EA040824B1 - METHOD AND DEVICE FOR CELL TRANSPLANTATION - Google Patents

Description

This application claims priority to U.S. Provisional Application No. 61/238011, filed Aug. 28, 2009, which is incorporated herein by reference in its entirety.

The present disclosure relates to the field of cell therapy, more specifically to methods and devices for transplanting cells into a host organism.

Recent discoveries in the field of cell therapy provide new opportunities for the use of cell transplantation in diseases where the urgent need for treatment remains unmet. Currently, there is no fully effective drug therapy for many acquired and congenital diseases, such as diabetes or Parkinson's disease, caused by the loss or damage of cells that produce biomolecules necessary to control physiological functions. Cellular therapy offers hope of replacing lost or damaged cells with donor cells or stem cells to restore impaired physiological functions. For example, cell transplantation of the islets of Langerhans would provide a means of restoring carbohydrate control in patients suffering from insulin-dependent forms of diabetes. Similarly, transplantation of dopaminergic neurons or neural stem cells has emerged as a promising cell therapy for the treatment of Parkinson's disease.

The main limiting factors in the use of cell therapy are the difficulty of transplanting cells into the host tissue and ensuring that the transplanted cells continue to function without triggering an immune response or other deleterious side effects in the host. Attempts have been made to introduce therapeutic cells directly into the host organism, for example, into the vascular system, or by implantation into an organ or tissue. However, with direct cell transplantation, the patient will require lifelong immunosuppressive therapy, while immunosuppressive drugs can cause toxicosis of the host organism and be toxic to implanted cells. In addition, the direct release of cells into the blood can lead to an immediate blood-mediated inflammatory response (IBMIR), which initiates the coagulation cascade and can destroy a significant proportion of the transplanted cells. In addition, cells can be deposited in microvessels and lead to vascular blockage and thrombosis, which can cause loss of functionality of transplanted cells and damage to local tissue.

The next therapeutic approach is to deliver cells using devices that provide the cells with a biologically suitable environment for living in the host organism. The main problems associated with this approach are the incorporation of blood vessels into the device to feed the cells and maintain an optimal environment in the device in order to ensure long-term cell survival. In the absence of an immediately available vascularized environment, the transplanted cells are not able to receive sufficient oxygen and easily dispose of waste products, and may quickly die or be damaged due to the effects of ischemia and hypoxia. In addition, even with the early growth of some vessels, the vessels may not receive the support of existence. In addition to this, the body's natural inflammatory cascade can also lead to cell death or damage. Some additional problems inherent in this approach include scratching and/or blocking of the device, incompatibility of the device material with the biological environment, difficulties in visualizing the device and the implantation environment, inappropriate device dimensions that adversely affect the biological functions of the cells, inability to load the appropriate number of cells for obtaining a stable therapeutic effect, the difficulty of removing the device when it needs to be moved. In addition, the configuration of the device may not conform to the outer contours of the body, which may result in abnormal protrusion of the device, making the device unacceptable to the patient from an aesthetic point of view.

Thus, there remains a need to develop an efficient technology for successful transplantation of therapeutic cells. The present disclosure provides methods and devices for delivering and maintaining cells in vivo over an extended period of time while addressing many of the problems associated with current device-based cell therapy approaches.

In one aspect of the present disclosure, a device is provided for transplanting cells into a host organism. The device contains a porous frame containing at least one chamber having a proximal end and a distal end, as well as at least one removable liner configured to be placed in at least one chamber. The porous frame contains a mesh with cells designed to promote the germination of vascular and connective tissues into at least one chamber. In some embodiments, the porous frame contains a polypropylene mesh.

Another embodiment of the present disclosure provides a device for implanting cells into a host organism, the device comprising a porous scaffold containing one or more chambers having a proximal end and a distal end, as well as a proximal opening.

- 1 040824 the proximal end or the distal end, or both the proximal end and the distal end. The porous frame contains a mesh with cells designed to facilitate the germination of vascular and connective tissues into one or more chambers. The device also comprises one or more dual-liner systems comprising an outer liner configured to be placed in one or more chambers, as well as an inner liner configured to be placed in an outer liner. In addition, the device contains at least one seal configured to close the system of liners in the chamber, as well as to close the hole at the proximal end or the distal end, or both at the proximal end and the distal end of the chamber.

In another aspect of the present disclosure, a method for transplanting cells into a host organism is provided. The method includes the steps of implanting a device to retain cells in a host body, the device comprising a porous scaffold containing at least one chamber having a proximal end and a distal end. The porous frame contains a mesh with cells designed to promote the germination of vascular and connective tissues into at least one chamber. In some embodiments, the porous frame contains a polypropylene mesh. The device further comprises at least one insert configured to be placed in at least one chamber, wherein at least one chamber has an opening at the proximal end or the distal end, or both the proximal end and the distal end. The method includes the steps of closing the opening at the proximal end or the distal end, or both the proximal end and the distal end of the chamber after implantation of the device. The method further includes maintaining the device in the host until vascular and connective tissues penetrate the porous scaffold, gaining access to the device through a surgical incision, reopening the proximal end or distal end or both the proximal end and the distal end of the chamber, removing the liner from the chamber to form a space in a porous scaffold encapsulated in a vascularized collagen matrix, delivering cellular material into the vascularized space, and reclosing the opening at the proximal end or distal end, or both the proximal end and the distal end of the chamber.

In another alternative manufacturing embodiment, in a method for implanting cells into a host body, an implantable device is provided for retaining cells in the host body, wherein the implantable device comprises a porous scaffold having pores configured to promote the growth of vascular and connective tissues into the porous scaffold, as well as at least at least one system with two liners, made with the possibility of placement in a porous frame. The porous frame of the implantable device contains at least one chamber having an opening at the proximal end or the distal end, or both at the proximal end and the distal end of the chamber. The device comprises a seal for closing an opening at the proximal or distal end, or both at the proximal and distal ends of at least one chamber. At least one insert system of the implantable device includes an outer insert configured to be placed in at least one chamber, as well as an inner insert configured to be placed in the outer insert. The method further includes the steps of implanting the device into the host, maintaining the device in the host until vascular and connective tissues penetrate the porous scaffold, and providing a cell delivery device comprising at least one cell infusion tube loaded with cell material, wherein the tube for cell infusion is configured to accommodate at least one insert system in the outer liner. In addition, the method includes gaining access to an implanted device through a surgical incision and opening a seal at the proximal end or distal end, or both the proximal end and the distal end of the device, removing the inner liner from the liner system, inserting the cell infusion tube into the outer liner, removing the outer liner from at least one chamber and the simultaneous infusion of cell material into the chamber, as well as re-closing the seal. It is to be understood that both the foregoing general description and the following detailed description are merely exemplary and illustrative, and do not limit the invention as expressed by the claims.

In a further aspect of the disclosure, a device for cell transplantation is provided, comprising a porous scaffold having pores configured to facilitate the ingrowth of vascular and connective tissues into a porous scaffold containing at least one chamber, but preferably 2-12 chambers, wherein the porous scaffold is coated from a biocompatible, biodegradable material, made with the possibility of temporarily filling the pores of the frame. In some embodiments, the porous frame contains a polypropylene mesh. Suitable biocompatible, biodegradable materials include, for example, collagen, fibronectin, extracellular matrix proteins, and cytoskeletal membrane proteins. The disclosure also provides a method for transplanting cells into a host organism, the method comprising implanting a transplant device comprising a porous scaffold having pores configured to facilitate the ingrowth of vascular and connective tissues into the porous scaffold containing at least

at least one chamber, but preferably 2-12 chambers, wherein the porous scaffold is coated with a biocompatible, biodegradable material that temporarily fills the pores of the scaffold, while at least one chamber is filled with transplantable cells and compacted.

The accompanying drawings, which form part of this description and form part of it, together with the description itself, illustrate methods and embodiments of the invention.

Brief description of the drawings

In FIG. 1A-1E show various embodiments of a single chamber cell transplant device according to the present disclosure;

in fig. 1F shows an embodiment of a multi-chamber cell transplant device according to the present disclosure;

in fig. 2A-2D show various mesh configurations that can be used to form a cell transplant device according to the present disclosure;

in fig. 3A shows a cell transplant device according to one embodiment of the present disclosure;

in fig. 3B shows the components of the cell transplant device shown in FIG. 1A;

in fig. 4 shows a porous frame of a cell transplant device according to one embodiment in the present disclosure;

in fig. 5A shows a cell transplant device seal according to one embodiment in the present disclosure;

in fig. 5B is a sectional view of the seal shown in FIG. 3A;

in fig. 6A shows a plurality of outer liners of a two-piece liner system of a cell transplant device according to one embodiment of the present disclosure;

in fig. 6B is a sectional view of the outer liner shown in FIG. 5A;

in fig. 6C shows a sectional view of the insert system and one porous scaffold assembly prior to implantation in the host;

in fig. 6D is a sectional view of the assembly shown in FIG. 4C, after incubation in the host;

in fig. 6E shows a sectional view of the porous scaffold implanted in the host after removal of the insert system;

in fig. 7 shows a plurality of inner liners of a two-piece insert system of a cell transplant device according to one embodiment of the present disclosure;

in fig. 8 shows a seal for closing cells in a vascularized chamber of a cell transplant device according to one embodiment of the present disclosure;

in fig. 9A shows a device for delivering cells to a cell transplant device according to one embodiment of the present disclosure;

in fig. 9B shows the cell infusion mechanism of the delivery device of FIG. 8A;

in fig. 9C shows additional steps in the cell infusion mechanism of the delivery device shown in FIG. 8A, 8B;

in fig. 10 is a flowchart showing the steps involved in implementing a cell transplantation method according to the present disclosure;

in fig. 11A-11D show a schematic overview of certain steps in a cell infusion procedure according to the present disclosure;

in fig. 12A shows line charts of blood glucose measurements after intraperitoneal implantation of cell transplant devices as described in Example 1;

in fig. 12B shows line charts of blood glucose measurements after subcutaneous implantation of the cell transplantation devices as described in Example 1;

in fig. 12C shows line charts of responses to an intravenous glucose tolerance test in Lewis rats that underwent islet cell transplantation at 40 days post-transplant, 80 days post-transplant, and also after device removal (110 days post-transplant) as described in Example 1;

in fig. 12D shows line charts of insulin levels in response to glucose loading in Lewis rats that have undergone islet cell transplantation;

in fig. 13A shows histological staining for insulin in the chamber of the implanted device as described in Example 2;

in fig. 13B shows histological staining for vascularization (microvascularization) in the chamber of the implanted device as described in Example 2;

in fig. 14 shows a table of average collagen thickness and total blood vessels/ ^cm2 calculated for four cell transplant devices according to manufacturing options.

- 3 040824 definitions in the present disclosure as described in example 3;

in fig. 15A shows tissue incorporation into a cell transplant device 2, 4 and 8 weeks after implantation as described in Example 3;

in fig. 15B shows the formation of blood vessels on various facets of an implanted device prior to cell transplantation as described in Example 3;

in fig. 16 shows bar graphs of levels of insulin produced by mature and immature islet cells as described in Example 4;

in fig. 17A shows histological staining for insulin and microvascularization in the chamber of the implanted device as described in Example 4;

in fig. 17B shows histological staining for microvascularization in the chamber of an implanted device after cell transplantation as described in Example 4;

in fig. 18 shows line charts of blood glucose levels after autologous islet transplantation as described in Example 4;

in fig. 19A shows line charts of blood glucose levels in response to glucose loading in Yorkshire Landrace pigs that underwent islet cell transplantation as described in Example 4;

in fig. 19B shows bar graphs of area under the curve (AUC) for blood glucose levels in response to glucose loading in Yorkshire Landrace pigs that underwent islet cell transplantation as described in Example 4;

in fig. 19C shows line charts of fold change in C-peptide levels in response to glucose loading in Yorkshire Landrace pigs that underwent islet cell transplantation as described in Example 4.

Description of examples of manufacturing options

Next will be discussed in detail the manufacturing options of the present disclosure, examples of which are presented in the accompanying drawings. Where possible, the same reference numbers will be used throughout the drawings to refer to the same or like parts. In the description, the terms cell infusion and cell transplantation are used interchangeably.

Proposed is a device for cell transplantation intended for keeping cells in vivo. In one example of a manufacturing variant, a cell transplant device comprises at least one porous scaffold containing a chamber and having an opening at the proximal end or distal end, or both at the proximal end and the distal end of the scaffold, as well as at least one liner made with the possibility of placing in the chamber. Holes at one or both ends of the chamber are configured to insert the liner into the chamber and withdraw back from the chamber. In one embodiment, at least one porous scaffold is tubular, with at least one liner being cylindrical and extending along the cavity of at least one porous scaffold. In some embodiments, the porous scaffold is only open at the proximal end. In one such embodiment, the distal end of the tubular porous scaffold comprises a rounded or flat bottom surface. In another embodiment, the edges at the distal end of the porous frame are tapered and come into contact with each other to seal the distal end.

In another exemplary embodiment, the cell transplant device comprises a porous scaffold containing one or more chambers having a proximal end and a distal end. One or more chambers contain an opening at the proximal end. The device also comprises one or more liner systems comprising an outer liner configured to be placed in one or more chambers, as well as an inner liner configured to be placed in an outer liner. In addition, the device comprises at least one seal configured to close the system of liners in the chamber and seal the opening at the proximal end of the chamber.

The porous framework is made of a biocompatible material and should cause only a moderate inflammatory response in the body. Moderate inflammation stimulates angiogenesis and promotes the incorporation of a vascularized collagen matrix into the device, but does not lead to significant inflammatory processes around the device. An example of such a biocompatible material is polypropylene. In exemplary embodiments, the porous scaffold comprises a woven polypropylene mesh having sufficient stiffness to allow the device to be fabricated. The polypropylene mesh is also configured to allow microvessels to penetrate into the device and support them as strong and healthy vessels, which is essential for the survival and normal functioning of therapeutic cells infused into the device.

By facilitating controlled outgrowth of vascularized tissue into the device, the porous scaffold prevents scar tissue from enveloping the device. Ingrown tissues also stabilize the implant and prevent accidental movement of the device in situ. In addition, in some embodiments, the porous scaffold is coated with biological or non-biological agents to promote tissue incorporation and angiogenesis, such as growth factors. Coating on the mouth

- 4 040824 The device can be applied by dipping into a polymeric drug composition or using other known device coating techniques. Examples of biological or non-biological agents for stimulating tissue incorporation and angiogenesis include, in particular: VEGF (vascular endothelial growth factor), PDGF (platelet growth factor), FGF-1 (fibroblast growth factor), NRP-1 (neuropilin-1 ), Ang-1, Ang-2 (angiopoietin-1,2), endoglin, MCP-1, ave3, ave5, CD-31, VE-cadherin, eprin, plasminogen activators, angiogenin, Del-1, aFGF (acid factor fibroblast growth), vFGF (basic fibroblast growth factor), follistatin, G-CSF (granulocyte colony stimulating factor), HGF (hepatocyte growth factor), IL-8 (interleukin-8), leptin, midkine, placental growth factor, PD-ECGF (platelet endothelial growth factor), PTN (pleiotropin), progranulin, proliferin, TGF-α and TNF-a.

In some embodiments, the outer surface of the porous scaffold is roughened to promote tissue penetration. In some embodiments, the porous framework includes various drug-eluting polymer coatings. In other embodiments, the porous scaffold may be coated with a biodegradable and non-biodegradable drug-free polymer. The frame may have a polymer coating partially or completely. Representative polymers that can be used to coat and/or release a drug include, but are not limited to, methacrylate polymers, polyethyleneimine and dextran sulfate, polyvinylsiloxane-polyethyleneimine copolymer (poly(vinylsiloxane)ecopolymerpolyethyleneimine), phosphorylcholine, polyethyl methacrylate, polyurethane, polyethylene glycol , copolymer of lactic and glycolic acids, hydroxyapatite, polylactic acid, polyhydroxyvalerate and its copolymers, polyhydroxybutyrate and its copolymers, polycaprolactone, polydioxanone, polyanhydrides, polycyanoacrylates, polyamino acids, polyorthoesters, polyesters, collagen, gelatin, cellulose polymers, chitosan, alginates, or combinations thereof. Other examples of materials that can be used as scaffold coatings include, inter alia: collagen, fibronectin, extracellular matrix proteins, vinculin, agar, and agarose. It should be understood that various mixtures of polymers can be used.

In view of drug release, in some exemplary embodiments, the porous scaffold contains an antibiotic coating to minimize infection. Representative antibiotics include, but are not limited to, ampicillin, tetracycline, nafcillin, oxacillin, cloxacillin, dicloxacillin, flucloxacillin, vancomycin, kanamycin, gentamicin, streptomycin, clindamycin, trimethoprim-sulfamethazole, linezolid, teicoplanin, erythromycin, ciprofloxacin, rifampin, penicillin , sulfonamides, nalidixic acid, norfloxacin, ciprofloxacin, ofloxacin, sparfloxacin, lomefloxacin, fleroxacin, pefloxacin, amifloxacin, 5-fluorouracil, chloramphenicol, polymyxin, mitomycin, chloroquine, novobiocin, nitroimidazole. In another embodiment, the porous framework contains bactericidal agents. Representative bactericidal agents include, but are not limited to, benzalkonium chloride, chlorhexidine gluconate, sorbic acid and its salts, thimerosal, chlorobutanol, phenethyl alcohol, and p-hydroxybenzoate.

In some other embodiments, parts of the cell transplant device are coated with anti-fibrotic drugs to inhibit capsule formation from fibrous tissue. Representative antifibrotic agents include, but are not limited to, paclitaxel, everolimus, tacrolimus, rapamycin, halofuginone hydrobromide, combretastatin, and their analogs and derivatives (such as combretastatin A-1, A-2, A-3, A-4, A -5, A-6, B-1, B-2, B-3, B-4, D-1, D-2 and combretastatin A-4 phosphate (Oxygen), docetaxel, vinblastine, vincristine, vincristine sulfate, vindesine , vinorelbine, camptothecin, topotecan, irinotecan, etoposide, teniposide, anthramycin, mitoxantrone, menogaryl, nogalamycin, aclacinomycin A, olivomycin A, chromomycin A3, plicamycin, methotrexate, edatrexate, trimetrexate, raltitrexide, pyrithrexim, denopterin, tomodex, pteropterin, and Derivatives and Analogues Them In some embodiments, the cell transplant device may also include poly(methyl methacrylate) or bone cement or other types of cyanoacrylates.

In some embodiments, the porous scaffold is made of a material that allows imaging of the implanted device using, for example, MRI, functional MRI, CT scanning, radiography, ultrasound, PET scanning, and so on. In one such manufacturing embodiment, the porous scaffold contains a polymeric mesh (eg, polypropylene, polytetrafluoroethylene (PTFE), polyurethane, polyester meshes, silk meshes, etc.) that are immunologically compatible and allow visualization of the newly formed vascular tissue. In another embodiment, the porous frame contains a combination of materials. In one such manufacturing embodiment, the porous frame contains woven polypropylene and silk threads.

The pore size of the scaffold material is chosen to promote incorporation and vascularization within the porous scaffold chamber. In some other embodiments, the pore size may be

- 5 040824 press in the range from about 50 nm to 5 mm. In one exemplary embodiment, the porous scaffold comprises a woven polypropylene mesh having a pore diameter of 0.53 mm.

In some embodiments, the pore size is chosen to prevent penetration of immune cells or immune agents into the implanted device. In some other embodiments, the pore size need not be such that immune cells or immune agents cannot be infiltrated into the device. This occurs when, for example, the device is used to transplant a combination of cells, including immunoprotective cells (eg, Sertoli cells, mesenchymal stem cells, etc.) capable of providing immune protection to co-transplanted cells. This also occurs when, for example, the device is used for transplantation of syngeneic cells or cells derived from the transplanted patient.

The insert or insert system of the cell transplantation device is designed to fit into a chamber in a porous frame. The liner or liner system may contain a non-porous material (eg, polytetrafluoroethylene (PTFE), polypropylene, etc.) that inhibits the ingrowth of biological tissue into the liner or liner system. The liner or liner system may be a hollow or one piece design. However, if a hollow liner is used, care should be taken to prevent collagen or any other biological material from infiltrating the liner cavity when the device is implanted in the host tissue. The insert system will be discussed in more detail below.

In some embodiments, the proximal end of the liner or liner system is connected to a seal. In such an embodiment, the seal is configured to close the proximal end of the chamber when the liner or liner system is fully inserted into the porous scaffold chamber. The seal is configured to hold the liner or liner system in place within the porous frame. In another embodiment, the seal is separate from the liner or liner system. In another embodiment, the seal is connected to a porous frame. In addition, in some exemplary embodiments, the proximal end of the chamber is closed using surgical sutures and/or vascular clips without the use of a separate sealant.

Once implanted in the host body, the porous scaffold of the device promotes ingrowth of vascular or connective tissue such that the insert or insert system located in the scaffold becomes encapsulated in a vascularized tissue matrix. When the insert or insert system is removed from the porous scaffold, a neovascularized chamber is formed in the device, which can then be used to retain the cellular material in the host.

The dimensions of the porous scaffold and the liner or liner system are sized to provide an optimal surface area to volume ratio for in vivo cell containment and to ensure long cell survival in the neovascularized chamber. Similarly, the number of chambers in the transplant device is determined based on the volume and/or number of cells to be transplanted. In some embodiments, the overall volume of the cell transplant device is adjusted by increasing or decreasing the number of chambers while maintaining an optimal surface area to volume ratio for each individual chamber. In other embodiments, the length of the chambers is adjusted to change the total volume. Alternatively, in various manufacturing embodiments, the cell transplant device contains a fixed number of chambers, however, cells are infused into only a selectable number of chambers, depending on the total required volume of the device. In other embodiments, both the length of the chambers and the number of chambers are adjusted to change the total volume required.

The described cell transplant device can be implanted either subcutaneously or intraperitoneally into the host body, including the omentum, or other suitable site. Alternatively, the described cell transplantation device can be implanted partially intraperitoneally into the host body, including the omentum, or other suitable site, and continue in the subcutaneous environment. In one embodiment, the cells may be loaded onto a portion of the device extending into the subcutaneous environment while the remainder of the device resides in the intraperitoneal environment. In another embodiment, the cell transplant device can be implanted in the brain, spinal cord region, or any other organ as needed to obtain a therapeutic effect from the transplanted cells. In most cases, the host organism is a human organism, but may also be other mammals or non-mammals. The cell transplantation procedure is a two-step process comprising a device implantation step followed by a cell infusion (cell transplantation) step. The cell infusion step occurs after an in vivo incubation period during which the vascularized collagen matrix infiltrates into the implanted device. In one embodiment, the incubation period is approximately thirty days, which is sufficient time for angiogenesis and collagen infiltration into the porous scaffold. The incubation period can be extended or shortened depending on the required or

- 6 040824 Degree of neovascularization or tissue formation (collagen and cells). For example, vascularization of transplant devices may proceed at different rates depending on device material, dimensions, or coatings available such as antibiotic coatings, growth factors, and so on. Vascularization of transplant devices can also occur at different rates in different host organisms or in different tissues of the same host organism. Determining the appropriate incubation period is within the skill of the art. For example, prior to cell delivery, imaging studies may be performed to ensure that sufficient vascular and/or connective tissue deposition has occurred around and within the walls of the porous scaffold during the incubation period. To perform the cell infusion step, a surgical incision provides access to the implant site, after which the liner or liner system is removed from the porous scaffold to form a pocket lined with collagen and blood vessels in the scaffold. Next, the cellular material is delivered to the vascularized pocket, after which the porous scaffold is recompacted. In another embodiment, the cell transplantation procedure is a one-step process in which device placement and cell implantation occur at the same time. In this case, the cells may be placed in the matrix so that they do not leak through the pores of the device, or alternatively, the device may be coated with a degradable polymer to prevent cell leakage from the device during collagen development and angiogenesis.

In some embodiments, the cells to be transplanted may be combined with a biocompatible viscous solution or biodegradable polymeric material prior to being loaded into the chamber of any of the transplant devices provided herein. The biodegradable polymer will provide cell protection until the device is fully vascularized in the host. These materials may be placed in the chamber before or after the device has been placed in the host, but before the collagen matrix and vascular structures have formed in the device. Cells combined with a biocompatible viscous solution or biodegradable polymeric material are particularly useful in devices capable of being loaded with cells prior to implantation of the device into the host. Representative polymers that can be used as biodegradable materials with cells include, but are not limited to, polyethyleneimine and dextran sulfate, polyvinylsiloxane-polyethyleneimine copolymer, phosphorylcholine, polyethylene glycol, lactic acid-glycolic acid copolymer, polylactic acid, polyhydroxyvalerate and its copolymers, polyhydroxybutyrate and its copolymers, polydioxanone, polyanhydrides, polyamino acids, polyorthoesters, polyesters, collagen, gelatin, cellulose polymers, chitosans, alginates, fibronectin, extracellular matrix proteins, vinculin, agar, agarose, hyaluronic acid, matrigel, and combinations thereof.

It should be noted that the cells can be placed in the device; however, cells can also be encapsulated. The following description is provided by way of example, but not in a limiting sense. Examples of polymeric systems for cell encapsulation include alginate shell encapsulation, polysaccharide hydrogels, chitosan, calcium or barium alginate, altinate and polylysine layered matrix, photopolymerizable polyethylene glycol polymer for single cell or cell cluster encapsulation, polyacrylates, including hydroxyethyl methacrylate, methyl methacrylate, silicone capsules, silicone nanocapsules, as well as polymembranes (copolymer of vinyl chloride with acrylonitrile).

In FIG. 1A-1E show various examples of embodiments of the cell transplant device 1. The device 1 contains a polymer mesh (eg polypropylene mesh, PTFE mesh or any other suitable material) forming a porous chamber 2 for containing cells in the host organism. In some embodiments, device 1 may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more porous chambers 2. The availability of multiple chambers allows any number or combination of chambers to be used, depending on the required volume of cellular material, the determination of which is within the competence of a person skilled in the art.

As shown in FIG. 1A, device 1 comprises a proximal end 3, a distal end 4, and an insert 5 placed in a porous chamber 2. In one embodiment, the porous chamber 2 is tubular in shape, with the insert 5 being cylindrical and extending along the cavity of the porous chamber 2 In another exemplary embodiment, the porous chamber 2 includes an opening at the proximal end 3. The opening at the proximal end 3 is configured to allow insertion of the liner 5 and retraction from the porous chamber 2. In one such exemplary embodiment, the opening at the proximal end 3 has a seal using surgical sutures and/or vascular clamps during device incubation and after cell infusion into the device. One of ordinary skill in the art will appreciate that any other surgical sealing element, such as microvascular clips, clamps, etc., can be used to seal the opening at the proximal end 3. In another embodiment, the device 1 includes a non-porous valve 6 at the proximal end 3 as shown in FIG. 1B. In one such manufacturing example, the valve 6 is made of silicone. Valve 6 can be sealed using a surgical

- 7 040824 sutures, clips, or any other suitable sealing mechanism during device incubation and after cell infusion into the device. In one exemplary embodiment, the distal end 4 of the device 1 comprises a rounded or flat bottom surface. In another embodiment, the device 1 includes an opening at the distal end 4 that can be sealed using surgical sutures, clips, or any other surgical sealing element during device incubation and after cell infusion into the device. In yet another example of a manufacturing embodiment, as shown in FIG. 1C, the distal end 4 includes a non-porous region 7 to prevent tissue ingrowth at the distal end of the device and to facilitate removal of the insert 5 from the device prior to cell infusion.

In some embodiments, as shown in FIG. 1D, the proximal end of the liner 5 is connected to the seal 8. In one such embodiment, the seal 8 is configured to close the opening at the proximal end 3 when the liner 5 is inserted into the chamber 5. The seal 8 is configured to hold the liner 5 in place within the porous chamber. . In another embodiment, the liner 5 of the long porous chamber 2 functions as a seal both at the proximal end 3 and at the distal end 4, as shown in FIG. 1E. The edges of the porous chamber 2 around the insert 5 are sealed using surgical sutures and/or surgical adhesive. After the insert 5 is removed prior to cell infusion, the openings at the proximal end 3 and distal end 4 may be sealed using surgical sutures, vascular clamps, or any other suitable sealing mechanism, as will be appreciated by one of ordinary skill in the art.

In some exemplary embodiments, device 1 comprises a plurality of porous chambers 2 that are laterally connected to one another. In one such manufacturing embodiment, a plurality of porous chambers 2 are formed, for example, by ultrasonic welding of the upper and lower surfaces of the porous material along a line substantially parallel to the longitudinal axis of the device. In FIG. 1F shows a cell transplant device having eight porous chambers 2. An insert 5 is placed in each chamber 2 during the incubation step of the device. Inserts 5 are removed from chambers 2 prior to infusion of cells into the chambers. In one embodiment, the device 1 contains eight porous chambers and has a total length of 50 mm by a width of 45 mm. Each porous chamber 2 has an internal diameter not exceeding 2.5 mm and accommodates an insert 5 which is approximately 40 mm long and 2.5 mm in diameter. In one such embodiment, the liner 5 is made from a non-porous biocompatible material such as polytetrafluoroethylene (PTFE).

In exemplary embodiments, the cell transplant device of the present disclosure is formed from polypropylene meshes suitable for medical use, such as propylene knitted mesh (PPKM) purchased from SURGICALMESH™, Brookfield, CT, USA. In exemplary embodiments, meshes are made from monofilament yarns with a diameter of 0.1 to 0.3 mm, with mesh pore sizes ranging from 0.3 to 1 mm, from 0.4 to 0.85 mm, and also from 0.5 up to 0.6 mm. In FIG. 2A-2D show various examples of mesh configurations that can be used to form cell transplant devices. In FIG. 2A shows a polypropylene mesh (PPKM601) with a pore size of 0.5 mm and a monofilament yarn thickness of 0.3 mm; in fig. 2B shows a polypropylene mesh (PPKM602) with a pore size of 0.53 mm and a monofilament yarn thickness of 0.18 mm; in fig. 2C shows a polypropylene mesh (PPKM404) with a pore size of 0.53 mm and a monofilament yarn thickness of 0.13 mm; and in fig. 2D shows a polypropylene mesh (PPKM604) with a pore size of 0.85 mm and a monofilament yarn thickness of 0.2 mm.

In FIG. 3A shows another exemplary embodiment of a cell transplant device 10. In FIG. 3B shows the components of a cell transplant device 10. The device 10 comprises a porous frame 12, a primary seal 14, at least one liner system comprising an outer liner 16 and an inner liner 18, and a secondary seal 20.

As shown in FIG. 4, the porous frame 12 of the cell transplant device 10 may comprise a polymeric mesh (eg, polypropylene mesh, PTFE mesh, or any other suitable material) defining one or more porous chambers 22 for containing cells in the host body. In some embodiments, the porous frame 12 may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more porous chambers 22.

The availability of multiple chambers allows any number or combination of chambers to be used depending on the volume of cell material required, the determination of which is within the skill of the art.

The porous chambers 22 can be created, for example, by joining the top and bottom surfaces of the porous frame 12 along a line substantially parallel to the longitudinal axis of the device. The plurality of porous chambers 22 may have the same or different cross-sectional dimensions and surface areas. In one embodiment, a plurality of porous chambers 22 is formed by ultrasonic welding of a polymer mesh from the proximal end 24 to the distal end 26 of the scaffold. The top and bottom surfaces of the porous scaffold 12 are continuous across one or more porous chambers 22, with continuity broken only by ultrasonic weld lines 28 extending substantially parallel to the longitudinal axis of the porous scaffold 12.

- 8 040824

The top and bottom surfaces may be slightly depressed at each weld line, which provides additional surface area for vascularization and physical stability of the device 10 in the host. In one embodiment, the edges at the distal end 26 are tapered and joined together by ultrasonic welding to seal the distal end 26.

As shown in FIG. 3B, primary seal 14 is configured to seal one or more porous chambers 22 during device incubation and after cell infusion. The primary seal 14 contains an inert biocompatible polymer film or any other suitable material. In one embodiment, the primary seal 14 is ultrasonically welded at the lateral edges and the tapered proximal end 31 as shown in FIG. 5A and 5B. The distal end 32 of the primary seal 14 is attached to the proximal end 24 of the porous frame 12. In one embodiment, the distal end 32 is ultrasonically welded to the proximal end 24 of the porous frame 12.

In various embodiments, the primary seal 14 includes a resealable seal 34 to assist in maintaining at least one outer liner 16 in the porous chamber 22 during the incubation period. The closure 34 also prevents leakage of cellular material during the cell infusion process. The closure 34 may be any suitable resealable closure mechanism. In one embodiment, the shutter 34 includes interlocking elements in the form of a groove and a ledge that form an airtight seal when compressed together and open when the upper and lower surfaces of the seal 14 are separated at the proximal end 31. After the incubation period of the device, the outer liner 16 is accessed by cutting the proximal end 31 of the primary seal 14 and opening of the resealable closure 34. After the cellular material has been delivered to the porous scaffold 12, the closure 34 is reclosed and the proximal end 31 is resealed using, for example, surgical sutures, staples or bioadhesives, or airtight seals.

The number of insert systems may correspond to the number of porous chambers 22 in the cell transplant device 10. The outer liner 16 is placed in the porous chamber 22 during the incubation period of the device. In some embodiments, the length of the outer liner 16 is approximately equal to the length of the corresponding porous chamber 22. As shown in FIG. 6A, in one embodiment, a plurality of outer liners 16 are attached at the proximal end 40 using a common core 42. The common core 42 may include one or more grooves 43 to assist in the removal of the outer liners 16 from the porous chambers 22. For example, the grooves 43 may allow the common core 42 to be grasped using forceps. In some embodiments, outer liner 16 has a hollow core 45 that receives inner liner 18. As shown in FIG. 6B, in one embodiment, the hollow core 45 is provided with restrictive internal projections 47 along the length of the inner surface of the liner. The inner projections 47 create an air space between the outer liner 16 and the inner liner 18 to allow trapped air bubbles to escape during the delivery of cellular material, which will be described in more detail below. The air space also prevents the formation of a vacuum during removal of the inner liner 18, thereby maintaining the integrity of the newly formed vascularized collagen matrix in and around the porous chamber. Thus, in some aspects, an liner system comprising an outer liner 16 and an inner liner 18 may facilitate the delivery of cells to the cell transplant device 10 and may also increase the chances of cells surviving in an intact collagen matrix.

In some embodiments, the proximal end 40 and distal end 41 of the outer liner 16 include sealing mechanisms, such as internal grooves or tapered surfaces, to provide effective sealing with the inner liner 18. As shown in FIG. 7, the proximal end 50 and distal end 51 of the inner liner 18 may include additional sealing mechanisms 53 to prevent infiltration of the collagen matrix into the hollow core 45 during the incubation period. For example, in one embodiment, the seal mechanism 53 includes a groove extending along the periphery of the proximal and distal ends of the inner liner 18, while the outer liner 16 includes a protrusion along the periphery of its distal and proximal ends. In this embodiment, the protrusion on the outer liner 16 and the groove on the inner liner 18 are interlocked when the inner liner 18 is inserted into the hollow core 45 of the outer liner 16, so as to form a complete seal between the inner and outer liner and prevent any biological material from entering the hollow core 45. In addition, in such embodiments, if the outer liner 16 includes one or more inner ridges 47, the height of the ridges at the proximal and distal ends of the outer liner 16 may exceed the height of the inner ridges 47.

In FIG. 6C and 6D show sectional views of the porous chamber assembly 22 and liners 16, 18 according to one embodiment of the present disclosure. In FIG. 6C shows a sectional view of the assembly prior to implantation in the host, and FIG. 6D shows a sectional view of the assembly

- 9 040824 nodes after incubation in the host organism. The inner diameter of the porous chamber 22 and the outer diameter of the outer liner 16 are selected to maintain a clearance 46 around the periphery of the outer liner 16 for tissue formation. For example, in one exemplary embodiment, the inner diameter of the porous chamber 22 does not exceed 4.5 mm and the outer diameter of the outer liner 16 does not exceed 3.5 mm. In another embodiment, the inner diameter of the porous chamber 22 does not exceed 3.5 mm and the outer diameter of the outer liner 16 does not exceed 2.5 mm. In these embodiments, for example, a gap of about 0.5 mm is provided around the outer liner 16 to form a vascularized collagen matrix. The gap around the outer liner 16 also creates sufficient space for insertion and removal of the outer liner into and out of the porous chamber.

When the cell transplant device 10 is implanted in the host body, vascular and connective tissues penetrate through the porous chamber 22 into the space 46 and form a vascularized tissue matrix 48 around the outer liner 16. The liner 16 prevents the tissue matrix 48 from penetrating deeper into the cavity of the porous chamber 22. After removal inner liner 18 and outer liner 16 from the porous chamber 22, a pocket 49 is formed in the porous chamber 22, which can be used to contain cells in the host organism. The pocket 49 is enclosed in a vascularized tissue matrix 48 as shown in FIG. 6E.

The number of inner liner 18 may correspond to the number of outer liner 16. The inner liner 18 is placed in the hollow core of the outer liner 16 during the device incubation step. In one embodiment, a plurality of inner bushings 18 are connected at the proximal end 50 using a common core 52. In some embodiments, the common core 52 includes a gripping element 54 to facilitate manipulation of the inner bushing 18 during removal from the outer bushing 16.

The secondary seal 20, as shown in FIG. 8 is configured to contain cellular material in the porous chambers when the primary seal is re-closed after delivery of the cellular material to the cell transplant device 10. The secondary seal 20 is positioned at the proximal end 24 of the porous scaffold 12 after the cellular material has been fully delivered into the porous chamber 22 and the outer liner 16 has been removed from the device 10. In some embodiments, the secondary seal 20 includes notches 60 to facilitate insertion into the device 10 with using forceps.

In another aspect of the present disclosure, a device and a method for delivering cells to a cell transplant device are described, which will be explained with reference to the cell transplant device 10. In FIG. 9A shows various components of the cell delivery device 70. The cell delivery device 70 includes at least one cell infusion tube 71, a connector cover 72 having a gripping member 73, and a connector spacer 74.

The cell infusion tube 71 may comprise a polymeric tube (eg, a polyethylene tube) or any other suitable material for delivering cellular material into the porous chamber 22 of device 10 during the cell infusion step. The number of cell infusion tubes in the delivery system may correspond to the number of porous chambers 22.

A connector spacer 74 is located at the distal end of the cell infusion tube 71 and connects or creates an interface with the proximal end 40 of the outer liner 16 during the cell delivery process. The connector spacer 74 includes one or more through holes through which the cell infusion tube 71 is inserted, as shown in FIG. 9A. The through holes are configured to fit the cell infusion tube 71 with a slight interference. The landing is configured to keep the cell infusion tube 71 in place during the cell delivery process. In addition, in certain embodiments, the connector spacer 74 includes vents 76 to vent air spaces in the outer liner 16 defined by the inner lip 47 during the cell delivery process, as will be described below. In one embodiment, the outer liner 16 has a sleeve 78 at the proximal end 40. In this embodiment, the connector spacer 74 is inserted into the sleeve 78 during the cell delivery process to attach the delivery device 70 to the cell transplant device 10.

The proximal end of the tube 71 for infusion of cells contains a cover 72 of the connector. As the tube is inserted into the outer liner 16, the connector cover 72 advances distally towards the connector spacer 74. When the tube 71 is fully inserted into the outer liner 16, the connector cap 72 is pushed onto the connector spacer 74 and/or sleeve 78, with the gripping member 73 engaging with the outer liner 16 and/or sleeve 78 along the common body 42 as shown in FIG. 9C. This allows the connector cap 72, connector spacer 74 and outer liner 16 to be removed as a single unit when the cellular material is infused into the porous chamber 22.

In a further aspect of the present disclosure, a method for cell transplantation is provided, the explanation of which will be given with reference to the cell transplant device 10 and the cell delivery device 70. The cell transplantation method is not limited to the device embodiments disclosed herein and can be used with any graft devices.

- 10 040824 cells and cell delivery.

In FIG. 10 is a flowchart showing the steps in a typical cell transplantation procedure.

The cell transplantation procedure is generally a two-step process comprising a device implantation step followed by a cell infusion step. Device 10 is implanted into the host prior to cell delivery to allow sufficient time for collagen and blood vessels to infiltrate porous scaffold 12. In some embodiments, device 10 is sterilized using ethylene oxide prior to implantation. The device 10 may be packaged in a self-sealing bag or any other sterilizable packaging along with a sterility test strip for the ethylene oxide sterilization process. In some other embodiments, gamma irradiation or oven sterilization is used to sterilize the device prior to implantation. The sterilization method used depends on the material of the frame, since oven sterilization is known to deform certain polymeric materials (eg polypropylene) due to low heat deformation resistance. Gamma radiation at sterilization doses of 6 Mrad can successfully sterilize devices intended for cell implantation; however, gamma radiation can reduce the shelf life of devices made from polypropylene.

Device 10 may be implanted subcutaneously or intraperitoneally. For example, to implant the device subcutaneously into the host body, an incision is made through the dermis and epidermis, followed by gentle blunt separation of connective tissue and adipose tissue to form a subcutaneous pocket caudal to the incision line (step 810). When sufficient space has been created (approximately the size of the device), the device 10 is implanted into the subcutaneous pocket, after which the incision is sutured (step 820). Alternatively, device 10 may be implanted into the peritoneal cavity through an abdominal incision. The device implantation steps (steps 810 and 820) are followed by a device incubation period (step 830) during which a vascularized collagen matrix is deposited in and around the porous scaffold 12.

After the incubation period, the device 10 is accessed through a second surgical incision. For example, the proximal end 31 of the primary seal 12 may be cut in situ to open the device 10. The inner liner 18 is then removed from the outer liner 16 and discarded (step 850). During the removal of the inner liner, the inner projections 47 assist in creating air movement to prevent the formation of a vacuum within the device that could cause rupture of newly formed blood vessels in and around the device. Removal of the inner liner 18 unhooks the proximal end 50 and distal end 51 of the inner liner 18 from the proximal end 40 and distal end 41 of the outer liner 16. The cellular material is then delivered to the device 10 using the cell delivery device 70.

In FIG. 11A-10 show a schematic overview of certain steps of a typical cell infusion procedure, which will be explained with reference to the flowchart shown in FIG. 10. To deliver cells to the device 10, the cell infusion tube 71 of the delivery device 70 is loaded with cell material 79, after which the tube is inserted into the hollow core 45 of the outer liner 16 as shown in FIG. 11A (block 860). The connector spacer 74 connects to the proximal end 41 and/or sleeve 78 of the outer liner 16. As the tube 71 advances into the outer liner, air is expelled through the inner lugs 4 7 of the outer liner 16 and the vents 76 of the connector spacer 74. When the tube 71 is fully pushed into the outer liner 16, the connector cover 72 is mated with the connector spacer 74. Clip 73 of connector cover 72 is then connected to collar 78 of outer liner 16 (step 870). In this case, the outer liner 16, connector cap 72, and connector spacer 74 are retracted slightly back from the porous chamber 22 as a single unit to form a space at the distal end of the porous chamber 22 (step 875). In some embodiments, the outer liner 16 may be retracted slightly from the porous chamber 22 before the delivery device 70 is connected to the outer liner 16. In other words, step 875 may be performed prior to step 870. On a syringe attached to the cell infusion tube 71, a light pressure to deliver the cells to the porous chamber 23 (step 880). Care is taken to ensure that the tube 71 remains in the porous chamber 22 when pressure is applied to deliver the cellular material. In one embodiment, the outer liner 16 is retracted approximately 5 mm prior to cell infusion, as shown in FIG. 11B. When pressure (P) is applied to the syringe connected to the cell infusion tube 71, the cell material 79 is infused into the porous chamber 22. When the cell material is delivered to the porous chamber 22, the outer liner 16 and the cell infusion tube 71 are removed from the device, as shown in FIG. 11C and 11D (step 885). When the device is completely filled with cell material 79, the cell infusion is stopped and the cell infusion tube 71 is completely withdrawn from the device 10 (step 890). Next, the porous chamber 22 is evaluated for remaining volume for cellular material, after which the remaining cellular material can be carefully added to the end of the porous chamber. cell

The material is retained in the porous chamber 22 by placing the secondary seal 20 at the proximal end 40 of the porous chamber 22, followed by closing the resealable closure 34 of the primary seal 12 and fixing the proximal end 31 of the primary seal 12 with surgical sutures or staples, or other suitable sealing mechanisms (step 895). Finally, the surgical incision is closed using surgical sutures, staples, or tissue glue, thereby completing the cell transplantation procedure.

The disclosed cell transplant devices and methods can be used to transplant any therapeutic cells or cell combinations into a host to deliver a therapeutic biological material to the host to treat a disease. The cells may be allogeneic, xenogenic or syngeneic donor cells, cells obtained from the patient, including stem cells, cord blood cells and embryonic stem cells. Stem cells can be converted into appropriate therapeutic cells. The cells may be immature, partially differentiated or fully differentiated, as well as mature cells when placed in the device. The cells may also be genetically engineered cells or cell lines. In one aspect, a manufacturing embodiment consistent with the present disclosure is used to transplant islet cells of Langerhans to provide a means for regulating blood glucose levels in the host. In another aspect, one embodiment of a cell transplantation device is used for co-transplantation of islets of Langerhans and Sertoli cells, wherein the Sertoli cells provide immune protection for the islet cells in the host. Immune protection provided by Sertoli cells in the host organism has been previously disclosed, for example, in US patent No. 5725854, which is fully incorporated into the present description by reference. Accordingly, the subject of this disclosure is also methods of treating various diseases by transplanting therapeutically relevant amounts of cells into subjects that need them, using an embodiment of a cell transplant device according to the present description.

The concentration of transplanted therapeutic cells or combinations of cells is determined based on the body weight of the host and the therapeutic effect of the cells. As noted earlier, the dimensions of the cell transplant device and the number of porous chambers used (in a multi-chamber device) are determined based on the number of cells required, the degree of vascularization that can be achieved during the incubation period of the device, and the diffusion characteristics of the nutrients and waste products of the cells, in and out of implanted devices.

Examples

The following examples are presented to illustrate various manufacturing options and should in no way be construed as limiting the scope of the present disclosure. The cell transplant devices used in these examples are made of polypropylene meshes and contain a single PTFE liner in each porous chamber of the devices.

1. Cell transplant devices containing islet cells are able to restore normoglycemia in Lewis rats.

Cell transplantation devices have been used to implant syngeneic islet cells in Lewis rats to restore normoglycemia. The change in glucose content during cell implantation was compared with the change in glucose content when islet cells were injected directly into the portal veins of rats. Lewis rats were divided into three study groups, nine rats in each group. In the first and second study groups, the devices were implanted in the intraperitoneal and subcutaneous cavities, respectively. In the third group, islet cells were injected directly into the portal veins.

The implanted devices were incubated in Lewis rats for at least one month to allow vascular germination. Next, the rats were chemically induced diabetes by injection of streptozotocin. Rats were considered to be diabetic if three consecutive blood glucose readings were at least 18.0 mM. Next, the isolated islet cells of Lewis rats (10,000 IEQ/kg body weight) were infused into implanted devices or directly into the portal veins of diabetic rats. Insulin pellets were removed 14 days after islet transplantation (denoted by closed boxes above the graphs in Figs. 11A and 11B). Blood glucose levels in rats were monitored for 100 days. At 100 days post-transplant, the devices were removed to confirm that the transplanted islets were responsible for reversing the diabetes.

In FIG. 12A and 12B show the results of glucose normalization in rats in which cell transplantation devices were implanted, respectively, intraperitoneally (into the omentum) and subcutaneously. Successful cell transplantation resulted in normalization of blood glucose (glucose readings were less than 8.0 mM) as indicated by solid lines. Transplantation cases in which normoglycemia was not achieved are indicated by dotted lines. The results show that normal glycemic levels were maintained in statistic

- 12 040824 a clinically significant number of diabetic rats injected with islet cells. Following removal of the implanted devices 100 days post-transplant, rats showing normal glycemic levels reverted to hyperglycemia, indicating that the devices contained a fully functional graft responsible for achieving normoglycemia prior to device removal. The rate at which blood glucose reached non-diabetic levels was statistically different between study groups (p<0.0001, t-test).

In FIG. 12C shows IVGTT (intravenous glucose tolerance test) responses in Lewis rats that have undergone islet cell transplantation. Intravenous glucose tolerance tests were performed 40 and 80 days after transplantation. The change in glucose in rats during intraperitoneal and subcutaneous transplantation was compared with the change in glucose in rats, which islet cages were injected intraportally. Intravenous glucose tolerance tests were performed on three rats in each category studied. At 40 and 80 days post-transplantation, blood glucose levels in islet-transplanted rats decreased below 8.0 mM within 50 minutes of receiving a glucose load, as shown in FIG. 12C. Cell transplant devices were removed after 100 days. After the glucose bolus on day 110, there was no decrease in blood glucose, indicating that the transplanted islet cells are responsible for the normoglycemia achieved in diabetic rats prior to removal of the implanted devices.

In FIG. 12D shows the change in insulin levels in Lewis rats transplanted with islet cells. Insulin levels were determined based on enzyme-linked immunosorbent assay (ELISA). The analysis was carried out three times. The results showed a significant difference in blood insulin levels in response to glucose loading (p<0.005, t-test). As shown in FIG. 12D, insulin levels in device-transplanted rats correlated well with insulin levels in rats receiving intraportal islet cages.

2. Histological detection of insulin and vascularization in porous chambers of cell transplantation devices

Following removal of the implanted devices one hundred days later, insulin was detected in the devices using specific primary anti-insulin antibodies. In FIG. 13A shows the results of insulin staining in a porous chamber of a subcutaneously implanted device. Insulin detection in the chamber indicates that the islet cells contained in the device are viable and functional 100 days after transplantation.

Histological evaluation of the implanted devices was also performed to confirm the formation of vascular tissue in the collagen matrix deposited in and around the devices. Immunohistochemical staining for Factor VIII-related endothelial cell antigen indicates a well-formed vascular structure deeply embedded in the connective tissue, as shown in FIG. 13B (dark structure indicates endothelium; cell nuclei indicated by arrows). Histological evaluation also indicates penetration of neovascularized tissue towards the core of the cell transplantation devices.

3. Evaluation of angiogenesis and collagen deposition in cell transplant devices

To determine the proper duration of the implantation phase (time between device implantation and islet cell engraftment), cell transplantation devices were subcutaneously implanted in 8-month-old Yorkshire Landrace pigs at 2, 4, and 8 weeks. After implantation for an appropriate period of time, the devices were explanted and examined to determine the level of angiogenesis and collagen deposition.

a) General assessment of angiogenesis and collagen deposition.

For a general analysis of the formation of blood vessels and tissue, photographs of the ventral and dorsal surfaces of the explanted devices were taken. To quantify the formation of microvessels and tissue (collagen and cells), a 1x1 cm grid was superimposed on the photographs. Each 1 ^cm2 square of the grid was taken into account to assess vessel formation, which made it possible to calculate the total number of vessels/ ^cm2 for the entire area of explanted devices. To assess the amount of collagen deposition, the average thickness was measured along the medial and lateral outer boundaries of the devices. In FIG. 14 shows a table of average collagen thickness and total blood vessels/cm ² calculated for four devices formed using different porous materials (mesh). Two weeks after implantation, sufficient formation of microvessels and tissues was observed for all four types of meshes. The results also showed that the amount of time required for microvessel formation and collagen deposition may vary depending on the material of the device (porosity, surface roughness, etc. of the meshes).

b) Histological analysis of angiogenesis and collagen deposition.

The degree of angiogenesis was determined by staining endothelial cells with hematoxylin and eosin (H&E) (Fig. 15A) and von Willebrand factor (Fig. 15B). In FIG. 15A shows tissue incorporation into devices 2, 4 and 8 weeks after implantation. In FIG. 15B shows the formation of blood vessels on various faces of the device prior to cell transplantation. Incorporation score

- 13 040824 tissues into devices showed that there was incorporation of collagen and microvessels in the devices at each time point of measurements prior to islet transplantation.

4. Evaluation of cell transplantation devices with autotransplanted porcine islet cells.

Eight-month-old Yorkshire Landrace pigs were implanted with cell transplant devices for four and eight weeks. To induce diabetes in animals, a 90% pancreatectomy was performed followed by an intravenous dose of streptozotocin 150 mg/kg the day after surgery. Before performing pancreatectomy, the islets were isolated from the pancreas. Animals were transplanted with an islet graft with immature cells five days after graft isolation and pancreatectomy to allow sufficient time for recovery and confirmation of diabetes.

Prior to transplantation, the insulin-producing abilities of immature islet cells were tested. As shown in FIG. 6, immature islet cells produced about 10% of the insulin normally expected from mature islet cells. This fact, combined with the low number of transplantable islets, around 3-5K IEQ/kg (5-10% of the insulin-producing islet cells commonly used in intraportal transplantation), creates a tough test environment for cell transplantation devices. Currently, in clinical therapy based on islet transplantation, adequate infusion of β-cells is an obstacle in the treatment of insulin-dependent diabetes. Insulin independence is usually achieved by delivering a sufficient number of islet cells, approximately 10,000 IEQ/kg body weight of the recipient. To provide this number of islet cells, according to current protocols for islet transplantation, more than one donor pancreas is required per recipient, which creates a shortage of already limited donor material. Thus, if glycemic control can be achieved using only 5-10% of the islet cells that are currently used in intraportal transplantation, the number of diabetic patients who can be therapeutically treated based on islet cell transplantation will increase significantly.

To test the long-term survival and function of the transplanted islets, histological analysis of the explanted devices was performed. The performance of the islet grafts was also monitored by biweekly blood glucose measurements and bimonthly intravenous glucose tolerance tests (IVGTT).

a) Histological analysis of the functioning of islet grafts.

After explanting the devices, 9 weeks later, insulin was detected in the devices using specific primary antibodies to insulin. In FIG. 17A shows the results of insulin staining in the porous chamber of the explant device. Insulin detection in the chamber indicates that the islet cells contained in the device are viable and functional 9 weeks after transplantation. Immunohistochemical staining of explant sections showed healthy, well-configured islets surrounded by robust microvessels (Figure 17B; microvessels are marked with arrows).

b) Glucose measurements.

To monitor the functioning of islet grafts, weekly fasting and non-fasting blood glucose measurements were performed following transplantation. These measurements helped to determine the overall effectiveness of cell transplantation devices in long-term monitoring of blood glucose. Fasting blood glucose measurements provided a monitored measure of graft function. Briefly, a drop of blood (several microliters) was taken from the vein of the recipient animal, after which the blood glucose level was determined using a Freestyle Lite glucometer or other glucose testing device.

As shown in FIG. 18, transplanted islets demonstrated long-term glucose control up to device explantation at 72 days. Animals in the glycemic control group (n=4) were non-insulin dependent, with blood glucose controlled only by islets in cell transplantation devices. Animals in this group showed long-term insulin independence after islet transplantation. However, some animals remained hyperglycemic (elevated daily blood glucose) after islet transplantation into devices (n=6). This was due to the poor metabolic quality of the islet grafts and the low dose of transplanted islet cells (IEQ/kg). Islet quality before transplantation correlated well with long-term islet function.

c) Glucose tolerance test.

Glucose tolerance tests are important in evaluating the performance of islet grafts by comparing IVGTT results before and after transplantation. To test the efficiency of cell transplantation devices, IVGTT tests were performed prior to pancreatectomy (baseline group) at various time points after islet transplantation into the devices, as well as after

-

Claims (29)

device scheduling. IVGTT tests were performed by administering a dose of dextrose and measuring the time it takes for endogenous insulin to bring glucose levels into line with the original group. In addition to blood glucose measurements, blood samples were taken at various time points to measure the content of C-peptide, which is a by-product of insulin synthesis by β-cells. IVGTT test results were interpreted based on absolute blood glucose values (FIG. 19A), area under the curve (AUC) of blood glucose (FIG. 19B), and fold change in C-peptide level (FIG. 19C). As shown in FIG. 19A and 19B, glucose increased significantly (p<0.001, by ANOVA) after explantation of the device, indicating that removal of the device results in a cessation of insulin function similar to diabetic animals that lack islet cells. Although the lowest glucose levels were found in animals not undergoing pancreatectomy, insular autograft recipients showed a significant decrease in glucose levels after dextrose injection, indicating that immature islet cells are able to survive and function after transplantation. Serum samples associated with IVGTT tests were analyzed using a porcine C-peptide radioimmunoassay kit (Linco), which uses an antibody designed specifically for testing synthetic porcine C-peptide. Serum samples 0, 5, 15, 30, 60 and 120 minutes after dextrose injection were examined for the presence of porcine C-peptide. Four groups were tested: non-pancreatectomy pigs (baseline group), islet autograft recipients (after islet transplantation), autograft recipients whose devices had been removed (after device removal), and diabetic control pigs. In the study of the fold change in the level of C-peptide among different study groups in the original group and in recipients after islet transplantation, the results were quite comparable, although the level of C-peptide in recipients after islet transplantation increased after 60 minutes, in contrast to the initial group, in which the increase had place after 30 minutes (Fig. 19C). In addition, the fold change in C-peptide level in the post-device removal group and the diabetic control group was similar, indicating that the transplanted islets were responsible for C-peptide release prior to device removal. Other embodiments of the invention will become apparent to those skilled in the art upon examination of the description and practice of the invention disclosed therein. The description and examples are to be regarded as illustrative only, the true scope and spirit of the invention being defined by the following claims. CLAIM 1. A device for implanting cells into a host organism, containing a porous frame containing a network of immunologically compatible polymeric material forming the walls of at least one chamber, where the chamber contains an opening at the proximal end or at the distal end, or both at the proximal end and at the distal the end of the chamber, while the proximal end and the distal end are separated by a space that is limited by the walls, and while the porous framework has pores configured to promote the germination of vascular and connective tissues around the walls and through the walls of at least one chamber; at least one seal configured to close the proximal or distal end or both the proximal and distal end of the chamber; at least one removable liner configured to accommodate at least one chamber in the cavity, with the liner extending along the cavity. 2. The device of claim 1, wherein the device further comprises a biocompatible, biodegradable material that temporarily fills the scaffold pores and temporarily prevents growth of vascular and connective tissue into the lumen of at least one chamber. 3. The device of claim 1, wherein at least a portion of the porous scaffold is coated with one or more of growth factors, an anti-fibrotic agent, and/or compounds to stimulate angiogenesis and incorporate tissue into at least one porous chamber. 4. The device of claim 3, wherein one or more of the compounds for stimulating angiogenesis and tissue incorporation comprises vascular endothelial growth factor (VEGF), collagen, fibronectin, cytoskeletal membrane proteins, polyethyleneimine and dextran sulfate, polyvinylsiloxane-polyethyleneimine copolymer, phosphorylcholine, polyethylene glycol, copolymer of lactic and glycolic acids, polylactic acid, polyhydroxyvalerate and its copolymers, polyhydroxybutyrate and its copolymers, polydioxanone, polyanhydrides, polyamino acids, polyorthoesters, polyesters, gelatin, cellulose polymers, chitosan, alginates, extracellular matrix proteins, vinculin, agar, agarose, hyaluronic acid, matrigel or combinations thereof. - 15 040824 5. A device according to any one of the preceding claims, wherein at least one seal is a polymer film that is ultrasonic welded to the porous frame. 6. The device according to any one of the preceding claims, wherein the device further comprises a cell delivery device comprising at least one cell infusion tube configured to be placed within the chamber and configured to deliver cells to the device chamber. 7. Device according to any one of the preceding claims, wherein the polymer mesh comprises polypropylene. 8. The device according to any one of the preceding claims, wherein the device comprises cells within the chamber. 9. The device of claim 8, wherein the cells contain islets of Langerhans. 10. The device according to claim 8 or 9, wherein the cells comprise islets of Langerhans cells and Sertoli cells. 11. Device according to any one of claims 8 to 10, wherein the cells contain stem cells. 12. The device according to any one of claims 8 to 11, wherein the cells contain one or more of the following: neural stem cells, stem cells, differentiated stem cells, dopaminergic neurons, cord blood cells, embryonic stem cells and/or mesenchymal stem cells. 13. The device according to any one of claims 8 to 12, wherein the cells comprise encapsulated cells. 14. Device according to claim 13, wherein cells are encapsulated in alginate, polysaccharide hydrogel, chitosan, calcium or barium alginate, layered matrix of alginate and polylysine, polyethylene glycol photopolymerizable polymer, polyacrylate, hydroxyethyl methacrylate, methyl methacrylate, silicone capsules, silicone nanocapsules, polymembranes, or copolymer vinyl chloride with acrylonitrile. 15. The device according to any one of claims 8 to 14, wherein the cells contain allogeneic, xenogenic or syngeneic donor cells. 16. The device according to any one of claims 8 to 15, wherein the cells contain cells or cell lines obtained through genetic engineering. 17. The device according to any one of claims 8 to 16, wherein the cells contain two or more types of cells selected from islets, Sertoli cells, stem cells, differentiated stem cells, embryonic stem cells, allogeneic cells, xenogeneic or syngeneic cells, and cells, or cell lines obtained through genetic engineering. 18. A device according to any one of the preceding claims, wherein the cells are included in a viscous solution or biodegradable polymer. 19. The device according to claim 18, where the biodegradable polymer is polyethyleneimine and dextran sulfate, a copolymer of polyvinylsiloxane and polyethyleneimine, phosphorylcholine, polyethylene glycol, a copolymer of lactic and glycolic acids, polylactic acid, polyhydroxyvalerate and its copolymers, polyhydroxybutyrate and its copolymers, polydioxanone, polyanhydrides, polyamino acids, polyorthoesters, polyesters, collagen, gelatin, cellulose polymers, chitosan, alginates, fibronectin, extracellular matrix proteins, vinculin, agar, agarose, hyaluronic acid, matrigel, and combinations thereof. 20. An apparatus according to any one of the preceding claims, wherein the liner comprises a two-liner system. 21. The device of claim 20, wherein the dual liner system comprises an outer liner and an inner liner. 22. The apparatus of claim 21 wherein the outer liner and the inner liner comprise additional sealing mechanisms. 23. An apparatus according to any one of the preceding claims, wherein the porous scaffold comprises one chamber, two chambers, three chambers, four chambers, five chambers, six chambers, seven chambers, eight chambers, ten chambers, twelve chambers, or more chambers, such as approximately eight chambers, approximately nine chambers, or approximately ten chambers. 24. An apparatus according to any one of claims 1 to 22, wherein the porous scaffold comprises a plurality of laterally connected chambers. 25. A method for transplanting cells into a host organism, including the steps: a) implanting the device according to any one of claims 1 to 24 into the host body, b) maintenance of the device in the host organism until infiltration into the device of vascular and connective tissues; c) evaluation of the implanted device; d) removing the liner and e) infusion of cells into the chamber. 26. The method of claim 25, wherein the cells comprise islets of Langerhans cells. 27. The method according to claim 25 or 26, wherein the cells comprise islets of Langerhans cells and Sertoli cells. 28. The method according to any one of claims 25-27, wherein the cells contain stem cells. 29. The method according to any one of claims 25-28, wherein the cells contain one or more of the following: -