Abstract: A method and apparatus for preparing biological cell samples for intracellular analysis. The invention is based upon the recognition that many of the steps of the conventional methods for such sample preparation can be eliminated, leading to a process that readily lends itself to automation and the advantages associated therewith. The method of the invention comprises the steps of (a) cell-fixation, (b) permeabilization and (c) staining (or labeling) of intracellular molecules of interest by probes that are readily detectable by flow cytometric techniques, all without any intervening cell-washing (and re-suspension) steps. Rather, the single cell-washing step is effected after these three steps have been carried out. Preferably, the washing step is carried out by passing the fixed, permeabilized and stained cell sample through a semi-permeable membrane that serves to filter out (by transmission) interferants to waste while retaining the cells of interest.

Abstract: The invention relates generally to devices for organ replacement and regenerative medicine providing a biocompatible and biodegradable scaffold capable of integral cell growth that forms a hollow chamber, as well as methods for producing such devices by melt-blowing a web of flexible, polymer fibers in the presence of a porogen to produce a seamless, three-dimensional shape.

Abstract: Described are compositions with tethered growth effector molecules, and methods of using these compositions for growing cells and tissues. Growth effector molecules, including growth factors and extracellular matrix molecules, are flexibly tethered to a solid substrate. The compositions can be used either in vitro or in vivo to grow cells and tissues. By tethering the growth factors, they will not diffuse away from the desired location. By making the attachment flexible, the growth effector molecules can more naturally bind to cell surface receptors. A significant feature of these compositions and methods is that they enhance the biological response to the growth factors. The method also offers other advantages over the traditional methods, in which growth factors are delivered in soluble form: (1) the growth factor is localized to a desired target cell population; (2) significantly less growth factor is needed to exert a biologic response.

Abstract: The invention relates to a method of in vitro testing of the efficacy of a potentially active substance comprising monitoring the effect of said potentially active substance on an artificial skin, comprising a composite product forming a collagen support comprising at least one porous collagen layer covered on at least one side with a collagen membrane component selected from the group consisting of a collagen membrane prepared by compression of a collagen sponge at a pressure of at least about 50 bar and of a collagen membrane comprising a collagen film prepared by drying a collagen gel separately from the porous collagen layer, thereby providing a reliable method for finding new potentially active substances.

Abstract: The present invention relates generally to growth factors and more particularly to growth factors which are capable of stimulating or otherwise facilitating formation of insulin-secreting cells. The identification of these growth factors permits the development of protocols to culture cells in vitro for transplantation into mammalian and in particular human subjects with insulin-dependent type 1 diabetes or related conditions. It is further contemplated that the endogenous expression of growth factors required for the development of insulin-producing cells may be manipulated in vivo, by the appropriate administration of agents including genetic agents capable of regulating the expression of growth factors in pancreatic duct epithelial cells. The growth factors ray also be administered to subjects with type 1 diabetes to stimulate the proliferation and differentiation of pancreatic cells into insulin-secreting cells.

Abstract: Implantable medical devices that include a non-woven framework are described, as well as methods of using such devices to deliver therapeutic compounds to a patient.

Abstract: A method of expanding/maintaining undifferentiated hemopoietic stem cells or progenitor cells by obtaining undifferentiated hemopoietic stem cells or progenitor cells; and either seeding the undifferentiated hemopoietic stem cells or progenitor cells into a stationary phase plug-flow bioreactor in which a three-dimensional stromal cell culture has been pre-established on a substrate in the form of a sheet, the substrate including a non-woven fibrous matrix forming a physiologically acceptable three-dimensional network of fibers, thereby expanding/maintaining undifferentiated hemopoietic stem cells or progenitor cells, or culturing the undifferentiated hemopoietic stem cells or progenitor cells in conditioned medium obtained from such a reactor.

Abstract: A scaffold for tissue culture and cell culture and for producing implant materials, in particular bone, cartilage or skin replacements or extra-corporal organ replacements or for other applications in medicine or biotechnology is made of biocompatible materials. It has at least one base material which is electrostatically flocked with fibers on at least one side. Through the electrostatic flocking the fibers are arranged almost perpendicularly on the surface of the base material and exhibits a high fiber pull-out resistance. The scaffold provides an elastic growth lattice, which is stable against compression, for cell colonization in vitro or the ingrowth of cells in vivo. Implants or implant materials can be produced with the scaffold.

Abstract: The present invention provides nano-porous fibers and protein membrane compositions. In certain embodiments, continuous fiber compositions are provided having nanometer sized diameters and surface pores. In another embodiment, a protein membrane composition is provided comprising a protein; and a polymer, wherein the protein and the polymer are electrospun to form a protein membrane composition. In certain instance, the protein is covalently bound to the fiber.

Abstract: The present invention provides a method for the propagation of lytic organisms which comprises the infection of the cells of a stable cell line within a hollow fibre bioreactor with a lytic organism, wherein after said infection, said organism multiplies within the cells and can be harvested, characteriscd in that the cell line can survive for at least ten days after said infection. The invention further provides a method as herein described wherein after harvest, the cell line is allowed to re-populate the bioreactor, and at least one subsequent harvest may be taken, with the cell line being able to repopulate the bioreactor after each harvest.

Abstract: Porous, bioabsorbable scaffolds for tissue engineering of human hair follicles, methods for their manufacture and methods of their use in creating new hair.

Abstract: A fully biodegradable fibre reinforced composite adapted for use as a medical implant which is shaped and processed by means of a resin reaction injection transfer molding process adapted for predetermining shape, physical properties and degradation profile, shaped preform and/or composition for preparation of the shaped composite, process for the production of the shaped composite comprising obtaining a shaped preform and impregnating with resin with simultaneous processing thereof, shaped composite comprising thermoplastic matrix and fibres adapted for use as a medical implant, characterised by a differential degradation of matrix with respect to fibres adapted to degrade via an intermediate shaped structure comprising residual porous matrix or residual fibre form respectively and selection of composite is made for primary growth of a preferred cell type, throughout voids created by degraded matrix or fibre respectively, according to the desired healing or reconstruction locus, the shaped composites for use

Abstract: A cell and substrate system and nerve regeneration implant are disclosed including a carbon nanotube and a neuron growing on the carbon nanotube. Both unfunctionalized carbon nanotubes and carbon nanotubes functionalized with a neuronal growth promoting agent may be utilized in the invention. A method is also disclosed for promoting neuronal growth.

Abstract: The present invention is directed to novel compositions and methods for the treatment of degenerative intervertebral disc disease. In some embodiments, the invention relates to a preparation of nucleus pulposus cells comprising purified nucleus pulposus cells. In some embodiments, the invention relates to methods of treating degenerative intervertebral disc disease in an individual comprising implanting nucleus pulposus cells into the nucleus pulposus space of a degenerated disc of the individual. Other embodiments of the invention relate to methods of generating nucleus pulposus cells.

Abstract: A device is provided containing cells or tissue distributed on a filamentous cell-supporting matrix which is encapsulated by a semi-permeable membrane which can be immunoisolatory. The matrix may be formed of a plurality of monofilaments twisted into yarn that is in non-woven strands, or of the monofilaments or yarn woven into a mesh. Configurations of the matrix include a hollow cylinder, tube, solid cylinder or cord. A coating of extracellular matrix molecules may be on the matrix, or the matrix may be treated with plasma irradiation to enhance cell adhesion. The device can be made by inserting the matrix in a hollow membrane tube, injecting cells or tissue into the tube and sealing ends of the tube. The device is particularly useful for implantation into a mammalian host to provide therapy resulting from a biologically active molecule produced by the cells and tissue.

Abstract: A bi-component microporous hollow fiber membrane structure is provided for in vivo propagation of cells and use in testing of the effect of medical treatments on cells within the structure. The structure has an inner structure fabricated from a first bio-compatible polymer and an outer structure fabricated from a different polymer that has a lower tendency for cell adhesion than the inner structure polymer. In this way, the inner structure can be selected to optimize cell propagation and the outer structure can be fabricated from a polymer which optimizes the removal of the bi-component structure from its implanted location. The inner and outer structures may have a pore size between 10 and 1000 Angstroms and 100 and 2000 Angstroms, respectively, and be formed from polysulfone or polyether sulfone and polyvinyledene difuoride, respectively. The membrane structure can form macrocapsules containing media and living cells for implanting.

Abstract: Use of collagen of aquatic origin for the production of supports for tissue engineering is disclosed. The collagen may be obtained from fish skin, preferably in its native form. Novel tissue engineering supports with a low risk of contamination are produced.

Abstract: The present invention describes methods for producing artificial fascial slings and their subsequent use in treating subjects with urinary incontinence. The invention is based, in part, on the discovery that mesenchymal cells that secrete elastin and collagen, extracellular proteins responsible for elasticity and strength, respectively, can be used to engineer artificial fascia in vitro.

Abstract: Cardiovascular components such as biocompatible heart valves and annular sewing rings are disclosed, as well as, methods for making the same. The heart valves include biodegradable polymer fiber scaffolds and collagen. Also disclosed are donor aortic heart valves processed without the use of crosslinking chemicals.

Abstract: The present invention describes methods for producing artificial fascial slings and their subsequent use in treating subjects with urinary incontinence. The invention is based, in part, on the discovery that mesenchymal cells that secrete elastin and collagen, extracellular proteins responsible for elasticity and strength, respectively, can be used to engineer artificial fascia in vitro.

Abstract: A method is provided whereby cells having a desired function are seeded on and into biocompatible, biodegradable or non-degradable porous polymer scaffolding matrix, previously implanted in a patient and infiltrated with blood vessels and connective tissue, to produce a functional organ equivalent. The resulting organoid is a chimera formed of parenchymal elements of the donated tissue and vascular and matrix elements of the host. The matrix should be compression resistant and a non-toxic, porous template for vascular ingrowth. The pore size, usually between approximately 100 and 300 microns, should allow vascular and connective tissue ingrowth throughout approximately 10 to 90% of the matrix, and the injection of cells such as hepatocytes without damage to the cells or patient. The introduced cells attach to the connective tissue and are fed by the blood vessels.

Abstract: A device is prepared having cells or tissue attached to a non-degradable filamentous matrix surrounded by a semi-permeable membrane. The matrix is preferably formed of a plurality of monofilaments twisted into a yarn or woven into a mesh, and can be in the form of a cylinder. When implanting the device, the semi-permeable membrane is preferably immunolsolatory, and the cells or tissue may produce a biologically active molecule to provide therapy. To enhance cell or tissue adhesion, the matrix is coated with extracellular matrix molecules or treated to provide a surface charge. The device can be made by inserting the matrix into a capsule formed of the semi-permeable membrane, distributing the cells or tissue on the matrix through an opening of the capsule, and sealing the opening of the capsule.

Abstract: The present invention provides genetically engineered cell lines that expresses bcl-2 in response to hypoxic conditions and are, therefore, resistant to apoptosis. In one embodiment, &bgr;Tc-tet cells are stably transformed with the bcl-2 gene operably linked to the hypoxia responsive PGK promoter. The cells may be provided directly to a patient or may be encapsulated to from a bioartificial organ.

Abstract: The invention relates to a bioreactor for a cell treatment of a medium. Said bioreactor comprises an element defining a chamber in which cells for treating the medium are located, a liquid permeable membrane separating the said chamber from a first channel in which flows the medium to be treated, and a gas permeable membrane separating the said chamber from a second channel in which flows a gas containing oxygen.

Abstract: A stromal cell-based three-dimensional cell culture system is provided which can be used to culture a variety of different cells and tissues in vitro for prolonged periods of time. The stromal cells along with connective tissue proteins naturally secreted by the stromal cells attach to and substantially envelope a framework composed of a biocompatible non-living material formed into a three-dimensional structure having interstitial spaces bridged by the stromal cells. Living stromal tissue so formed provides support, growth factors, and regulatory factors necessary to sustain long-term active proliferation of cells in culture and/or cultures implanted in vivo. When grown in this three-dimensional system, the proliferating cells mature and segregate properly to form components of adult tissues analogous to counterparts in vivo, which can be utilized in the body as a corrective tissue.

Abstract: A matrix for tissue culture comprising two kinds of sponges having at least one different physical property and/or at least one different chemical property; a method for culturing tissue using said matrix for tissue culture comprising inoculating and culturing first cell on a first sponge, laminating a second sponge thereon, and inoculating and culturing second cell on said second sponge; a method for fixing a cultured tissue comprising placing a cultured tissue in gelatin solution solated by elevating temperature, lowering temperature to gelatinize gelatin to fix the cultured tissue by said gelatinated gelatin; and an artificial skin fixed comprising dermis layer fixed by gelatin and epidermis layer laminated on the dermis layer.

Abstract: Disclosed are compositions with tethered growth effector molecules, and methods of using these compositions for growing cells and tissues. Growth effector molecules, including growth factors and extracellular matrix molecules, are flexibly tethered to a solid substrate. The compositions can be used either in vitro or in vivo to grow cells and tissues. By tethering the growth factors, they will not diffuse away from the desired location. By making the attachment flexible, the growth effector molecules can more naturally bind to cell surface receptors. A significant feature of these compositions and methods is that they enhance the biological response to the growth factors. The new method also offers other advantages over the traditional methods, in which growth factors are delivered in soluble form: (1) the growth factor is localized to a desired target cell population; (2) significantly less growth factor is needed to exert a biologic response.

Abstract: The present invention provides an apparatus and methods for incubating cells which rely on the use of a semipermeable membrane placed between two planar members, at least one planar member having an aperture.

Abstract: A stromal cell-based three-dimensional cell culture system is prepared which can be used to culture a variety of different cells and tissues in vitro for prolonged periods of time. The stromal cells and connective tissue proteins naturally secreted by the stromal cells attach to and substantially envelope a framework composed of a biocompatible non-living material formed into a three-dimensional structure having interstitial spaces bridged by the stromal cells. The living stromal tissue so formed provides the support, growth factors, and regulatory factors necessary to sustain long-term active proliferation of cells in culture and/or cultures implanted in vivo. When grown in this three-dimensional system, the proliferating cells mature and segregate properly to form components of adult tissues analogous to counterparts in vivo, which can be utilized in the body as a corrective tissue.

Abstract: Improved hollow fiber bioreactor systems and cell culture methods are described. The improvement includes means which may be a novel basket bioreactor for extending the extracapillary space of a conventional hollow fiber bioreactor.

Abstract: A method and apparatus for forming a permanent, composite skin replacement consisting of an epidermal component and a porous, resorbable, biosynthetic laminated dermal membrane component for use in wound repair. The dermal membrane is formed in the apparatus, which permits regulation of variables affecting membrane structure. The dermal membrane may be modified to incorporate biologically active molecules to enhance wound repair and to reduce infection when the skin replacement is applied to a wound.

Abstract: Plant tissues which have been cultured in vitro are packaged with a nutritive medium for survival of the tissues in a sealed sterile container and with support members so that the plant tissues are positioned between surfaces of the support members. The support members have opposing surfaces for supporting the tissues and for support by a surface of the container and are made of a material suitable for being impregnated by the nutritive medium, and the container has a surface sized suitably for supporting the surface of the support member. At the time of packaging, at least one of the two support members is impregnated with the nutritive medium in an amount so that the tissues attach, by capillarity, to one surface of the at least one impregnated support member and so that the opposing surface of the at least one impregnated support member, when packaged, attaches, by capillarity, to the container surface.

Abstract: A method for maintaining hepatocytes in culture includes providing the hepatocytes with a support, the support including extracellular matrix, the support having a configuration that permits each of at least a portion of the hepatpocytes to form at least one apical surface and at least two discrete basal surfaces.

Abstract: Disclosed are compositions with tethered growth effector molecules, and methods of using these compositions for growing cells and tissues. Growth effector molecules, including growth factors and extracellular matrix molecules, are flexibly tethered to a solid substrate. The compositions can be used either in vitro or in vivo to grow cells and tissues. By tethering the growth factors, they will not diffuse away from the desired location. By making the attachment flexible, the growth effector molecules can more naturally bind to cell surface receptors. A significant feature of these compositions and methods is that they enhance the biological response to the growth factors. The new method also offers other advantages over the traditional methods, in which growth factors are delivered in soluble form: (1) the growth factor is localized to a desired target cell population; (2) significantly less growth factor is needed to exert a biologic response.

Abstract: The invention relates to the field of biology and, in particular, to the field of cellular biology. The invention concerns a novel glucose-sensitive cell line designated .beta. cell line (INS-I) expressing glucokinase and the glucose carrier Glut 2 at levels comparable with those of normal .beta. cells but which is, in addition, incapable of IGF-II expression-dependent proliferation because of genetic manipulation. The invention also concerns a method for the production of said novel cell line, its aggregation in the form of a pseudoislet, its immobilization in a biocompatible hydrogel and its hardening by means of a hardening solution. Application in insulin-secreting .beta.-cell transplants.

Abstract: A stromal cell-based three-dimensional cell culture system is provided which can be used to culture a variety of different cells and tissues in vitro for prolonged periods of time. The stromal cells along with connective tissue proteins naturally secreted by the stromal cells attach to and substantially envelope a framework composed of a biocompatible non-living material formed into a three-dimensional structure having interstitial spaces bridged by the stromal cells. Living stromal tissue so formed provides support, growth factors, and regulatory factors necessary to sustain long-term active proliferation of cells in culture and/or cultures implanted in vivo. When grown in this three-dimensional system, the proliferating cells mature and segregate properly to form components of adult tissues analogous to counterparts in vivo, which can be utilized in the body as a corrective tissue.

Abstract: The present invention relates to a three-dimensional cell and tissue culture system. In particular, it relates to this culture system for the long term culture of liver cells and tissues in vitro in an environment that more closely approximates that found in vivo. The culture system described herein provides for proliferation and appropriate liver cell maturation to form structures analogous to tissue counterparts in vivo. The resulting liver tissues survive for prolonged periods, perform liver-specific functions, and maintain hepatic tissue architecture following in vivo implantation.The liver cultures have a variety of applications ranging from transplantation or implantation in vivo, to screening cytotoxic compounds and pharmaceutical compounds in vitro, to the production of biologically active molecules in "bioreactors" and to the construction of extracorporeal liver assist device.

Abstract: A matrix structure containing attached cells such as endocrine cells, fibroblasts, endothelial cells or genitourinary cells is implanted in a patient adjacent tissue having a high surface area and vasculature such as mesentery, omentum or peritoneum tissue. Large volumes of cells can be attached to the matrix and the matrix implanted with minimum trauma and blood loss into a patient to produce a functional organ equivalent. Multiple matrix structures containing cells can be implanted to functionally resemble naturally occurring organs. Implanting multiple matrices between folds of the mesentery is particularly well suited for growth of endocrine structures, including liver, pancreas, and adrenal gland. The matrix structure is preferably formed from a biodegradable artificial polymer. Collagen and non-biodegradable materials can also be used, and the matrix structure can be overlaid with a material that enhances cell attachment.

Abstract: A method is provided for preparing a low density porous rigid fused-fiber matrix having a density of between about 3.5 and 5.5 pounds/cubic foot and a free volume of between about 90-98 volume percent for use as a cell-culture substrate or implant material, or in chromatographic separation of blood cells. The method is carried out by forming a slurry containing (I) silica, alumina or silica and alumina fibers having thicknesses between about 0.5 and 20 .mu.m and lengths between about 1 and 10 mm, and having a fiber:liquid weight ratio of between about 1:25 to 1:70, (ii) a thickening agent to give the slurry a viscosity between about 1,000 and 25,000 centipoise, (iii) boron nitride particles between about 2-12 percent by weight of the total fiber weight, and (iv) a dispersing agent when the slurry contains silica fibers, allowing the slurry to settle in a mold to produce a fiber block, drying the fiber block, and heating the dried fiber block to at least about 2200.degree. F.

Abstract: Fibers of a biocompatible, biodegradable or non-biodegradable, synthetic polymer are provided, and are formed into a three-dimensional scaffold. The fibers of the scaffold may have a branched configuration extending outwardly from a central stem. The fibers provide sufficient surface area to permit attachment to the scaffold in vitro of an amount of cells effective to produce functional vascularized organ tissue in vivo. Fibers of the scaffold are spaced apart such that the maximum distance over which diffusion of nutrients and gases must occur through a mass of cells attached to the fibers is between 200 and 300 microns. The diffusion provides free exchange of nutrients, gases and waste to and from cells proliferating throughout the scaffold in an amount effective to maintain cell viability throughout the scaffold in the absence of vascularization.

Abstract: A cell-scaffold composition is prepared in vitro for implanting to produce functional organ tissue in vivo. The scaffold is three-dimensional and is composed of hollow or solid fibers of a biocompatible, synthetic polymer which is biodegradable or non-biodegradable. The fibers of the scaffold may have a branched configuration extending outwardly from a central stem. Fibers of the scaffold are spaced apart such that the maximum distance over which diffusion of nutrients and gases must occur through a mass of cells attached to the fibers is between 200 and 300 microns. The diffusion provides free exchange of nutrients, gases and waste to and from cells proliferating throughout the scaffold in an amount effective to maintain cell viability throughout the scaffold in the absence of vascularization.

Abstract: A composition and method are provided for inhibition of vascular smooth muscle cell proliferation following injury to the endothelial cell lining of a blood vessel such as resulting from angioplasty, vascular bypass surgery or organ transplantation. The composition is a matrix such as a biodegradable hydrogel made of a synthetic polymer, protein or polysaccharide seeded with vascular endothelial cells which can be xenografts, allografts or autografts, or genetically engineered cells. Attachment of cells to the matrix can be enhanced by coating with collagen, laminin, fibronectin, fibrin, basement membrane components or attachment peptides. Biologically active compounds such as anti-inflammatory agents may also be contained in the matrix. In the method, the matrix containing endothelial cells is implanted in a patient at a site adjacent the injury such as by wrapping the matrix around the blood vessel.

Abstract: A cell-scaffold composition is prepared in vitro for implanting to produce functional organ tissue in vivo. The scaffold is three-dimensional and is composed of fibers of a biocompatible, biodegradable, synthetic polymer. Cells derived from vascularized organ tissue are attached in vitro to the surface of the fibers uniformly throughout the scaffold in an amount effective to produce functional vascularized organ tissue in vivo. Fibers of the scaffold are spaced apart such that the maximum distance over which diffusion of nutrients and gases must occur through a mass of cells attached to the fibers is between 100 and 300 microns. The diffusion provides free exchange of nutrients, gases and waste to and from cells proliferating throughout the scaffold in an amount effective to maintain cell viability throughout the scaffold in the absence of vascularization.

Abstract: Cells for implantation into a patient are packaged within a barrier of immunoprotective tissue prior to implantation to obviate or minimize rejection of the cells. The preferred immunoprotective tissue for forming the barrier is cartilage. The tissue is formed into a layer that is thin enough to allow diffusion of nutrients and gases into the center of a cell mass packaged within the immunoprotective tissue. Typically the layer is less than 300 microns, preferably between 5 and 20 microns. Cells to be implanted, typically dissociated parenchymal cells including hepatocytes, Islets of Langerhans, or other cells having metabolic functions, are then placed on the tissue layer, and the layer is folded to seal the cells to be implanted within the tissue layer. In the preferred embodiment, the dissociated cells are first seeded onto a polymeric fiber matrix.

Abstract: Methods and artificial matrices for the growth and implantation of cartilaginous structures and surfaces and bone are disclosed. In the preferred embodiments, chondrocytes are grown on biodegradable, biocompatible fibrous polymeric matrices. Optionally, the cells are proliferated in vitro until an adequate cell volume and density has developed for the cells to survive and proliferate in vivo. One advantage of the matrices is that they can be cast or molded into a desired shape, on an individual basis, so that the final product closely resembles a patient's own ear or nose. Alternatively, flexible matrices can be used which can be manipulated at the time of implantation, as in a joint, followed by remodeling through cell growth and proliferation in vivo. The cultured cells can also be maintained on the matrix in a nutrient media for production of bioactive molecules such as angiogenesis inhibiting factor.

Abstract: A method of growing complete vertebrate skin in vitro, which comprises obtaining a segment of vertebrate skin, positioning the skin segment in an artificial cell-growth medium containing sufficient nutrients to maintain growth of cells of the skin, and subjecting the skin segment to stretching forces while the skin segment is in the medium. Skin produced by the method and an apparatus for carrying out the method are also part of the present invention.

Abstract: A high performance hollow fiber bioreactor having concentric hollow fiber bundles: a central hollow fiber bundle supplies media, and an outer array supplies oxygen needed for cell culture. Useful to expand therapeutic cells such as stem cells ex vivo, and as an extracorporeal device such as an artificial liver.