Immunoprotected implantable cell delivery device

Abstract

The present invention relates to an immunoprotective implantable cell delivery device for implanting cells in a subject, comprising a closed space between an upper and a lower porous membrane and a mesh. The mesh is used to prevent cell clustering. The present invention further relates to a method for constructing the closed implantable cell delivery device. The present invention also relates to said device for use in a method of treatment.

Description

[Technical field]

[0001] The present invention relates to the technical field of implantable cell delivery devices, in particular immunoprotective implantable cell delivery devices that allow for isolating implanted cells from host immune cells. The present invention particularly relates to an immunoprotective device with a mesh for embedding cells. Such a device can be used for implanting cells, such as pancreatic islet cells, in a subject. The present invention further relates to a method for constructing the device and a method for loading the device with cells. Furthermore, the present invention relates to the use of a closed implantable cell delivery device containing cells in the treatment of a disease or disorder by implanting the device in a subject. Technical Background

[0002] Transplantation of donor cells into patients is a promising tool for treating a variety of diseases. For example, diabetes patients could be transplanted with pancreatic islet cells.

[0003] Type 1 diabetes (T1D) is an autoimmune disease in which the insulin-producing beta cells in the islets of Langerhans are destroyed by the immune system. It is estimated that 20 to 40 million people worldwide have T1D. Insulin administration must be tightly controlled to prevent hypoglycemia or hyperglycemia, which, if left untreated, can lead to life-threatening tissue and organ damage. Most T1D patients are prescribed daily exogenous insulin, but achieving tight control of blood glucose levels remains difficult due to poor dose adjustments and poor patient compliance. One way to achieve normoglycemic blood glucose levels without insulin is through clinical islet transplantation (CIT). CIT is a minimally invasive treatment with few complications and has the benefit of allowing patients to become insulin independent for approximately 5 years. Islets are isolated from a donor pancreas and transplanted into the hepatic portal vein of the recipient. However, due to the shortage of donors and the need for long-life immunosuppressants to prevent any alloimmune or autoimmune relapse, CIT is only available for patients with poorly controlled diabetes. The efficiency of CIT is limited because 50-70% of transplanted islets are lost early after transplantation due to instant blood-mediated inflammatory reaction (IBMIR). IBMIR is triggered by direct exposure of islets to blood, resulting in rapid activation and binding of platelets to the islets. The islets then become encapsulated in a blood clot, which ultimately leads to the loss of transplanted cells. Cell delivery can be improved by using extrahepatic islet delivery devices such as closed (immunoprotected) devices. Because the immune system cannot access the cells inside the device, IBMIR is avoided and the recipient does not need immunosuppressive drugs.

[0004] A disadvantage of existing closed (immunoprotective) devices is that cells or cell clusters tend to aggregate inside the device. For example, US Patent Application Publication No. 2021 / 022846 describes a closed device for implanting cells, but does not describe any means to prevent the cells from forming clumps. Cell aggregation tends to cause necrosis of cells in the center of the cell cluster due to nutrient and oxygen depletion. A method to prevent aggregation is to embed the cells (or cell clusters) in a hydrogel inside the immunoprotective device. However, a disadvantage of embedding in a hydrogel is that although it prevents cluster formation and the resulting necrosis, it still impedes the diffusion of nutrients, gases and proteins. This reduces the diffusion capacity of such immunoprotective devices too much, resulting in a device that cannot encapsulate enough cells for the intended treatment (e.g., islet-like cell transplantation in type 1 diabetes). In addition, the hydrogel impedes the diffusion of secreted factors (e.g., insulin in the case of islet cells) out of the implanted device. For these reasons, among others, there is a need for improved immunoprotection devices.

[0005] Furthermore, WO 2018 / 114098 discloses an open device in which a mesh may be used to prevent cells from migrating or clumping. US 6008049 discloses a cell culture device in which a mesh is used. US 2020 / 360566 describes a closed device for implantation in hypoxic sites, which uses beads to which the cells adhere to prevent the formation of clumps. Although these documents describe methods to prevent cells from clumping, such as by using a mesh, none of these documents describes a suitable method for implementing them in a closed device that allows the diffusion of oxygen and nutrients and allows for uniform seeding of cells in the device. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] US Patent Application Publication No. 2021 / 022846 [Patent Document 2] International Publication No. 2018 / 114098 [Patent Document 3] U.S. Patent No. 6,008,049 [Patent Document 4] US Patent Application Publication No. 2020 / 360566

[0007] The present invention addresses, among other things, the above problems through the immunoprotection devices and methods set forth in the claims. Summary of the Invention

[0008] In a first aspect, the present invention relates to an immune-protected implantable device for implanting cells or cell aggregates in a subject, the device comprising a lower membrane and an upper membrane, the lower membrane and the upper membrane being joined to a support structure such that the lower membrane, the upper membrane and the support structure when implanted form an enclosed interior space that is not accessible by the immune system, the lower membrane and the upper membrane comprising a plurality of pores that allow diffusion of nutrients and oxygen through the membranes to the interior space, the device further comprising a mesh disposed in an interior space within the device to provide support for the cells or cell aggregates when the cells or cell aggregates are inserted into the interior space of the device such that the cells do not form clumps, the mesh having a pore size of 60-300 μm.

[0009] In a second aspect, the present invention relates to an implantable device according to any one of the first aspects of the invention, comprising cells or cell aggregates, for use as a medicament.

[0010] In a third aspect, the present invention relates to a method of constructing an implantable device according to the first aspect of the invention, comprising providing a lower membrane, positioning a support structure generally along an edge of the lower membrane leaving a central space, positioning a mesh on top of the lower membrane in the central space, positioning an upper membrane on top of the support structure covering the central space with the mesh to completely seal it creating an internal space, and fixing the upper and lower membranes to the support structure, preferably by ultrasonic welding, heat sealing, pressure sealing, adhesive, more preferably by ultrasonic welding.

[0011] In a fourth aspect, the present invention relates to a method for loading cells or cell aggregates into an implantable device, the method comprising providing a device according to the first aspect of the invention or a device obtained or obtainable by the second method of the invention, providing a suspension of cells or cell aggregates in a container, connecting a tube, one end in open contace with the interior of the container containing the suspension of cells or cell aggregates and the other end for insertion through an opening into the interior space of the implantable device, and allowing the cells or cell aggregates in the container to flow out through the tube into the interior space of the implantable device, and optionally closing the opening in the implantable device after removing the tube, preferably by ultrasonic welding, heat sealing, pressure sealing, adhesive, more preferably by heat sealing.

[0012] definition With respect to the present invention, the following terms are defined below. As used herein, the singular terms "A," "an," and "the" include plural referents unless expressly indicated otherwise. Thus, for example, reference to "a cell" includes a combination of two or more cells, and the like. As used herein, the term "cell" or "cells" specifically includes a "cell aggregate" or "cell aggregates."

[0013] As used herein, the term "and / or" refers to a situation in which one or more of the stated cases may occur alone or in combination with at least one of the stated cases up to and including all of the stated cases.

[0014] As used herein, the term "at least" a particular value means greater than or equal to the particular value. For example, "at least 2" is understood to be the same as "2 or greater", i.e., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, ..., etc. As used herein, the term "at most" a particular value means less than or equal to the particular value. For example, "at most 5" is understood to be the same as "less than or equal to 5", i.e., 5, 4, 3, ..., -10, -11, etc.

[0015] As used herein, the word "comprise" or variations thereof, such as "comprises" or "comprising," will be understood to include a stated element, integer or step, or group of elements, integers or steps, but not to exclude any other elements, integers or steps, or group of elements, integers or steps. The verb "comprising" encompasses the verbs "essentially consisting of" and "consisting of."

[0016] The term "conventional technique" used herein refers to a situation where the method of carrying out the conventional technique used in the method of the present invention would be obvious to a person skilled in the art. The practice of conventional techniques in molecular biology, biochemistry, computational chemistry, cell culture, tissue engineering, regenerative medicine, recombinant DNA, bioinformatics, genomics, sequencing and related fields is well known to a person skilled in the art and is discussed, for example, in the following references: Sambrook et al., "Molecular Cloning.A Laboratory Manual", 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989; Ausubel et al., "Current Protocols in Molecular Biology", John Wiley&Sons, New York, 1987 and periodic revisions, and "Methods in Enzymology" series, Academic Press, San Diego; Mozafari et al., "Handbook of Tissue Engineering Scaffolds", Vol. 2, 2019 Elsevier; Atala et al., "Principles of Regenerative Medicine", 3rd Edition, 2019.

[0017] As used herein, the term "in vitro" refers to experiments or measurements performed with components of living organisms isolated from their natural conditions.

[0018] As used herein, the terms "subject" or "individual" or "animal" or "patient" or "mammal" are used interchangeably and refer to any subject for which diagnosis, prognosis or treatment is desired, particularly a mammalian subject. Mammalian subjects include humans, livestock, farm animals, and zoo, sport or pet animals, such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, dairy cows, bears, etc. Subjects as defined herein may be living or dead. Samples may be taken from subjects post-mortem, i.e. after death, and / or samples may be taken from living subjects.

[0019] As used herein, the terms "treatment", "treat", "alleviate", "alleviate" or "ameliorate" are used interchangeably and refer to an approach to obtain a beneficial or desired result, such as, but not limited to, therapeutic benefit. Therapeutic benefit refers to eradication or amelioration of the underlying disease being treated, or reduction (or delay) in its progression. Therapeutic benefit is also achieved by eradication or amelioration of one or more of the physiological symptoms associated with the underlying disease, or reduction (or delay) in its progression, in such a way that improvement or slowing down or reduction in decline is observed in the patient, even though the patient may still be affected by the underlying disease.

[0020] As used herein, the term "implantable cell delivery device" is used interchangeably with "implantation device", "cell delivery device", "implantable device", "macroencapsulation implant", "cell carrier" or simply "device" and refers to an enclosure suitable for holding cells and intended for implantation into a subject. The device thus serves as a vehicle for implanting cells into a subject. It can therefore be envisioned that the device is made of a material suitable for implantation into a subject, and that the device is constructed in a manner suitable for containing living cells.

[0021] The term "closed" as used herein, when referring to an implantable cell delivery device, implies that the device does not have openings large enough to allow immune cells or host vascularization to enter the device after sealing the cells inside. Thus, the term closed does not necessarily exclude the presence of pores or openings that are present to allow material transport of essential molecules such as nutrients, gases, small proteins or peptides, metabolites, cytokines, hormones, and other biomolecules. As used herein, the term pores refers to openings that have a diameter that allows the diffusion (or material transport) of nutrients, gases, small proteins or peptides, metabolites, cytokines, hormones, and other biomolecules, but whose diameter is too small to allow vascularization inside the device and to allow immune cells to enter the device.

[0022] As used herein, "pore size," "pore dimension," or "mesh size," when referring to a mesh, should be interpreted as referring to the size of the openings in the mesh, which is the average diameter of the openings if the openings are substantially circular, and the average size of a side if the openings are substantially square or rectangular.

[0023] Detailed Description of the Invention The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0024] Portions of this invention include material that is subject to copyright protection, such as, but not limited to, diagrams, device photographs, or any other aspect of this disclosure for which copyright protection is or may be available in any jurisdiction. The copyright owner has no objection to the reproduction by anyone of the patent document or the patented invention as it appears in the Patent and Trademark Office patent files or records, but otherwise reserves all copyrights.

[0025] Throughout this specification and claims, various terms are used in connection with the methods, compositions, uses and other aspects of the present invention. Such terms shall be given their ordinary meaning in the art to which the invention pertains, unless otherwise indicated. Other specifically defined terms shall be interpreted in a manner consistent with the definitions provided herein. Although preferred materials and methods are described herein, any methods and materials similar or equivalent to those described herein may be used in the practice of testing the present invention.

[0026] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0027] The present invention relates to an immunoprotected implantable cell delivery device, or closed device, intended for implanting cells into a subject, e.g., as a means of treating a disease. Exemplary cells that could be used in implantation therapy are islet cells in the treatment of diabetes, although other cells that could be used in implantation therapy methods are known to those skilled in the art.

[0028] Encapsulating cells, such as islet cells, prior to transplantation may overcome the challenges faced by current clinical cell transplantation therapies (e.g., clinical islet transplantation). However, it is understood that the device can be used for any type of procedure that would benefit from transplantation of cells or cell aggregates, including novel therapies based on, for example, induced pluripotent stem cells or transdifferentiation-induced cell sources. Here, we describe the development of an immunoprotective islet encapsulation device made of a porous membrane that protects cells from immune cells while allowing the diffusion of nutrients to keep the cells viable and functional. There are three major challenges in the development of current cell encapsulation devices. First, by selecting a biocompatible material to avoid or reduce the foreign body response, we screened potential biomaterials and, based on those results, selected polyvinylidene fluoride (PVDF) as a typical candidate biocompatible material. Second, by enabling the transport of nutrients, gases, small proteins or peptides, metabolites, cytokines, hormones and other biomolecules to the encapsulated cells, by the production of thin and porous membranes and finally protect the encapsulated cells against the immune system by using immunoprotective membranes with pores that do not allow passage of immune cells.

[0029] Third, a common challenge is that cells sequestered in implanted devices tend to aggregate and form large aggregates within which necrosis occurs due to lack of oxygen and nutrients. This challenge has been addressed, for example, by using hydrogels in the lumen of the device to encapsulate the cells or cell clusters. Although this solution prevents the cells from aggregating into large aggregates to some extent, it was found that the presence of the hydrogel still impedes the diffusion of oxygen and nutrients, as well as preventing the efficient diffusion of factors produced by the cells (e.g. insulin) out of the device.

[0030] As used herein, the terms membrane and film or thin film are used interchangeably and refer to a flat, sheet-like material.

[0031] Thus, in a first aspect, the present invention relates to an immunoprotected implantable device for implanting cells or cell aggregates in a subject, the device comprising a lower membrane and an upper membrane, the lower membrane and the upper membrane being joined to a support structure such that the lower membrane, the upper membrane and the support structure form an enclosed internal space that is not accessible by the immune system when implanted, the lower membrane and the upper membrane comprising a plurality of pores that allow diffusion of nutrients and oxygen through the membranes to the internal space, the device further comprising a mesh disposed in the internal space within the device to provide support for the cells when contained in the internal space of the device so that the cells do not form clumps, the mesh having a pore size of 60-300 μm. Thus, the incorporation of the mesh prevents or reduces the formation of large cell clumps, resulting in the cells or cell aggregates surviving longer and functioning better. As used herein, the term clumping or clump forming refers to (or the formation of) large cellular aggregates where the large size of the cellular aggregates can result in negative effects, such as, but not limited to, necrosis, impaired growth or impaired function.

[0032] It has been found that the use of meshes can effectively address the challenge of cell aggregate formation. Although meshes have been described in open cell delivery devices (e.g., US Patent Application Publication No. 2017 / 182139), to the inventors' knowledge, there is no similar solution in closed (immunoprotective) devices. The reason is that it is extremely difficult to properly contain cells or cell aggregates in such devices. Such devices are preferably assembled and provided without cells, and in use, cells are seeded with cells just before implanting the device in a subject. However, it is difficult to seed cells in closed devices through narrow openings to allow for uniform distribution of cells (or cell clusters) throughout the mesh in the device, and cells will generally end up in large aggregates in the device.

[0033] Theoretically, the device can undergo final assembly prior to implantation: cells are seeded onto a mesh, which is then sealed between the outer layers (membranes) of the device, allowing a uniform distribution of cells on the mesh. In practice, however, this solution does not work: assembling and fixing the components together is too disruptive for the cells, causing them to die or become displaced within the device. The inventors have now developed a new method of seeding cells into the device, as described below. This allows for uniform seeding of cells (or cell aggregates) into an already assembled device, thus enabling a practical solution for closed devices with meshes.

[0034] As a concern in any transplantation site or implantation, proper nutrient diffusion is essential to ensure optimal survival and function of the transplanted graft. For example, pancreatic islets exhibit high metabolic activity and therefore require rapid access to oxygen and nutrients to survive. Therefore, it is crucial that the delivery device is as thin and porous as possible to reduce the diffusion path length and minimize material transport time to and from the cells inside the implant. Therefore, the present invention provides, among other things, a method for fabricating an immunoprotective cell delivery device to achieve cell delivery and allows for implant design with clinically relevant device dimensions.

[0035] The implantable cell delivery device of the present invention is intended to implant cells into a subject. The examples shown in this patent illustrate that the delivery device is not limited to beta cells and islets, but can be used for cell delivery in general, such as human mesenchymal stem cells, alpha cells, or any other relevant cell type. It is understood that the implantable cell delivery device can incorporate other cell types, mixtures of cell types, organoids, or (parts of) tissues or organs, according to the examples. Since the device is intended for implantation into a subject, there are certain limitations, such as the materials used must be biocompatible, and the dimensions of the device. It is understood that the dimensions may depend on the subject in which the device is intended to be implanted. As used herein, the term subject may refer to animals, such as rodents or mammals, or humans. Thus, the size limitations for an implantable cell delivery device are different for, for example, mice compared to humans, but even within the same species, differences such as body size may dictate the size of the implantable cell delivery device. The size of the device will also depend on the cell type to be incorporated into the implantable cell delivery device and the function or purpose of implanting the cells. One skilled in the art can estimate the approximate desired size of the implant based, inter alia, on the subject and the cell type to be implanted.

[0036] The implantable cell delivery device of the present invention comprises a lower membrane and an upper membrane, the lower membrane and the upper membrane being joined to a support structure such that the lower membrane, the upper membrane and the support structure form a sealed internal space that is not accessible by the immune system when implanted, and the lower membrane and the upper membrane are provided with a plurality of pores that allow the diffusion of nutrients and oxygen through the membranes to the internal space. The device further comprises a mesh disposed in the internal space of the device. The mesh is intended as a support and spacer structure. The inventors have found that the use of the mesh effectively prevents the aggregation of cells or cell clusters, both in cell culture and when used within the device (see, for example, FIG. 12). The aggregation of cells or cell clusters is undesirable and is a problem with currently known cell implantation devices. The aggregation of cells or cell clusters leads to oversized cell aggregates, in which the cells inside are deprived of nutrients and oxygen, resulting in, among other things, necrosis and loss of function, potency and efficacy. Therefore, additional measures are required to keep the cells or cell clusters apart from each other. This patent describes a method to prevent cells from clumping inside the cell delivery device by using a mesh. This advantageously allows easy diffusion of nutrients while keeping the cells and cell aggregates apart from each other. Both the upper and lower membranes have numerous pores that allow diffusion of nutrients and oxygen to the cells, and optionally diffusion of secreted factors from the cells out of the device. A non-limiting example of a secreted factor is insulin.

[0037] As used herein, a membrane refers to a thin, flat material. As used herein, a surface area of ​​a membrane refers to the upper and lower surfaces, where the upper and lower surfaces are the surfaces facing each other with the smallest thickness between them. As used herein, a pore refers to an opening or cavity in the membrane that passes completely through the membrane and allows the passage of molecules, etc., from one surface of the membrane to the other, preferably from the upper surface to the lower surface.

[0038] The implantable cell delivery device of the present invention further comprises a support structure disposed substantially around the surface area of ​​the lower membrane and the surface area of ​​the upper membrane, the support structure being adapted to be disposed in the plane of the surface area of ​​the upper membrane and the lower membrane. The support structure may be, for example, elliptical or circular, but it is understood that the support structure may have any kind of shape. Ideally, the shape of the support structure follows the contour of the upper and lower membranes. For example, if the lower and upper membranes have an elliptical shape, the support structure is preferably an elliptical ring that follows the edge of the lower and upper membranes. It is understood that the support structure may have a break or gap, for example, the support structure may be U-shaped.

[0039] It is understood that the device may include additional support structures. For example, an immunoprotected implantable cell delivery device of the present invention may include one or more additional support structures, where the one or more additional support structures are preferably more centrally positioned with respect to the lower and upper membranes.

[0040] The function of the support structure is to provide some rigidity to the device. Although some flexibility is desirable for an implantable cell delivery device, structural integrity must be guaranteed. Because the lower and upper membranes must allow for the diffusion of nutrients and oxygen, there is a limit to the thickness of the membrane, which is generally quite thin and therefore fragile. The support structure helps to avoid tearing or rupturing of the membrane. Furthermore, the incorporation of the support structure prevents the device from folding or bending, which would otherwise lead to an increase in internal space, resulting in an increase in diffusion paths and reduced cell function that is undesirable for the recipient of the device. Thus, the support structure generally has a thickness that exceeds that of the lower and upper membranes. For example, the thickness of the upper and lower membranes may be 10-250 μm thick, preferably 35-200 μm, more preferably 40-160 μm, respectively, while the support structure may be around 75-500 μm thick, preferably 100-400 μm or more, such as around 200 μm thick.

[0041] The support structure further provides a scaffold for attachment of the lower and upper membranes. The membranes may be attached to the support structure such that the support structure is sandwiched between the edges of the membranes, or the membranes may be attached together to one surface of the support structure, for example the top or bottom surface. The membranes may be attached, for example, by ultrasonic welding, heat sealing pressure sealing or adhesives, although ultrasonic welding is preferred.

[0042] The implantable cell delivery device of the present invention further comprises a lower membrane and an upper membrane attached at one or more points to the support structure so as to substantially, preferably completely, seal the internal space when implanted. Thus, when the device is ready to be implanted, it only allows the diffusion of molecules such as nutrients and oxygen through the pores, and thus the diffusion of nutrients can be controlled by controlling the pore size of the membrane. However, since cells need to be loaded into the device, the device is constructed to leave an opening between the upper and / or lower membrane and the support structure to allow cells to be inserted into the device as described below. Once the cells have been inserted into the device, the diffusion of nutrients can only occur through the pores, and thus the openings will be sealed so as to create a barrier for immune cells that allows the diffusion of nutrients. Thus, a pore as used herein should be interpreted as an opening in the device that does not allow the passage of immune cells into the device due to its size.

[0043] It has been found that the device is preferably constructed from PVDF, as it has improved porosity compared to other suitable materials while maintaining mechanical strength. An additional advantage is that PVDF is biocompatible, which significantly reduces the immune response, i.e., the cells in the device are less affected by the significantly reduced immune response compared to devices that are not biocompatible and therefore exhibit a significantly stronger immune response. It is understood that PVDF may be combined with suitable materials, such as, but not limited to, polyvinylpyrrolidone (PVP).

[0044] Thus, in certain embodiments, the lower and / or upper membranes, and optionally the support structure, are made of a water-insoluble polymer, preferably polyvinylidene fluoride (PVDF), polyethersulfone (PES), poly(ethylene oxide terephthalate) / poly(butylene terephthalate) (PEOT / PBT), or a water-insoluble polymer mixed with a water-soluble copolymer, and preferably the membrane further comprises a pore-forming agent, more preferably the pore-forming agent is polyvinylpyrrolidone (PVP).

[0045] A further advantage of using PVDF as a material for manufacturing the device is that it allows spot welding. Thus, in one embodiment, the upper and lower membranes are attached to the support structure by spot welding. Spot welding has the advantage of not requiring the use of additional materials such as glue, which may trigger an immune response, may not be biocompatible, may even be toxic, or may dissolve over time, resulting in structural failure of the device.

[0046] It is further envisaged that a marker for imaging is incorporated into or on the device. This may be advantageous as it allows the device to be located in a subject without the need for surgical procedures. Thus, in an embodiment, the device comprises one or more markers for imaging, preferably the one or more markers comprise a radiopaque agent that is infused into or coated on the PVDF of the upper membrane, the lower membrane and / or the support structure, more preferably the radiopaque agent is barium-based, such as barium sulfate, bismuth-based, such as bismuth trioxide, bismuth subcarbonate or bismuth oxychloride, or the radiopaque agent is tungsten or graphene oxide.

[0047] As used herein, radiopaque agents, also called radio contrast agents, are substances that are opaque to the radio and x-ray portions of the electromagnetic spectrum (i.e., such electromagnetic radiation is relatively unable to penetrate the particular material). Non-limiting examples of radio contrast agents include titanium, tungsten, barium sulfate, bismuth oxide, and zirconium oxide. Some solutions involve the direct attachment of heavy elements, such as iodine, to the polymer chains to obtain a more uniform material with less interface criticality.

[0048] It is further envisaged that it may be advantageous to include drugs or compounds in the device. Thus, in one embodiment, the device includes drugs or compounds injected into or coated on the PVDF membrane, such as the upper and lower membranes and / or support structures. For example, the device may be coated with drugs to avoid fibrous capsule formation or drugs to regulate foreign body response. Alternatively, therapeutic drugs may be included as co-treatment when the device is implanted as a treatment option in subjects. Non-limiting examples are chemotherapeutic agents for treating cancer, immune checkpoint inhibitors, cell stress inhibitors that help cell clusters and / or organoids survive early after surgery, or imaging markers for tracking implants after surgery. It is further envisaged to incorporate angiogenic factors to promote blood vessel growth within the device.

[0049] The pores in the upper and lower membranes allow for the diffusion of nutrients and oxygen to the cells in the device. Ideally, the average pore size is chosen to allow maximum diffusion while maintaining structural integrity and preventing the cells from exiting the device. In an embodiment, the pores are created by incorporating a soluble polymer into the film, which allows for the creation of pores in the film. Thus, in an embodiment, the (average) pore size of the lower and upper membranes is 10-500 nm, preferably 50-450 nm, more preferably 100-400 nm. The pore size may be altered, for example, by controlling the ratio of (water) soluble polymer to non-soluble polymer used during film manufacture. It is understood that other methods known to those skilled in the art may also be used. Furthermore, it is understood that the pore size is limited by the immune cells, since the pores must not allow the immune cells to enter the device. The average pore size in the upper and lower membranes may be the same or different.

[0050] The thickness of the membrane is assumed to be a trade-off between structural integrity and the ability to diffuse nutrients, with thicker membranes being stronger but allowing less diffusion, and vice versa. Thus, in one embodiment, the lower and upper membranes have a thickness of 10-200 nm, preferably 35-200 nm, more preferably 40-160 nm. These values ​​have been found to provide a good balance between strength and diffusion capacity.

[0051] It has been found that the mesh can be constructed from any non-degradable polymer, although biocompatible polymers are preferred. Thus, in certain embodiments, the mesh is constructed from a non-degradable polymer, preferably the non-degradable polymer is selected from nylon, PVDF, PTFE, PP, PE or PET. In certain embodiments, the mesh has a pore size of 50-300 μm, preferably 60-200 μm, more preferably 70-150 μm, and most preferably 75-120 μm to accommodate islet cells, although it is understood that for different cell types or organoids, meshes with different pore sizes are desirable. One skilled in the art can determine the appropriate pore size for each cell type or organoid.

[0052] In an embodiment, the device comprises cells in the interior space, preferably the cells are cell aggregates and / or organoids, preferably the cell aggregates are endocrine cells or cytokine producing cells or aggregates thereof, preferably the cell aggregates are selected from islet cells, human mesenchymal stem cells, kidney cells, thyroid cells, thymus cells, testicular cells, pancreatic cells, endocrine cells, liver cells, cytokine producing cells, or preferably the organoids are kidney organoids, intestinal organoids, pancreatic organoids, neural organoids, hepatic organoids, thyroid organoids, stomach organoids, ovarian organoids, prostate organoids, or the like. The organoids may be selected from organoids of the spleen, esophagus, breast, bladder, lung, eye, inner ear, heart, gallbladder, salivary gland, pituitary, lymphoid tissue, bladder, tongue, cerebral / brain, spinal cord, fallopian tube, lacrimal gland, skin, thymus, testicular, epithelial, gastruloid (embryonic organoid), blastoid (blastocyst-like organoid), retina, and hippocampus. Cell aggregates may also refer to a piece of tissue or organ that has been excised, such as from a donor organism. It is understood that the size of the device can be scaled depending on the intended application (e.g., treatment method or type of cells contained in the device) and based on the subject. It will be apparent to one skilled in the art that a device intended for implantation in a human subject will need to be larger than a device intended for implantation in a rodent. Since the device is essentially two-dimensional, i.e., there is a single plane with indentations, it is expected that for some applications the device will need to be scaled to an impractical size in a larger mammal such as a human. Therefore, it is further envisioned that multiple smaller versions of the device may be stacked on top of each other or implanted separately in a subject. Thus, in one embodiment, the present invention relates to an implantable device of the first aspect of the present invention, comprising two or more implantable devices stacked on top of each other and separated by a spacer such that an open space is created between each stacked implantable device. The additional support structure essentially serves as a spacer between the individual devices.

[0053] Two or more devices can in principle be stacked to obtain a more compact design, provided that proper diffusion of nutrients and oxygen to the cells is guaranteed. This becomes especially important for the middle layers (devices) when three or more layers are stacked. Therefore, the inventors have found that stacked layers (devices) should be separated by spacers. Preferably, the spacers are constructed such that the space between the layers is not completely blocked by the spacers, allowing easy diffusion of molecules between the devices. Thus, several small spacers may be used or the spacers may have openings. The spacers may be considered as additional support structures, and therefore the spacers referred to herein may be referred to as "additional support structures". Preferably, the spacers are also constructed from PVDF to ensure biocompatibility. It is further envisaged that the support structure and the spacer (additional support structure) are one continuous structure. The spacers may be attached to the support structure or membrane by ultrasonic welding, heat sealing, pressure sealing or by adhesive, more preferably by ultrasonic welding.

[0054] It is envisaged that the device may be used in a medical method or a method of treatment. Thus, in a second aspect, the present invention relates to an immunoprotected implantable cell delivery device of the present invention for use as a medicament. The present invention further relates to an immunoprotected implantable cell delivery device of the first aspect of the present invention for use in treating, preventing or ameliorating a disease. The device preferably comprises cells, more preferably cell aggregates or organoids. Thus, in one embodiment, the present invention relates to an immunoprotected implantable cell delivery device comprising cells, preferably cell aggregates and / or organoids, of the present invention for use in treating, preventing or ameliorating a disease. Alternatively, the present invention relates to a method of treating, preventing or ameliorating a disease or condition in a subject in need thereof, comprising implanting a device comprising cells, preferably cell aggregates or organoids, into the subject.

[0055] Several treatment options are envisioned for this device. For example, the device may be used to treat diabetes. Thus, in one embodiment, the treatment is a treatment for diabetes, preferably a treatment for type 1 diabetes. Preferably, when the treatment is a treatment for diabetes, the device comprises insulin secreting cells, such as islet cells or cells engineered to secrete insulin.

[0056] However, it will be clear to those skilled in the art that the use of the device is not limited to the treatment of diabetes. Because the device can accommodate the incorporation of any type of cell, cell aggregate or organoid. The device can be used to implant cells that excrete extracellular factors that may be beneficial in some treatments. Non-limiting examples of extracellular factors include peptides and proteins such as insulin, glucagon, cytokines, growth factors, hormones, carbohydrates, and coagulation factors.

[0057] The device may therefore be used to treat immune-related disorders, including, but not limited to, multiple myeloma, melanoma, rheumatoid arthritis, inflammatory bowel disease, lupus, scleroderma, hemolytic anemia, vasculitis, type 1 diabetes, Graves' disease, multiple sclerosis, Goodpasture's syndrome, pernicious anemia, myopathy, Lyme disease, severe combined immunodeficiency (SCID), DiGeorge syndrome, hyperimmunoglobulin E syndrome (also known as Job's syndrome), common variable immunodeficiency (CVID), chronic granulomatous disease (CGD), Wiskott-Aldrich syndrome (WAS), autoimmune lymphoproliferative syndrome (ALPS), hyper-IgM syndrome, leukocyte adhesion deficiency (LAD), NF-κB essential regulator (NF-κB essential regulator), and other conditions. The device may be used to treat NEMO (Neuromuscular Ovary Modifier) ​​mutations, selective immunoglobulin A deficiency, X-linked agammaglobulinemia (XLA; also called Bruton's agammaglobulinemia), X-linked lymphoproliferative disease (XLP), ataxia-telangiectasia, or acquired immune deficiency syndrome (AIDS). In addition, the device may be used to treat growth factor-related diseases, such as cancer. In addition, the device may be used in hormone or endocrine-related disorders, such as renal insufficiency, Addison's disease, Cushing's disease, Cushing's syndrome, gigantism (acromegaly), hyperthyroidism, Graves' disease, hypothyroidism, hypopituitarism, multiple endocrine neoplasia types I and II (MEN I and MEN II), polycystic ovarian syndrome (PCOS), or precocious puberty.Additionally, the device is designed to treat coagulation disorders such as factor V Leiden, prothrombin gene mutations, deficiencies of natural proteins that prevent clotting (e.g., antithrombin, protein C and protein S), elevated homocysteine ​​levels, elevated levels of fibrinogen or dysfunctional fibrinogen (dysfibrinogenemia), elevated levels of factor VIII and other factors including factors IX and XI, fibrinolytic system abnormalities such as hypoplasminogenemia, dysplasminogenemia and plasminogen activator inhibitors. The device may also be used to treat elevated levels of progesterone activator (PAI-1), cancer, obesity, pregnancy, use of supplemental estrogen, such as oral contraceptive pills (birth control pills), hormone replacement therapy, prolonged bed rest or immobility, heart attack, congestive heart failure, stroke and other diseases that lead to reduced activity, heparin-induced thrombocytopenia, antiphospholipid syndrome, history of deep vein thrombosis or pulmonary embolism, myeloproliferative disorders, such as polycythemia vera or essential thrombocytosis, paroxysmal nocturnal hemoglobinuria, inflammatory bowel syndrome, HIV / AIDS or nephrotic syndrome. Additionally, the device may be used in regenerative medicine indications, including but not limited to spinal cord injury, type 1 diabetes, Parkinson's disease, amyotrophic lateral sclerosis, Alzheimer's disease, heart disease, stroke, burns, cancer and osteoarthritis. In a third aspect, the present invention relates to a method of constructing an implantable device according to any one of claims 1 to 8, comprising providing a lower membrane, placing a support structure roughly along the edge of the lower membrane leaving a central space, placing a mesh on top of the lower membrane in the central space, placing an upper membrane on top of the support structure covering the central space with the mesh to completely seal it creating an internal space, and fixing the upper and lower membranes to the support structure, preferably by ultrasonic welding, heat sealing, pressure sealing, adhesive, more preferably by ultrasonic welding. In an embodiment, the upper and lower membranes are fixed to the support structure leaving a single opening for loading cells into the device.

[0058] As used herein, the term "spot welding" preferably refers to ultrasonic spot welding, an industrial process in which high frequency ultrasonic acoustic vibrations are locally applied under pressure to work pieces held together to create a solid weld. It is commonly used with plastics.

[0059] To the best of our knowledge, there are no other implantable cell delivery devices with meshed immune protection. This may be because there are several barriers to constructing such devices, such as the lack of straightforward methods to load the device with cells. One method would be to seed the bottom layer with cells prior to device assembly, but this has several impracticalities, since the device is preferably shipped in assembled form to the surgeon or researcher who will use it, or to the patient or subject who will receive it. Moreover, welding a polymer layer close to the cells would lead to a severe loss of cell viability. To overcome these problems, we have developed a method to seed the device that does not require assembly of the device after seeding, i.e., the device is fully assembled and then seeded. Thus, in a fourth aspect the present invention relates to a method for loading cells into an implantable device comprising providing a device according to the first aspect of the invention or a device obtained or obtainable by the method of the second aspect of the invention, providing a cell suspension in a container, connecting a tube such that one end is in open contact with the interior of the container containing the cell suspension and the other end is inserted through an opening into the interior space of the implantable device and allowing the cells in the container to flow out through the tube into the interior space of the implantable device and optionally closing the opening in the implantable device after removing the tube, preferably by ultrasonic welding, heat sealing, pressure sealing or adhesive, more preferably by heat sealing.

[0060] The assembled device is completely closed except for the seeding inlet. Cells are seeded through the seeding inlet by holding the device approximately horizontally. A tube containing a cell suspension is inserted into the single opening inside the device, between the top membrane and the mesh. In this way, cells, cell clusters or organoids are trapped inside the mesh structure.

[0061] Thus, in one embodiment, the cells move by gravity through the tube and into the interior space of the immunoprotected implantable cell delivery device. [Brief description of the drawings]

[0062] [Figure 1] Representative SEM images of surface morphology and cross-sections of fabricated PVDF and PVDF / PVP membranes. 15% (w / w) PVDF membranes cast at 200 μm (A-C) and 15% (w / w) PVDF + 5% (w / w) PVDF membranes cast at 200 μm (D-F). (A-F) Views of glass side, 5,000x and 25,000x magnification (A,D), air side, 5,000x and 25,000x magnification (B,E), and cross-sections (C,F). Scale bars in A,B,D, and E represent 4 μm. Scale bars in C and F represent 50 μm. (G) Thickness of fabricated membranes measured from SEM cross-sections. (H) Pore diameter of fabricated membranes measured at air-exposed site. Data are presented as mean ± SD (n=3). *P<0.05 by t-test. [Diagram 2]Physical properties of the fabricated PVDF pure membranes and PVDF / PVP membranes. (A) Water contact angles from both sides of the fabricated membranes. Data are presented as mean ± SD (n=9). *P<0.05 by Student's t-test. (B) Representative images of water contact angle measurements of the fabricated membranes. (C-F) Mechanical properties of the fabricated membranes, characterized by Young's modulus (C), peak stress (D), breaking strain I, and breaking stress (F). Data are presented as mean ± SD (n=5). *P<0.05 by Student's t-test. [Diagram 3] Indirect cytotoxicity test and chemical composition of the prepared PVDF and PVDF / PVP membranes. (A) Level of metabolic activity of INS-1E cells contacted with different exposure media during MTT assay. Data are presented as mean ± SD (N=3). *P<0.05, one-way ANOVA. (B) Measurement of LDH release of INS-1E cells contacted with different exposure media. Data are presented as mean ± SD (N=3). *P<0.05, one-way ANOVA. (C) FTIR spectrum of the prepared membranes. The green line at about 1660 cm-1 indicates the C=O peak present in the PVDF / PVP membrane. The red and black lines at about 1400 and about 1180 cm-1, respectively, correspond to CH2 and CF2 present in the PVDF basic structure. [Figure 4]Diffusion characteristics of various molecules through fabricated PVDF and PVDF / PVP membranes in two-chamber diffusion apparatus (A-J) or assembled devices (K-L). (A-C) Diffusion of 2 mg / mL 3-5 kDa fluorescently labeled dextran beads over 200 min (A), their calculated flux (B) and diffusion coefficient (C). Data are presented as mean ± STD (n=5). *P<0.05 by Student's t-test. (D-F) Diffusion of 20 mM D-glucose over 200 min (D), their calculated flI (E) and diffusion coefficient (F). Data are presented as mean ± STD (n=5). *P<0.05 by Student's t-test. (G-I) Diffusion of FITC-labeled insulin as a percentage after a total of 24 h, their calculated flux (H) and diffusion coefficient (I). Data are presented as mean ± STD (n=5). *P<0.05 by Student's t test. (J) Diffusion of IgG as a percentage after a total of 3 hours. Data are presented as mean ± STD (n=4). (K-L) Diffusion of oxygen from 21% oxygen to 5% oxygen (K) and area under the curve (AUC) of oxygen diffusion (L). Data are presented as mean ± STD (N=3). [Diagram 5] Immunoprotective properties of fabricated membranes PVDF and PVDF / PVP. (A) Representative SEM images of fixed human primary macrophages seeded on various porous transwell inserts (0.4, 1, 3 and 8 μm pores) or on top of fabricated membranes. Cells that were able to migrate through the pores can be seen in the representative bottom images. The scale bar in all images represents 10 μm. (B) Quantification of migrated human primary macrophages on the bottom of 24-well plates. Data are presented as mean ± STD (N=3). (C) Representative light microscopy images of the well plate bottom of 3 μm and 8 μm transwells after 24 hours. The scale bar represents 200 μm. [Figure 6]Schematic of device fabrication and islet seeding. (A) The three layers of the device, two membrane sheets (light grey) and the middle support ring (dark grey). (B) Sealing the three layers of the device by ultrasonic welding. (C) Assembled device with seeding inlet. (D) Seeding of islets through the seeding inlet. (E) Closing the seeding inlet by heat sealing. (F) Cultivation of the cell-loaded device. [Figure 7] Fabrication and testing of encapsulated devices. (A) Schematic of assembly of encapsulated device. (B) Assembled encapsulated device. (C) Handling of assembled encapsulated device. Camera images of device testing for leakage (D-E). (D) Leak-free and leaky devices (leakinlevice) (E). Mechanical properties of heat and ultrasonic welded seals (F-G). (F) Break stress and (G) break strain of seals. Data are presented as mean ± std (n=5). *P<0.05 by Student's t-test. [Figure 8] Human islets cultured inside the immunoprotective device. (A) Representative image of human islets inside the encapsulation device after 8 days of culture. Red channel represents dead cells and green channel represents live cells. Scale bar represents 200 μm in all pictures. (B) Quantification of human islet viability after 8 days of culture inside the encapsulation device or without the device as control. Data are presented as mean ± STD (N=3). (C) Insulin release profile of human islets during GSIS after 8 days of culture inside the device, normalized to total insulin. *P<0.05 by one-way ANOVA. (D) Stimulation index at day 8. Data are presented as mean ± SEM (N=3). *P<0.05 by Kruskal-Wallis test. [Figure 9]Schematic overview of different mesh sizes. This figure depicts different mesh sizes diagrammatically. The indicated sizes in micrometers are a guideline for islet cells; other cell types or organoids will require different sized meshes. Schematic depictions are shown of situations where the mesh is too small (left) and cells can still form clusters, the ideal mesh size where cells are isolated from each other (center), or where the mesh is too large (right) and cells fall off the mesh and can still form clusters (top row: plan view, bottom row: cross section). [Figure 10] Schematic cross-section of the device, depicting the upper and lower membranes, the mesh, and the cells inside the device embedded on the mesh. Additionally, the support structure (in this case the support ring) is shown. [Figure 11] Figure 1 shows a cross-section of stacked devices. Two devices are depicted stacked on top of each other and separated by an additional support structure to allow for nutrient diffusion. [Figure 12] Islet cells cultured on a mesh do not aggregate. Pancreatic islet cells cultured in medium without (left) or with (right) a mesh in a tissue culture dish. [Figure 13] Islet cells cultured on mesh do not aggregate. Pancreatic islet cells cultured without (left) or with (right) a mesh inside an implantable device described herein. [Figure 14] hMSC cell clusters can be cultured within the device. Illustrated are clusters of hMSC cells cultured on the outside or inside of a device described herein. Shown are viable cell clusters as determined by staining (not shown). [Figure 15] Alpha-TC cell clusters can be cultured within the device. Illustrated are clusters of alpha-TC cells cultured on the outside or inside of the device described herein. Shown are viable cell clusters as determined by staining (not shown). [Figure 16]Seeding of human islets inside devices with mesh (pore size 400 μm) and without mesh. Top: Photographs of the overall view of devices without mesh (left) or with mesh (right) after cell seeding. Devices with pore size 400 μm were used. Bottom: Photographs of enlarged images showing individual cell clusters. [Figure 17] Seeding of beta cell aggregates onto a device with a mesh of approximately 50 μm. The left photo shows a general view of the device with a 50 μm mesh after cell seeding. The right photo shows a magnified image showing individual cell clusters. [Figure 18] Seeding of pseudo-islets on a mesh with 80 μm pores. A) Cells are seeded at two different concentrations (left: 1000 clusters per 2 mL, right: 3000 clusters in 1.5 mL) on a device without a mesh and grown for 4 days. B) Cells are seeded at four different concentrations (from left to right: 1000 clusters per mL, 1000 clusters per 2 mL, 1000 clusters per 5 mL, and 3000 clusters in 1.5 mL) on a device with an 80 μm mesh and grown for 4 days. Photographs are taken from below the mesh (top row) and above the mesh (bottom row). EXAMPLES

[0063] Example 1 material and method Fabrication of porous membrane A casting solution was made by dissolving 15% (w / w) polyvinylidene fluoride (PVDF) (Kynar 720, Foster, Germany) in N,N-dimethylacetamine (DMA) (Sigma-Aldrich, The Netherlands) and, for polyvinylpyrrolidone (PVP)-containing membranes, 5% (w / w) PVP (40 kDa) (Kollidon® 30, BTC, The Netherlands) was added. The solution was mixed overnight at room temperature (RT) on a roller bank until a homogenous solution without bubbles was obtained. The solution was then cast (at RT, relative humidity <30%) on a glass plate using an automatic film applicator (Elcometer, The Netherlands) equipped with a 200 μm casting knife, which was then immediately immersed directly in a coagulation bath (distilled water at RT). The membrane was collected from the coagulation bath and dried between tissue papers for 2 days before use.

[0064] Scanning Electron Microscope (SEM) Membrane samples were fixed onto metal scanning electron microscope stubs using carbon tape and gold coated using a Cressington sputter coater (Cressington 108 auto, UK). Prior to imaging, membranes were cross-sectioned by immersing them in liquid nitrogen and then breaking each sample in half. Electron microscopy images were acquired using a scanning electron microscope (SEM) (Teneo, FEI, The Netherlands) under high vacuum using secondary electron mode with ETD detection, a working distance of 10 mm and an accelerating voltage of 5.00 kV.

[0065] Fabrication of the support ring The PVDF pellets were sandwiched between a mould (200 μm thick, made in-house) and preheated in a hot press (Specac, UK) at 180° C. for 1 min. Then, 20 kN was applied to soften the pellets for 1 min to create a solid film. Finally, the mould was removed from the hot press and cooled at RT for 5 min before removing the solid PVDF film from the mould.

[0066] Membrane Wettability The membranes were mounted horizontally on a glass slide. A 4 μL droplet of distilled water (room temperature) was dispensed onto the surface of the membrane at a rate of 10 μL / min, and high-resolution images were taken. Contact angles were measured by using Drop Shape Analysis4 software (Kruss, Germany) equipped with a drop shape analyzer. Nine droplet measurements were taken for each membrane.

[0067] Mechanical Tensile Test The mechanical tensile properties of the fabricated films and sealing methods were determined by a mechanical tensile tester (45kN load cell, Electroforce3230-ES series III, UK). The dimensions of the test specimens were 35x10mm with an active area between the clamps of 15x10mm. The ramp rate was 0.05mm / s. Each condition was tested five times. The resulting stress-strain curves were used to calculate the peak strain, break stress, break strain and Young's modulus. Different device components were annealed to each other using two different methods for comparison: heat sealing with a flat wire impulse sealer (Durapak, USA) and ultrasonic welding (LPX, Branson Ultrasonic, Netherlands). The strength of the seal was then determined in mechanical tension.

[0068] cell culture INS-1E rat insulinoma cells (Addexbio Technology, USA) were cultured in Roswell Park Memorial Institute (RPMI) medium containing L-glutamine (Sigma-Aldrich, Netherlands) supplemented with 10% (v / v) fetal bovine serum (FBS) (Sigma-Aldrich, Netherlands), 10 mM HEPES (Thermo Fisher Scientific, Netherlands), 1 mM sodium pyruvate (Thermo Fisher Scientific, Netherlands), 5 mM glucose (Thermo Fisher Scientific, Netherlands), 23.8 mM sodium bicarbonate (Thermo Fisher Scientific, Netherlands) and 50 mM beta-mercaptoethanol (Thermo Fisher Scientific, Netherlands). INS-1E from P38 to P43 were used. Human primary macrophages (Celprogen, USA) were cultured in Dulbecco's modified Eagle's medium (DMEM) high glucose (Thermo Fisher Scientific, Netherlands) supplemented with 10% (v / v) FBS (Sigma-Aldrich, Netherlands). Macrophages from P6 to P10 were used. All cells were maintained at 37 °C and 5% CO 2 and cultured.

[0069] Human islet culture Human islets were provided by the Human Islet Isolation Laboratory at Leiden University Medical Centre (LUMC, The Netherlands), which is licensed by the Dutch government to isolate human islets for clinical purposes. The mean age of the organ donors (2 males, 1 donor of unknown gender) was 43 ± 14 years. The mean islet purity was 75 ± 5%. Islets were cultured in CMRL-1066 medium (Pan Biotech, Germany) supplemented with 10% (v / v) FBS (Sigma-Aldrich, The Netherlands), 10 mM HEPES (Thermo Fisher Scientific, The Netherlands), 1% penicillin-streptomycin (Thermo Fisher Scientific, The Netherlands) and 10 μg / mL ciprofloxacin (Sigma-Aldrich, The Netherlands). Islets were cultured at 37 °C and 5% CO 2 and cultured.

[0070] MTT assay The metabolic activity of the cells was assessed by measuring the conversion of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to formazan, a process that occurs only in metabolically active cells. Extract samples were generated by culturing the test materials in RPMI cell culture medium (see Cell Culture) for 24 h. 1% Triton X-100 in RPMI (VWR, The Netherlands) was used as a negative control, while untreated medium served as a positive control. INS-1E cells were plated in 96-well plates at 5 × 10 5Cells / mL were seeded (100 μL) and incubated overnight to form a 50% confluent monolayer in RPMI medium. After 24 h, the medium was removed and 100 μL of the extract sample was added and incubated overnight. After 24 h, the medium was replaced with 100 μL of fresh cell culture medium supplemented with 10 μL of 12 mM MTT (Thermo Fisher Scientific, Netherlands) solution in phosphate-buffered saline (PBS) (Sigma-Aldrich, Netherlands) and incubated for 4 h. All but 25 μL of the medium was removed, mixed with 100 μL of DMSO (VWR, Netherlands) and incubated for 10 min at 37 °C. The absorbance was measured at 540 nm using a CLARIOstar microplate reader (BMG Labtech, Germany). Cell viability was calculated according to the formula 1 (OD 540e = Optical density reading of the extract of the test sample, OD 540b = blank optical density reading).

[0071]

number

[0072] Lactate dehydrogenase (LDH) cytotoxicity assay Cytotoxicity was assessed by measuring LDH levels in the cell culture medium. INS-1E cells were plated at 5 × 10 5 Cells / mL were seeded (100 μL) and incubated overnight. Then, 10 μL of the extracted sample was added (see MTT assay) and incubated for 45 min. 50 μL of each sample was transferred to a 96-well plate and 50 μL of reaction mixture (Pierce LDH, Thermo Fisher Scientific, The Netherlands) was added and incubated for 30 min at RT in the dark. Then, 50 μL of stop solution was added, mixed and the absorbance was measured at 490 nm and 680 nm on a CLARIOstar microplate reader. Cytotoxicity was calculated using Equation 2:

[0073]

number

[0074] Fourier Transform Infrared Spectroscopy (FTIR) The chemical composition of the prepared membranes was analyzed using Fourier transform infrared spectroscopy (FTIR) (Nicolet iS50-FT-IR, Thermo Scientific, The Netherlands). Infrared spectra were recorded from 400 to 4000 cm -1 The results were analyzed using SpectraGryph (version 1.2.13, Germany) to identify peaks related to chemical bonds.

[0075] Assembly of the encapsulated device The membrane and the support ring were cut into an oval shape (44 × 28 mm) using a cutting machine (Curio, Silhouette, The Netherlands). The device was fabricated by placing the support ring between two membrane sheets and annealing them by ultrasonic welding (LPX, Branson Ultrasonic, The Netherlands) inside a custom-made mold (IDEE, The Netherlands). The assembled device was tested for leakage by the air bubble test. Briefly, the assembled device was submerged in water while blowing air (~4 L / min) into the device through the inlet. If air bubbles were observed escaping from the device, leakage was detected.

[0076] Membrane diffusion properties The membrane was pre-wetted in 70% ethanol for 2 min and then washed with distilled water for 2 min to remove the ethanol. The wetted membrane was placed in an in-line equilibrium diffusion cell (Sigma-Aldrich, The Netherlands) with a donor chamber and a receiver chamber. The donor chamber was filled with a PBS solution containing 2 mg / mL 3–5 kDa FITC-dextran (Sigma-Aldrich, The Netherlands), 20 mM D-glucose solution (Sigma-Aldrich, The Netherlands) or 0.5 mg / mL FITC-labeled human insulin (Sigma-Aldrich, The Netherlands), or a NaCl solution (150 mM) containing 1 mg / mL IgG solution (Sigma-Aldrich, The Netherlands). The receiver chamber was filled with PBS for dextran, glucose or insulin diffusion and with 150 mM NaCl for IgG diffusion. Samples of 10 μl were removed from both chambers at 0, 5, 10, 20, 30, 60, 120, and 180 min, and in the case of insulin diffusion, an additional sample was taken at 1440 min. Quantitative analysis of diffused FITC-dextran and FITC-insulin was determined based on fluorescence intensity (excitation and emission wavelengths = 480 nm and 530 nm) using a CLARIOstar microplate reader. The amount of glucose was measured by using a colorimetric glucose assay (Bio-connect Diagnostic, The Netherlands) according to the manufacturer's protocol. Briefly, samples were collected and diluted in PBS. 50 μL of glucose solution was incubated with 50 μL of reaction mixture for 20 min at 37 °C. The absorbance was measured using a CLARIOstar microplate reader (excitation wavelength = 555 nm). The amount of IgG was measured by IgG ELISA (Thermo Fisher Scientific, The Netherlands) according to the manufacturer's protocol. To gain insight into how much of the molecule of interest diffuses through the membrane at a particular time, the flux value was calculated using Equation 3 (C レシーバー = concentration in the receiver chamber, V レシーバー= volume of the receiver chamber, A = area of ​​the membrane, t = time. The diffusion coefficient was calculated using Equation 4 (ΔC = concentration difference between donor and receiver, I = membrane thickness). Each condition was tested at least four times.

[0077]

number

[0078]

number

[0079] Oxygen diffusion characteristics inside the device Prior to filling the device with medium, the assembled device was pre-wetted with 70% ethanol for 2 min and then washed with distilled water for 2 min to remove the ethanol. Oxygen diffusion was measured by filling the device with 500 μL of RPMI cell culture medium. The medium-filled device was heat-sealed and placed inside a 100 mL beaker containing 100 mL of fresh cell culture medium. The beaker containing the device was then heated in 5% O2 and placed in a 100 mL beaker containing 100 mL of fresh cell culture medium. 2 The devices were placed in a humidified incubator at 37 °C. Oxygen concentrations inside and outside the devices were recorded over a 24-h period using a calibrated needle-type oxygen microsensor (NTH-PSt7, Presens, Germany) connected to an oxygen meter (OXY-10, Presens, Germany). The devices were kept submerged in the medium at all times during the oxygen measurements. Each condition, control and both devices were tested independently in triplicate.

[0080] Membrane immunoprotection 0.33cm 2Membranes were mounted on empty transwell inserts (Corning, The Netherlands) with glue (Bison, The Netherlands) and O-rings (Erik, The Netherlands). After the glue was allowed to dry overnight, the inserts were sterilized with 70% ethanol. Human primary macrophages were seeded (100,000 cells in 100 μL) in the upper compartment of each insert in a 24-well plate, and 600 μL of medium was added to each well. Cells were cultured for 24 h (37 °C, 5% CO 2 ). Cells that migrated to the lower compartment were counted manually using a Neubauer hemocytometer. To observe cell attachment and invasion through the membrane, the membrane was fixed with 3.6% formaldehyde in PBS (VWR, The Netherlands) for 30 min at RT and then rinsed multiple times with fresh PBS before the membrane was excised from the insert. Samples were dehydrated in an ascending ethanol series (30, 50, 70, 90, 96, 100 and 100% ethanol) (VWR, The Netherlands) for 10 min each. Dehydrated samples were processed in a critical point drying apparatus (Leica, The Netherlands). Dry membrane samples were then prepared and imaged using a scanning electron microscope as previously described.

[0081] Islet seeding into the containment device The devices were sterilized by overnight incubation in 70% ethanol and then washed with sterile PBS. Before seeding the islets into the devices, the devices were incubated overnight in culture medium (37°C, 5% CO). 2 ). 3,000 human islet equivalents (IEQ) in 500 μL of medium were seeded inside each device. For control samples, 30 IEQ were seeded on 12 μm cell culture inserts (Merck, The Netherlands). The seeding inlets were closed with a heat seal (Durapak, USA). The devices were cultured for 8 days (37 °C, 5% CO 2 ), and the medium was refreshed every other day.

[0082] Human islet viability Human islet viability was assessed using the live-dead fluorescent dyes calcein AM and ethidium homodimer 1 (Thermo Fisher Scientific, The Netherlands) for live and dead cells, respectively. After 8 days of culture, the devices were dissected and the islets were flushed out of the devices with PBS. The islets were then incubated with 2.5 μM calcein AM and 4 μM ethidium homodimer 1 in PBS for 30 min (protected from light) and visualized under a fluorescent microscope (Eclipse Ti-S inverted microscope, Nikon, The Netherlands). The percentage of viability was quantified by dividing the area of ​​live cells by the total area of ​​cells (live and dead).

[0083] Human islet function during glucose stimulation To evaluate insulin release of human islets inside the device, a glucose stimulated insulin secretion (GSIS) assay was performed. Krebs buffer was prepared by dissolving 25 mM HEPES (Sigma-Aldrich, Netherlands), 115 mM sodium chloride (Sigma-Aldrich, Netherlands), 26 mM sodium bicarbonate (Sigma-Aldrich, Netherlands), 5 mM potassium chloride (Sigma-Aldrich, Netherlands), 1 mM magnesium chloride dihydrate (Sigma-Aldrich, Netherlands), 25 mM calcium chloride dihydrate (Sigma-Aldrich, Netherlands) together with 2% bovine serum albumin (BSA) (VWR, Netherlands) in MilliQ, pH 7.3-7.5, and sterile filtered. Low and high glucose solutions contained 1.67 and 16.7 mM D-glucose, respectively, in Krebs buffer. After 8 days of culture, the devices and islets were washed three times in low glucose solution and then preincubated in low glucose for 1 h (37°C, 5% CO 2 The devices and islets were then exposed to low-high-low glucose solutions for 1 h each (37°C, 5% CO 2), after which the supernatant was collected and stored at -30°C until analysis. After high glucose exposure, the devices and islets were washed three times with low glucose solution. After the last incubation step, the devices were opened and the islets were exposed to acid ethanol to release all the insulin inside the cells. Insulin secretion was determined by human insulin ELISA kit (Mercodia, Sweden or Sanbio, The Netherlands) according to the manufacturer's protocol. The glucose stimulation index was calculated by dividing insulin secretion in high glucose solution by basal insulin secretion in low glucose solution.

[0084] 4. Data Analysis Results are expressed as mean ± standard deviation (STD) unless otherwise specified. Experiments were performed in triplicate unless specified. Three samples were taken per experimental condition for each experiment. Statistical analysis was performed using GraphPad Prism9 (GraphPad Software, USA). Statistical differences between two groups were determined using unpaired Student's t-test, between multiple groups using Kruskal-Wallis test, or one-way ANOVA test with multiple comparisons. p<0.05 was considered statistically significant.

[0085] result Membrane fabrication and physicochemical characterization Thin porous membranes were fabricated by casting 15% (w / w) PVDF + 5% (w / w) PVP (PVDF / PVP) and 15% (w / w) PVDF (PVDF alone) solutions onto glass plates using a solvent / nonsolvent casting method. The surfaces of the PVDF membranes, both with and without PVP, have a smooth and uniform appearance. Larger pores were observed on the glass side of the membrane (Figures 1A and 1D) compared to the air side of the membrane (Figures 1B and 1E). The pores on the glass side in the PVDF / PVP membranes had an average diameter of 0.51 ± 0.15 μm, whereas the pores on the glass side in the PVDF membranes had an average diameter of 0.25 ± 0.12 μm (data not shown). The presence of PVP increased the pore size on the air-exposed side (Fig. 1B and E), more than doubling the mean diameter to 113 ± 4 nm (Fig. 1H) compared with the pure PVDF membrane (51 ± 2 nm). The cross-section of the pure PVDF membrane was denser and thinner (45 ± 4 μm) compared with the PVDF / PVP membrane (137 ± 19 μm) (Fig. 1C, F, G).

[0086] To determine the hydrophilicity / hydrophobicity of the fabricated membranes, contact angle measurements were performed by the static sessile drop method. Unlike the glass-exposed side (both approximately xx), the surface wettability of the air-exposed side of the PVDF / PVP membrane was significantly lower compared to the PVDF membrane (80 ± 3° vs. 99 ± 4°; Figures 2A and 2B).

[0087] The mechanical properties of the fabricated membranes were determined by mechanical tensile tests. PVDF / PVP membranes exhibited significantly lower mechanical strength than PVDF membranes, as determined by Young's modulus measurements (0.32 ± 0.04 MPa for PVDF / PVP vs. 0.87 ± 0.11 MPa for PVDF alone; Fig. 2C) and peak stress measurements (0.97 ± 0.03 MPa for PVDF / PVP vs. 4.95 ± 0.16 MPa for PVDF alone; Fig. 2D). Furthermore, both the breaking strain (Fig. 2E) and breaking stress (Fig. 2F) were significantly lower for PVDF / PVP membranes compared to PVDF alone: ​​0.92 ± 0.06 MPa v. 4.78 ± 0.07 MPa and 11 ± 1% v. 84 ± 8%, respectively.

[0088] Membrane cytotoxicity LDH and MTT assays were performed to determine the cytotoxicity of the fabricated PVDF membranes and support rings against INS-1E cells. Cell viability was slightly but significantly lower for the PVDF / PVP membranes compared to the PVDF-only membranes (92 ± 8 vs. 106 ± 9%; Fig. 4A). Neither membrane showed significant differences in cytotoxicity compared to the positive control (-7 ± 15 and 10 ± 14% for the PVDF and PVDF / PVP membranes, respectively; Fig. 4B). Although PVP is water-soluble, residual potentially cytotoxic PVP particles may have remained after membrane fabrication, which may have influenced the cytotoxicity measurements. Therefore, FTIR analysis was performed to determine whether PVP residues were still present (Fig. 3C). For the PVDF-only membranes, the CHs present in the basic structure of PVDF were not observed. 2 Stretching groups and CF 2 Approximately 1400 cm corresponding to the stretching group -1 and about 1180cm -1 In the case of PVDF / PVP, a peak was observed at approximately 1660 cm, which corresponds to the C=O group present in PVP. -1 An additional peak was observed (green dotted line in Figure 3C).

[0089] Diffusion of small molecules across membranes To gain insight into the transport of different molecules across the membrane, we tested four types of molecules: dextran, glucose, insulin, and IgG. The membrane was placed between two chambers: donor and receiver chambers. The diffusion kinetics were calculated by performing concentration measurements of different molecules in each chamber at a given time point. The diffusion of dextran (3-5 kDa) through PVDF / PVP was almost three times higher than that through the PVDF membrane (Figure 4A), and the calculated flux and diffusion coefficients were significantly higher (Figures 4B and 4C). Similarly, glucose molecules also diffused faster through the PVDF / PVP membrane, reaching equilibrium (equal in the two chambers) after 3 hours, whereas only 30% of glucose diffused through the PVDF membrane at the same time point (Figure 4D). This observation was consistent with the glucose flux of 10*10 through the PVDF / PVP membrane. -4 g m -2 seconds -1 and diffusion coefficient 4.0*10 -7 cm 2 s -1 In the case of PVDF alone (4*10 -4 g m -2 seconds -1 and 1.1*10 -7 cm 2 s -1 ) (Figure 4E and 4F). Glucose was not absorbed by the membrane, as the total glucose concentration did not change over time. FITC-labeled insulin reached equilibrium with 47 ± 2% of insulin diffused through the PVDF / PVP membrane after 24 h, compared with 28 ± 5% diffused through the PVDF membrane at the same time point (Figure 4G). This insulin diffusion capacity was significantly higher than the flux through the PVDF / PVP membrane of 1.91*10 -5 g m -2 seconds -1 and the diffusion coefficient is 1.31*10 -8 cm 2 s -1, and in the case of PVDF membrane (0.35*10 -5 g m -2 seconds -1 and 0.08*10 -8 cm 2 s -1 ) (Figure 4H and Figure 4I). For IgG, only 1.6 ± 0.7% penetrated the PVDF / PVP membrane, but was not detected in the receiver chamber with a PVDF membrane alone, suggesting that no diffusion occurred (Figure 4J).

[0090] Oxygen transport in free solution (cell culture medium) was not significantly different when compared to either the PVDF-based or PVDF / PVP-based membrane devices, as the areas under the curve were comparable (Figures 4K and 4L).

[0091] Immune-protective function of PVDF membrane To evaluate the immune-protective properties of the fabricated membranes (PVDF and PVDF / PVP), we assessed the migration of macrophages from the top to the bottom of transwell inserts immobilized with specific membranes. As shown by SEM images (Figure 5A), no macrophages were detected on the bottom side of PVDF and PVDF / PVP membranes and those with 0.4 μm pores. Macrophages were detected on the bottom side of membranes with 3 and 8 μm pores. However, only in the case of 8 μm pores were macrophages able to migrate completely to the bottom well (Figure 5B and Figure 5C). Although human macrophage nuclei could not migrate through 1 μm pores, those pores were large enough to allow the movement of filopodia, the cellular antennae that sense the extracellular environment (Figure 5A).

[0092] Assembly of immune protection encapsulation devices To assemble the encapsulation device, two membrane sheets were welded together by high-frequency ultrasonic welding or heat sealing with a support ring between them, as illustrated in Figure 6A. A small portion was left unwelded to serve as an inlet for loading cells into the device (Figure 6D). Each device was inspected for leakage with a pressurized bubble test. Welding defects were detected by visual inspection for bubble leakage (Supplementary figure 4D). Devices with welding defects were not used for subsequent experiments.

[0093] Viability and function of human islets inside containment devices Human islets were cultured inside the closed immunoprotective devices for 8 days to assess their viability and islet function using live-dead staining and GSIS assay, respectively. No significant difference in viability was observed between islets cultured inside PVDF and PVDF / PVP devices compared to controls (Figures 8A and 8B). 92 ± 4% of human islets were viable after 8 days of culture inside PVDF / PVP encapsulated devices. In control conditions, islets responded to changes in glucose levels by increasing insulin release under high glucose conditions after 8 days, with a stimulation index of 2.3 ± 0.4 (Figures 8C and 8D). Increased insulin secretion during high glucose conditions was observed in the PVDF / PVP devices (Figure 8C). The stimulation index of the PVDF / PVP devices was significantly higher than that of the PVDF devices, 3.2 ± 0.7 and 1.3 ± 0.2, respectively (Figure 8D).

[0094] Example 2 Use of mesh (80um) for better cell distribution Pancreatic beta cell clusters were grown in the presence or absence of mesh. In the absence of mesh, it can be observed that all cell aggregates are close to each other, which leads to further cell aggregates with increasing culture time. In the presence of mesh, it can be observed that the cell aggregates are more dispersed. This may be to prevent the cell aggregates from coming together and forming clusters with increasing culture time. We conclude that the mesh enhances the distribution of cells to prevent cluster formation. The results are illustrated in Figure 12. The images were made at the same magnification and the same concentration of aggregates was seeded on the same culture area.

[0095] Testing in Encapsulated Devices The effect of the mesh when placed inside a closed containment device, compared to the Petri dish experiments (see above), gives similar results. In the device without the mesh, the cell aggregates are close to each other (see circles). In the device with the mesh, the cell clusters are more evenly distributed inside the device and there is less cluster formation. We conclude that the use of a mesh can be beneficial for a better cell aggregate distribution inside the device. The results are illustrated in Figure 13. The images were made at the same magnification and the same concentration of aggregates was seeded on the same culture area.

[0096] Viability of cell clusters after 7 days of culture inside a closed containment device Both clustered cells (alpha-TC cells and hMSCs) remained viable after 7 days of culture inside the closed containment device. The viability level was comparable to cell aggregates that were not cultured inside the containment device. It can be concluded that the containment device can not only help to keep the islets alive, but also that it is possible to culture different cell types inside the closed containment device without affecting the viability of the cell clusters. The results are illustrated in Figures 14 and 15.

[0097] Example 3 - Various mesh sizes Reference Experiment 1 Goal: To observe the distribution of islands inside the device. method: Equivalent sized devices were made with and without mesh. The mesh used had a pore diameter of 400 μm. The mesh was placed between one membrane sheet and a support structure. Approximately 3,000 islet equivalents (IEQ) were seeded into the device in a volume of 500 μL. For devices containing mesh: islet cells were seeded on top of the mesh (so that the islets could become trapped inside the mesh). Observations: The device and cells are illustrated in FIG. Conclusion: The presence of this mesh inside the device did not aid in islet distribution because the mesh structure was very open and the islets fell through the openings, as shown in the magnified image, and the islets were not retained.

[0098] Reference Experiment 2 Goal: To create a transparent device with a ~50 μm mesh on the inside. The aim is to verify whether the mesh helps to prevent clump formation of pseudo-islands seeded in the device and whether the pores are small enough to prevent the pseudo-islands from falling out of the pores. Methods: Two devices were made using approximately 50 μm mesh. The mesh was placed between one membrane sheet and a support structure. Approximately 2,500 pseudo-islets were seeded into the device in a volume of 500 μL. For devices containing mesh: islet cells were seeded on top of the mesh (so that the islets could become trapped inside the mesh). Observations: The device and cells are illustrated in Figure 17. The seeded pseudo-islands did not fall out of the pores. The pseudo-islands were not distributed evenly on the surface. Conclusion: The seeded pseudo-islets did not fall off the mesh; this is probably because the pore diameter was smaller than the diameter of the seeded pseudo-islets. The distribution of the pseudo-islets was better in the device with mesh compared to the device without mesh. However, the pseudo-islets tended to settle close to each other and were not trapped inside the pores. Thus, only a portion of the seeded cells remained apart from each other. For better distribution, slightly larger pores are needed.

[0099] Experiment 3 Objectives: To assess pseudo-islet distribution in well plates using an 80 μm mesh and compare with floating controls.

[0100] Part 1: Method: Mesh (80 μm pores) was placed in 6-well plates. Seed pseudo-islets on top of pseudo-islets on top of a mesh with 80 μm pores. Pseudo-islets have a diameter of approximately 150 μm. Note: Non tissue treated 6 well plates were used as controls. Seeding density: C1: 1,000 aggregates in 2 mL C2: 3,000 aggregates in 2 mL M1: 1,000 aggregates in 1 mL M2: 1,000 aggregates in 2 mL M3: 1,000 aggregates in 5 mL M4: 3,000 aggregates in 1.5 mL Results: The aggregates in the control are free to move and tend to gather in the center. With the mesh present, some of the aggregates are trapped inside the mesh structure and some of the aggregates are still floating. A larger volume results in more aggregates floating and concentrating in a few areas on the mesh.

[0101] Part 2: After 4 days of culture, live / dead staining was performed to visualize the viability of pseudo-islets. Observation and image-results are illustrated in Figure 18. C1: Somewhat larger clusters were seen, which were floating. Some of the clusters attached to the well plate. C2: Most of the clusters were floating and did not attach to the well plate. Viability was slightly lower. The clusters were not as round as C1. M1: Most of the clusters attached to the mesh and dispersed. Only a few aggregates moved under the mesh. M2: Some of the clusters moved under the mesh and started to aggregate. But most of them were still on the mesh, attached to the mesh and separated by the mesh. M3: A relatively large amount of clusters moved under the mesh and started to aggregate, but no large clusters were formed. The clusters on the mesh remained separated from each other. M4: A relatively large amount of clusters moved under the mesh and started to aggregate, but no large clusters were formed. The clusters on the mesh remained separated from each other. Conclusion: In control conditions the pseudo-islets coalesced over the 4-day culture period, resulting in the formation of large clusters (C1 and C2). In mesh conditions the pseudo-islets remained in situ and did not form large clusters. The mesh did not affect the viability of the encapsulated pseudo-islets, as viability remained high. Thus the mesh showed a positive effect in keeping the pseudo-islets apart from each other.

[0102] From the experiments in this example, it can be concluded that the mesh size is preferably less than 400 μm, such as 350, 300, 250, 200 or 150 μm or less. Furthermore, it can be concluded that the mesh size is preferably greater than 50 μm, such as 60, 70, 75 or 80 μm or more.

Claims

1. An immunoprotective implantable device for implanting cells or cell aggregates in a subject, the device comprising a lower membrane and an upper membrane, the lower membrane and the upper membrane being joined to a support structure such that the lower membrane, the upper membrane and the support structure, when implanted, form an enclosed interior space that is not accessible by the immune system; the lower membrane and the upper membrane comprising a plurality of pores that permit diffusion of nutrients and oxygen through the membranes to the interior space; The device is characterized by the presence of a mesh disposed in the interior space of the device to provide a support for the cells or cell aggregates when the cells or cell aggregates are contained in the interior space of the device so that the cells do not form clumps, the mesh having a pore size of 60 to 300 μm. Implantable devices.

2. the lower membrane and / or the upper membrane, and optionally the support structure, are made of a water-insoluble polymer, preferably polyvinylidene fluoride (PVDF), polyethersulfone (PES), poly(ethylene oxide terephthalate) / poly(butylene terephthalate) (PEOT / PBT), or PVDF mixed with a water-soluble copolymer, preferably the membrane further comprises a pore-forming agent, more preferably the pore-forming agent is polyvinylpyrrolidone (PVP); The implantable device of claim 1 .

3. The pore size of the lower membrane and the upper membrane is 10 to 500 nm, preferably 50 to 450 nm, more preferably 100 to 400 nm; 3. An implantable device as claimed in claim 1 or 2.

4. The lower membrane and the upper membrane have a thickness of 10 to 200 nm, preferably 35 to 200 nm, more preferably 40 to 160 nm; An implantable device according to any one of claims 1 to 3.

5. The mesh is constructed from a non-degradable polymer, preferably the non-degradable polymer is selected from nylon, PVDF, PTFE, PP, PE or PET; 5. An implantable device according to any one of claims 1 to 4.

6. the mesh has a pore size of 60 to 200 μm, preferably 70 to 150 μm, more preferably 75 to 120 μm; 6. An implantable device according to any one of claims 1 to 5.

7. The device comprises cells in an internal space, preferably the cells are cell aggregates and / or organoids, preferably the cell aggregates are endocrine cells or cytokine producing cells or aggregates thereof, preferably the cell aggregates are selected from islet cells, human mesenchymal stem cells, kidney cells, thyroid cells, thymus cells, testicular cells, pancreatic cells, endocrine cells, liver cells, cytokine producing cells, or preferably the organoids are kidney organoids, intestinal organoids, pancreatic organoids, neural organoids, hepatic organoids, thyroid organoids, stomach organoids, ovarian organoids, prostate organoids, or the like. organoids, spleen organoids, esophageal organoids, breast organoids, bladder organoids, lung organoids, eye organoids, inner ear organoids, heart organoids, gallbladder organoids, salivary gland organoids, pituitary gland organoids, lymphoid tissue organoids, urinary bladder organoids, tongue organoids, cerebral / brain organoids, spinal cord organoids, fallopian tube organoids, lacrimal gland organoids, skin organoids, thymus organoids, testicular organoids, epithelial organoids, gastruloid (embryonic organoids), blastoid (blastocyst-like organoids), retinal organoids and hippocampal organoids, 7. An implantable device according to any one of claims 1 to 6.

8. comprising two or more implantable devices stacked on top of one another and separated by spacers such that an open space is created between each of the stacked implantable devices; 8. An implantable device according to any one of claims 1 to 7.

9. For use as a medicament, wherein the implantable device contains cells.

9. An implantable device according to any one of claims 1 to 8.

10. For use in the treatment of diabetes, wherein the implantable device comprises pancreatic islet cells or cells engineered to secrete insulin.

9. An implantable device according to any one of claims 1 to 8.

11. The diabetes is type 1 diabetes. The implantable device of claim 10.

12. said use comprising implanting said device in a subject in need thereof, preferably said implantation being at an extrahepatic site.

12. An implantable device as claimed in claim 10 or 11.

13. 9. A method for constructing an implantable device according to any one of claims 1 to 8, comprising the steps of: Providing a lower membrane; placing a support structure generally along an edge of the lower membrane to leave a central space; disposing a mesh in the central space over the lower membrane; placing an upper membrane over the support structure to cover and completely enclose the central space comprising the mesh to create an interior space; and fastening said upper and lower membranes to said support structure, preferably by ultrasonic welding, heat sealing, pressure sealing, adhesive, or more preferably by ultrasonic welding; method.

14. The upper and lower membranes are secured to a support structure so as to leave a single opening for loading cells into the device. The method of claim 13.

15. 1. A method for loading cells into an implantable device, comprising: Providing a device according to any one of claims 1 to 8 or obtained or obtainable by a method according to claim 13 or 14, Providing a cell suspension in a container; connecting a tube such that one end is in open contact with the interior of the vessel containing the cell suspension and the other end is inserted through an opening into the interior space of the implantable device; and allowing the cells in the container to flow through the tube and into the interior space of the implantable device, and optionally closing the opening in the implantable device after removing the tube, preferably by ultrasonic welding, heat sealing, pressure sealing, adhesive, or more preferably by heat sealing. method.

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