example, Figure 1A: Device (EN20B1) has a nonwoven ring 51, a membrane ring 52, and a membrane layer where two layers of nonwoven fabric are thermally laminated to a cell-exclusion membrane (53), with the nonwoven fabric layer facing outward toward the host, and the cell-exclusion membrane layer facing inward toward the chamber or implanted cells, and a membrane ring (54) at the periphery or weld. This pattern is repeated for the other device walls. The other side of the device has a nonwoven ring 58, a membrane ring 57, and two layers of nonwoven fabric are thermally laminated to a cell-exclusion membrane (56), with the nonwoven fabric layer facing outward toward the host, and the cell-exclusion membrane layer facing inward toward the chamber or implanted cells. As previously mentioned, there is a membrane ring (55) at the periphery or weld. That is, the two sides or walls of the chamber are mirror images of each other, with a port (85) for adding cells between them forming a lumen (86) where therapeutic agents reside. See Figure 1A. In this embodiment, no mesh layer is included. In some embodiments, the entire surface of the delivery device is covered in nonwoven fabric rather than a nonwoven ring (compare Figures 1A, 1B, 51 and 59).

[0092] Figure 1B shows one embodiment of a delivery device (EN20B2) having a nonwoven fabric layer (59), a membrane ring (60), a mesh layer (61), and a cell-exclusion membrane layer in which the nonwoven fabric layer is thermally laminated to the membrane (62) and membrane ring (63) at the periphery and welds. The two sides or walls of the chamber are mirror images of each other, with a port (85) for adding cells between them forming a lumen (86) in which the therapeutic agent resides. Thus, the opposite side of the device includes a nonwoven fabric layer (68), a membrane ring (67), a mesh layer (66), and a cell-exclusion membrane layer in which the nonwoven fabric layer is thermally laminated to the membrane (65) and membrane ring (64) at the periphery and welds. As in Figure 1A, the two sides of the chamber are mirror images of each other.

[0093] FIG. 1C shows one embodiment of a delivery device (EN20B3) having a cell-excluded membrane layer in which a mesh layer (69), a membrane ring (70), and a nonwoven fabric layer are thermally laminated to a membrane (71) and a membrane ring (72) at the periphery and welds. Thus, a cell-excluded membrane layer is shown in which another mesh layer (76), a membrane ring (75), and a nonwoven fabric are thermally laminated to a membrane (74) and a membrane ring (73). The two sides or walls of the chamber are mirror images of each other, with a port (85) between them for adding cells and a lumen (86) where the therapeutic agent is present. 86). As in Figures 1A-B, the two sides of the chamber are mirror images of each other.

[0094] Figure 1D shows an embodiment of a delivery device (EN20B4) that has the same structure as EN20B3, but the nonwoven layers (79 and 82) have a different density than the nonwoven layers of EN20B3. The mesh layers (71 and 84), membrane rings (78, 80, 81, and 83) are similar to the respective elements shown in Figure 1C.

[0095] In one embodiment, the nonwoven fabric and cell exclusion membrane can be laminated using a heat laminator or heat press (e.g., an ARB Arbor Press manufactured by Plastic Assembly Systems). The press is heated to approximately 305-320°F. A pressure of 0-6 PSI is applied to the nonwoven fabric and membrane at a speed of 3 ft / min or 10 ft / min. However, the nonwoven fabric and cell exclusion membrane do not need to be laminated.

[0096] In one embodiment, the nonwoven layer faces outward toward the host and the cell-exclusion membrane layer faces inward toward the chamber or implanted cells, although one of skill in the art can envision different configurations using this disclosure, e.g., where the nonwoven layer faces inward toward the chamber or implanted cells and the cell-exclusion membrane layer faces outward toward the host. In some embodiments, the polyester nonwoven is on the outside of the cell-exclusion membrane and is laminated to the membrane.

[0097] In some embodiments, the cell exclusion membrane and / or nonwoven are laminated together and perforated. In some embodiments, the cell exclusion membrane is first perforated and then laminated to the nonwoven. In some embodiments, the nonwoven is perforated and then laminated to the cell exclusion membrane. When the cell exclusion membrane is perforated, the mammalian host is immunocompromised or treated with immunosuppressive drugs.

[0098] The nonwoven fabrics used in the cell delivery devices shown in Figures 1A-D are substantially flat, but may be further manipulated to provide thickness. For example, the nonwoven fabric may be pleated, contoured, or embossed. Additionally, tufting may be used to create fabrics with pile and other three-dimensional structures, such as in the manufacture of pile fabrics, knitwear, and carpets. See, e.g., U.S. Patent No. 7,754,937, incorporated herein by reference in its entirety.

[0099] The devices contemplated herein can have many different configurations and different therapeutic agent holding capacities. An ENCAPTRA EN20 or EN20 device or small delivery device refers to a device with a functional volume of approximately 20 μL and can contain approximately 2,500-3,500 IEQ or over 80,000 IEQ of beta cell mass per kg of mouse. An ENCAPTRA EN250, EN250, or EN250 device or large delivery device has a functional volume of approximately 250 μL, which is approximately 12.5 times (12.5X) larger than an EN20 device and can contain up to approximately 30,000-45,000 IEQ per kg of mouse. An ENCAPTRA EN100, EN100, or EN100 device has a functional volume of approximately 100 μL, which is approximately 6.5 times (6.5X) larger than an EN20 device and can contain up to approximately 16,250-22,750 IEQ per kg of mouse. The EN Large Capacity or EN-LC device is about 48.4 times (48.4X) larger than the EN20. An EN-LC device containing four cell chambers can contain up to about 121,000-169,400 IEQ. Therefore, at least about four, about five, about six, about seven, or about eight EN250 devices or about two EN-LC devices are required to deliver a sufficient PEC volume to deliver a therapeutically effective dose to a patient.

[0100] In addition to increasing the size of the device to increase dosing capacity, perforating the device can also increase dosing capacity. The perforated EN20 device has approximately five times the delivery capacity (representing the beta cell mass achieved at maturity) than the non-perforated EN20 device. Another delivery method for the described perforated device is approximately one-fifth that of the intact device.

[0101] Perforated cell delivery device To promote angiogenesis shortly after implantation, cells are implanted into a perforated cell delivery device that provides direct cell-to-cell contact between the host vasculature and the encapsulated cells. In some embodiments, not all layers of the device are perforated. For example, a perforated cell delivery device provides perforations in one layer, such as the cell-exclusion membrane, or the cell-exclusion membrane and nonwoven fabric layer. This allows for retention of the implanted cells / tissue while simultaneously allowing exchange with the host, such as the ingress of vasculature and macrophages.

[0102] Laser drilling of the perforations allows for selection of the size, number, and location of the perforations. They are large enough to allow host vascular tissue (e.g., capillaries) and stromal cells supporting pancreatic cell types to enter the lumen of the device. In one embodiment, the perforations are sized to allow host macrophages and other phagocytic cells to enter the device and clear necrotic debris from the lumen of the perforated device. In one embodiment, the perforations are also sized to allow therapeutic agents, such as insulin, produced by the implant to exit the cell delivery device. Perforations that allow vasculature to grow into the lumen of the device help anchor the device to the host and inhibit device migration. In one embodiment, the perforations are also sized based on the diameter of the cell aggregates that maximizes cell retention.

[0103] In some embodiments, the device comprises a cell enclosure made from a biocompatible material adapted to be implanted into a host and to substantially contain a therapeutic agent that may be immunologically compatible or incompatible with the host, the chamber having a cell-exclusion membrane and optionally a mesh layer and membrane weld, the wall having pores passing through the cell-exclusion membrane, the pores having an inner diameter at their narrowest point large enough to allow host capillaries to pass through the thickness of the wall, and the number of pores is sufficient to allow the host capillaries to support the viability of the therapeutic agent that may be contained therein.

[0104] In one embodiment, a perforated delivery device is provided, in which one or more layers of the delivery device are perforated. In one embodiment, a perforated delivery device is provided, in which one or more layers of the delivery device are not perforated. In one embodiment, only the cell-exclusion membrane is perforated. In one embodiment, a cell delivery device is provided, in which the perforations do not pass through each wall of the device. In one embodiment, the perforations in the cell delivery device are composed of holes that do not pass through each wall of the device, but allow host vasculature to still grow into the luminal interior surface of the cell delivery device. In one embodiment, a cell delivery device is disclosed that does not include a nonwoven fabric. In one embodiment, a cell delivery device is disclosed that does not include a nonwoven fabric, but in which the cell-exclusion membrane is perforated. In such embodiments, the diameter of the pores in the cell-exclusion membrane is used to retain cells, i.e., the pores of the device are smaller than the cell aggregates contained therein.

[0105] In one embodiment, the cells of the perforated delivery device are comprised of PDX1 / NKX6.1 co-positive pancreatic progenitor cells. In one embodiment, the cells of the perforated delivery device are comprised of immature beta cells that express insulin (INS) and NKX6.1, or immature beta cells that express INS, NKX6.1, and MAFB. In one embodiment, the cells of the perforated delivery device are comprised of mature beta cells that express INS and MAFA, or INS, NKX6.1, and MAFA. In one embodiment, the cells of the perforated delivery device are comprised of pancreatic endocrine cells. In one embodiment, the cells of the perforated delivery device are comprised of pancreatic insulin-secreting cells. In one embodiment, the cells of the perforated delivery device are comprised of pancreatic beta or insulin cells that can secrete insulin in response to blood glucose levels.

[0106] Perforated device surrounded by nonwoven fabric In these embodiments, the nonwoven fabric is external to the cell delivery device. Rather than affecting the implanted cells, the nonwoven fabric enhances host vascularization surrounding the cell enclosure.

[0107] In one embodiment, a cell delivery device is disclosed that includes a nonwoven fabric. In one embodiment, a cell delivery device is disclosed that includes a polyester nonwoven fabric (NWPF). Polypropylene, polyethylene, nylon, polyurethane, and polyamide are some examples of polyester nonwoven fabrics that can be used. In one embodiment, the cell exclusion membrane is surrounded (or covered) by the nonwoven fabric, i.e., the nonwoven fabric is external to the cell exclusion membrane. Alternatively, the nonwoven fabric faces the host rather than the transplanted cells. In one embodiment, the nonwoven fabric forms a jacket around the cell exclusion membrane. In one embodiment, only the cell exclusion membrane is perforated, and the outer layer of the device, including the nonwoven fabric, is not perforated. In one embodiment, the cell exclusion membrane and the nonwoven fabric are perforated, and the other layers of the device are not perforated.

[0108] In one embodiment, the pores are smaller than the cell aggregates, such as hPSC-derived aggregates, contained in the device, e.g., the definitive endoderm cell aggregates contained therein. In one embodiment, the pores are smaller than the PDX1-positive pancreatic endoderm cell aggregates contained therein. In one embodiment, the pores are smaller than the pancreatic progenitor cell aggregates contained therein. In one embodiment, the pores are smaller than the endocrine cell aggregates contained therein. In one embodiment, the pores are smaller than the mature beta cell aggregates contained therein.

[0109] In one embodiment, the pore diameter is small enough to retain cells, but large enough to ensure that a therapeutic effect is achieved, e.g., in a diabetic patient, the pore diameter is determined by the ability of the transplanted cells to mature and / or produce insulin in response to blood glucose levels.

[0110] In one embodiment, the perforated cell delivery device is implanted into a rat or human. In one embodiment, the perforated cell delivery device implanted into a rat or human comprises holes in the cell exclusion membrane and polyester nonwoven fabric (the other layers of the device are not perforated), the holes are about 2 mm or more apart (measured center to center), and the diameter of the holes is less than 100 microns. In one embodiment, the perforated cell delivery device implanted into a rat or human comprises holes in the cell exclusion membrane and polyester nonwoven fabric (the other layers of the device are not perforated), the holes are about 2 mm or more apart. In one embodiment, the perforated cell delivery device implanted into a rat or human comprises holes in the cell exclusion membrane and polyester nonwoven fabric (the other layers of the device are not perforated), and the diameter of the holes is less than 100 microns.

[0111] In one embodiment, the perforated cell delivery device implanted in a rat or human contains holes in the cell-exclusion membrane (the other layers of the device are not perforated), the holes are spaced at least about 2 mm apart, and the hole diameter is less than about 100 microns.

[0112] In one embodiment, a cell delivery device comprises a perforated cell exclusion membrane and PDX1-positive pancreatic endoderm cells, and can be implanted into a human patient, where the PDX1-positive pancreatic endoderm cells mature into insulin-producing cells in vivo. In one embodiment, a cell delivery device comprises a perforated cell exclusion membrane, a perforated NWF layer, and PDX1-positive pancreatic endoderm cells, and the cell delivery device can be implanted into a human patient, where the PDX1-positive pancreatic endoderm cells mature into insulin-producing cells in vivo.

[0113] Stacked Devices In one embodiment, the cell exclusion membrane is laminated to a nonwoven fabric. The outer membrane is laminated to the NWF. When the cell exclusion membrane is laminated to the nonwoven fabric, the cell exclusion membrane remains flat. If not laminated, the cell exclusion membrane may deform out of plane, creating a dam that can result in uneven cell distribution. Uneven cell distribution can induce necrotic areas, cell death, and other issues, reducing efficacy. Controlling cell distribution within a chamber or lumen of a cell delivery device is also referred to as the spatial location of cells within the cell delivery device. In one embodiment, a method for controlling cell distribution (cell location) within a cell delivery device includes laminating a cell exclusion membrane to a nonwoven fabric. In one embodiment, a method for controlling cell distribution (cell location) within a cell delivery device includes laminating a cell exclusion membrane to a NWF.

[0114] By achieving a uniform distribution of cells within the lumen of the cell delivery device, fewer cells need to be transplanted. Using fewer cells results in less cell debris. An additional benefit is increased diffusion of nutrients to the cells because the cells are uniformly distributed and in closer contact with the membrane. Increased diffusion of nutrients to the cells leads to improved cell viability.

[0115] As more laminated membranes are incorporated into the device, the lumen can be filled completely, maximizing the therapeutic effectiveness of the transplanted cells. Longer and larger lumens can be filled with devices incorporating laminated membranes.

[0116] Perforated lamination device Perforations are also called holes, holes, openings, punctures, apertures or channels.

[0117] The foregoing devices are a non-limiting disclosure, and other devices in accordance with the embodiments described herein are embodied by the present disclosure. The present disclosure contemplates combinations of the foregoing devices. For example, in one embodiment, a cell-exclusion membrane and a nonwoven fabric are laminated together and perforated. In one embodiment, a perforated cell-exclusion membrane and a non-perforated NWF nonwoven fabric are laminated together.

[0118] The potential therapeutic value of perforated devices loaded with therapeutic cells is of great value to the type 1 diabetes (T1D) population, where the side effects of chronic immunosuppressive medications are tolerated or already required due to a previous organ transplant (e.g., kidney transplant). Importantly, therapeutic cells derived from pluripotent stem cells overcome the limitations associated with the use of deceased organ donors, primarily (1) the very limited supply of pancreatic islets from eligible cadavers and (2) the risks to patients associated with donated organs (e.g., donor-derived pathogens with limited ability to test donated tissue prior to transplantation). Furthermore, the delivery device and subcutaneous implantation route of administration offer several advantages over current clinical islet transplantation, including the ability to noninvasively monitor and image the transplant site, ease of graft and explant / biopsy procedures, and elimination of portal vein thrombosis events. Indeed, the islet transplantation field has long sought an alternative to intraportal vein transplantation sites. See Cantarelli et al., Alternative transplantation sites for pancreatic islet grafts Curr Diab Rep. 2011 Oct;11(5):364-74.

[0119] To determine the desired perforation geometry of the device, the diameter, quantity, and distribution of the pores were characterized. Figures 2A-D each depict an embodiment of a device having a particular density of pores. While the pore shapes and sizes shown in the figures are uniform, it is important to note that: i) the pore size or shape need not be uniform; ii) the pores on both sides of the device need not be aligned (in regular rows) when the device is assembled; iii) the number or density of pores in each device may be minimal, promoting direct host-to-graft cell-to-cell contact and vascularization; iv) the pore diameter depends on the size of the encapsulated cells or tissue, e.g., cell clusters or aggregates, but encapsulated cells do not necessarily leak out of the device through the pores; rather, there will be more host-derived cells inside the device than grafted cells outside the device; and v) a cell-exclusion membrane. The holes in the nonwoven fabric do not have to be aligned when the device is assembled, but may form irregular paths from the outside to the inside of the chamber of the device. Furthermore, although the device is shown as oval, the shape of the device may be any of circular, rectangular, square, or triangular shapes.

[0120] In one embodiment, the perforations are circular, oval, or elliptical. It should be noted that the perforations may have other shapes, such as rectangular, hexagonal, polygonal, or slit shapes. In one embodiment, the perforations have a uniform shape. In one embodiment, the perforations do not have a uniform shape. In one embodiment, the perforations are uniformly distributed across the cell-exclusion membrane. In one embodiment, the perforations are spaced at various intervals across the cell-exclusion membrane, and may be clustered, for example, at the center of the device or at the edge of the device. In one embodiment, the perforations are spaced apart in a series of rows and columns, forming a grid array, concentric circles, or any other geometric configuration or combination of configurations. In one embodiment, the perforations are randomly distributed. In one embodiment, perforations are not present in each cell-exclusion membrane, but only on one side of the device.

[0121] In one embodiment, the cell delivery device includes a layer in which only the cell exclusion membrane is perforated. In one embodiment, the cell delivery device includes a membrane ring, a mesh, and a cell exclusion membrane, and only the cell exclusion membrane is perforated. In one embodiment, the cell delivery device includes a membrane ring, a mesh, a nonwoven fabric, and a cell exclusion membrane, and only the cell exclusion membrane and the nonwoven fabric layer are perforated. In one embodiment, the cell delivery device includes a nonwoven fabric and a cell exclusion layer, and only the cell exclusion membrane and the nonwoven fabric layer are perforated. In one embodiment, the cell delivery device includes a nonwoven fabric on the outside of the cell exclusion membrane, and only the cell exclusion membrane and the nonwoven fabric layer are perforated. In one embodiment, the cell delivery device includes a nonwoven fabric and a cell exclusion layer laminated together, and only the cell exclusion membrane and the nonwoven fabric layer are perforated. In one embodiment, the cell delivery device includes a nonwoven fabric on the outside of the cell exclusion layer, laminated to the cell exclusion membrane, and only the cell exclusion membrane and the nonwoven fabric layer are perforated.

[0122] In one embodiment, the cell delivery device comprises a cell-exclusion membrane and a nonwoven layer, where only the layers are perforated, and the holes are made with a laser.

[0123] Perforation diameter The use of perforated cell delivery devices has certain drawbacks, including cell escape, cell deficiency, and tumorigenicity. The perforated openings allow the cell-exclusion membrane to retain encapsulated elements while simultaneously allowing exchange with the host, such as the ingress of vasculature, macrophages, and other phagocytes, which can remove necrotic debris from the lumen of the perforated device and the stromal cells supporting the pancreatic cell population. In one embodiment, the perforations are less than approximately 100 μm in diameter, allowing for capillary ingrowth. Applicant has previously demonstrated that for pancreatic progenitor cell aggregates with a mean diameter of approximately 180 μm and a quartile diameter of 100-200 μm (Schulz et al. (2012) supra), a pore diameter of approximately 100 μm or less substantially retains the cell product and achieves the other benefits described above, thereby facilitating both cell delivery and recovery, as well as capillary ingrowth. While cells are exposed to host tissue, e.g., host vasculature, larger sizes minimize the risk of cell escape. In one embodiment, the pores have an inner diameter large enough to allow host capillary ingrowth and release and to allow hormones produced from the therapeutic agent to exit the lumen / chamber of the device.

[0124] Pore ​​size (diameter) can vary depending on the cell function. For example, if complete cell containment is not required, there are lesser restrictions on pore diameter and density. The pores of a particular device may have the same diameter or different diameters in different parts of the device. For example, if the majority of encapsulated cells, cell aggregates, organoids, clusters, clumps, and tissues tend to be located mostly in the center of the device, more pores may be required for cell survival in areas of the device compared to the proximal and distal ends of the device, which may have fewer and / or smaller pores. Thus, as long as host-to-graft cellular vascularization is established shortly after implantation, the size of the perforations may be increased. There is a lot of flexibility in size, density and distribution. Again, Figures 2A and 2B show embodiments of perforated devices. Figure 2B shows dual lumens and dual ports, which reduces the area for cell pooling.

[0125] In other embodiments, the pancreatic progenitor cell aggregates are larger than the average diameter of the pores in the device. In one embodiment, the cell delivery device is perforated with pores having a diameter of less than about 300 microns, less than about 200 microns, less than about 150 microns, less than about 100 microns, less than about 75 microns, less than about 60 microns, or less than about 50 microns. In one embodiment, the cell delivery device is perforated with pores having a diameter of about 300-50 microns, about 200-50 microns, about 200-75 microns, or about 70-80 microns. In one embodiment, the pore size is greater than about 200 microns. In one embodiment, the pore size is about 200-400 microns.

[0126] In one embodiment, the perforations have a diameter of about 40 to 150 μm. In one embodiment, the perforations have a uniform shape. In one embodiment, the perforations do not have a uniform shape.

[0127] Perforation Density In one embodiment, less than 0.4% of the device's surface area is perforated, with the perforations spaced about 2 mm apart (measured center-to-center), although perforations may be spaced less than or greater than 2 mm apart to promote host-to-graft cell angiogenesis. In some embodiments, less than about 5.0%, less than about 4.0%, less than about 3.0%, less than about 2.0%, less than about 1.0%, less than about 0.8%, less than about 0.3%, less than about 0.2%, less than about 0.1%, or less than about 0.05% of the device's surface area is perforated. In some embodiments, between about 5.0 and 0.5%, between about 5.0 and 3.5%, or between about 4.0 and 2.0% of the device's surface area is perforated.

[0128] In one embodiment, perforation is avoided by replacing the cell-exclusion membrane with a highly permeable membrane, for example, a membrane consisting of 80-120 micron pores, with such pores fabricated at a density similar to the pores described herein.

[0129] Example 1 demonstrates that relatively few perforations provide the desirable benefit of direct host vascularization while improving cell viability. Glucose-Stimulated Insulin (GSIS) studies (described in Example 1) at 12 and 34 weeks post-implantation demonstrate that perforations approximately 100 μm in diameter and spaced approximately 1, 1.5, or 2 mm apart (measured center-to-center) produce functional grafts in athymic nude mice. Indeed, even the sparsest perforations, spaced 2 mm apart, demonstrated direct vascularization and robust C-peptide levels. From a clinical safety perspective, fewer perforations reduce the risk of cells escaping the cell delivery device, and the sparsest perforations (2 mm spacing) of the perforated device are preferred.

[0130] In one embodiment, a cell delivery device comprises a perforated cell exclusion membrane having holes spaced about 0.5 mm, 1.0 mm, 1.5 mm, 2 mm, 4 mm, 8 mm or more apart (measured center-to-center). In one embodiment, a cell delivery device comprises a perforated cell exclusion membrane having holes spaced about 0.5 mm, 1.0 mm, 1.5 mm, 2 mm, 4 mm, 8 mm or more apart and a perforated NWF layer. In one embodiment, a cell delivery device comprises a perforated cell exclusion membrane and a perforated NWF layer laminated to a perforated NWF layer having holes spaced about 0.5 mm, 1.0 mm, 1.5 mm, 2 mm, 4 mm, 8 mm or more apart. In one embodiment, a cell delivery device is provided that comprises holes or perforations spaced about 0.5 mm to 4 mm, about 0.5 mm to 2 mm, or about 1.0 mm to 2 mm apart.

[0131] The number / density of holes may be 5-200 or 20-100 per device, depending in part on the size (luminal surface area) of the device. Indeed, the number / density of holes may be 20-50-100 per lumen of the device. Indeed, the number / density of holes may be 20-50-100 per lumen of the device. The number of holes may be 5 to 200, or 20 to 100. Those skilled in the art can determine the number / density of holes to achieve the desired effect. In the case of diabetic patients, the number / density of holes is determined by the ability of the transplanted cells to mature and / or produce insulin in response to blood glucose levels.

[0132] In one embodiment, a cell delivery device is provided that comprises holes or perforations that are about 0.5 mm, 1.0 mm, 1.5 mm, 2 mm or more apart, and the pore size is less than about 200 microns, less than about 150 microns, less than about 100 microns, or less than about 75 microns. In one embodiment, a cell delivery device is provided that comprises perforations that are about 2 mm or more apart and the pore size is less than about 200 microns. In one embodiment, a cell delivery device is provided that comprises holes or perforations that are about 2 mm or more apart and the pore size is less than about 100 microns (measured center-to-center).

[0133] Manufacturer of porous cell delivery devices The perforated devices of the present disclosure can be fabricated using manufacturing methods well known in the art. Historically, the device was assembled, cells were added, and then holes were manually created in the intact device using a needle. In this manner, all layers of the device were perforated. See U.S. Patent Application No. 12 / 618,659 and PCT Application No. WO / 1993 / 002635. Furthermore, because the cells were inside the device when the perforations were created, some of the encapsulated cells were in the path of the needle when the perforations were made, and the needle could potentially damage some of the encapsulated cells. This method also has the potential for accidental contamination (cells leaving the device) as the needle is inserted, creates a hole, and then removed.

[0134] Embodiments herein use a laser to control the size and distribution of the pores, do not perforate each layer of the device, and do not perforate the device after the cells have been added. In this way, forming perforations does not harm or destroy the cells, reduces potential contamination, and the cell-exclusion membrane (or cell-exclusion membrane and nonwoven layer) can be perforated while the other layers help retain the encapsulated cells in the delivery device upon implantation.

[0135] Porous cell delivery devices can be constructed in many size configurations, including preclinical rodent models (typically 20 μL volume) and larger devices (EN250) for clinical trials. Porous and non-porous cell delivery devices use the same materials, manufacturing techniques, and thicknesses.

[0136] We disclose the fabrication of perforated cell delivery devices in which only the cell-exclusion membrane is perforated by using a laser instead of a needle. In some embodiments, a nonwoven fabric is laminated to the cell-exclusion membrane, and only these two layers are perforated. In one embodiment, the fabrication of holes in the device layers is automated.

[0137] In one embodiment, the perforations are circular, oval, or elliptical. It should be noted that the perforations may have other shapes, such as rectangular, hexagonal, polygonal, or slit shapes. In one embodiment, the perforations have a uniform shape. In one embodiment, the perforations do not have a uniform shape. In one embodiment, the perforations are uniformly distributed across the cell-exclusion membrane. In one embodiment, the perforations are spaced at various intervals across the cell-exclusion membrane, and may be clustered, for example, at the center of the device or at the edge of the device. In one embodiment, the perforations are spaced apart in a series of rows and columns, forming a grid array, concentric circles, or any other geometric configuration or combination of configurations. In one embodiment, the perforations are randomly distributed. In one embodiment, perforations are not present in each cell-exclusion membrane, but are present on only one side of the device.

[0138] In one embodiment, the device has multiple distinct cell populations. In one embodiment, the device has multiple chambers, each separated by acellular regions or islets. In one embodiment, the device has multiple chambers, each separated by acellular regions, and not all chambers are perforated. In one embodiment, pancreatic progenitors are encapsulated in one chamber and a different therapeutic agent is encapsulated in another chamber. In this example, only the chamber containing the pancreatic progenitors is perforated.

[0139] Improved Dosage Profile One advantage of using perforated devices to deliver pancreatic endodermal cells is increased cell proliferation capacity. The pores allow cell-to-cell contact with the host vasculature, which improves viability. The pores allow the device chamber to expand, allowing room for more cells inside the perforated device. Increased cell proliferation capacity increases cell mass, and increased cell mass correlates with increased cell volume. Applicant has shown that when appropriate cell volumes are added to devices of the same size (e.g., EN20, EN100, EN250, etc.), the final cell volume (at approximately 12-16 weeks post-implantation, the cells mature) of perforated devices is significantly greater than that of intact or non-perforated devices. In fact, the final (mature) cell volume is approximately 5-6 times greater in perforated devices compared to non-perforated or intact devices. This improves the dosing capacity of otherwise equivalent sized cell delivery devices.

[0140] In islet transplantation, the number or volume of islets is often expressed as islet equivalents (IEQ). One IEQ is considered equivalent to an islet with a diameter of 150 μm. A healthy human has approximately 1 million IEQ, and patients diagnosed with T1D have lost approximately 80% of their islet cells. Treatment does not necessarily restore islet mass or function completely. Restoring approximately 20% of islet mass or function, or approximately 200,000 IEQ, would be functionally curative for T1D patients. Gillard et al., "Minimal functional β-cell mass in intraportal implants that reduces glycemic variability in type 1 diabetic recipients," Diabetes Care 2013(11)3483-8, showed that recipients with functional β-cell grafts exhibited a mean functional β-cell mass equivalent to 18% (interquartile range 10-33%) of that of normal controls.

[0141] Applicant attempted to calculate IEQ based on C-peptide released by transplanted mature PEC grafts, as previously described in Kroon et al. (2008), supra. Applicant demonstrated a linear scale where C-peptide produced by the device correlated with relative IEQ numbers. See Figure 3. To generate this graph, Applicant purchased specific aliquots of cadaveric islets. Six different islet preparations were mashed and the total C-peptide was measured from each sample. The C-peptide measurements for each islet sample were graphed, demonstrating a linear relationship between C-peptide levels and the amount of human islet equivalents. See Figure 3. The following equation defines the linear relationship between C-peptide levels (picomoles) and human islet numbers (IEQ): C-peptide = 0.3889x + 245.53 This formula has a quantitative R of 0.9852 2 The correction is made by the quantification R 2 The coefficient is a statistical measure of how well the regression line fits the actual data points. 2 A ρ of 1 indicates that the regression line fits the data perfectly. Therefore, the correlation between C-peptide and IEQ shown in the formula has a strong or high confidence level.

[0142] In one embodiment, the IEQ number can be extrapolated or estimated by the linear relationship of C-peptide levels to islet IEQ, where the IEQ number is a relative measure of the cell mass achieved in either a perforated or non-perforated device containing therapeutic cells. Thus, for example, 2000 picomoles of C-peptide is approximately 4500 IEQ, or less, for human islets. 2000=0.3889x+245.53→(2000-245.53) / 0.3889 =x→x=4511.36IEQ

[0143] In one embodiment, a perforated cell delivery device improves dosing capacity by at least two-fold, at least three-fold, at least four-fold, or at least five-fold or more compared to a fully (non-perforated) cell delivery device. For example, a perforated EN20 device or small delivery device has a nominal fill volume of approximately 20 μl and contains a beta cell mass of approximately 7,000-47,000 IEQ (average of approximately 23,000) or over 250,000 IEQ per kg of rat. A larger delivery device with a nominal fill volume of 300 μl can contain a beta cell mass of up to 700,000 IEQ. Therefore, it is estimated that one or two large perforated devices may be required to deliver a sufficient amount of PECs to deliver a therapeutically effective dose to a patient.

[0144] In one embodiment, C-peptide production in a mammal following implantation with a perforated cell delivery device is not steady-state until about 15 weeks, about 16 weeks, about 17 weeks, about 18 weeks, about 19 weeks, about 20 weeks, about 21 weeks, about 22 weeks, about 23 weeks, about 24 weeks, about 25 weeks, about 26 weeks, about 27 weeks, about 28 weeks, about 29 weeks, about 30 weeks, about 31 weeks, about 32 weeks, about 33 weeks, about 34 weeks, or about 35 weeks.

[0145] In one embodiment, C-peptide production in a mammalian animal after implantation with a perforated cell delivery device reaches a steady state at about 30 weeks post-implantation, about 31 weeks post-implantation, about 32 weeks post-implantation, about 33 weeks post-implantation, about 34 weeks post-implantation, about 35 weeks post-implantation, about 36 weeks post-implantation, about 37 weeks post-implantation, about 38 weeks post-implantation, about 39 weeks post-implantation, or about 40 weeks post-implantation. In one embodiment, C-peptide production in a mammalian animal after implantation with a perforated cell delivery device reaches a steady state at about 30-40 weeks post-implantation. In one embodiment, C-peptide production in a mammalian animal after implantation with a perforated cell delivery device reaches a steady state at about 35-45 weeks post-implantation, or about 30-45 weeks post-implantation.

[0146] It is hypothesized that perforated cell delivery devices may provide a type of protective niche in the lumen of the device, shielding the transplanted cells from what becomes the harsh subcutaneous host environment immediately after implantation. Indeed, when applicants compared perforated devices with the complete absence of cell encapsulation (i.e., no device / "naked cells" implanted under the epididymal fat pad), cell function was improved in perforated devices compared to naked cells (data not shown). Thus, more direct host-to-graft cell-to-cell contact (direct vascularization) alone is not the solution to improved cell function in vivo, and it is expected that naked cells will have improved function over cells in perforated devices. See applicant's U.S. Patent No. 12 / 618,659, supra.

[0147] The delivery device can be used for tissue or cell replacement in correcting a pathological condition. Currently, the most common method is allotransplantation of organelles or free cells by injecting them into the portal circulation to correct a pathological condition, allowing the cells to remain in the liver. The present invention allows for the use of alternative, easily accessible transplantation sites, and the delivery device with its contents can be removed if necessary. The delivery device allows the host to provide sufficient trophic support to the transplanted tissue to correct the pathological condition. Thus, the delivery device can be used for allotransplantation of human tissue.

[0148] The delivery device can also be used to transplant a patient's own genetically modified cells to produce a therapeutic product. Once the cells are transformed, express the gene, and secrete the therapeutic product, a removable container for the cells is desirable. The delivery device of the present invention can also be used to construct hybrid bioartificial organs. The delivery device can provide a bioartificial organ with a detailed vasculature to deliver nutrients to the organ, remove metabolic waste products, and deliver therapeutic products made by the organ to the host.

[0149] Further development of biological immune protection strategies may in the future eliminate the need for chronic immunosuppression. See vent immune rejection of human ESC-derived allografts Cell Stem Cell 2014 14(1):121-130, incorporated herein by reference in its entirety.

[0150] Unless otherwise specified, a non-perforated device or intact device means a device that has no perforations (holes).

[0151] Combination Products The embodiments described herein disclose combination products, which refer to devices loaded with cells or therapeutic agents, each of which alone could be a potential medical device or cell product, but which together create a combination product. In one embodiment, the combination product refers to a perforated device into which cells are loaded. This is referred to as a "perforated combination product." The device (perforated or non-perforated) can be a macro cell delivery device described herein, including, but not limited to, EN20, EN100, EN250, or EN large capacity. The combination product may specify the size of the device; for example, VC-01-20 refers to an EN20 loaded with cells. The cells added to the device (perforated or non-perforated) may be, but are not limited to, definitive endoderm, PDX1-positive endoderm, PDX1-positive foregut endoderm, pancreatic endoderm, pancreatic endoderm cells expressing PDX1 and NKX6.1, endocrine precursors, endocrine precursors expressing NKX6.1 and INS, immature beta cells, immature beta cells expressing NKX6.1, INS and MAFB, mature endocrine cells, mature endocrine cells expressing INS, GCG, SST and PP, mature beta cells, and mature beta cells expressing INS and MAFA.

[0152] A perforated delivery device loaded with mature pancreatic endoderm cells ("perforated combination product"), when implanted in vivo, is intended to reduce insulin dependence and / or hypoglycemia in high-risk type 1 diabetic patients who are hypoglycemic unaware, unstable (fragile), have undergone organ transplants, may be resistant to immunosuppressive therapy, or are already undergoing treatment. The primary method of action is contained in a permeable, durable implantable medical device that facilitates direct vascularization of the host via human pancreatic endoderm cells (PECs) or pancreatic progenitor cells. After implantation, the PEC cells differentiate and mature into therapeutic glucose-responsive insulin-releasing cells. In this way, the perforated combination product supports the secretion of human insulin. The perforated combination product limits the distribution (release) of PEC cells in vivo. The perforated combination product is implanted in a location that maintains a population of therapeutic cells within the device and allows for sufficient vascularization to facilitate the distribution of insulin and other pancreatic products into the bloodstream. The perforated combination product is intended to be implanted or explanted with conventional surgical instruments and provide a therapeutic dose for at least two years. The device maintains a sufficient dose of PEC cell product in the formulation, shelf life, manipulation, and surgical implantation to achieve clinical efficacy, and the cell product is securely positioned within the tissue capsule to meet safety requirements.

[0153] The perforated combination product is comprised of a perforated device (PD) containing a dose of PEC, a human pancreatic progenitor cell therapy product. After implantation into a patient, the perforated combination product is designed to allow for the differentiation and maturation of the PEC cells into glucose-responsive, insulin-producing cells for the treatment of patients in need of insulin, through device integration and direct vascularization of the implanted cell product.

[0154] A perforated device (PD) is defined as a durable, biocompatible, easily removable implantable device composed of deposited layers of material bonded together to form a cell-containing lumen. The device is constructed of biocompatible and biostable materials intended for long-term implantation. The semipermeable membrane allows diffusion of nutrients into the lumen immediately after implantation, while the perforations in the membrane allow host vascular growth into the lumen of the device and directly to the implanted cells, improving perfusion and release of implanted cell products, including insulin, into the bloodstream.

[0155] PD contains fenestrations large enough to allow invasion or ingrowth of host blood vessels and other host cells, including immune cells, into the lumen containing the cells of the device, necessitating the use of immunosuppressive drugs.

[0156] The perforated combination product is expected to be implanted for five years, but must meet at least a two-year service life. The design intent of the PD is to provide a defined, protected space for the initial survival and differentiation / maturation of transplanted cells during capsule formation and to retain the majority of cells throughout the entire implantation period. The device components must be biocompatible. Two device configurations are being developed for clinical trials: a device with a sufficient dose to potentially achieve therapeutic administration, and a smaller unit suitable for easy implantation and explantation to evaluate the transplant and host tissue response via tissue diagnosis at an intermediate time point (sentinel). The size and number of perforations in the device should be selected to allow a sufficient amount of host blood vessels to be directly ingrowth-into the transplanted cells without prejudice to the perforated device's ability to maintain sufficient cell mass and provide efficacy. Furthermore, the perforated device ensures that a sufficient amount of the transplanted cells, including the surrounding host tissue capsule, is removed from the body during product explantation to meet safety requirements.

[0157] The design of a PD and perforated combination product has additional benefits related to the similarity to an intact (non-perforated) cell delivery device, including:

[0158] The PD supports the same materials, similar manufacturing processes, and comprehensive biocompatibility testing established for intact cell encapsulation devices previously disclosed in U.S. Patent Application No. 8,278,106 and U.S. Patent Application No. 14 / 201,630.

[0159] Because the intact and perforated devices offer similar geometry and handling characteristics, it is intended that the surgical method for both implantation and explantation will be the same for both. In summary, the perforated combination product is designed to support existing manufacturing processes and clinical experience with intact cell encapsulation devices for delivery of cellular products.

[0160] The following paragraphs describe other embodiments.

[0161] A cell delivery device comprising a nonwoven fabric.

[0162] 25. The cell delivery device of paragraph 24, further comprising a cell exclusion membrane, the nonwoven fabric being external to the cell exclusion membrane.

[0163] A cell delivery device comprising a cell exclusion membrane and a nonwoven fabric external to the cell exclusion membrane, wherein only the cell exclusion membrane is perforated.

[0164] 27. The cell delivery device of paragraph 26, wherein the host's blood vessels directly contact the lumen of the cell delivery device.

[0165] A cell delivery device comprising a cell-exclusion membrane and a nonwoven fabric external to the cell-exclusion membrane, wherein only the nonwoven fabric is perforated.

[0166] 29. The cell delivery device of paragraph 28, wherein the host's blood vessels directly contact the outer surface of the cell delivery device.

[0167] A cell delivery device comprising a cell exclusion membrane, a nonwoven fabric exterior to the cell exclusion membrane, and either a mesh layer or a membrane weld, or both, wherein the nonwoven fabric and the cell exclusion membrane are perforated.

[0168] 31. The cell delivery device of paragraph 30, wherein the host's blood vessels directly contact the lumen of the cell delivery device.

[0169] A cell delivery device comprising a cell exclusion membrane and a nonwoven fabric external to the cell exclusion membrane, wherein only the nonwoven fabric and the cell exclusion membrane are perforated.

[0170] 33. The cell delivery device of paragraph 32, wherein the host's blood vessels directly contact the outer surface of the cell delivery device.

[0171] 33. The cell delivery device of paragraph 32, wherein the host's blood vessels form throughout the cell delivery device and come into direct contact with the therapeutic agent added to the cell delivery device.

[0172] 33. The cell delivery device of paragraph 32, wherein the nonwoven fabric is laminated to a cell-exclusion membrane.

[0173] 33. The cell delivery device of paragraph 32, wherein the cell delivery device is implanted into a mammalian host undergoing treatment with at least one immunosuppressant drug.

[0174] 37. The cell delivery device of paragraph 36, wherein the immunosuppressant is selected from the group consisting of a calcineurin inhibitor, antimetabolite immunosuppressant, and combinations thereof.

[0175] 38. The cell delivery device of paragraph 37, wherein the immunosuppressant is selected from the group consisting of cyclosporine A (CsA), mycophenolate mofetil (MMF), tacrolimus (TAC), and combinations thereof.

[0176] A cell delivery device comprising a perforated nonwoven fabric that is implanted into a host being treated with an immunosuppressant drug.

[0177] A cell delivery device comprising a nonwoven fabric on the outside of a cell-exclusion membrane that is implanted into a host treated with an immunosuppressant drug.

[0178] 41. The cell delivery device of paragraph 39 or 40, wherein the nonwoven is perforated.

[0179] 42. The cell delivery device of paragraph 41, wherein the cell exclusion membrane is perforated.

[0180] 43. The cell delivery device of paragraph 42, wherein the cell-exclusion membrane and nonwoven are perforated.

[0181] 41. The cell delivery device of paragraph 39 or 40, wherein the immunosuppressant is selected from the group consisting of a calcineurin inhibitor, antimetabolite immunosuppressant, and combinations thereof.

[0182] 45. The cell delivery device of paragraph 44, wherein the immunosuppressant is selected from the group consisting of cyclosporine A (CsA), mycophenolate mofetil (MMF), tacrolimus (TAC), and combinations thereof.

[0183] 41. The cell delivery device of paragraph 39 or 40, comprising a cell exclusion membrane, wherein the nonwoven fabric is laminated to the cell exclusion membrane.

[0184] A method for promoting the survival of cells transplanted in vivo into a mammalian animal, comprising: a) adding cells to a perforated cell delivery device; and b) implanting the perforated device containing the cells into a mammalian host, thereby promoting the survival of the transplanted cells.

[0185] 48. The method of paragraph 47, wherein the cells are pancreatic endoderm cells.

[0186] 48. The method of paragraph 47, wherein the mammal is not a mouse.

[0187] 48. The method of paragraph 47, wherein the mammal is a human or a rat.

[0188] A method for lowering blood glucose in a mammalian animal, comprising: 1) adding cells to a cell delivery device, the device comprising a perforated cell-exclusion membrane and a perforated nonwoven fabric external to the cell-exclusion membrane, and no other perforated layers; b) implanting the cell delivery device into a mammalian host; and c) allowing the implanted cells to mature, thereby lowering blood glucose in the mammalian animal.

[0189] A cell delivery device comprising a cell exclusion membrane and a nonwoven fabric on the exterior of the cell exclusion membrane, wherein the nonwoven fabric is laminated to the cell exclusion membrane.

[0190] 53. The cell delivery device of paragraph 52, wherein the nonwoven fabric and the cell-exclusion membrane are perforated.

[0191] A cell delivery device comprising a cell-exclusion membrane and no NWF, wherein only the cell-exclusion membrane is perforated.

[0192] A cell delivery device that includes a cell-exclusion membrane, does not include a NWF, is not implanted into a mammalian animal to be treated with an ISD, and only the cell-exclusion membrane is perforated.

[0193] A cell delivery device containing a cell exclusion membrane, no NWF, and implanted into an ISD-treated rat or human, in which only the cell exclusion membrane is perforated.

[0194] A cell delivery device that includes a cell exclusion membrane, does not include a NWF, and is implanted into a mammalian animal not treated with an ISD, such that only the cell exclusion membrane is perforated.

[0195] A cell delivery device containing a cell exclusion membrane, not containing a NWF, implanted into a rat or human not treated with an ISD, with only the cell exclusion membrane being perforated.

[0196] A cell delivery device comprising a cell-exclusion membrane and a NWF, wherein only the cell-exclusion membrane and the NWF are perforated.

[0197] A cell delivery device comprising a cell-exclusion membrane and a NWF, which is implanted into a mammalian animal undergoing treatment with an ISD, and in which only the cell-exclusion membrane and the NWF are perforated.

[0198] A cell delivery device comprising a cell-exclusion membrane and a NWF, implanted into a rat or human being treated with ISD, in which only the cell-exclusion membrane and the NWF are perforated.

[0199] A cell delivery device comprising a cell-exclusion membrane and a NWF, which is implanted into a mammalian animal not undergoing treatment with an ISD, and in which only the cell-exclusion membrane and the NWF are perforated.

[0200] A cell delivery device comprising a cell-exclusion membrane and a NWF, implanted into a rat or human not treated with an ISD, in which only the cell-exclusion membrane and the NWF are perforated.

[0201] A cell delivery device containing an intact cell-exclusion membrane and no NWF.

[0202] A cell delivery device comprising an intact cell-exclusion membrane, no NWF, that is implanted into a mammalian animal to be treated with an ISD.

[0203] A cell delivery device containing a cell-exclusion membrane but no NWF, which is implanted into a rat or human being treated with ISD.

[0204] A cell delivery device that includes a cell-exclusion membrane, does not include a NWF, and is implanted into a mammalian animal that is not treated with an ISD.

[0205] A cell delivery device containing an intact cell-exclusion membrane, no NWF, and implanted into rats or humans not treated with an ISD.

[0206] A cell delivery device comprising an intact cell-exclusion membrane and an intact NWF.

[0207] A cell delivery device comprising an intact cell-exclusion membrane and an intact NWF, which is implanted into a mammalian animal to be treated with an ISD.

[0208] A cell delivery device containing an intact cell-excluding membrane and an intact NWF is implanted into rats or humans to be treated with ISD.

[0209] A cell delivery device comprising an intact cell-exclusion membrane and an intact NWF, which is implanted into a mammalian animal not treated with an ISD.

[0210] A cell delivery device containing an intact cell-excluding membrane and an intact NWF is implanted into rats or humans not treated with ISD.

[0211] immunosuppression Transplantation of mature pancreatic islets while treating the host with immunosuppressive compounds has been previously described. See U.S. Patent Application No. 9,062,290, which is incorporated by reference in its entirety. However, calcineurin inhibitors (1) are diabetogenic (result in diabetes) (reviewed in Crutchlow MF, Transplant-associated hyperglycemia: a new look at an old problem Clin J Am Soc Nephrol. 2(2):343-55 (2007)) and (2) negatively affect endogenous pancreatic cell regeneration in mice (Heit J, Calcineurin / NFAT signaling regulates pancreatic beta-cell growth and function Nature 21;443(7109):345-9 (2006) and Nir T, Recovery from diabetes in mice by beta cell regeneration J Clin Invest 117(9):2553-61 (2007). Thus, it was not known and could not be predicted whether immature pancreatic precursors would mature in vivo in the presence of calcineurin inhibitors, and if so, whether the mature grafts would be able to survive and function if the host was treated with immunosuppressive drugs.

[0212] Even treatment of type 1 diabetes with islet transplants has not been effective in freeing many patients from exogenous insulin injections for the long term. One problem has been that the immunosuppressive agents required to prevent allograft rejection can significantly impair the function of the transplanted islet cells. Cellular and Molecular Approaches to Achieving Euglycemia (1997), NIH See Guide 26(38).

[0213] Embodiments described herein relate to implanted perforated devices containing cells, wherein the host is receiving immunosuppressant therapy. In one embodiment, the perforated device includes at least one layer of a nonwoven fabric, such as a NWF, facing the host, wherein the host is receiving immunosuppressant therapy. In the method, the perforated device comprises at least one layer of NWF laminated to an extracellular membrane and facing the host, and the host is treated with an immunosuppressant. In one embodiment, the immunosuppressant is selected from the group consisting of a calcineurin inhibitor, an antimetabolite immunosuppressant, and combinations thereof. In one embodiment, the immunosuppressant is selected from the group consisting of cyclosporine A (CsA), mycophenolate mofetil (MMF), tacrolimus (TAC), and combinations thereof. In one embodiment, the immunosuppressant is administered to a host implanted with the non-perforated device.

[0214] In one embodiment, a method for administering a therapeutically effective amount of a therapeutic agent to a host undergoing treatment with an immunosuppressant drug via a perforated cell delivery device is disclosed.

[0215] The following paragraphs describe other embodiments.

[0216] The method includes a) administering an immunosuppressant to a mammalian host; b) implanting a perforated device containing pancreatic endoderm cells into the mammalian host; and c) maturing a population of pancreatic endoderm cells in the perforated device in vivo, whereby the population of progenitor cells matures into insulin-secreting cells, thereby producing insulin in the mammal. This involves producing insulin in vivo.

[0217] 63. The method of paragraph 62, wherein the immunosuppressant is selected from the group consisting of a calcineurin inhibitor, antimetabolite immunosuppressant, and combinations thereof.

[0218] 64. The method of paragraph 63, wherein the immunosuppressant is selected from the group consisting of cyclosporine A (CsA), mycophenolate mofetil (MMF), tacrolimus (TAC), and combinations thereof.

[0219] 63. The method of paragraph 62, wherein the perforated device comprises a cell exclusion membrane and at least one layer of nonwoven fabric external to the cell exclusion membrane.

[0220] 666. The method of paragraph 665, wherein the nonwoven fabric is laminated to a cell exclusion membrane.

[0221] A perforated cell delivery device containing pancreatic endoderm cells that is implanted into a host being treated with an immunosuppressant drug.

[0222] 68. The perforated cell delivery device of paragraph 67, wherein the immunosuppressant is selected from the group consisting of a calcineurin inhibitor, antimetabolite immunosuppressant, and combinations thereof.

[0223] 69. The perforated cell delivery device of paragraph 68, wherein the immunosuppressant drug is selected from the group consisting of cyclosporine A (CsA), mycophenolate mofetil (MMF), tacrolimus (TAC), and combinations thereof.

[0224] 68. The perforated cell delivery device of paragraph 67, wherein the perforated device comprises at least one layer of nonwoven fabric.

[0225] 71. The perforated cell delivery device of paragraph 70, wherein the perforated device comprises a cell exclusion membrane and the nonwoven fabric is laminated to the cell exclusion membrane.

[0226] A method for improving the viability of therapeutic cells in a mammalian subject, comprising administering to the subject an effective amount of therapeutic cells from a perforated cell delivery device and an effective amount of an immunosuppressant, wherein the effective amount of the immunosuppressant does not impair the ability of the therapeutic cells to survive and mature in vivo.

[0227] A method for producing insulin in a mammal, comprising transplanting pancreatic endoderm into a host and administering an effective amount of to a host, thereby allowing pancreatic endoderm to mature into insulin-producing cells in the mammal, thereby producing insulin in the mammal.

[0228] A method for suppressing or modulating a host's immune or inflammatory response to transplanted xenogeneic or allogeneic cells, comprising delivering cells in a perforated cell delivery device and administering to the host an effective amount of an anti-inflammatory factor or immunosuppressant, wherein the cells are pancreatic endoderm.

[0229] A method for treating diabetes in a subject, comprising: (a) administering an immunosuppressant to the diabetic subject; and (b) administering pancreatic endoderm cells to the subject with a perforated cell delivery device, wherein the pancreatic endoderm cells mature into insulin-producing cells, thereby treating diabetes in the subject.

[0230] Additional features and advantages of the embodiments described herein will become apparent from the detailed description, figures, and examples. Examples of embodiments are as follows. (1) A combination product comprising a nonwoven layer and a cell delivery device comprising PDX1-positive pancreatic endoderm cells. (2) The combination product of (1), wherein the cell combination product further comprises a cell exclusion membrane, and the nonwoven fabric layer is external to the cell exclusion membrane of the cell delivery device. (3) The combination product according to (1) or (2), wherein the nonwoven fabric layer and the cell-exclusion membrane are laminated together. (4) (5) The combination product according to (2) or (3), wherein the nonwoven fabric layer and the cell-exclusion membrane contain perforations. The combination product according to (2) or (3), wherein the nonwoven fabric layer is not perforated and the cell-exclusion membrane is perforated. (6) The combination product according to any one of (1) to (5), wherein the combination product is transplanted into rats treated with immunosuppressants. (7) The combination product according to any one of (1) to (6), wherein the PDX1-positive pancreatic endoderm cells are pancreatic progenitor cells. (8) The combination product according to any one of (1) to (6), wherein the PDX1-positive pancreatic endoderm cells are pancreatic endocrine cells. (9) The combination product according to any one of (1) to (6), wherein the PDX1-positive pancreatic endoderm cells are pancreatic beta cells. (10) A combination product according to any one of (4) or (6) to (9), wherein the perforations in the nonwoven fabric layer and the cell exclusion membrane have a diameter of 150 microns or less. (11) A combination product according to any one of (4) or (6) to (9), wherein the perforations in the nonwoven fabric layer and the cell exclusion membrane have a diameter of 100 microns or less. (12) A combination product according to any one of (4) or (6) to (9), wherein the perforations in the nonwoven fabric layer and the cell exclusion membrane have a diameter of 75 microns or less. (13) A combination product according to any one of (4) or (6) to (9), wherein the perforations in the nonwoven fabric layer and the cell exclusion membrane have a diameter of 50 microns or less. (14) A combination product according to any one of (4) or (6) to (13), wherein the perforations in the nonwoven fabric layer and the cell-exclusion membrane are spaced apart by approximately 1.0 mm. (15) A combination product according to any one of (4) or (6) to (13), wherein the perforations in the nonwoven fabric layer and the cell-exclusion membrane are spaced apart by approximately 1.5 mm. (16) A combination product according to any one of (4) or (6) to (13), wherein the perforations in the nonwoven fabric layer and the cell-exclusion membrane are spaced apart by approximately 2 mm. (17) The combination product of any of (4) or (6) to (16), wherein the nonwoven fabric layer and the cell-exclusion membrane each contain less than about 40 perforations per device. (18) The combination product of any of (4) or (6) to (16), wherein the nonwoven fabric layer and the cell-exclusion membrane each contain less than about 20 perforations per device. (19) a) administering to a mammalian host an immunosuppressant; b) implanting a perforated device containing pancreatic endoderm cells into said mammalian host; and c) maturing said pancreatic endoderm cell population in said perforated apparatus of said mammalian host, and said progenitor cell population producing insulin-secreting cells, thereby producing insulin in said mammal. (20) The method according to (19), wherein the mammalian host is a human or a rat.

[0231] The use of perforated devices in typical human patients requires chronic immunosuppressive drug (ISD) therapy. Due to adverse events caused by ISDs, it was unclear whether Applicant's proprietary PDX1-positive pancreatic endoderm cells or pancreatic precursors (also referred to as "PECs") would (1) mature when transplanted into hosts receiving ISDs, and (2) whether the mature cells would survive, remain biologically active, and produce insulin in response to blood glucose levels, as previous studies have shown that certain calcineurin inhibitors are diabetogenic (cause diabetes) (see Crutchlow et al. (2007) supra) and negatively affect endogenous pancreatic cell regeneration in mice (Heit (2006) supra). Applicant made the surprising discovery that PECs transplanted with perforated devices continue to differentiate and function in nude rats receiving ISDs, as evidenced by the fact that serum levels of human C-peptide in animals treated with cyclosporine A were similar to those measured in untreated control rats. Furthermore, we observed a reversal of the initial hyperglycemia observed in CsA-treated rats. This continued for at least 30 weeks after transplantation, indicating abolition of graft sensitivity to the aforementioned therapeutic levels of calcineurin inhibition with cyclosporine A. In subsequent studies, we further tested the pancreatic endoderm from perforated devices by transplanting them into hosts receiving a combination of CsA or tacrolimus (TAC) with mycophenolate mofetil (MMF). This study established pancreatic endoderm and graft tolerance to all ISD regimens tested. In summary, pancreatic endoderm and graft function did not appear to be negatively affected by the combined presence of hyperglycemia and calcineurin inhibition, contrary to expectations from previous literature reports. Thus, one skilled in the art would expect that pancreatic endoderm and graft function would not be negatively affected by most commonly used drugs to maintain immunosuppression.

[0232] In one embodiment, the device is implanted preperitoneally.

[0233] Related literature Encapsulation of human pluripotent stem cell-derived pancreatic cells is described in Martinson et al., U.S. Patent Application No. 8,278,106, filed October 2, 2012. Implantable encapsulation devices and tools and instruments for use are described in U.S. Patent Application No. 14 / 254,844, filed April 16, 2014, and U.S. Design Patent Application Nos. 29 / 488,209, 29 / 488,217, 29 / 488,191, and 29 / 488,204. Instruments and methods for loading cells into implantable devices are described in PCT / US2014 / 060306, filed October 13, 2014. A "LOADING SYSTEM FOR AN ENCAPSULATION DEVICES" is described in U.S. Patent Application No. 14 / 000,864, filed August 21, 2013. "3-DIMENSIONAL LARGE CAPACITY CELL ENCAPSULATION DEVICE ASSEMBLIES" are described in U.S. Patent Application No. 14 / 201,630, filed March 7, 2014, and U.S. Design Patent Applications Nos. 29 / 447,944, 29 / 509,102, 29 / 484,363, 29 / 484,360, 29 / 484,359, 29 / 484,357, 29 / 484,356, 29 / 484,355, 29 / 484,362, and 29 / 484,358. "CELL ENCAPSULATION DEVICES" are described in U.S. Design Patent Application Nos. 29 / 408,366, 29 / 517,319, 29 / 408,368, 29 / 518,513, 29 / 518,516, 29 / 408,370, 29 / 517,144, 29 / 423,365, and 29 / 530,325. "CULTURING OF HUMAN EMBRYONIC STEM CELLS INTO PANCREATIC ENDOCRINE CELLS" are described in U.S. Design Patent Application Nos. 13 / 998,884 and 62 / 352,968. "FORAMINOUS IMPLANT" are described in International Publication No. WO 1993 / 02635. Each of the above referenced patents / applications is incorporated herein by reference in its entirety.

[0234] All publications and patents mentioned herein are incorporated by reference in their entirety. [Example]

[0235] It will be appreciated that the foregoing relates to the described embodiments and that numerous modifications may be made thereto without departing from the scope of the present invention. The present invention is further illustrated by the following examples, which should not be construed as limiting its scope. To the contrary, it is clearly understood that various other embodiments, modifications, and equivalents thereof exist and may be suggested to those skilled in the art after reading the description herein without departing from the spirit of the described embodiments and / or the scope of the appended claims.

[0236] Example 1: Cell delivery devices with nonwoven fabrics of varying densities The EN20 device generally consists of three layers: 1) an inner cell-exclusion membrane, 2) a central membrane ring, and 3) an outer layer of woven mesh. During the manufacturing process, when the three components are ultrasonically welded (or sealed) together, the cell-exclusion membrane can be compressed by the tightly woven mesh, especially near the transition area between the weld and the lumen. Extreme flexing of the device can also compress areas of the membrane. These compressed areas could potentially cause the membrane to crack and compromise the integrity of the device (e.g., cell leakage from the device). Additional materials and / or layers were tested to improve the structural integrity of the device while maintaining its functionality (e.g., cell exclusion, angiogenesis, biocompatibility). Nonwoven fabrics were identified as a potential intervening buffer layer.

[0237] A novel device was fabricated using a nonwoven fabric. As previously mentioned, the device components (nonwoven fabric, membrane mesh, and membrane) can be arranged in a variety of patterns. Here, a nonwoven fabric was added between the outer woven mesh layer and the outer surface of the cell-exclusion membrane using various fiber densities: 0.40, 0.75, and 1.00 oz / yd^2. See Figure 4, which shows a cross-section of a delivery device with the added nonwoven fabric. Please refer to.

[0238] Either no nonwoven fabric was laminated to the cell exclusion membrane, or a custom polyethylene, dry adhesive woven fabric (e.g., Part Number Spunfab PO4605, SpunFab, Inc.) was heat laminated to the cell exclusion membrane. See Table 5. A low basis weight (0.145 oz / yd^2) adhesive was selected to inhibit closure of the pores of the cell exclusion membrane during the lamination process.

[0239] This study was conducted in two groups of SCID-Bg mice. Cryopreserved pancreatic endoderm cells (PECs) were thawed and cultured in Dulbecco's medium containing DMEM / HI glucose (27 mm) and B27, and each cohort was added separately to an EN20-sized device.

[0240] [Table 2]

[0241] [Table 3]

[0242] To determine the functionality of the various constructs, glucose-stimulated insulin secretion (GSIS) assays were performed approximately 8, 13, 16, and 22 weeks after implantation, as described in Kroon et al., 2009, supra, and Agulnick et al., 2015, supra. Specifically, prior to the GSIS assay, mice were fasted for approximately 15–18 h. Glucose was administered at approximately 3.0 g / kg body weight via intraluminal infusion of approximately 30% glucose (Hospira) solution. Blood was collected before (fasting), 30, and / or 60 min after glucose administration. Approximately 50 μL of blood samples were collected by puncturing the retroorbital venous plexus under isoflurane anesthesia and transferred to microtiter tubes (BD Biosciences, cat#365956) containing blood / serum separator gel. These tubes were spun at 4000–6000 × g for 10 min, after which serum was collected. ELISA assays (Mercodia Ultrasensitive Human Immunosorbent Assay) were performed. Blood samples were analyzed for human C-peptide in serum using c-peptide ELISA, cat#10-1141-01).

[0243] Table 8 shows that the device incorporating the NWF provided improved performance compared to the control device (no NWF) as measured by human C-peptide in serum after glucose stimulation.

[0244] For example, maximum C-peptide was achieved in mice implanted with the EN20-NWF(2) (0.75 oz / yd^2, not laminated to a cell-exclusion membrane). C-peptide levels were approximately 2.5-fold higher than those of the EN20 Control-1 device at both 13 weeks (1945 pM vs. 841 pM) and 16 weeks (3697 pM vs. 1326 pM) after implantation. Mice implanted with the EN20-NWF(2)-SB (nonwoven PET material laminated to a cell-exclusion membrane with a polyethylene adhesive woven fabric) had approximately 1.5-fold higher human C-peptide serum levels at both 13 weeks (1314 pM vs. 841 pM) and 16 weeks (1696 pM vs. 1326 pM) after implantation.

[0245] Similarly, when other densities of nonwoven PET materials were used (NWF(3) at 0.40 oz / yd^2 and NWF(1) at 1.00 oz / yd^2, without a cell-exclusion membrane), C-peptide levels were approximately 2.5-fold higher in mice implanted with the EN20 Control-2 device at 13 weeks (1603 pM in the NWF(3) device vs. 644 pM in the EN20 Control-2 device, 1482 pM in the NWF(1) device vs. 644 pM in the EN20 Control-2 device) and 16 weeks (2186 pM in the NWF(3) device vs. 826 pM in the EN20 Control-2 device, 2285 pM in the NWF(1) device vs. 826 pM in the EN20 Control-2 device) after implantation than in mice implanted with the EN20 Control-2 device (see Table 8).

[0246] [Table 4]

[0247] Histological analysis of the explanted implants showed increased vascularization around devices containing NWF between the cell-exclusion membrane and mesh layer compared with control devices without NWF, suggesting that NWF contributes to improved functional performance of the device, as demonstrated by significantly higher human C-peptide serum levels compared with controls after glucose stimulation.

[0248] Although an NWF between the cell-exclusion membrane and the outer mesh improved the functional performance of the EN20 device in vivo, varying the density (basis weight) of NWF(1), (2), or (3) did not appear to significantly affect functional performance (compare EN20-NWF(3) with EN20-NWF(1) in Cohort 2 in Table 8). Surprisingly, devices in which the NWF was not layered on the cell membrane layer (EN20-NWF(2) in Cohort 1, EN20-NWF(3) in Cohort 2, EN20-NWF(1) in Cohort 2) did not significantly affect functional performance. The devices (EN20-NWF(3), EN20-NWF(1)) showed improved functional performance (2.5-fold and 1.5-fold higher human C-peptide) compared to devices in which a nonwoven PET layer was laminated to a cell-exclusion membrane with a polyethylene adhesive woven fabric (EN20-NWF(2)-SB in Cohort 1). From this, we conclude that the presence of the NWF (laminated or not) has the greatest effect on the functionality of delivered cells.

[0249] Example 2: Lamination of a nonwoven fabric to a cell-exclusion membrane of a cell delivery device The effect of laminating an NWF layer to a cell-exclusion membrane layer was further investigated. At least three configurations of small-sized 20 μL cell delivery devices were constructed ("EN20"). A control device consisted of an innermost cell-exclusion membrane and an outermost mesh ("EN20 Control"). Experimental devices consisted of an innermost cell-exclusion membrane, a central NWF, and an outermost mesh. In one configuration, the NWF was not laminated to the cell-exclusion membrane ("EN20-NWF(2)"). In another configuration, the NWF was laminated to the cell-exclusion membrane using heat and pressure ("EN20-NWF(2)-HL"). Thermal lamination (i.e., heat stacking) of the cell-exclusion membrane and NWF was performed using a standard heat press machine (e.g., ARB Arbor Press, Plastics The press was set at 305-320°C. o The tube was heated to 100°F and pressure of 0 to 6 PSI was applied at a rate of 3 ft / min or 10 ft / min.

[0250] All completed devices were sterilized, aseptically loaded with research-grade pancreatic progenitor cells derived from human pluripotent stem cells, and implanted into SCID-Bg mice as described at least in Kroon et al., 2008, supra, and Agulnick et al., 2015, supra. To characterize the functionality of the various configurations, glucose-stimulated insulin secretion (GSIS) assays were performed approximately 12 and 16 weeks after implantation, as described in Example 1.

[0251] Maximum C-peptide values ​​obtained from mice implanted with EN20-NWF(2) (non-thermolaminated) devices were approximately 2.4-fold higher at 12 weeks and 1.9-fold higher at 16 weeks (compare 241% vs. 100% and 185% vs. 100%), while mice implanted with EN20-NWF(2)-HL (thermolaminated) were approximately 2.8-fold higher at 12 weeks and 2.30-fold higher at 16 weeks (compare 278% vs. 100% and 229% vs. 100%) compared to controls.

[0252] This demonstrates that laminating a nonwoven to a cell-exclusion membrane has an improved effect on the cellular functionality of the device (compare 241% vs. 278% at 12 weeks and 185% vs. 229% at 16 weeks). However, as previously mentioned, it was the presence of the nonwoven (laminated or not) that had the greatest effect on cellular functionality: devices with the nonwoven had C-peptide levels that were at least 1.9-fold higher than control devices without the nonwoven.

[0253] [Table 5]

[0254] The nonwoven fabric appears to improve device implantation by improving the host vascularization of the device, thereby improving cell viability, proliferation, development, maturation and function within the device.

[0255] Example 3: Optimization of hole density in perforated devices Applicant attempts to characterize the optimal number (density) of perforations in a macrocell delivery device. Table 2 below lists perforated cell delivery devices with and without a perforated nonwoven fabric, varying in density (number of perforations per approximately 100 microns of device). It should be noted that the NWF layer generally has varying pores or gaps due to its nonwoven structure. Therefore, there may be pores or gaps less than 100 microns, and there may also be pores or gaps greater than 100 microns. The NWF layer may have a basis weight of approximately 0.4 to 0.75 oz / yd², a nominal thickness of approximately 127 to 228 μm, and a fiber diameter of 26 μm. Perforations in the device can be created in a variety of ways, including by first stacking all layers on top of each other and then perforating each layer on either side or on the walls of the device. Alternatively, the cell-exclusion layer may be perforated and then combined with the other non-perforated layers of the device, including the NWF layer. Laser perforation allows considerable control over the hole size (diameter) and number (density) of holes. In Groups 1-3 in Table 2 below, the cell-exclusion membranes were laser perforated to create holes approximately 50-120 μm in diameter with an average diameter of approximately 87 μm, spaced approximately 1 mm, 1.5 mm, or 2 mm apart. A small-gauge hypodermic needle was used to manually create holes approximately 2 mm apart in each layer of the control groups (Groups 4 and 5).

[0256] [Table 6]

[0257] All animals (athymic nude rats) were implanted with two subcutaneous implants of test or control EN20 devices, each containing approximately 20 μL of fixed pancreatic progenitor cell aggregates.

[0258] All device configurations (laser-generated holes spaced approximately 1 mm, 1.5 mm, or 2 mm apart and needle-generated holes spaced approximately 2 mm apart) allowed pancreatic progenitors to survive, proliferate, develop, and mature into functional pancreatic endocrine cells in athymic nude rats, as indicated by human C-peptide levels shown in Figure 5. GSIS assays (Kroon et al. (2008) supra; Agulnick et al. (2015) supra) were performed 34 weeks after implantation in all perforated devices, independent of hole density, and in control devices at 15 and 30 minutes after glucose challenge. See Figure 6.

[0259] Those skilled in the art would assume that increased cell-to-cell contact between transplanted cells and host cells, particularly in the host vasculature, would increase cell viability, proliferation, and maturation, and that the more pores in the device, the better. Therefore, it was surprising to discover that cells in devices with low pore density performed as well as devices with higher pore density. For example, devices with pores spaced 2 mm apart (larger pore spacing, meaning fewer pores and lower density) performed as well as devices with pores spaced less than 2 mm apart (closer pore spacing, meaning more pores and higher density). Thus, Applicant discovered that even low pore density or a small number of perforations (approximately 20 perforations per wall of the small EN20, with less than about 0.4% of the device's surface area perforated) provided desirable benefits to host vascularization and cell viability. This was surprising, since those skilled in the art would expect that more pores (higher density) would increase and / or speed host vascularization, aiding in the delivery of oxygen and other nutrients to transplanted cells, thereby increasing cell viability and differentiation.

[0260] A lower density device may be preferred because fewer pores will reduce or prevent cells from escaping from the device, thereby improving safety. Additionally, it is desirable to retain cells within the device from which the entire graft can be retrieved, should the entire graft be removed.

[0261] Example 4: Perforated Devices with Improved Dosage Profiles Compared to Non-Perforated Devices Rats implanted with the perforated delivery devices described above in Example 3, Table 2, showed increased C-peptide content over time compared to intact mouse devices (see, e.g., the dotted line in Figure 7 for intact devices). Figure 7 shows the C-peptide produced by various perforation configurations, demonstrating that perforated devices with an NWF layer increased C-peptide (indicating insulin content) up to approximately 30, 35, or 40 weeks. At approximately 15 weeks, cells in perforated devices produced approximately 50% more C-peptide than cells implanted in non-perforated devices. At 16 weeks, the average C-peptide produced by cells in perforated devices was 2,696 pmol (Figure 7, SP-2016-149), which, based on the linear relationship of C-peptide levels to IEQ as described in Figure 3, is approximately 6,300 IEQ per device, i.e., a cell mass of approximately 6,300 IEQ. By approximately 39 weeks, cells in the perforated devices had an average C-peptide level of 9,244 pmol (Figure 7, SP-2015-128), again based on Figure 3, a dose of approximately 23,100 IEQ per device.

[0262] Rats implanted with cell-retaining perforated devices produced more C-peptide than those with intact cells, and it took a longer time (approximately 35 weeks) for C-peptide to plateau in the perforated devices (Figure 7, SP-2015-128). That is, perforated and nonperforated (intact) cell devices had similar levels of human C-peptide until approximately 16 weeks, after which C-peptide plateaued in the intact devices and continued to increase in the perforated devices.

[0263] The higher C-peptide concentrations achieved with the perforated device are not reached until approximately 16 weeks. See Figure 7, SP-2016-149 to SP-2015-128. In contrast, C-peptide levels in mice implanted with intact devices plateau below 2,000 pmol at approximately 16 weeks. See Figure 7 (dotted horizontal line).

[0264] The cell mass or IEQ of perforated devices implanted in rats for 36 or 39 weeks was compared to intact devices implanted in mice for the same period. Compared to intact mouse devices, there was an approximately five-fold increase in cell mass in the rat perforated devices. See Figure 8. Figure 8 shows that intact mouse devices had an IEQ of less than approximately 5,000, while perforated rat devices had an IEQ of approximately 23,100 picomoles, a five-fold difference. This may be because the cells in the perforated devices were able to proliferate and thus retain more insulin-producing cells and / or the cells were producing more insulin per cell. See Figure 9, which shows photomicrographs of histological cross-sections of cells from intact (top) and perforated (bottom) delivery devices of the same size and with the same initial amount of cells added to each device. After maturation, the cells in the perforated devices are more proliferative than the cells in the intact devices, i.e., they have a higher cell mass, or preferably a higher beta cell mass. The C-peptide data described above and shown in Figure 7 indicate that mature cells in perforated apparatus produce more C-peptide than cells in intact apparatus. As shown in Figure 7, higher C-peptide levels indicate a higher number of islets or IEQ, and higher IEQ indicates a higher cell mass.

[0265] Because cells in perforated devices have 5-6 times greater IEQ values ​​than cells in intact or non-perforated devices, therapeutic cell doses can be achieved with fewer or smaller perforated devices than cells in intact devices. Thus, in one embodiment, cells in perforated devices improve delivery capacity for the same size volume as intact or non-perforated devices.

[0266] Example 5: Pancreatic endoderm can mature in nude rats treated with calcineurin inhibitors For pancreatic islet replacement therapy, transplantation of therapeutic cells into perforated devices requires chronic immunosuppressive therapy. Maintenance immunosuppression of cadaveric islet transplants involves the use of calcineurin inhibitors such as tacrolimus (TAC) and, less frequently, cyclosporine (CsA), antiproliferative agents, and mycophenolate mofetil (MMF). Calcineurin inhibitors (1) are diabetogenic (causing diabetes; Crutchlow et al. (2007) supra) and (2) negatively affect endogenous pancreatic regeneration in mice (Heit (2006) supra and Nir (2007) supra). Thus, adverse events resulting from the administration of immunosuppressive drugs can be expected for maturing pancreatic endoderm, as proliferation is a key component of in vivo encapsulated pancreatic endoderm maturation, and for mature beta cells arising from pancreatic endoderm cell grafts.

[0267] To test the effects of ISD, five nude rats were fed a normal diet and five nude rats were fed a diet formulated with 250 mg / kg of cyclosporine A for 18 weeks. This method has the advantage of avoiding the stress of daily administration by intraperitoneal injection and / or gavage. The 12-hour area under the blood concentration curve (AUC 0-12hr The resulting drug exposure level, shown as AUC ), was estimated to be approximately 16 μg hr / mL. This exposure is within the clinical target AUC of cyclosporine A in kidney and liver transplant recipients. 0-12hr This is higher than the 6-9 μg·hr / mL.

[0268] In response to calcineurin inhibitors, rats in the treatment group developed diabetes. In particular, rats receiving 250 mg / kg of cyclosporine A in the diet became hyperglycemic after approximately 10 weeks, i.e., they did not produce endogenous C-peptide. Serum C-peptide levels in the rats 30 and 60 minutes after glucose challenge were below 500 pM (Figure 10A). See

[1999] . This effect was temporarily managed by administration of exogenous insulin (Linbit pellet and Lantus). Otherwise, the cyclosporine A diet was well tolerated by rats; indeed, body weight and food consumption rates were more normal than in rats fed the control diet.

[0269] Perforated devices containing pancreatic progenitors (or PECs) were implanted into rats in the small-sized EN20 device as described above. The devices did not contain a NWF layer. At 18 weeks after implantation, serum human C-peptide levels in cyclosporine A-treated rats were not substantially different from those in the untreated control group (Figure 10B). CsA-treated rats exhibited robust insulin levels, as observed with serum human C-peptide levels ranging from 1069 to 4098 pM at 30 minutes and 1995 to 4144 pM at 60 minutes. Importantly, the rats were not hyperglycemic or required exogenous insulin. For example, at 15 weeks, mean blood glucose was 283 mg / dL (hyperglycemic) and 128 mg / dL (euglycemic) at 20 weeks. This indicated that the perforated device implants regulated normal blood glucose levels. The data demonstrate that PECs from perforated devices continued to differentiate and function in rats receiving CsA, continuing for at least 36 weeks after transplantation (data not shown), demonstrating the lack of graft sensitivity to the aforementioned therapeutic levels of PEC and calcineurin inhibition by cyclosporine A.

[0270] The foregoing demonstrates that even in diabetic (hyperglycemic) rats treated with calcineurin inhibition, pancreatic endoderm transplanted into perforated devices is able to mature, generate insulin-producing cells, and reverse diabetic pathology.

[0271] Example 6: Pancreatic endoderm can mature in nude rats treated with calcineurin inhibitors and antimetabolite immunosuppressants The ability of pancreatic endoderm encapsulated in perforated devices to mature into insulin-producing cells upon exposure to a calcineurin inhibitor (antimetabolite) immunosuppressant was evaluated. Table 12 outlines the study protocol. Nude rats were fed a normal chow (control), a chow containing 250 mg / kg cyclosporine A (CsA-250), a chow containing 250 mg / kg cyclosporine A and 500 mg / kg mycophenolate mofetil (CsA-250 + MMF500), or a chow containing 150 mg / kg tacrolimus and 500 mg / kg mycophenolate mofetil (TAC-150 + MMF500). After two weeks of acclimation to the chow, rats were implanted with PECs delivered via the perforated devices described in Examples 1-4.

[0272] Diet reformulation with the desired ISD content was performed by Bio-Serv (Flemington, NJ). Grain-based PicoLab 5053 diet, ½" pellets, was the basis of the diet and was identical to the control diet and was fed ad libitum to the rats.

[0273] [Table 7]

[0274] The estimated ISD dose is based on a typical rat food consumption rate of approximately 70 g / kg body weight. The actual food consumption rate and ISD dose were measured.

[0275] [Table 8]

[0276] * Approximately 7 x 10 delivered with a device manually perforated with a needle 6 All animals were administered subcutaneously with a pancreatic endoderm cell graft (one per mouse, two per nude rat). Note that the delivery device does not include a NWF layer.

[0277] At 9 weeks post-transplant, rats treated with ISD (either CsA-MMF or TAC-MMF) had higher human C-peptide levels than controls (no ISD). See Figure 11. The transiently elevated human C-peptide levels are due, in part, to the fact that these animals became diabetic as a result of the ISD treatment and had higher blood glucose levels than control animals due to a lack of endogenous insulin secretion. Thus, rats treated with ISD are expected to have lower rat C-peptide levels than controls.

[0278] Example 7: Polyester nonwoven layer of perforated cell delivery device improves pancreatic progenitor development and function Examples 1-4 demonstrate that the incorporation of NWFs increases the structure and function of encapsulated cells and delivery devices, independently of whether the NWF is laminated to a cell-excluding membrane layer and independently of the pore density (number of pores) of the device. Example 5 demonstrates that pancreatic progenitors derived from human pluripotent stem cells can, in fact, survive, develop, and mature into functional pancreatic endocrine cells when the host is treated with an immunosuppressive regimen of calcineurin inhibitors (e.g., CsA), a finding that has not been previously described and was unknown until the applicant's disclosure. Example 6 further demonstrates that pancreatic progenitors can survive not only in immunosuppressive regimens of calcineurin inhibitors but also in immunosuppressive regimens of antimetabolite immunosuppressants.

[0279] In this study, the teachings of Examples 1-6 were combined to characterize the function of pancreatic progenitors in perforated and non-perforated delivery devices incorporating at least one NWF layer (per device wall or side) treated with a combination calcineurin inhibitor and antimetabolite immunosuppressive regimen.

[0280] Overall, control rats implanted with perforated NWF delivery devices had higher human C-peptide levels than rats implanted with cells from non-perforated NWF encapsulated devices. See Figure 12 (comparing control, intact delivery device, non-perforated device, and no CsA-MMF treatment). This is due to direct vascularization of host vessels with the implanted cells due to the perforations (holes) in the perforated device, improving cell survival. Furthermore, in rats treated with ISD, rats implanted with perforated NWF delivery devices had higher human C-peptide levels than rats implanted with non-perforated NWF devices. The levels were higher in the intact and non-perforated NWF delivery devices compared with the control group. See Figure 12 (comparing intact and non-perforated devices, without treatment with CsA-MMF). Interestingly, human C-peptide levels in animals receiving CsA-MMF and implanted with a perforated NWF delivery device were approximately 2-fold (1.6-fold) higher than in animals implanted with the same perforated NWF delivery device but not receiving the CsA-MMF immunosuppressant. See Figure 12. This increase in human C-peptide may be the result of the synergistic effect of the combination of the NWF delivery device and the CsA-MMF immunosuppressive regimen. This improved human C-peptide level may be the result of improved host vascularization, which is mitigated by the NWF alone and / or in combination with an ISD.

Claims

1. a lumen configured to receive cells; a cell-exclusion membrane forming the lumen; and a nonwoven layer in contact with the exterior surface of the cell-exclusion membrane; the nonwoven fabric layer and the cell exclusion membrane have perforations; each of said perforations having a diameter of 40 to 150 microns; the density of the perforations is such that less than 5.0% of the surface area of ​​the device is perforated; Cell encapsulation device.

2. a woven mesh on the exterior of the nonwoven layer; and a membrane ring disposed between the woven mesh and the nonwoven layer; The cell encapsulation device of claim 1 further comprising:

3. 10. The cell encapsulation device of claim 1, wherein the nonwoven fabric layer and the cell exclusion membrane are welded together.

4. 2. The cell encapsulation device of claim 1, wherein the perforations comprise 5 to 200 perforations.

5. 2. The cell encapsulation device of claim 1, wherein the perforations comprise 20 to 100 perforations.

6. 2. The cell encapsulation device of claim 1, wherein the perforations are spaced apart from one another by 0.5 to 2.0 mm measured from the center of a perforation to the center of its adjacent perforation.

7. 7. The cell encapsulation device of claim 6, wherein the perforations are spaced 1.0 mm apart from one another, measured from the center of a perforation to the center of its adjacent perforation.

8. The cell encapsulation device of any one of claims 1 to 7, further comprising a port leading to said lumen for loading said lumen with cells.

9. 9. The cell capsule of claim 8, wherein the cell-exclusion membrane comprises two layers bonded together to form the lumen therebetween, and the port is disposed between the two layers of the cell-exclusion membrane. Capsuleization device.

10. 10. The cell encapsulation device of claim 1, further comprising pancreatic endoderm cells, pancreatic progenitor cells, pancreatic endocrine cells, endocrine progenitor cells, immature beta cells, immature endocrine cells, mature endocrine cells, or pancreatic beta cells packed into the lumen of the cell exclusion membrane.

11. 11. The cell encapsulation device of any one of claims 1 to 10, wherein the cell exclusion membrane forms two lumens, and the cell encapsulation device further comprises two ports, each of which leads to one of the two lumens for loading cells into the two lumens.

12. 12. The cell encapsulation device of any one of claims 1 to 11, wherein the cell exclusion membrane and the nonwoven fabric layer are joined at their periphery to form the cell encapsulation device.

13. The cell encapsulation device according to any one of claims 1 to 12, wherein the nonwoven fabric layer comprises a nonwoven fabric, and the filament cross section of the nonwoven fabric is trilobal.

14. The cell encapsulation device according to any one of claims 1 to 13, wherein the nonwoven fabric layer is made of polytetrafluoroethylene (PTFE).

15. A cell encapsulation device according to any one of claims 1 to 14 for a method of producing insulin in a mammal.

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