A transplantable bioreactor, and a method for manufacturing and using the same.

Abstract

The present invention provides an implantable bioreactor comprising cells enclosed within an enclosure, wherein the cells are capable of producing paracrine factors, the enclosure being collapsible, expandable, collapsible and expandable, or neither collapsible nor expandable, the enclosure providing containment of the cells and preventing escape of the cells and being semi-permeable to further provide an immunological barrier to protect the cells from immunological attack, the enclosure being permeable to the entire cellular selectome, including exosomes, nucleic acids, and proteins. The implantable bioreactor may have a variety of configurations and may house a cell culture matrix therein, which may include hydrogels, microbeads, and nanofiber matrices along with other active agents. [Selection diagram] None

Description

Cross-reference of related applications

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 568,348, filed on 5 October 2017, the contents of which are incorporated herein by reference for any purpose as if they were fully described herein. [Background technology]

[0002] Stem cells and the products they generate have the potential to regenerate damaged tissue and improve post-injury healing. Such treatments may limit adverse remodeling after tissue injury, such as myocardial infarction (MI). Adverse remodeling, namely an increase in left ventricular volume assessed by changes in end-systolic volume (ESV) and end-diastolic volume (EDV) after MI, is a strong predictor of mortality and the development of heart failure. However, the effectiveness of stem cell therapy against adverse remodeling in many clinical trials has been modest to date. One reason may be that many of the methods currently used to deliver stem cells to patients, whether intravenous, intra-coronary infusion, or direct myocardial infusion, likely result in only a small percentage of cells remaining in the heart, and of those that remain, very few survive for a significant period. At the same time, there is growing evidence from preclinical studies that many of the favorable effects of stem cell therapy stem from the release of growth factors, cytokines, chemokines, nucleic acids, exosomes, and other molecules and vesicles. These and other components of the cellular secretome are collectively called "paracrine factors." Therefore, if cells delivered to the heart remain there and survive for only a limited time, the time available for those cells to exert beneficial effects through the paracrine mechanism is similarly limited, which may explain the modest beneficial effects seen in most clinical trials.

[0003] Clinical trials using intracoronary and intramyocardial injections of stem cells to promote healing and regeneration of infarcted myocardium have yielded modest results to date. Potential reasons for these modest results include insufficient levels of paracrine factors, which lead to poor cell retention due to cell death, removal by immunological mechanisms, and simple "washout" after delivery, leaving only a short opportunity for cells to exert beneficial effects.

[0004] Therefore, there remains an unmet need for improved methods of delivering paracrine factors to organs that require repair and healing. [Overview of the Initiative]

[0005] To improve the survival and retention of stem cells, thereby increasing the time during which stem cells can exert beneficial paracrine-mediated effects, the inventors have developed a transplantable bioreactor. The bioreactor is an enclosure for containing a population of cells, such as stem cells, and in some embodiments comprises other non-stem cell types, having a semipermeable membrane that allows for the free exchange of paracrine factors, nutrients, and waste, but not the free exchange of cells, relating to the inventors' first-generation bioreactor disclosed in U.S. Patent Application No. 13 / 251,910, filed October 3, 2011, the contents of which are incorporated herein by reference as if described in whole. By preventing cell escape from the bioreactor and invasion by host immune cells, the bioreactor provides a protected environment in which the contained cells can survive for extended periods in vivo while releasing paracrine factors, thus extending the time during which beneficial effects can be exerted. The transplantable bioreactor may be for systemic or topical delivery of paracrine factors. The bioreactor may optionally be attached to another medical device.

[0006] According to several embodiments, the present invention provides an implantable bioreactor comprising cells in a cell culture matrix within an enclosure, which are capable of producing paracrine factors, wherein the enclosure is foldable, expandable, foldable and expandable, or neither foldable nor expandable, and the enclosure is semipermeable to provide containment of the cells, prevent leakage of the cells, and also provide an immunological barrier.

[0007] According to several embodiments, the present invention provides a transplantable bioreactor in which the enclosure of the bioreactor comprises a non-cell-supporting structure that allows for the proliferation of cells on its surface. Examples of such supporting structures include, but are not limited to, microbeads, nanofibers, and hydrogels, and can also be foldable and expandable.

[0008] In a further embodiment, the present invention provides an implantable bioreactor comprising cells sealed within a three-dimensional network of nanofibers in an enclosure, the cells being capable of producing paracrine factors, wherein the enclosure is foldable, expandable, foldable and expandable, or neither foldable nor expandable, and the enclosure is semipermeable to provide containment of the cells, prevent leakage of the cells, and also provide an immunological barrier.

[0009] According to one embodiment, the present invention provides an implantable bioreactor comprising cells sealed within an enclosure, capable of producing paracrine factors, wherein the enclosure is foldable, expandable, foldable and expandable, or neither foldable nor expandable, and the enclosure includes at least one semipermeable material layer in contact with or adjacent to at least one permeable material layer, the permeable material layer being on the side facing outward from the enclosure, and the enclosure being semipermeable to provide containment of the cells, prevent leakage of the cells, and also provide an immunological barrier.

[0010] In another embodiment, the present invention provides an implantable bioreactor comprising cells in an enclosure capable of producing paracrine factors, wherein the enclosure is foldable, expandable, foldable and expandable, or neither foldable nor expandable, and the enclosure includes at least one semipermeable material layer positioned in contact with or adjacent to at least first and second permeable material layers, the first permeable material layer being on the side facing outward from the enclosure, and the second permeable material layer being on the side facing inward from the enclosure, and the enclosure being semipermeable to provide containment of the cells, prevent leakage of the cells, and also provide an immunological barrier.

[0011] According to one embodiment, the present invention provides a transplantable bioreactor comprising cells in an enclosure capable of producing paracrine factors, wherein the enclosure includes at least one semipermeable material layer in contact with or adjacent to at least one cell layer in a cell culture matrix, the first permeable material layer being on the side facing outward from the enclosure and the cell layer being on the side facing inward from the enclosure, and the cell layer being rolled, stacked, or folded so that the structure can provide containment of the cells, prevent leakage of the cells, and also provide an immunological barrier.

[0012] According to some embodiments, the present invention provides a transplantable bioreactor comprising cells in an enclosure, wherein one or more enclosures or lumens are present within the bioreactor. [Brief explanation of the drawing]

[0013] [Figure 1] This figure shows a first-generation (G1) stem cell transplantable bioreactor (SCIB) for in vitro paracrine factor production and cell viability experiments. [Figure 2] This figure shows the release of paracrine factors from a prototype G1-SCIB in vitro. The G1-SCIB, containing 106 human MSCs, released PF related to the surrounding medium when cultured for 7 days. [Figure 3] This figure shows an in vivo prototype G1-SCIB. A) Fluoroscopic image of a prototype G1-SCIB transplanted via the right internal jugular vein. The interspecies human MSC B) continued to release associated PF from the prototype SCIB after 1 week in vivo in a pig, and C) showed normal MSC morphology and growth characteristics in culture. [Figure 4]As measured by the reduction in the increase in ESV (p=0.036 cells vs placebo) and EDV (p=0.059 cells vs placebo), G1-SCIB therapy reduced adverse remodeling during the 4 weeks after MI. [Figure 5] Scar size measured by delayed gadolinium-enhanced MRI 3 days and 4 weeks after MI. Scar size varied in both the cell group and the placebo group, but SCIB-based cell therapy resulted in an increase in heterogeneous gray scars, along with a reduction in dense core scars. [Figure 6] Scanning electron microscope images of track-etched (poly)ethylene terephthalate (PET) film, showing a dense pattern of uniform cylindrical pores (low magnification: left; high magnification: right). [Figure 7] Figure 7A is a schematic diagram of one embodiment of the G2 SCIB device, showing a dual-lumen pouch on a catheter having the following dimensions: a 0.064-inch catheter shaft with a shaft length of 30 cm, a 2 mm tapered catheter tip, a single 0.014-inch OTW guidewire port, a single cell injection port, a Luer-compatible connection, and two radiopaque markers. Figure 7B is a photograph showing an embodiment of the bioreactor pouch. The photograph shows a G2-SCIB cell chamber design with the following specifications: a polyethylene terephthalate structure with a wall thickness of approximately 25 μm, a diameter of approximately 6.0 mm, a length of approximately 120 mm, and track-etched pores of approximately 2 μm in diameter, resulting in approximately 7 × 10⁶ to 8 × 10⁶ pores / cm². The chamber or pouch may include non-cell support structures such as internal microbeads (customizable) and / or internal hydrogels (customizable). [Figure 8] This figure shows a series of photographs of the dual-lumen pouch or chamber of the present invention, with micrographs at two different magnifications showing in detail the track-etched pores with a diameter of approximately 2 μm and their high uniform density. [Figure 9]This figure shows that mesenchymal stem cells (MSCs) can adhere to polystyrene beads coated with fibronectin, which provide an internal cell adhesion microsurface that enhances cell volume and viability within cell pouches or chambers in bioreactors. Micrographs taken after 7 days of culture in a cell chamber using green: live cells / red: dead cells staining show living MSCs adhering to polystyrene beads, with a single bead showing living MSCs at higher magnification. Cell viability of over 90% was achieved within the cell chamber at 7 days, and live cells were distributed throughout the chamber or pouch. [Figure 10] This figure shows the permeability of a track-etched PET membrane pouch with pores of 0.44 μm in diameter. (A) FITC-labeled bovine serum albumin is rapidly released from the SCIB; (B) exosomes (shown in red) also pass freely through the membrane and are readily taken up by H9c2 rat cardiomyocytes (green). [Figure 11] Bioluminescence imaging (BLI) (A) of mouse MSCs in G2-SCIB pouches is significantly higher when MSCs are loaded into SCIB pouches with a hydrogel matrix (B, blue) compared to when MSCs are loaded into SCIB pouches without a hydrogel matrix (B, orange). Higher bioluminescence is associated with higher cell viability. [Figure 12] A) VEGF release from G2-SCIB (orange) is more than 10 times greater than VEGF release from G1-SCIB (blue). B) Consistent with this, endothelial tubule formation induced by G2-SCIB also exceeds that induced by G1-SCIB. [Figure 13] This figure shows an example of an embodiment of the bioreactor enclosure of the present invention, having a permeable membrane and a semipermeable membrane for encapsulating a cell culture matrix or a nanofiber cloud. [Figure 14] This figure shows an example of an embodiment of a roll-type enclosure bioreactor having alternating layers of nanofiber mesh and cell layers. [Modes for carrying out the invention]

[0014] Embodiments of the implantable bioreactor disclosed in the present specification can solve the problems of diffusion or washout by providing appropriate long-term delivery of paracrine factors secreted from the bioreactor while protecting the contents of the bioreactor from washout or immunological clearance in a sealed housing. The present invention includes, in one embodiment, a minimally invasive percutaneous bioreactor, and in another embodiment, an implantable device, both of which are capable of appropriately producing and releasing paracrine factors. The bioreactor can also be used to promote healing and regeneration in other tissues or organs through the release of paracrine factors.

[0015] As used herein, "bioreactor" refers to a collection of cells within a housing or enclosure, or a collection of cells sandwiched within a semipermeable membrane that permits the release of paracrine factors produced by the cells in the pouch. The bioreactor can include a plurality of housings, enclosures, or lumens. The bioreactor can be placed in situ in a mammal with or without the use of a catheter. In some embodiments, the bioreactor can be placed in situ within the vascular space of a mammal using a catheter or a similar device.

[0016] As used herein, a "paracrine factor" is a diffusible component produced by one cell to affect another cell. The diffusible component can be any component of the cellular secretome, including any protein, growth factor, nucleic acid, nuclear material, virus, viral vector (including vectors used in gene therapy), lipid, biomolecule, cytokine, chemokine, vesicle, exosome, nutrient or fluid produced by cells housed within a bioreactor. The permeability characteristics of the pouch of the present invention are an important feature of the present invention, as they are designed to allow paracrine factors, nutrients, waste products, and signaling factors to move into and out of the enclosure, but not to allow the entry of immune cells, or the entry or egress of stem cells or other types of cells. Those skilled in the art will appreciate that "paracrine factors" include many factors, including exosomes, which are typically vesicles 50 nm to 200 nm in diameter. Without being bound by any particular theory, it is also contemplated that these factors are one of the primary mediators of any protective effect attributed to stem cells or other cell types introduced in vivo for the purpose of supporting tissue repair. Examples of paracrine factors include factors that promote angiogenesis (e.g., VEGF, HGF, ANG), factors that promote cytoprotection (e.g., IGF-1, LIF, TMSB4), factors that promote cell proliferation (e.g., FGF, PGF, SCG), factors that promote cell migration (e.g., THBS1, SDF-1, PDGF), as well as secreted exosomes incorporating one or more of these factors.

[0017] As used herein, the term “semipermeable” means, in some embodiments, that the outer membrane of an enclosure allows molecules, proteins, peptides, nucleic acids, vesicles, etc. of a certain size to pass through the membrane, but larger objects such as cells cannot. These types of membranes are commercially available and have various molecular weight cutoff values ​​or pore sizes. In other embodiments, the term “semipermeable” is used to mean the presence of micropores of a certain diameter fabricated on a non-permeable membrane or surface to allow proteins, peptides, nucleic acids, vesicles, etc. of a certain size to pass through the membrane, but larger objects such as cells cannot.

[0018] As used herein, “stem cells” may include, but is not limited to, embryonic stem cells, adult stem cells, and induced pluripotent stem cells. Embryonic stem cells include, but are not limited to, totipotent, pluripotent, and multipotent stem cells, and adult stem cells include, but are not limited to, mesenchymal stem cells, cardiac stem cells, adipose-derived stem cells, and endothelial stem cells. In some embodiments, any non-stem types of cells and any combination thereof may also be included, for example, fibroblasts, cardiomyocytes, endothelial cells, other paracrine factor-secreting cells (e.g., hepatocytes, islet cells, myeloid cells, etc., without limitation), or engineered cells. In some embodiments, cell clusters such as cardiospheres, as well as mixtures of different stem cells and non-stem cells, may also be included. In some embodiments, the cells may include "modified" or "engineered" cells, such as genetically modified cells (e.g., cells transfected with genes encoding growth factors which may or may not be specific to the cell or cell population), cells transformed from another cell type (e.g., induced pluripotent stem cells and cells derived from these cells), or cells labeled with a detectable portion (e.g., cells having a fluorescent tag or transfected cells having genes encoding bioluminescence).

[0019] Various embodiments of the present invention include: (a) an implantable bioreactor that enhances the recovery of damaged cardiomyocytes and other tissues using a cellular strategy; (b) a percutaneously implantable bioreactor (including an easily retrievable percutaneous bioreactor that allows removal after the intended treatment period is completed); (c) a transient implantable device that releases de novo paracrine factors generated by cells; (d) a de novo paracrine factor generated by cells. (i) a permanent, implantable device that releases paracrine factors produced; (e) a bioreactor implanted via a standard vascular sheath; (f) an implantable bioreactor that locally releases paracrine factors in a target tissue; (g) an implantable bioreactor comprising a barrier having pores, the pores enabling the release of cell-derived biomolecules but not large enough to allow intrusion of immune cells and other cells or outflow of cells contained within the bioreactor; (h) an implantable bioreactor comprising a barrier composed of the materials described herein, the barrier being designed to enable the release of cell-derived biomolecules but not large enough to allow intrusion of immune cells and other cells or outflow of cells contained within the bioreactor; (j) an implantable bioreactor that systemically releases paracrine factors; (g) an implantable bioreactor having a multi-tube and multi-lumen cell chamber or pouch within a catheter; and (k) an implantable bioreactor having a non-cell-supporting structure such as microbeads or nanofibers or a hydrogel matrix within the cell chamber or pouch.

[0020] According to one or more embodiments, the bioreactor of the present invention may be designed for either systemic or local delivery of paracrine factors. The bioreactor encompasses embodiments of implantation by any clinical technique, including but not limited to surgical, laparoscopic, percutaneous, endoscopic, arthroscopic, or bronchoscopy techniques. In various embodiments, the bioreactor can be attached to a medical device.

[0021] In some embodiments, a transplantable bioreactor for systemic delivery comprises an enclosure containing stem cells or other cell types that produce and release paracrine factors. The cell enclosure includes, in one embodiment, a physical enclosure fabricated from a semipermeable membrane, and in another embodiment, a physical enclosure fabricated from a microporous polymer matrix that encapsulates the stem cells. The cell enclosure is impermeable to the cells but contains micropores large enough to allow the free passage of fluids, paracrine factors, waste, and nutrients, so that they can be transported efficiently and without interference. In some embodiments, the transplantable bioreactor is placed in intravascular spaces (such as central veins or aorta); implantation into other body cavities, openings, tissues, blood vessels, organs, or skin (e.g., in wounds) is also envisioned. In some embodiments, the transplantable bioreactor is temporarily implanted and later retrieved. In other embodiments, the transplantable bioreactor remains indefinitely as a permanent implant.

[0022] The implantable bioreactor of the present invention may be designed for systemic or local delivery of bio-products produced at a target. Both types encompass embodiments of implantation by any clinical technique, including but not limited to surgical, laparoscopic, percutaneous, endoscopic, arthroscopic, or bronchoscopy. Both types can be attached to medical devices in various embodiments.

[0023] In one embodiment, a transplantable bioreactor for systemic delivery comprises an enclosure for housing stem cells and / or other cell types that produce and release paracrine factors. In one embodiment, the cell enclosure includes a physical enclosure made of a semiporous membrane for encapsulating the stem cells, and in another embodiment, a physical enclosure made of a microporous polymer matrix for encapsulating the stem cells. The cell enclosure is impermeable to the cells but contains micropores large enough to allow the free passage of fluids, paracrine factors, waste, and nutrients, so that they can be transported efficiently and without interference.

[0024] In another embodiment of a whole-body delivery bioreactor, the implantable bioreactor enclosure is an enclosure containing stem cells, other cell types, and / or culture media in its lumen. In various embodiments, the enclosure may be (a) standalone, or (b) attached to a wire or catheter, or (c) attached as part of another implantable device, in which case the enclosure may be implanted together with the other device. In any of these examples, the bioreactor may be implanted by any clinical technique, including but not limited to surgical implantation, percutaneous implantation, insertion through any body opening or wound, or placement via laparoscopy, endoscopy, arthroscopy, or bronchoscopic techniques. In any of these examples, the bioreactor may be removed. In one embodiment, the enclosure may be pre-filled with the desired contents, or, if attached to a catheter, may be filled during and after implantation, as well as potentially emptied and refilled. When implanted percutaneously, the device can be passed directly or through a standard vascular sheath for placement in the intravascular space, for example (possible embodiments are shown in Figures 1, 7, and 8).

[0025] According to some embodiments, the bioreactor of the present invention comprises one or more cell chambers or pouches that function as enclosures for cells. (a) Structure In some embodiments, the cell chamber / pouch is attached to the body or distal end of one or more catheter tubes and optionally spans at least one open port to allow for the injection, sampling, and / or circulation of material into the cell pouch via the catheter. There are no restrictions on the geometric shape of the cell pouch; for example, the cell pouch may be smooth and cylindrical, or it may be formed by multiple recesses and surface undulations. The maximum diameter of the cell pouch is such that it does not completely occlude the anatomical structure in which it is placed. To assist in the positioning of the pouch, radiopaque markers may be placed at either end of the pouch, inside the pouch, or on the attached catheter.

[0026] In some embodiments, the bioreactor catheter may be closed and not have an open port.

[0027] In summary, there may be multiple variations of embodiments of the bioreactor of the present invention. Such variations may include, for example, one or more catheter tubes; each catheter tube may include one or more cell chambers or pouches; and each cell chamber or pouch may span one or more catheter tubes.

[0028] (b) Semi-transparent In some embodiments, the cell pouch wall of the bioreactor enclosure is semipermeable. There are at least two broad possible embodiments. In the first exemplary embodiment, the wall is an immunoprotective barrier and is therefore impermeable to cells but permeable to components of the cellular secretome (including growth factors, proteins, lipids, exosomes, nuclear material, and extracellular vesicles), as well as waste products and nutrients. A specific range of pore sizes that can satisfy these conditions includes pore diameters of 50 nm to 5000 nm.

[0029] In a second exemplary embodiment, the wall functions solely as a washout barrier. In this embodiment, the cell pouch wall of the bioreactor enclosure is impermeable to the immunoprotective structure containing the cells within the cell pouch (details below), but otherwise permeable. In this case, the pore diameter only needs to be smaller than the minimum diameter of the internal immunoprotective structure.

[0030] (c) internal immune protective structures; According to several embodiments, cells are placed in cell pouches within bioreactor enclosures, either standalone or within immunoprotective structures. These immunoprotective structures are described in detail in the literature and include hydrogel microspheres (e.g., based on chitosan, alginates, collagen, gelatin, agarose, and various derivatives thereof) and enclosures, cellulose-based enclosures, nanofiber-based matrices, or microfabricated cell enclosures. A feature of these immunoprotective structures is semipermeability; that is, they are impermeable to cells but permeable to nutrients, waste products, and components of the cellular secretome. Placing cells within immunoprotective structures in cell pouches suggests that the cell pouches themselves do not need to be immunoprotective.

[0031] (d) Material composition According to several embodiments, the cell chamber / pouch of a bioreactor enclosure may be composed of one or a combination thereof of a wide range of biocompatible synthetic and non-synthetic materials. Examples of synthetic materials include polymers and copolymers of polyesters and their derivatives, particularly poly(ethylene terephthalate) and its derivatives; fluoropolymers, particularly polytetrafluoroethylene and its derivatives; polycarbonates and their derivatives; poly(ethylene-co-vinyl acetate) and its derivatives; poly(n-butyl methacrylate) and its derivatives; poly(styrene-b-isobutylene-b-styrene) and its derivatives; polycaprolactone and its derivatives; polyimides and their derivatives; polyurethanes and their derivatives; polysulfones and their derivatives; polyacrylonitrile and its derivatives; polymethyl methacrylate and its derivatives; poly(lactic acid); poly(lactide-co-glycolide); and silicon. Non-polymer materials may include silicon and its derivatives. Examples of non-synthetic materials include cellulose-based materials and their derivatives; collagen-based materials and their derivatives; and extracellular matrix-based materials and their derivatives. The material can be porous to begin with (e.g., cellulose or stretched polytetrafluoroethylene), or it can be made porous through various manufacturing methods (such as track etching, laser microdrilling, micromachining, photolithography etching, and chemical etching).

[0032] coating The inner surface of the bioreactor enclosure of the present invention may be coated with molecules that enhance cell adhesion and function. Specific examples include fibronectin, polylysine, proteins having an Arg-Gly-Asp sequence, and any derivatives. The inner surface may also be surface-modified to enhance cell adhesion and function. Specific examples of this process include corona treatment and plasma treatment. The outer surface of the bioreactor enclosure may be coated or surface-modified to minimize thrombosis and / or fibrosis, reduce immunogenicity, improve biocompatibility, or improve the sustained delivery of other substances. The modification of coatings or surface modifications is well known to those skilled in the art. Specific examples include the attachment of heparin chains or polyethylene glycol chains to the outer surface, or fluoropassivation treatment of the outer surface. Furthermore, drug delivery devices may be attached to the cell chamber / pouch to enable the delivery of any drug or agent to the internal microenvironment or to the outside of the cell pouch. Such devices may include, for example, hydrogels, polymer matrices, beads, and nanoparticles containing drugs or bioactive agents.

[0033] shape The physical shape of the bioreactor enclosure can be designed to be smooth, or it can be designed to incorporate surface irregularities and depressions to maximize the surface area for mass transfer.

[0034] Bioreactor contents Based on the type of injury and organ targeted for healing and / or regeneration, any number of natural or genetically modified cell types or stem cell lines may be used in the device. These cells may be used standalone or immersed in a culture medium containing any number of proteins, growth factors, nucleic acids, or other molecules or vesicles. Specific and non-exclusive examples of bioreactor contents are outlined in the following paragraphs.

[0035] Internal environment of cell chamber / pouch The internal microenvironment of a cell pouch includes at least one of the following components: (a) one or more paracrine factor-producing cells; (b) non-cell support structures; (c) one or more support cells. The internal microenvironment of a cell pouch may also include cell culture medium. All of these components can be customized at any time by the end user to control the amount of paracrine factor production, release dynamics, and type of paracrine factor. Furthermore, all components of the internal microenvironment can be modified to include drugs that enhance cell adhesion and proliferation (e.g., polylysine, fibronectin, or proteins with Arg-Gly-Asp sequences), drugs that reduce thrombosis (e.g., heparin, direct thrombin inhibitors, or antiplatelet agents), drugs that reduce inflammation (e.g., using steroids), drugs that reduce infection (e.g., using antibiotics or antivirals), drugs that modulate cell proliferation (e.g., using growth factors), or to allow any drug or agent to be delivered into or outside the internal microenvironment. Furthermore, to enable delivery of any drug or pharmaceutical agent only to the internal microenvironment, or similarly to delivery to the outside of the cell pouch, the internal microenvironment may include drug delivery devices. Such devices may include, for example, hydrogels, polymer matrices, beads, and nanoparticles containing drugs or bioactive agents.

[0036] In some embodiments, the bioreactor is an enclosure attached to a catheter tube, as described above, and the catheter tube has a port connecting the lumen of the pouch to the catheter portion outside the patient, allowing for the injection, sampling, and circulation of cells and culture media. In other embodiments, multiple lumens within the catheter housing can provide additional options for continuous circulation of cells in the pouch, and the open distal port can be used as a venous line or for central venous pressure. Alternatively, the bioreactor may be attached directly to the wire.

[0037] As used herein, the term “non-cell-supporting structure” may include, without limitation, physical structures such as beads, microparticles, nanofibers, and cell culture matrices. Cell culture matrices may include, for example, hydrogels, scaffolds, nanofibers, and microparticles, and may be based on biological materials, synthetic materials, or any combination of different biological and / or synthetic materials. Cell culture matrices may be biodegradable or non-biodegradable. Alternatively, cell culture matrices may be made of materials that do not degrade unless they are in contact with certain agents, such as enzymes. Examples of such materials are collagen or hyaluronan matrices, which do not degrade unless they are arranged to come into contact with collagenase or hyaluronidase, respectively. The cell chamber or pouch of the bioreactor's internal microenvironment may include one or a combination thereof of culture media, microbeads (e.g., polystyrene-based microbeads, poly(lactic acid-coglycolic acid)-based microbeads, or poly(lactic acid)-based microbeads), hydrogels (e.g., hyaluronan-based hydrogels, poly(ethylene glycol)-based hydrogels, or collagen-based hydrogels), polymer matrices, tissue engineering scaffolds, nanofibers (e.g., poly(lactic acid-coglycolic acid)-based nanofibers), and biological extracellular matrices. Support structures can be used to promote cell adhesion and proliferation of primary paracrine factor-producing cells or supporting cells, or both. To promote cell adhesion or cell proliferation, components of the support structures may also be coated (e.g., with fibronectin, polylysine, integrins, cadherins, syndecanes, proteins having Arg-Gly-Asp sequences, or other agents) or surface modified (e.g., by corona treatment or plasma treatment). Support structures can also be used to control the release dynamics of paracrine factors by imposing a diffusion barrier (for example, by filling pouches with hydrogels of varying densities). The material of the support structure may be synthetic or non-synthetic, and may be biodegradable or non-biodegradable.Cells can be injected into the cell pouch before, during, or after the injection of the cell support environment. Alternatively, cells can be grown first in a support environment outside the pouch (e.g., on the surface of microspheres or nanofibers) and then placed inside the pouch.

[0038] According to some embodiments, the cell culture matrix or non-cell support structure may be pre-formed before or after being encapsulated in the bioreactor enclosure along with cells that have been pre-loaded and stored for later use; or alternatively, its individual components may be stored in the reactants and prepared only when necessary by direct addition into the bioreactor enclosure; or alternatively, it may be formed outside the bioreactor enclosure and then injected into the bioreactor enclosure in its formed state.

[0039] In some embodiments, non-cellular support structures can be formed, modified, or both during the implantation of the device. For example, during the implantation of the bioreactor, non-cellular extracellular matrix can be formed, modified, or both in situ within the bioreactor enclosure by fibroblasts. Non-cellular support structures can be modified by drugs or by changes in conditions inside or outside the bioreactor. For example, a hyaluronan-based matrix placed inside the bioreactor enclosure can be degraded over time by exposure to host hyaluronidase. Cells can be grown in a cell culture matrix inside the bioreactor enclosure, or they can be grown first in a cell culture matrix outside the bioreactor enclosure and then injected into the bioreactor enclosure together with the cell culture matrix.

[0040] As a specific example, mesenchymal stem cells are seeded on polystyrene microbeads coated with fibronectin (e.g., approximately 100 μm in diameter) before being placed in a pouch. The seeded cells are grown on the microbeads for a certain period before being placed in a cell pouch, or seeded onto the microbeads immediately before being placed in a cell pouch, or alternatively, blank microbeads are placed in a pouch before the cells are directly injected into the cell pouch, or a combination of some of the aforementioned methods. Each 100 μm diameter microbead may have a capacity of approximately 10 to 100 cells, for example, containing 15 to 100, 20 to 100, 25 to 100, 30 to 100, 40 to 100, 50 to 100, 60 to 100, 70 to 100, 80 to 100, or 90 to 100 cells, and therefore the end user can control the number of cells in the cell pouch by controlling the number of microbeads injected.

[0041] The intention is to enable seeding of any cell type or combination of cell types, including but not limited to human or non-human cells, stem cells, non-stem cells, genetically modified cells, tagged cells, surface-modified cells, engineered cells, cell clusters, and prokaryotic or eukaryotic cells, within the bioreactor enclosure.

[0042] supporting cells Paracrine factor-producing cells and non-cell-supporting structures may be supported by one or more supporting cell types arranged within a cell pouch, potentially enabling longer-lasting benefits provided by paracrine factor-producing cells. While these supporting cells may also produce paracrine factors, their primary purpose is to support primary paracrine factor-producing cells and, possibly in response to signals from primary paracrine factor-producing cells or an external host, to generate or modulate non-cell-supporting structures. There are no restrictions on the ratio of supporting cells to paracrine factor-producing cells. Examples of supporting cell-to-paracrine factor ratios range from 1:1 to 1:20. Examples of supporting cell types include (but are not limited to) fibroblasts, endothelial cells, endothelial progenitor cells, stem cells, induced pluripotent stem cells, and bone marrow stromal cells. Any other suitable cell type can be used as supporting cells. Cell types may be human or non-human cells, stem cells, non-stem cells, genetically modified cells, tagged cells, surface-modified cells, engineered cells, and prokaryotic or eukaryotic cells.

[0043] As a first exemplary embodiment, fibroblasts can be used to enhance and regulate the formation of the extracellular matrix within the pouch over time, thereby enhancing the adhesion, viability, and proliferation of paracrine factor-producing cells within the pouch.

[0044] In a second exemplary embodiment, endothelial cells and endothelial progenitor cells are used to create a primitive vascular capillary system within the pouch. Such a system can enhance the efficiency of the transport of nutrients, waste, signaling molecules, and paracrine factors across a semipermeable enclosure and allow for denser three-dimensional packing of paracrine factor-producing cells within the pouch.

[0045] In a third exemplary embodiment, bone marrow stromal cells are used to produce signaling molecules and growth factors that regulate and stimulate the proliferation of stem cells within the pouch. Since the target host and the supporting cells themselves can sense signals from paracrine factor-producing cells proliferating within the pouch, the supporting cells can alter their activity based on the needs of the proliferating cells and the target host, and thus continuously regulate the internal microenvironment within the cell pouch.

[0046] Possible variations The bioreactor is designed to allow end users to customize the internal environment of the cell pouch at any point, from manufacturing to use.

[0047] In a first exemplary embodiment, an end user may modify paracrine factor-producing cells to change the type of paracrine factor released for different diseases (for example, an end user may use hepatocytes to produce factors for liver disease or mesenchymal stem cells to produce factors for heart disease).

[0048] In a second exemplary embodiment, the end user may modify the non-cell-supporting structure to alter the amount of paracrine factor produced or the rate of paracrine factor release (for example, the end user may inject fewer cell-coated microbeads into pediatric patients than into adult patients to reduce the amount of paracrine factor produced; or, to accommodate different patients or the same patient with different needs at different stages of recovery, the end user may decrease or increase the crosslinking density of the hydrogel to increase or decrease the rate of paracrine factor release, respectively).

[0049] As a third exemplary embodiment, the end user may change the type of supporting cells to accommodate different types of paracrine factor-producing cells or different transplantation periods.

[0050] The end user may also make any of these changes while the device is implanted in the patient. For example, using embodiments that include a distal port to a bioreactor, the end user may change the type of paracrine factor-producing cells, supporting cells or structure, or the ratio of supporting cells to paracrine factor-producing cells, based on changes in the patient's condition. The end user may want to do this for different disease types to control the type of paracrine factor released, the total amount of paracrine factor produced, or the release dynamics of paracrine factor.

[0051] A "hydrogel" refers to a water-swellable polymer matrix that can absorb water and form an elastic gel. When placed in an aqueous environment, a dry hydrogel swells by incorporating liquid to the extent permitted by the degree of crosslinking.

[0052] The term "polymer" is used to refer to molecules composed of repeating monomer units, including homopolymers, block copolymers, heteropolymers, random copolymers, and graft copolymers. "Polymers" also include linear and branched polymers, with branched polymers encompassing highly branched polymers, dendritic polymers, and star polymers.

[0053] A monomer is the basic repeating unit (or multiple units) in a polymer. A monomer may be a monomer itself, or it may be a dimer or oligomer of at least two different monomers, each dimer or oligomer being repeated in the polymer.

[0054] In some embodiments, the cell culture matrix may comprise polymers, a matrix, and a gel, and this disclosure includes methods for preparing and using the matrix, polymer, and gel. One of the polymers comprises an imide. Desired gels, networks, scaffolds, films, etc., prepared from the desired composition promote the integration and growth of cells, tissues, and organs. In other embodiments, the cell culture matrix may include any structures (e.g., microbeads, nanofibers, etc.) that support cell culture.

[0055] Biocompatible polymers, biocompatible crosslinked polymer matrices, and biocompatibility are recognized in the art. For example, biocompatible polymers include polymers that are not toxic to a host (e.g., an animal or a human) and do not decompose at a rate that produces monomer or oligomeric subunits or other byproducts at concentrations toxic in the host (if the polymer decomposes). In certain embodiments of the present invention, biodegradation generally includes the decomposition of a polymer in a living organism, for example, decomposition to its monomer subunits which may be known to be non-toxic. However, intermediate oligomeric products resulting from such decomposition may have different toxicological properties, or the biodegradation may include oxidation or other biochemical reactions that produce molecules other than the monomer subunits of the polymer. Therefore, in certain embodiments, the toxicology of a biodegradable polymer intended for in vivo use, such as transplantation or injection into a patient, may be determined after one or more toxicity analyses. It is only necessary that the composition is biocompatible as described above. Therefore, the composition may also contain polymers that are biocompatible, including, for example, the polymers and other materials and excipients described herein, and that are still biocompatible.

[0056] In this specification, "crosslinked material" generally refers to a composition that includes intermolecular crosslinks and optionally intramolecular crosslinks resulting from the formation of covalent bonds. The covalent bonds between two crosslinkable components may be direct (in which case atoms in one component are directly bonded to atoms in the other component) or indirect via linking groups. In addition to covalent bonds, the crosslinked gel or polymer matrix may also include intermolecular and / or intramolecular non-covalent bonds such as hydrogen bonds and electrostatic (ionic) bonds.

[0057] A "gel" refers to a state of matter between liquid and solid, and is generally defined as a cross-linked polymer network that swells in a liquid medium. Typically, a gel is a two-phase colloidal dispersion containing both solid and liquid, with the amount of solid in the gel being greater than the amount of solid in a two-phase colloidal dispersion called a "sol." Therefore, a "gel" possesses some of the properties of a liquid (i.e., its shape is elastic and deformable) and some of the properties of a solid (i.e., its shape is discrete enough to maintain three dimensions on a two-dimensional surface).

[0058] Hydrogels can consist of hydrophilic polymers crosslinked to form an insoluble polymer network that swells with water. Crosslinking can be initiated by a number of physical or chemical mechanisms. Photopolymerization is a method of covalently crosslinking polymer chains, where a photoinitiator and polymer solution (called a "pregel" solution) are exposed to a light source specific to the photoinitiator. Once activated, the photoinitiator reacts with specific functional groups in the polymer chains, crosslinking them to form a hydrogel. The reaction is rapid (3-5 minutes) and proceeds at room temperature and body temperature. Photo-induced gelation allows for spatial and temporal control of scaffold formation and enables shape manipulation during gelation after injection and in vivo. Cells and bioactive factors can be readily incorporated into the hydrogel scaffold simply by mixing them with the polymer solution before photogelation.

[0059] Alternatively, the reactants may contain complementary reactive groups, such as imides and amides, that result in crosslinking without the need for an external initiator.

[0060] The desired hydrogel may be a semi-interpermeable network that promotes cell proliferation. Viscosity can be adjusted by the monomers and polymers used, the level of water trapped in the hydrogel, and the incorporated thickeners, such as biomacromolecules like proteins, lipids, and sugars. Examples of such thickeners include hyaluronic acid or collagen.

[0061] In some embodiments, the cell culture matrix may contain at least one monomer unit of a biocompatible polymer functionalized with an imide, such as chondroitin sulfate, hyaluronic acid, heparin sulfate, or keratan sulfate. Their starting molecules are natural components of the extracellular matrix. However, generally, any biocompatible polymer can be used as the polymer, and that polymer has at least an imide. Other suitable polymers include naturally occurring ones, such as GAGs, mucopolysaccharides, collagen, or proteoglycan components (e.g., hyaluronic acid, heparin sulfate, glucosamine, dermatan, keratan, heparan, hyaluronan, aggrecan, etc.).

[0062] The crosslinked polymer matrix of the present invention may contain and may form a hydrogel. The water content of the hydrogel provides information about its pore structure. Furthermore, the water content may be a factor that affects, for example, the survival of cells encapsulated within the hydrogel. The amount of water that the hydrogel can absorb may be related to the crosslink density and / or pore size. For example, the percentage of imides on a functionalized macromer such as chondroitin sulfate, hyaluronic acid, dextran, carboxymethyl starch, keratin sulfate, or ethylcellulose may determine the amount of water that can be absorbed.

[0063] According to one or more embodiments, a bioactive agent can also be incorporated into the cell culture matrix in the bioreactor enclosure of the present invention. The terms “incorporated,” “encapsulated,” and “confined” are recognized in the art when used in reference to therapeutic agents, dyes, or other materials and polymer compositions such as cell culture matrix compositions. In certain embodiments, these terms include incorporating, formulating, or containing such agents in a composition that enables the sustained release of such agents in a desired application. These terms may, for example, contemplate any method by which a therapeutic agent or other material is incorporated into a polymer matrix, including, for example, attaching such monomers to such polymer monomers (by covalent bonds or other bonding interactions) to make such monomers part of the polymerization to give a polymer formulation, distributing throughout the polymer matrix, attaching to the surface of the polymer matrix (by covalent bonds or other bonding interactions), or encapsulating within the polymer matrix. The terms “co-incorporated” or “co-encapsulated” refer to the incorporation of a therapeutic agent or other material, and at least one other therapeutic agent or other material, in the composition.

[0064] More specifically, the physical form in which any therapeutic agent or other material is encapsulated in a polymer and / or enclosure may vary depending on the particular embodiment. For example, in some embodiments, the therapeutic agent or other material may first be encapsulated in microspheres and then combined with the polymer such that at least a portion of the microsphere structure is maintained. Alternatively, the therapeutic agent or other material may be sufficiently immiscible with the polymer of the present invention and dispersed as small droplets rather than dissolved in the polymer. Any form of encapsulation or incorporation is contemplated by the present invention, insofar as the sustained release of any encapsulated therapeutic agent or other material determines whether the encapsulation form is sufficiently acceptable for any particular application.

[0065] According to some embodiments, the cell culture matrix used within the enclosure can be immersed in a liquid medium containing any number of proteins, growth factors, lipids, nucleic acids, salts, other molecules, cells, or extracellular vesicles.

[0066] While not limited to these, biological motifs such as arginine-glycine-aspartate oligopeptides, collagen, and fibronectin can be incorporated into cell culture matrices to enhance cell adhesion. Examples of the class of cell adhesion molecules include, but are not limited to, cadherins, integrins, and syndecans, as well as their parts and fragments.

[0067] In one aspect of the present invention, a cell culture matrix can be prepared comprising a crosslinked polymer matrix or gel and one or more bioactive agents. The bioactive agents can vary considerably depending on the intended purpose of the composition. The term "active" is recognized in the art and refers to any part that is biologically, physiologically, or pharmacologically active in a subject, either locally or systemically. Examples of bioactive agents, sometimes called "drugs," are listed in well-known literature such as the Merck Index, Physicians' Desk Reference, and The Pharmacological Basis of Therapeutics, and include, but are not limited to, pharmaceuticals; vitamins; mineral supplements; substances used to treat, prevent, diagnose, cure or alleviate diseases or illnesses; substances that affect the structure or function of the body; or prodrugs that become biologically active or more active after being placed in a physiological environment. A specific example of a "drug" is heparin, which may be used for its antithrombotic effect. For example, various forms of bioactive agents that can be released into adjacent tissues or body fluids upon administration to a subject may be used. In some embodiments, bioactive agents may be used in the crosslinked polymer matrix of the present invention, for example, to promote angiogenesis. In other embodiments, bioactive agents may be used in the crosslinked polymer matrix of the present invention, for example, in conjunction with promoting angiogenesis or healing after injury or damage, to treat, improve, inhibit, or prevent a disease or condition.

[0068] Further examples of bioactive agents include, but are not limited to, enzymes, receptor antagonists or agonists, hormones, growth factors, autologous bone marrow, antibiotics, antimicrobial agents, and antibodies. The term “bioactive agent” is also intended to encompass the various cell types and genes that may be incorporated into the compositions of the present invention.

[0069] In certain embodiments, the composition contains a bioactive agent in an amount of about 1% to about 75% or more by weight of the total composition, or about 2.5%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, or 70%.

[0070] Non-exclusive examples of bioactive agents include: adrenergic blockers, anabolic agents, androgenic steroids, anticholesterol and antilipid agents, anticholinergic and sympathomimetic agents, anticoagulants, antihypertensive agents, anti-infective agents, anti-inflammatory agents such as steroids, non-steroidal anti-inflammatory drugs, antipyretics and analgesics, antithrombotic agents, antianginal agents, biological agents, cardiac activators, vasodilators, coronary artery dilators, diagnostic agents, hematopoietic agents, estrogens, growth factors, peripheral vasodilators, progesterones, prostaglandins, vitamins, antigenic substances, and prodrugs.

[0071] Furthermore, recombinant or cell-derived proteins such as recombinant beta-glucan; bovine immunoglobulin concentrate; bovine superoxide dismutase; preparations containing fluorouracil, epinephrine, and bovine collagen; recombinant hirudin (r-Hir), HIV-1 immunogen; recombinant human growth hormone, recombinant EPO (r-EPO); gene-activated EPO (GA-EPO); recombinant human hemoglobin (r-Hb); recombinant human mecasermin (r-lGF-l); recombinant interferon α; lenograstim (G-CSF); olanzapine; recombinant thyroid-stimulating hormone (r-TSH); and topotecan may be used.

[0072] Furthermore, the following peptides, proteins, and other large molecules may also be used: interleukins 1-18 (including variants and analogues); interferons α and γ, hormone-releasing hormone (LHRH) and its analogues, gonadotropin-releasing hormone, transforming growth factor (TGF), which may be useful for cartilage regeneration; fibroblast growth factor (FGF); tumor necrosis factor-α; nerve growth factor (NGF); growth hormone-releasing factor (GHRF), epidermal growth factor (EGF), connective tissue-activating osteogenic factor, fibroblast growth factor homolog (FGFHF); hepatocyte growth factor (HGF); insulin growth factor (IGF); invasion inhibitor-2 (IIF-2); bone morphogenetic proteins 1-7 (BMP1-7); somatostatin; thymosin-α-γ-globulin; superoxide dismutase (SOD); and complement factors, as well as biologically active analogues, fragments, and derivatives of such factors (e.g., growth factors).

[0073] Members of the transforming growth factor (TGF) supergene family, which are multifunctional regulatory proteins, can be incorporated into the polymer matrix of the present invention. Members of the TGF supergene family include beta-transforming growth factors (e.g., TGF-131, TGF-132, TGF-133); bone morphogenetic proteins (e.g., BMP-1, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8, BMP-9); heparin-binding growth factors (e.g., fibroblast growth factor (FGF), epidermal growth factor (EGF), platelet-derived growth factor (PDGF), insulin-like growth factor (IGF)), (e.g., inhibin A, inhibin B), growth differentiation factors (e.g., GDF-1); and activins (e.g., activin A, activin B, activin AB). Growth factors can be isolated from natural or natural sources such as mammalian cells, or they can be synthetically prepared by recombinant DNA technology or various chemical processes. Furthermore, analogs, fragments, or derivatives of these factors may be used, provided that they exhibit at least some of the biological activity of the native molecules. For example, analogs may be prepared by the expression of genes modified by site-directed mutagenesis or other genetic engineering techniques.

[0074] According to one embodiment, a thiol-modified hyaluronan hydrogel incorporating collagen can be used as a cell culture matrix for stem cells in a bioreactor enclosure. In this particular embodiment, a stem cell suspension is prepared in a thiol-modified hyaluronan solution, injected into a bioreactor enclosure, and crosslinked with thiol-reactive polyethylene glycol diacrylate to form a hydrogel.

[0075] According to another embodiment, cells can be grown on the surface of microparticles on the outside of a bioreactor enclosure; the cell-coated microparticles can then be injected into the bioreactor enclosure to fill part or most of the internal volume of the bioreactor enclosure.

[0076] Catheter design (a) Structure According to several embodiments, the present invention may include a catheter. The catheter of the present invention may include a single tube, or alternatively, a plurality of tubes joined at one or more points. Each catheter tube may have one or more lumens that allow options for injecting, sampling, or circulating cells, culture media, and other materials. In some embodiments, one or more catheter tubes may have one or more additional ports, some of which open into an internal lumen of a cell pouch, and others which communicate directly with circulation to allow its use as an intravascular line. For example, one of the lumens may be dedicated to the passage of a guidewire in an "over-the-wire" or monorail "rapid exchange" configuration to facilitate intravascular placement and positioning. Another lumen may be connected to an inflatable balloon filled with air or fluid, located at the distal end of the catheter tube, to aid in positioning. The catheter may be attached to one or more external ports to allow for the injecting, sampling, and circulation of cells, culture media, and other materials, as needed. (b) Material The catheter housing can be manufactured from any suitable biocompatible material (e.g., polyvinyl chloride, polyester, polycarbonate, polyurethane, polyamide, polyimide, fluoropolymer, polyolefin, or polyetheretherketone). The outside of the catheter can be coated with molecules that help enhance cell adhesion and function. Some of these molecules include poly-L-lysine, fibronectin, or other proteins with an Arg-Gly-Asp sequence. This can be achieved by first oxidizing the catheter surface in a plasma reactor or with a chemical oxidizing agent such as potassium permanganate, and then reacting the surface with the appropriate molecular functional groups. The outside of the catheter can also be spray-coated or dip-coated with an anticoagulant (such as heparin) to minimize thrombus formation during implantation. The outside of the catheter may also be surface-modified (e.g., using fluoropassivation) to minimize fibrosis or to facilitate delivery. Alternatively, a portion of the catheter (e.g., the segment of the catheter extending inside the cell pouch) can be modified to improve cell adhesion, while another portion of the catheter (e.g., the segment of the catheter outside the cell pouch) can be modified to reduce fibrosis and thrombosis and facilitate delivery. Furthermore, drug delivery devices can be attached to the catheter. Such devices may include, for example, hydrogels, polymer matrices, beads, and nanoparticles containing drugs or bioactive agents.

[0077] According to several embodiments, including catheters, the catheter housing may be made from a polyvinyl chloride tube (or other suitable biocompatible material) consisting of multiple lumens, a proximal access port, and a distal opening, similar to a standard multi-lumen central venous catheter. These ports can be used for the circulation of cells, culture media, therapeutic agents, gases, or other drugs recognized as beneficial by those skilled in the art. Several arbitrary modifications to the basic tubular configuration will be obvious to those skilled in the art.

[0078] In some embodiments, the surface of the catheter to which the bioreactor is attached may be coated with molecules that help enhance stem cell adhesion and function. Some of these molecules include polylysine, fibronectin, or other proteins having an Arg-Gly-Asp sequence. This can be achieved by first oxidizing the catheter surface in the plasma reactor or with a chemical oxidizing agent such as potassium permanganate, and then reacting the surface with the appropriate molecular functional groups. The outside of the catheter may also be spray-coated or dip-coated with an anticoagulant (such as heparin) to minimize thrombus formation during and after transplantation.

[0079] Guiding balloon A small balloon (approximately 1 cm in diameter) can be placed near the tip of the catheter, and when filled with gas or fluid, it can help guide its placement within the blood vessel.

[0080] Guide wire guidance The individual lumens within the catheter can be used to guide the guidewire through in an "over-the-wire" or monorail "rapid exchange" configuration, guiding its intravascular placement. The device also allows for the injection, intermittent recycling, or continuous circulation of cells, cell-conditioned media, concentrated paracrine factors, or other therapeutic fluids that may or are determined to have beneficial effects.

[0081] In some embodiments, the catheter-based device may be removed if necessary. The enclosure is first deflated by drawing it back through the injection port. The entire device can then be withdrawn from the body. The vascular sheath is removed, and manual compression or a vascular occlusion device is used to achieve hemostasis.

[0082] Bioreactor with secondary device In one embodiment, the bioreactor enclosure described above may be attached to and implanted with various other secondary devices. The bioreactor enclosure may be miniaturized as needed for attachment to secondary devices. In the cardiovascular field, secondary devices include, but are not limited to, stents, balloons, intra-aortic balloon pumps, percutaneously and surgically implanted ventricular assist devices, percutaneously and surgically implanted prosthetic valves and valve clips or rings, intravascular grafts, thrombus filters, pacemaker or defibrillator surface or lead, septal defect closure devices, atrial appendage closure devices, pulmonary artery catheters, venous catheters, and arterial catheters. Outside the cardiovascular field, any device that provides direct or indirect access to target tissue is a possible candidate for attaching the bioreactor enclosure.

[0083] According to several embodiments, cells in a bioreactor may be loaded into a three-dimensional network of nanofibers called a “nanofiber cloud.” Nanofibers are defined as fibers with a typical diameter of about 1000 nm and can be fabricated by known methods such as electrospinning. Nanofibers can be fabricated from most biocompatible polymers, including, for example, poly-L-lactic acid copolymers. The polymer is then electrospun onto a matrix or surface to collect the fibers. The inventors have published data showing that stem cells readily adhere to a matrix composed of nanofibers and proliferate vigorously on the matrix (Biomaterials June 2015; 52:318-26), which is incorporated herein by reference.

[0084] Accordingly, according to one embodiment, the present invention provides an intravascular bioreactor comprising cells sealed within a three-dimensional network of nanofibers in an enclosure, the cells being capable of producing paracrine factors, the enclosure being foldable and expandable so as to be implantable intravascularly, and the enclosure being semipermeable so as to result in containment of the cells, preventing their leakage, and also providing an immunological barrier.

[0085] In some embodiments, the nanofiber cloud may be formed inside the bioreactor enclosure, or alternatively, it may be formed outside the bioreactor enclosure and then later injected or placed inside the bioreactor enclosure. The nanofiber cloud may fill all or part of the bioreactor enclosure.

[0086] Those skilled in the art will understand that the nanofiber density or nanofiber orientation of the nanofiber cloud within the bioreactor pouch can be adjusted according to the desired cloud permeability, cell seeding density, and cell seeding pattern.

[0087] According to several embodiments, the nanofibers of the present invention may be based on biodegradable polymer materials including, but not limited to, poly(lactic acid-coglycolic acid), polycaprolactone, polylactic acid, or polydioxanone. Nanofibers may also be composed of non-biodegradable materials including, but not limited to, polyurethane, polytetrafluoroethylene, polyethylene terephthalate, or polysulfone. Nanofibers may also be composed of biological materials including, but not limited to, cellulose, collagen, lipids, nucleic acids, and proteins. Nanofibers may also be composed of any combination of one or more biodegradable materials, non-biodegradable materials, and biological materials.

[0088] "Biodegradable" is a term recognized in the art and includes monomers, polymers, polymer matrices, gels, compositions, and formulations, such as those described herein, that are intended to degrade during use, such as in vivo. Biodegradable polymers and matrices typically differ from non-biodegradable polymers in that the former may degrade during use. In certain embodiments, such use includes in vivo use, such as in vivo therapy, and in other specific embodiments, such use includes in vitro use. Generally, degradation due to biodegradability includes the breakdown of a biodegradable polymer into its constituent subunits, or digestion of the polymer into smaller non-polymeric subunits, for example, by biochemical processes. In certain embodiments, two different types of biodegradation may generally be identified. For example, one type of biodegradation may involve the cleavage of bonds (whether covalent or not) in the polymer backbone. Such biodegradation typically yields monomers and oligomers, and more typically, such biodegradation occurs by the cleavage of bonds connecting one or more subunits of the polymer. In contrast, another type of biodegradation may involve the cleavage of bonds (whether covalent or not) within side chains, or the cleavage of bonds connecting side chains, functional groups, etc., to the polymer backbone. For example, therapeutic agents, bioactive agents, or other chemical parts attached to the polymer backbone as side chains may be released by biodegradation. In certain embodiments, one or the other or both of the common types of biodegradation may occur during the use of the polymer. As used herein, the term “biodegradation” encompasses both common types of biodegradation.

[0089] The degradation rate of biodegradable polymers often depends in part on a variety of factors, including the chemical identity of the bonds responsible for degradation, the molecular weight, crystallinity, biostability, and degree of crosslinking of such polymers, the physical properties, shape, and size of the implant, and the method and location of administration. For example, higher molecular weight generally leads to higher crystallinity and / or higher biostability. The term "biodegradable" is intended to cover materials and processes also known as "bioerodible."

[0090] In certain embodiments, the biodegradation rate of such polymers may depend on the presence of enzymes, such as chondroitinase. In such situations, the biodegradation rate may depend not only on the chemical identity and physical properties of the polymer matrix, but also on the identity of such enzymes.

[0091] In certain embodiments, the polymer formulations of the present invention biodegrade within a period acceptable for the desired application. In certain embodiments, such as in vivo therapy, such degradation typically occurs within a period of less than 5 years, less than 1 year, less than 6 months, less than 3 months, less than 1 month, less than 15 days, less than 5 days, less than 3 days, or less than 1 day when exposed to a physiological solution having a pH of 6–8 and a temperature of about 25°C–37°C. In other embodiments, the polymer degrades within a period of about 1 hour to several weeks, depending on the desired application. In some embodiments, the polymer or polymer matrix may contain a detectable agent released upon degradation.

[0092] In some embodiments, adhesion molecules such as arginine-glycine-aspartate oligopeptide, collagen, and fibronectin may be incorporated into the nanofiber cloud to enhance cell adhesion.

[0093] Cells for use in the bioreactor of the present invention may be seeded in the nanofiber cloud of the present invention immediately after electrospinning and stored for later use, or alternatively, seeded in the nanofiber cloud immediately before use.

[0094] According to some embodiments, any cell type or combination of cell types, including but not limited to human or non-human cells, stem cells, non-stem cells, genetically modified cells, tagged cells, surface-modified cells, engineered cells, prokaryotic or eukaryotic cells, can be seeded into a nanofiber cloud within a bioreactor enclosure.

[0095] It will be understood by those skilled in the art that nanofiber clouds can be immersed in liquid culture media containing any number of proteins, growth factors, lipids, nucleic acids, salts, any other molecules, cells, or extracellular vesicles. Nanofiber clouds can also be immersed in culture matrices, including but not limited to those described herein, such as hydrogels. Nanofiber clouds can also be immersed in any combination of liquid culture media and culture matrices such as hydrogels.

[0096] Those skilled in the art will also understand that one or more cell types that do not directly contribute to the paracrine factor production of the bioreactor may be incorporated into the bioreactor pouch. For example, such cells may be used within the bioreactor enclosure to enhance the formation of the extracellular matrix within the enclosure or to support the viability of other cells in the bioreactor enclosure. It is intended that any cell type may be used. For example, cell types may be human or non-human cells, stem cells, non-stem cells, genetically modified cells, tagged cells, surface-modified cells, engineered cells, cell clusters, prokaryotic cells or eukaryotic cells.

[0097] According to one embodiment, the present invention provides, for example, a bioreactor enclosure comprising fibroblasts to support the formation of an extracellular matrix within a bioreactor pouch, the formation of which enhances the adhesion, viability, and proliferation of primary paracrine factor-producing bioreactor cells.

[0098] Alternative compositions for bioreactor enclosures

[0099] As described above, bioreactor enclosures can be constructed from a wide range of biological or synthetic semipermeable membranes with predetermined molecular weight cutoffs designed to restrict cell movement but allow the free movement of paracrine factors, nutrients, and waste.

[0100] According to one or more alternative embodiments, the present invention provides novel compositions for bioreactor enclosures that do not require an intrinsically semi-permeable membrane having a suitable predetermined molecular weight cutoff.

[0101] Machined materials

[0102] The bioreactor enclosure of the present invention can be manufactured from any impermeable material by machining the material to form pores of appropriate diameter and density. The bioreactor enclosure can also be manufactured from any essentially semi-permeable material (but having an inappropriate pore diameter or pore density) by machining the material to form pores of appropriate diameter and density.

[0103] As used herein, “appropriate pore diameter” is defined as a diameter that prevents the passage of cells while allowing the free passage of paracrine factors, including exosomes, as well as nutrients, waste, and fluids. In some embodiments, those skilled in the art will understand that it is possible to place cells within an immunoprotective structure (e.g., hydrogel beads or an immunoprotective micropouch) before placing them in the pouch of the present invention. In such embodiments, the pouch itself does not need to be immunoprotective. The appropriate pore diameter in that scenario only needs to prevent passage through the immunoprotective structure.

[0104] As used herein, “appropriate pore density” is defined as a pore density sufficient to support the viability of cells within a bioreactor enclosure and may vary depending on the maximum cell population and metabolic requirements of the cells within the bioreactor pouch. For example, pore diameters in the range of 100 nm to 5000 nm can meet these criteria. For example, 100,000 / cm² 2 ~100,000,000 / cm 2 Pore ​​densities within this range can satisfy these criteria. These pore diameters apply only if the cells were not initially placed within the immunoprotective structure of the pouch. In embodiments where the cells are placed within an immunoprotective structure (e.g., hydrogel beads or an immunoprotective micropouch) before being placed within the pouch of the present invention, much larger pouch pore diameters can satisfy this criterion, as long as the pore diameter of the pouch is smaller than the minimum diameter of the internal immunoprotective structure.

[0105] According to some embodiments, a continuous or intermittent outward-flowing fluid flux can be applied across the bioreactor enclosure to facilitate the outflow of material from the pouch and reduce the ingress of material into the enclosure. A continuous or intermittent inward-flowing fluid flux across the bioreactor enclosure may be applied for the opposite effect. Such fluid flux may be applied to any enclosure described herein. Specifically, a continuous outward-flowing flux of saline at a rate of 10 cc / hour is applied by connecting the lumen of the bioreactor enclosure to an external fluid source. Furthermore, optionally, the fluid may include any medication required by the patient (e.g., drugs, antithrombotic agents, anti-inflammatory agents, nutrition) or any medication required by the cells or supporting structures within the bioreactor pouch. The fluid flux can be generated using peristaltic pumps, intravenous infusion pumps (IV infusion pumps), and other pumps used in medical devices known in the art.

[0106] According to several embodiments, the methods used to machine the pouch of the present invention include, but are not limited to, track etching, microdrilling, laser drilling, photolithography etching, or other suitable means for forming pores. Track etching is a technique that uses ions to impact a material and form damaged tracks; pores are then created by expanding the damaged tracks using a chemical etching solution such as sodium hydroxide. Microdrilling and laser drilling are techniques that use a microdrill or laser to physically form pores. Photolithography etching is a technique established in the semiconductor industry in which a geometric pattern of pores is transferred through a photomask to an enclosure surface coated with photoresist; pores are then generated using a liquid wet etching or plasma dry etching process.

[0107] In some embodiments, the enclosure material may consist of non-biodegradable materials including, but not limited to, polyurethane, polytetrafluoroethylene, polysulfone, or polyethylene terephthalate. The enclosure material may also consist of biodegradable materials including, but not limited to, poly(lactic acid-coglycolic acid), polycaprolactone, polylactic acid, or polydioxanone. The enclosure material may also consist of biological materials including, but not limited to, cellulose, collagen, lipids, nucleic acids, and proteins. The enclosure material may also consist of any combination of one or more biodegradable materials, non-biodegradable materials, and / or biological materials. The enclosure material may be coated with molecules that enhance cell adhesion and function. Specific examples include fibronectin, polylysine, integrins, cadherins, syndecanes, and proteins having Arg-Gly-Asp sequences. The enclosure material may also be surface-modified to enhance cell adhesion and function. Specific examples of this process include corona treatment and plasma treatment. Enclosure materials may also be coated or surface-modified to minimize thrombosis or fibrosis, reduce immunogenicity, improve biocompatibility, or enhance the delivery of other substances. Specific examples include the attachment of heparin chains or polyethylene glycol chains, or fluoropassivation treatment. The modification of coatings or surface modifications is well known to those skilled in the art. Enclosure materials may have drug delivery capabilities (e.g., through the use of a hydrogel or polymer matrix incorporating a drug, biologic, or other agent) or may be attached to a drug delivery device.

[0108] As described above for unmachined bioreactor enclosures, it will be understood by those skilled in the art that machined bioreactor enclosures may be formed using surface ridges, recesses, or both, or without using either. In some embodiments, the machined enclosure may be expandable or foldable, or expandable and foldable, or neither expandable nor foldable. In some embodiments, the outer surface of the enclosure may be coated with an agent that inhibits cell adhesion or thrombus formation, and the inner surface of the enclosure may be coated with an agent that enhances cell adhesion, or the outer surface of the enclosure may not be coated with an agent that inhibits cell adhesion or thrombus formation and the inner surface may not be coated with an agent that enhances cell adhesion.

[0109] According to one embodiment, an expandable and foldable semi-permeable bioreactor enclosure has a capacity of approximately 3 million / cm³. 2 ~10 million / cm 2 It can be formed from an impermeable polyethylene terephthalate film by track etching pores with a diameter of approximately 1000 nm to 2500 nm at a given pore density.

[0110] Composite material of a permeable film and a semipermeable material

[0111] According to one embodiment, the present invention provides a transplantable bioreactor comprising cells in a cell culture matrix within an enclosure, which are capable of producing paracrine factors, the enclosure may be foldable and expandable, and the enclosure comprises at least one semipermeable material layer in contact with or adjacent to at least one permeable material layer, the permeable material layer being on the side facing outward from the enclosure, and the enclosure being semipermeable so as to result in containment of the cells, preventing leakage of the cells, and also providing an immunological barrier.

[0112] In another embodiment, the present invention provides a transplantable bioreactor comprising cells in a cell culture matrix within an enclosure, the cells being capable of producing paracrine factors, the enclosure may be foldable and expandable, and the enclosure includes at least one semipermeable material layer positioned in contact with or adjacent to at least first and second permeable material layers, the first permeable material layer being on the side facing outward from the enclosure, and the second permeable material layer being on the side facing inward from the enclosure, the layers being semipermeable to provide containment of the cells, prevent leakage of the cells, and also provide an immunological barrier (Figure 13).

[0113] According to some embodiments, the present invention can provide a appropriately semipermeable bioreactor enclosure, which can be manufactured by sandwiching an appropriately semipermeable material or machined material between layers of more permeable membranes, or alternatively, by attaching one semipermeable material or machined material to a permeable membrane to form a composite enclosure.

[0114] According to another embodiment, a nanofiber mesh with an average pore diameter of less than 5000 nm in the inner layer can be sandwiched between two permeable membranes, each having an average pore diameter of more than 5000 nm in the outer layer (Figure 10). The nanofiber mesh may be composed of one or more biodegradable materials, non-biodegradable materials, biological materials, or non-biological materials to constitute a composite enclosure.

[0115] According to another embodiment, a composite enclosure can be constructed by using two permeable outer layers, each having an average pore diameter greater than 5000 nm, to sandwich a hydrogel having an inner layer with an average pore diameter of less than 5000 nm (Figure 10). The hydrogel may be composed of a biodegradable material, a non-biodegradable material, a biological material, or a non-biological material.

[0116] According to some other embodiments, one, two, or more layers of such permeable films can be used within a composite enclosure. The composite enclosure may be formed using surface undulations, recesses, or both, or without using either.

[0117] In some embodiments, the composite enclosure may be expandable or foldable, or expandable and foldable, or neither expandable nor foldable.

[0118] In some embodiments, the outer surface of the composite enclosure may be coated with an agent that inhibits cell adhesion or thrombus formation, and the inner surface of the composite enclosure may be coated with an agent that enhances cell adhesion; the outer surface of the composite enclosure may be coated with an agent that inhibits cell adhesion or thrombus formation and the inner surface of the composite enclosure may be coated with an agent that enhances cell adhesion; and the outer surface of the composite enclosure may not be coated with an agent that inhibits cell adhesion or thrombus formation and the inner surface of the composite enclosure may not be coated with an agent that enhances cell adhesion.

[0119] Foldable enclosure bioreactor

[0120] According to one embodiment, the present invention provides a transplantable bioreactor comprising cells sealed within an enclosure, capable of producing paracrine factors, wherein the enclosure includes at least one semipermeable material layer in contact with or adjacent to at least one cell layer in a cell culture matrix, the first permeable material layer being on the outward-facing side of the enclosure, and the cell layer being on the inward-facing side of the one or more enclosures, and the cell layer being able to be rolled or folded over itself so that the semipermeable layer provides containment of the cells, prevents the cells from escaping, and also provides an immunological barrier. Such embodiments can enable the bioreactor to be placed in any body cavity, organ, blood vessel, tissue, or on the skin, or in an external or internal wound, or in a surgical wound.

[0121] In some embodiments, the folded enclosure bioreactor may consist of layers of machined material having a predetermined degree of porosity.

[0122] In some embodiments, the bioreactor comprises one or more different semipermeable and / or machined material layers and / or permeable layers, and the bioreactor may be manufactured from one or more different permeable and semipermeable sheets, either by stacking the sheets on top of each other and rolling them up or arranging them around a central axis so as to form a spiral pattern in cross-section, similar to a “Swiss roll” structure as shown in Figure 14. In a variation, instead of rolling up the sheets, the sheets may be folded into one of any desired configurations so that cells are sandwiched between the semipermeable sheets.

[0123] In some embodiments, different permeable and / or semipermeable sheets of the bioreactor can be rolled or folded to incorporate or surround a cavity or tubular structure around a central axis, and can contain cells with or without culture media, culture matrix, or nanofiber cloud. Alternatively, different permeable and / or semipermeable sheets of the bioreactor can be rolled or folded directly onto or around the surface of a medical device such as a stent or catheter.

[0124] For example, in one embodiment, different permeable and / or semi-permeable sheets of the bioreactor are wound up using a catheter located at the center of a central cavity. In some embodiments, the catheter can be made of a porous material.

[0125] According to some embodiments, one or more cell types can be grown directly on one or more different permeable and / or semipermeable sheets of a bioreactor before being rolled up or folded together with the permeable and / or semipermeable sheets.

[0126] In one embodiment, a cell layer can be grown on a flat sheet of biodegradable nanofibers, rolled up, and incorporated into a spiral structure that can constitute part of a bioreactor.

[0127] In another embodiment, a flat sheet of semipermeable nanofiber mesh can be cultured with stem cells until confluence, then wrapped around a catheter to form a spiral of alternately arranged stem cells and semipermeable nanofiber mesh layers in a concentric pattern surrounding the catheter at its center (Figure 11).

[0128] In some embodiments, the roll-type bioreactor may be formed using surface irregularities, recesses, or both.

[0129] In some embodiments, the roll-type bioreactor may be expandable, foldable, or both expandable and foldable.

[0130] In some embodiments, the roll-type bioreactor may be coated with a drug that affects cell adhesion or thrombus formation.

[0131] According to another embodiment, a roll-type bioreactor may contain a specific cell type that is grown in a particular portion of the permeable and / or semipermeable sheet, such that a desired three-dimensional structure containing cells in a particular configuration is formed when the permeable and / or semipermeable sheet is rolled up or folded. This configuration can provide a convenient method for fabricating a three-dimensional tissue engineering scaffold from a permeable and / or semipermeable sheet.

[0132] For example, one embodiment of the present invention can provide a useful structure that can be used to fabricate an artificial blood vessel. The strip is a long, flat strip of a semipermeable material having at least enough length to include three or more segments, each segment having enough length to encircle a helical axis once, the strip having endothelial cells seeded in the first segment, smooth muscle cells seeded in the middle segment, and fibroblasts seeded in the last segment. The composite structure is then rolled up, thereby creating a blood vessel-like structure having endothelial cells in the innermost layer, smooth muscle cells in the middle layer, and fibroblasts in the outermost layer.

[0133] In all of these embodiments, it will be understood by those skilled in the art that cells can be seeded on the permeable and / or semipermeable sheet either before or after the permeable and / or semipermeable sheet is folded or rolled up. Alternatively, cells can be seeded during the folding or rolling process. [Examples]

[0134] The examples presented herein illustrate the scope of the present invention, but are not intended to limit it.

[0135] (Example 1) In vitro and in vivo studies of first-generation (G1) stem cell transplantable bioreactors (SCIBs).

[0136] A prototype of a first-generation SCIB (G1-SCIB) with a cylindrical stem cell chamber (Figure 1) attached to a vascular catheter shaft was fabricated and tested. The G1 prototype was constructed from a semipermeable cellulose ester membrane with a molecular weight cutoff of 100 kilodaltons and was impermeable to the paracrine factor (PF) exosome fraction. A 10 cm membrane segment was attached to a 20 cm modified 6-French vascular catheter (Boston Scientific, Marlborough, Massachusetts) with a heat-sealed distal end and a shaft with 1 cm-slotted windows, using an 18-gauge needle. The membrane was secured to the central portion of the modified catheter using cyanoacrylate medical device adhesive (Henkel, Rocky Hill, Connecticut), 2-0 silk surgical suture, and 0.125-inch medical-grade heat-shrink tubing (InsulTab, Woburn, Massachusetts). The SCIB was sterilized in 70% ethanol under UV light for 12 hours and washed with phosphate-buffered saline (PBS; Mediatech, Manassas, Virginia) and cell culture medium before cell culture experiments and in vivo transplantation. As designed, the device is placed in the lumen of a large blood vessel along with the stem cell chamber, allowing for free exchange of PF, signaling molecules, nutrients, and waste products with the bloodstream.

[0137] MSC survival rate and paracrine factor (PF) release in G1-SCIBs To evaluate PF release in vitro, we increased the number of human mesenchymal stem cells (MSCs) into SCIBs. 5 , 10 6 , and 5×10 6The culture medium was loaded and immersed in culture medium in separate flasks for 7 days under normal culture conditions (37°C, 5% CO2). The culture medium consisted of Alpha-MEM (Mediatech, Manassas, Virginia) supplemented with 20% fetal bovine serum (FBS; Hyclone, Logan, Utah), L-glutamine (350 μg / mL; Mediatech, Manassas, Virginia), and penicillin / streptomycin (50 iU / mL / 50 μg / mL; Mediatech, Manassas, Virginia). Samples of the medium outside the SCIB were collected on days 1, 3, and 7, centrifuged for pelletizing the residue, and then flash-frozen at -80°C. The concentrations of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF), and interleukin-8 (IL-8) in this conditional medium (CM) were evaluated using multiplex ELISA (Quansys, Logan, Utah). SCIBs loaded with only medium (i.e., no cells) were used as controls. MSCs showed high viability over 7 days of culture (75.4 ± 11.6%, n=6) and produced the relevant PFs as evaluated by ELISA (Figure 2). MSCs in SCIBs placed in a hypoxic chamber increased the release of angiogenic PFs, including VEGF, confirming the ability of contained cells to modify their PF release based on external conditions within the SCIB. To determine the MSC capacity of G1-SCIB, MSCs of varying doses were cultured in stem cell chamber pouches for 7 days, and PF production was evaluated. PF production in G1-SCIB peaked at a cell dose of 25 million cells, and this dose was adopted as the maximum capacity of G1-SCIB.

[0138] Biocompatibility and immunoprotection of G1-SCIBs To confirm the biocompatibility of the device in vivo, the SCIB was surgically inserted into the right internal jugular vein of Yorkshire pigs (20-30 kg) and advanced under fluoroscopy to the junction between the SVC and the right atrium. Once positioned, the proximal end of the SCIB was fixed and the wound was closed. 10 6G1-SCIBs loaded with individual human MSCs were transplanted into the superior vena cava of pigs (Figure 3A), and despite the use of xenogeneic MSCs, there was no evidence of an immune or transfusion reaction. After one week, the G1-SCIBs were explanted and returned to the culture medium. MSCs recovered from the explanted G1-SCIBs showed continuous growth, continuous release of representative PFs such as VEGF over the following 7 days (Figure 3B), and normal morphology (Figure 3C).

[0139] Placebo-controlled pig myocardial infarction trial To determine whether SCIB-based therapy altered adverse cardiac remodeling after myocardial infarction (M), G1-SCIBs containing 25 million porcine MSCs and G1-SCIBs containing only culture medium (placebo) were placed in the superior vena cava (SVC) of pigs three days after anterior MI induced by 90-minute left anterior descending artery occlusion (n=8 in each group). To assess left ventricular (LV) function, chamber size, and scarring prior to bioreactor implantation, baseline cardiac MRI was acquired using a whole-body 3.0T MR scanner immediately before SCIB insertion three days after MI. Cine images were acquired using a radiofrequency spoiled gradient recalled echo (SPGR) pulse sequence (repetition time, TR=4.3 msec; echo time, TE=2.1 msec; flip α=12°; in-plane resolution 1.3 × 1.3 mm; 30 phases / cardiac cycle). Following peripheral injection of 0.2 mmol / kg of Magnevist (Bayer, Wayne, New Jersey), delayed gadolinium-enhanced (LGE) images were acquired using an inversion recovery-prepared T1-weighted gradient echo sequence (TR=5.3; TE=2.6; 1 RR; flip α=20°; in-plane resolution 1.4×1.5 mm). Short-axis images from base to vertex were acquired with a slice thickness of 6 mm, and there were no gaps between the cine and LGE images. Cardiac MRI at week 4 using the same imaging parameters revealed that, while G1-SCIB was in place, cell-treated pigs showed significantly reduced adverse left ventricular cardiac remodeling compared to placebo pigs (by blind analysis), and slightly increased end-systolic volume (ESV, 2.1±4.1 mL / 11.4±2.9 mL, cell / placebo, p=0.036) and end-diastolic volume (EDV, 0.3±4.3 mL / 10.4±3.8 mL, cell / placebo, p=0.059) (Figure 4). Left ventricular ejection fraction decreased in placebo pigs (-5.8±1.7%, p=0.03), but did not decrease in cell-treated pigs (-2.3±2.9%, p=0.33).As assessed by delayed gadolinium-enhanced cardiac MRI, changes in overall scar size were observed in both groups (Figure 5). Scar size was further divided into core dense scars and heterogeneous gray scars. The core area included all pixels with an SI greater than 50% of the maximum SI within the hyper-enhanced region. Areas of microvascular occlusion were included as part of the core. The gray zone area included all pixels with an SI greater than the peak SI in normal myocardium but less than 50% of the maximum SI within the hyper-enhanced region. When the components of heterogeneous scar masses (i.e., “gray” masses and dense “core” masses) were analyzed in this way, animals treated with SCIB-based cell therapy showed a significant increase in heterogeneous gray scars, along with a significant decrease in dense core scars that were not present in placebo-treated animals. G1-SCIBs removed from the animals were placed in culture medium, and G1-SCIBs with MSCs continued to release PF (787 ± 257 pg / mL of VEGF at 7 days). Importantly, all animals survived throughout the study period; there were no late or unexpected deaths suggestive of lethal arrhythmias, device thrombosis, or pulmonary embolism.

[0140] safety All pigs transplanted with SCIB survived all trials. Despite the use of allogeneic porcine MSCs, there was no evidence of fever or leukocytosis suggestive of an immune response. The pigs were observed for 4 weeks after SCIB removal and remained asymptomatic. Necropsy revealed no evidence of neoplastic activity. Interestingly, there was no pericarditis in the cell therapy group, compared to 50% of the placebo group, suggesting a reduced inflammatory response after MI in the cell therapy group.

[0141] summary MSCs proliferated within G1-SCIB are viable and freely release associated PFs; G1-SCIB is safe and well-tolerated, providing up to 4 weeks of immune protection for the housed cells when placed in the superior vena cava of pigs; SCIB-based cell therapy using allogeneic MSCs beneficially reduced adverse remodeling at 4 weeks post-MI in pigs.

[0142] (Example 2) In vitro and in vivo testing of second-generation (G2) stem cell implantable bioreactors (SCIBs) with enhanced paracrine factor permeability and internal hydrogel matrix

[0143] A prototype of the second-generation SCIB (G2-SCIB) was constructed as a stem cell pouch, which was based on a 25 μm thick polyethylene terephthalate (PET) film and incorporated a highly porous hyaluronan hydrogel to enclose mesenchymal stem cells within the pouch (Figures 7A-7B). The pores in the PET film were fabricated using track etching, a technique that enables the creation of a high-density field of highly uniform circular pores (Figure 6, Figure 8). The PET film is first irradiated under high vacuum with single-energy heavy ions accelerated by a cyclotron to create linear damage tracks through the film. Next, the damage tracks in the irradiated film are treated in successive alkaline hydrolysis baths prior to acid neutralization and washing with demineralized water, etching uniform cylindrical pores with pore size and density that can be strictly controlled by varying irradiation time, base and acid concentrations for etching, etching temperature, and etching time. The stem cell pouch of the prototype G2-SCIB has a pore diameter of 0.44 μm to 2.0 μm and a density of 2.4 million pores / cm 2 ~8 million pores / cm 2Although fabricated within this range, as detailed above, smaller or larger pore diameters and lower or higher pore densities are achievable. Since exosomes are generally less than 0.2 μm in diameter and cells are generally more than 8 μm in diameter, the pores of G2-SCIBs allow the pouch to be completely permeable to the exosome fraction of PF (unlike G1-SCIBs) while maintaining impermeability to cells.

[0144] Permeability of G2-SCIB to proteins and exosomes

[0145] The permeability of the G2-SCIB pouch to FITC-albumin was measured over 2 hours. Albumin with a molecular weight of 66 kD (comparable to or larger than many growth factor paracrine factors) traversed the track-etched PET membrane with 0.44 μm pores without obstruction and entered the surrounding bath. 20% was released from the SCIB within 30 minutes, and nearly 100% was released in less than 2 hours (Figure 10A). Exosomes also readily traversed the G2-SCIB pouch membrane. Rat H9c2 cardiomyocytes greedily took up exosomes that moved freely across the track-etched PET membrane (Figure 10B). Simultaneously, no H9c2 cardiomyocytes were observed traversing the membrane, which is consistent with the significantly larger cell diameter compared to the track-etched pore diameter.

[0146] Enhancement of stem cell survival in G2-SCIBs using a hydrogel matrix.

[0147] Luciferase-expressing mouse MSCs were subjected to 14 days in track-etched PET pouches of cylindrical SCIBs measuring 4.0 mm in diameter and 32 mm in length, with or without HyStem HP hyaluronane hydrogel injected into the pouch. 6Cells were grown according to the specified dose. Flow cytometry using markers CD44, Sca-1, CD31, CD45, and CD34 showed that MSCs retained the expression profiles of these cell surface markers. MSC viability was assessed up to 14 days of culture using luciferase bioluminescence imaging (BLI). Mouse MSCs in G2-SCIB pouches incorporating an internal hydrogel matrix maintained significantly higher viability compared to mouse MSCs in G2-SCIB pouches without an internal hydrogel matrix (Figure 11A). The four-fold increase in mouse MSC viability with hydrogel matrix in the pouch persisted up to 14 days of culture (Figure 11B).

[0148] PF production and tubule formation in G2-SCIB

[0149] Human MSCs grown in G2-SCIBs incorporating hyaluronane hydrogel and human MSCs grown in G1-SCIBs of similar size, as well as relative PF production and release, were evaluated. The production of VEGF, a representative angiogenic PF, was measured by ELISA. VEGF production in G2-SCIBs was uniformly more than 10 times higher than in G1-SCIBs throughout the 7-day culture period (Figure 12A). Consistent with this, in vitro endothelial cell tubule formation induced by paracrine factors released from G2-SCIBs after 8 days of culture was also significantly greater than that induced by G1-SCIBs (Figure 12B). The improved in vitro tubule formation potential of G2-SCIBs can be explained by the presence of an internal hydrogel matrix. When G2-SCIBs without an internal hydrogel matrix were compared to G1-SCIBs, there was no longer a significant difference in the degree of tubule formation.

[0150] Biocompatibility and immunoprotection of G2-SCIBs

[0151] 5×10⁻⁶ cells expressing luciferase 6G2-SCIBs loaded with mouse MSCs were transplanted into the superior vena cava of pigs for two weeks; control G2-SCIBs loaded with the same number of cells were maintained in culture medium. Despite the use of xenogeneic cells, the animals tolerated the transplant well, and there was no evidence of immune responses, transfusion reactions, or thrombosis. Post-explantation, bioluminescence imaging showed a 35% higher MSC survival rate in transplanted G2-SCIBs compared to in vitro G2-SCIBs maintained in culture medium. These data demonstrated that immunoprotection and biocompatibility are maintained by G2-SCIBs; furthermore, the higher MSC survival rate in transplanted G2-SCIBs suggests that the efficiency of nutrient and waste product migration across the SCIB stem cell pouch membrane is improved by circulating it through the bloodstream.

[0152] Cell viability of G2-SCIBs using microbeads

[0153] MSCs were loaded onto polystyrene microbeads (diameters of 106 μm–125 μm were used, but larger or smaller diameters are also possible). The microbeads were sterilized and coated with fibronectin, and MSCs were seeded on them. The MSCs were cultured on the microbeads for 3 days. The microbeads coated with MSCs were then injected into cell chambers of SCIB at a density of approximately 600,000–1,200,000 microbeads per 3.4 ml of cell chamber volume. Higher or lower microbead densities can also be used to achieve different cell loading levels. After 7 days in the cell chamber, the MSCs remained attached to the microbeads, and the cell viability on the microbeads after 7 days of culture in cell pouches exceeded 90% according to live / green fluorescence and dead / red fluorescence viability assays (Figure 9).

[0154] summary

[0155] Tests of G2-SCIBs with a novel track-etched PET pouch incorporating an internal hyaluronane hydrogel matrix demonstrated permeability to large proteins and exosomes, impermeability to cells, and superior stem cell viability through high levels of PF production and endothelial tubule formation, while maintaining biocompatibility and immunoprotection in vivo.

[0156] All references cited herein, including publications, patent applications, and patents, are incorporated herein by reference to the same extent as if they were included herein, provided that each reference is indicated to be incorporated individually and specifically by reference.

[0157] Unless otherwise indicated herein, or unless clearly inconsistent with the context, the use of the terms “a,” “an,” and “the” and similar referents in the context of the description of the Invention (in particular, in the context of the following claims) should be construed to cover both singular and plural. The terms “equip,” “have,” “include,” and “contain” should be construed as unrestrictive terms (i.e., “include but not limited to”) unless otherwise specified herein. The enumeration of value ranges herein is intended only to serve as abbreviations for each individual value included in the range individually, unless otherwise indicated herein, and each individual value is incorporated herein as if it were individually described herein. All methods described herein may be performed in any suitable order unless otherwise indicated herein, or unless clearly inconsistent with the context. The use of any examples or illustrative language provided herein (e.g., “etc.”) is intended merely to better illustrate the Invention and, unless otherwise requested, does not limit the scope of the Invention. The language used herein should not be construed as indicating that any element not claimed is essential to the implementation of the invention.

[0158] Preferred embodiments of the Invention, including the best mode known to the inventors for carrying out the Invention, are described herein. Variations of these preferred embodiments may become apparent to those skilled in the art by reading the foregoing description. The inventors expect that those skilled in the art will appropriately use such variations, and the inventors intend that the Invention may be carried out in ways other than those specifically described herein. Accordingly, the Invention includes all modifications and equivalents of the subject matter enumerated in the claims appended herein, as permitted by applicable law. Furthermore, any combination of the above elements in all possible variations thereof is included in the Invention unless otherwise indicated herein or unless it is clearly inconsistent with the context.

Claims

1. a) One or more enclosures defining a sealed space having an inner and outer surface, wherein the one or more enclosures include at least one semipermeable material layer positioned in contact with or adjacent to at least first and second permeable material layers, the first permeable material layer being on the outer surface of the enclosure, the second permeable material layer being on the inner surface of the enclosure, the one or more enclosures being semipermeable to result in the containment of cells in the sealed space and to prevent the outflow of the cells, the one or more enclosures being able to allow the outflow of paracrine factors from the enclosure, the at least one semipermeable material layer having pores with a diameter of 50 nm to 5,000 nm, and the first and second permeable material layers having pores with a diameter greater than 5,000 nm; and b) Cells in a cell culture matrix within one or more enclosures, wherein the cells are capable of producing paracrine factors, and the cell culture matrix comprises a hydrogel. A portable bioreactor equipped with [specific features / equipment].

2. The portable bioreactor according to claim 1, wherein one or more enclosures are foldable and expandable.

3. The implantable bioreactor according to claim 1 or 2, wherein the enclosure is made of a biological material and / or a synthetic material, the biological material being selected from cellulose, collagen, lipids, nucleic acids and proteins.

4. The transplantable bioreactor according to claim 3, wherein the biological material is cellulose acetate.

5. The transplantable bioreactor according to claim 3, wherein the synthetic material is selected from the group consisting of polysulfone, polyamide, polyacrylonitrile, copolymers thereof, polyethylene terephthalate (PET), polymethyl methacrylate, polytetrafluoroethylene and derivatives thereof, polycarbonate and derivatives thereof, poly(ethylene-co-vinyl acetate) and derivatives thereof, poly(n-butyl methacrylate) and derivatives thereof, poly(styrene-b-isobutylene-b-styrene) and derivatives thereof, polycaprolactone and derivatives thereof, polyimide and derivatives thereof, polyurethane and derivatives thereof, poly(lactic acid), poly(lactide-co-glycolide), and silicon.

6. The transplantable bioreactor according to any one of claims 1 to 5, wherein the cells are stem cells.

7. The transplantable bioreactor according to claim 6, wherein the stem cells are selected from the group consisting of embryonic stem cells, mesenchymal stem cells, adipose-derived stem cells, and endothelial stem cells.

8. The transplantable bioreactor according to any one of claims 1 to 7, wherein the cell culture matrix comprises one or more compositions selected from the group consisting of cell culture medium, microbeads, polymer matrix, tissue engineering scaffold, nanofibers (e.g., PLGA-based nanofibers), and biological extracellular matrix.

9. The implantable bioreactor according to any one of claims 1 to 8, wherein the hydrogel comprises a hyaluronan-based, PEG-based, or collagen-based hydrogel.

10. The implantable bioreactor according to claim 1 or 2, wherein the bioreactor enclosure is attached to a wire, attached to a catheter, or attached as part of another implantable medical device.

11. The transplantable bioreactor according to any one of claims 1 to 5, wherein the cells comprise stem cells and supporting cells, the supporting cells being selected from fibroblasts, endothelial cells, endothelial progenitor cells, and bone marrow stromal cells.

12. The portable bioreactor according to claim 1 or 2, wherein the enclosure defines a port configured to be accessible in order to allow at least one of the containment space defined by the enclosure to be emptied, filled, or refilled.

13. The present invention further comprises a catheter having a distal end and a proximal end, wherein the distal end of the catheter is attached to the enclosure via the port. The implantable bioreactor according to claim 12, wherein the catheter is at least partially implantable, while the proximal end of the catheter is adapted to extend outside the patient's body during use, and the distal end of the catheter is implantable.

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