WO2002024107A2 - Fabrication of thin sheet bio-artificial organs - Google Patents
Fabrication of thin sheet bio-artificial organs Download PDFInfo
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- WO2002024107A2 WO2002024107A2 PCT/US2001/029586 US0129586W WO0224107A2 WO 2002024107 A2 WO2002024107 A2 WO 2002024107A2 US 0129586 W US0129586 W US 0129586W WO 0224107 A2 WO0224107 A2 WO 0224107A2
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/14—Macromolecular materials
- A61L27/20—Polysaccharides
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
- A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
Definitions
- the present invention concerns methods for making thin sheet bio-artificial organs for use in treatment of disease.
- Thin sheet bio-artificial organs are devices for surgical implantation which entrap cells or tissue producing desirable substances or having desirable properties.
- the field relates to thin sheets containing cells and which have dimensions and physicochemical properties allowing maintenance of tissue viability through rapid diffusion of nutrients and oxygen, and affording protection from contact of said cells with cells of the recipient' s immune system, said sheets optionally having the further properties of substantially excluding factors necessary for humoral immune destruction of the entrapped cells and having the additional properties of bioco patibility, mechanical strength and chemical stability sufficient that the entrapped cells or tissue can function in vivo for a long time .
- bioartificial organ implants to treat disease has been long recognized.
- the exemplary bioartificial organ is the bioartificial pancreas containing insulin- producing islets of Langerhans, for which there is great clinical need in the treatment of diabetes.
- Evidence that islet transplantation can normalize diabetic blood sugars and arrest and reverse vascular decay continues to mount 1 .
- the bioartificial pancreas would make possible the benefits of islet cell transplantation without the need for immunosuppression.
- the failure of any bioartificial pancreas to be commercialized after decades of research emphasizes the difficulty of the task.
- Some devices do not cover the entire islet surface. If even a small bit of the islet is uncovered, macrophages can infiltrate and destroy the entire islet. The cellular attack and destruction sensitize the immune system, leading to a humoral (antibody) response, which may then destroy even those cells that are completely covered. Thus, complete coverage of all of the islets is required to protect the islet cells from both cellular and humoral immune responses .
- a successful bioartificial implant must have dimensions that permit sufficient diffusive flux of nutrients into the implant and secretion of bioactive agents out of the implant. Yet the vast majority of bioartificial implants described in the literature have dimensions too large to permit sufficient diffusion.
- the nutrient limiting cell viability and functionality in bioartificial organs is usually oxygen.
- the oxygen available at the center of the bioartificial organ (where oxygen is at its lowest concentration) is governed by the density of oxygen consuming tissue in the bioartificial pancreas, the geometry of the device, diffusivity of oxygen through the bioartificial organ and oxygen tension in the surrounding tissues 6 .
- Calafiore' s group 15 also recognized the seriousness of this problem ("it was, in fact, found that a viable human islet cell quantity . . . would take, upon microencapsulation [in 700 ⁇ m microcapsules] a final graft volume of approximately 180 mL, thereby creating quite serious technical implant problems” ) and worked to make much smaller microcapsules.
- Islet cell mass, viability and functionality may be improved and enhanced by adding cells and/or other substances in the bioartificial implant composition.
- Recent publications include various trophic cells and substances in the core of the bioartificial organ to enhance the viability and functionality of the cells, including Sertoli Cells 16 .
- Islet and other primary cells are characterized by very slow rates of cell division, usually limited to replacement of cells that die naturally.
- Embodiments of the thin sheets described herein can optionally be made that comprise rapidly dividing cell lines entrapped in rigid capsules, for example alginate- poly-lysine. This can be done, for example, by the method of Cochrum, Dorian and Jemtrud 17 . Previous Approaches
- the Ohgawara paper greatly expanded the information available on the device compared with the earlier paper cited in patent No. 5,855,613.
- the planar chamber is 40 mm diameter and 5 mm thick, fabricated from two membranes made by Nucleopore Corp.
- the tissue density reported was 8X10 6 cells/1.5 mL.
- the relevant micrograph shows a fibrotic mass about 40-50 ⁇ m thick.
- Suzuki et al 23 reported on the Baxter double-membrane design, but in greater detail than ever before.
- the Suzuki paper described a flat device substantially similar to the Scharp device (from the same supplier, Baxter) discussed in the prior patent.
- Baetge et al. 24 described a flat sheet sealed double-membrane with loading port. Each membrane was 100 ⁇ m thick with a 200 ⁇ m core, and thus a total sandwich of 400 ⁇ m thickness. The disc was 10mm diameter. Thus, with 3.5 X 10 6 cells the packing efficiency in the core is 30%, or 15% in the total sheet.
- Dionne 25 describes imunoisolatory vehicles with a core and permselective jacket, including flat sheets.
- the methods described may be distinguished from the present invention in that the flat sheet fabrication method of Dionne does not work with alginate.
- the membranes are not laminated to the core allowing changes in dimensions post implant.
- Tatarkiewicz at al. 26 describe a polylysine coated alginate slab, 1mm thick, 0.8 cm 2 . They report that only 4000 islets survive when up to 8000 are incorporated. The calculated tissue density is 8%.
- Usala 27 describes an implant in the form of a plate with multiple wells, each filled with a cell-collagen matrix, the entire implant coated with poly-para-xylene.
- U.S. Patent No. 5,855,613 discloses making the bioartificial implant using a series of molds constructed with frit materials that can be molded or milled, and membranes. This allows diffusion of chelating agents (e.g., sodium citrate) or ultivalent cation gelling agents (e.g., calcium, barium or strontium chlorides) to liquify and gel, respectively, the core, coat and overcoat.
- chelating agents e.g., sodium citrate
- ultivalent cation gelling agents e.g., calcium, barium or strontium chlorides
- the cross-linked core and coats are bonded together by simply contacting the cross-linked surfaces with a small amount of chelating agent (e.g., sodium citrate).
- the chelating agent diffuses into the gelled layer and partially liquefies it.
- the layers are brought into contact with each other.
- a 5 cationic cross-linking agent e.g., calcium, barium or strontium chlorides
- a tight bond is formed between the layers .
- the outer surface is made very smooth through the simple step of wetting the mold with cross- linking agent solution before contacting the mold with the 10 cross-linked coat or overcoat. This assures that the outer surface is as smooth as the mold surface, limited only by machining of the frit.
- VEGF Vascular Endothelial Growth Factor
- VEGF Vascular Endothelial Growth Factor
- a method for making a physiologically active and biocompatible cellular implant for implantation into a host body. The method includes the steps of:
- the method includes the steps of
- objects of the present invention include a bioartificial implant in a thin sheet configuration that:
- tissue cells for implantation in a device which is physiologically acceptable to the host and which effectively provides prolonged protection of the tissue cells, after implantation, from destruction by the host immune system;
- the sheet may contain a mesh or support polymer to improve physical properties of the sheet
- trophic agents such as nurse cells, nutrients, hormones or oxygen carriers to support the cellular health, longevity and effective function of the implant after implantation.
- An object of the present invention is to ease scaling up methods for fabrication of larger sheets.
- a single simple device may be used to make sheets ranging from very small up to a limit imposed only by the spacing of shims .
- Another object of the present invention is to reduce the processing time for making a bioartificial implant.
- Prior methods relied on many steps to liquify gelled alginate, time for the liquids to diffuse, then addition of chelating agent to gel the alginate again. The new process does not contain any chelation step.
- Another object of the present invention is to fabricate the coat and overcoat of a bioartificial implant in a single step.
- an overcoat may be added in an additional step.
- Another object of the present invention is incorporation of rapidly dividing cells into thin sheets without risk of the cells bursting out of the sheet.
- Another object of the present invention is to control the thickness of the coat/overcoat by physicochemical means, not relying on a mold. This is accomplished by controlling the composition of the first polymer and cross-linking solution so that the layers are substantially cross-linked to a depth of only several microns or by sweeping a straightedge over shims.
- the methods of the present application allow for excellent cross-linking between layers, smoother outer surface, easier scale up and more rapid sheet fabrication.
- the utility of this invention is not limited to encapsulation of the islets of Langerhans in a sheet ("islet sheet").
- Any cell that secretes a substance with therapeutic value may be used.
- primary parathyroid cells may be used to treat parathyroid hormone deficiency, or erythropoietin secreting cells may be used to treat anemia or cytokine secreting cells may be used to modulate immunity.
- Another form of utility would be encapsulation of cells that transform or metabolize substances found in the body.
- hepatic cells may detoxify toxic compounds, or cells may be used to oxidize compounds such as ethanol when they are present in undesirable amounts.
- cells may be, for example, primary cells, cultured cells, or genetically engineered cells. Mammalian and non-mammalian cells including prokaryotes may be used.
- the dimensions of the bioartificial implant are such that cell viability may be maintained by passive diffusion of nutrients, and preferably, such that a high cell density can be maintained.
- the dimensions of the bioartificial implant are also such that the bioartificial implant is macroscopic and is easily retrievable from the host and is large enough to contain a significant fraction of the tissue required to achieve the desired therapeutic effect. Such high cell density makes practical surgical use of bioartificial organs possible.
- the permeability of the bioartificial implant is such that passive diffusion of secreted cell products permits rapid response to changing physiological conditions.
- the permeability of the membrane ' sufficiently impedes diffusion of antibody and complement to prevent killing of the implanted cells, even when the tissue is a xenograft or the host is presensitized to the implant tissue.
- the bioartificial implant is biocompatible, meaning it produces minimal foreign body reaction. We have found that only implants that are neutral (causing neither fibrosis nor neovascularization) have been shown to , last over a year with minimal decay of function.
- the dimensions of the present bioartificial implant when in a thin sheet configuration, are such that the surface area of a side of a sheet is at least 30 mm 2 , preferably at least 2.5 cm 2 and more preferably at least 10 cm 2 , as defined by either (a) the diameter (if the sheet is circular) or (b) the area determined by the method of converging polygons.
- a suitable surface area of a face of the present thin sheet implant may be 400 cm 2 , more preferably 300 cm 2 and most preferably 250 cm 2 (for a human patient with type 1 diabetes) . (A smaller sheet would be sufficient for a more potent hormone such as erythropoietin.)
- the cell density is that which can be contained within the entire implant.
- the cell density is at least 10%, more preferably 20% and most preferably at least 30% by volume.
- the bioartificial implant is sometimes described using the terms "core,” “coat” and “overcoat.”
- the core comprises the living tissue, optional trophic factors or nurse cells, alginate polymer cross-linked with a multivalent cation such as calcium, and an optional support polymer such as collagen or fiber mesh for strength.
- the coat comprises alginate polymer (optionally of different chemical composition) cross-linked with a multivalent cation that partly serves to control permeability.
- the optional overcoat comprises alginate polymer (optionally of different chemical composition) cross-linked with a multivalent cation that serves to render the bioartificial implant biocompatible.
- Use of polymers of different chemical composition may require other methods for cross-linking, such as covalent bonding or phase change by cooling.
- the sum of the core, coat and overcoat thicknesses preferably is less than 500 ⁇ m, more preferably 350 ⁇ m or less, and most preferably no more than 300 ⁇ m.
- the coat and optional overcoat thickness preferably is minimized so that the tissue quantity may be maximized.
- preferable coat and overcoat thicknesses may be from 5 to 100 ⁇ m thick, more preferably from 10-80 ⁇ m, and most preferably from 10-50 ⁇ m.
- the length and width of the bioartificial implant on the other hand preferably is maximized to permit the greatest possible volume of living tissue to be included in the bioartificial implant and to permit easy retrieval but not so large as to be surgically impractical.
- the thin sheet bioartificial organ made according to the present invention typically includes an implant core having a thin sheet configuration comprising viable, physiologically active tissue or cells and a cross-linked alginate gel and optionally, trophic factors and nurse cells, and optionally, a fiber mesh support, being completely covered by an acellular biocompatible coat and optional overcoat of alginates.
- the alginates are preferably free from fibrogenic concentrations of impurities.
- the bioartificial implant may have a coat and overcoat to control permeability and enhance biocompatibility.
- the implant sheet is thin and may be permeable enough to provide a physiologically acceptable oxygen tension at the center of the sheet when implanted in a suitable site in a human or animal subject.
- the thinness and permeability of the implant allow diffusion of nutrients, especially oxygen, metabolic waste products and secreted tissue products.
- the implant preferably inhibits diffusion of antibody and complement .
- improved methods are provided for making thin sheet bioartificial implants without the necessity of a series of molds constructed with frit materials or membranes that allow diffusion of chelating agents to liquify the gelled core, coat and overcoat.
- the use only of gelling agents and ions known to be compatible with the implanted cells has been retained.
- the use of membranes that allow diffusion of gelling agents to gel the core, coat and overcoat has been made optional, with diffusion of gelling agents to gel the core, coat and optional overcoat now possible from a single solution.
- the shapes of the core, coat and overcoat are molded while the polymer solutions are uncross- linked and in a flowable liquid form preferably at a viscosity less than that of a gum but more viscous than water (e.g., from about 100 to 100,000 centipoise) .
- the coat can be formed by spreading as by sliding a straightedge over two shims to form a liquid layer of uniform thickness.
- the liquid core can be formed by gently suspending cells into a second liquid polymer solution and sweeping this suspension over the first.
- the first polymer solution is more viscous than the second.
- the second layer is formed with thicker shims defining its thickness.
- the layered sheet can be formed and the core and coat are simultaneously cross-linked by simply contacting the two liquid polymer solutions formed in the desired dimensions using a system of shims and straightedges, then adding a cross-linking agent to form a tight bond between the layers .
- the outer surface can be made smooth during the coat formation step through the simple means of gelling the outer surface of the coat by first wetting a permeable membrane on which it is formed with cross-linking agent solution or by leaving the coat liquid against a smooth impermeable mold until the final cross- linking step.
- the thickness of the thin sheet is easily controlled through the use of an impermeable mold of one or two surfaces with shim spacers.
- the outer surface can be made smoother and thus more biocompatible.
- the new methods do not require the manufacture of custom frit molds for each sheet size and geometry.
- Fig. 1 shows the apparatus for the optical glass casting method.
- Fig. 2 is a diagram of the optical glass casting method showing initial diffusion of calcium ions into the layered liquid alginate and islets.
- Fig. 3 is a diagram of the optical glass casting method showing complete diffusion of calcium ions into the Islet Sheet.
- Fig. 4 shows the apparatus of the hydrophilic membrane method for making sheets at the beginning of a cycle.
- Fig. 5 shows the apparatus of the hydrophilic membrane method for making sheets as alginate for the coat/overcoat layer is applied.
- Fig. 6 shows the apparatus of the hydrophilic membrane method at the addition of the core, cells suspended in alginate.
- Fig. 7 shows the apparatus of the hydrophilic membrane method closing.
- Fig. 8 shows the apparatus of the hydrophilic membrane method completely closed.
- Fig. 9 shows the apparatus of the hydrophilic membrane method as the finished sheet is withdrawn wrapped in a protective membrane, about to be transferred to a bath of cross-linking agent.
- Fig. 10 is a diagram of the hydrophilic membrane method; the membrane is prepared by soaking in a solution of calcium gluconate.
- Fig. 11 is a diagram of the hydrophilic membrane method; the membrane is covered with a layer of liquid sodium alginate at the instant of application.
- Fig. 12 is a diagram of the hydrophilic membrane method; calcium diffuses from the membrane into the liquid alginate, gelling the alginate.
- Fig. 13 is a diagram of the hydrophilic membrane method; two membranes layered with partially gelled alginate squeeze a dollop of liquid alginate containing cells or islets.
- Fig. 14 is a diagram of the hydrophilic membrane method; the cell/alginate suspension is flattened and the liquid alginate from the membrane layer is displaced by the advancing liquid alginate from the cell dollop (arrows) .
- Fig. 15 is a diagram of the hydrophilic membrane method; at the completion of the squeezing process the sheet is of uniform thinness and the liquid portion of the membrane alginate has been displaced from view.
- Fig. 16 is a diagram of the hydrophilic membrane method; the calcium diffuses in through both membranes and gels all the liquid alginate into a uniform sheet.
- Fig. 17 is a photograph of a thin sheet comprising erythrocytes encapsulated in alginate.
- the U.S. quarter dollar coin gives scale.
- Fig. 18 is an electron micrograph of the surface of an islet containing thin sheet. The surface is smooth. The bumps show that the surface of the sheet is convex over the islets.
- Fig. 19 is an electron micrograph of the surface of a thin sheet reinforced with a non-woven fabric. The sheet is fractured to reveal the fabric.
- Fig. 20 is a micrograph of an Islet Sheet that has been retrieved after 2 ⁇ months sutured on to the omentum of a dog. The islets are stained with dithizone.
- Fig. 21 is a photograph of an Islet Sheet as it is being sutured on to the omentum of a diabetic dog described in Example 8.
- Fig. 22 is a chart showing the blood sugars of a canine allograft described in Example 8.
- Fig. 23 is a chart showing intravenous glucose tolerance tests of the canine allograft described in Example 8.
- a sheet is fabricated as illustrated in Figs. 1-3.
- Figs. 2 and 3 show the beginning and final stages of diffusion of calcium ions into the sheet.
- a first substantially uncross-linked polymer solution is formed by substantially uniformly spreading the polymer solution onto a solid support surface, preferably one that is flat and smooth, e.g., optical quality glass.
- a suitable polymer solution is a substantially uncross-linked soluble alginate salt (such as sodium alginate or another monovalent cation salt) in a viscosity (e.g., less than about 50,000 centipoise) which permits it to be readily flowable and spreadable in contrast to a hydrogel (which is cross-linked).
- the polymer solution (42) is spread as a thin layer on a smooth surface, such as optical glass (40) .
- a convenient means of sweeping out this and the subsequent layers described below is by spreading the alginate across the smooth surface with a straightedge (43) while controlling the thickness by means of guide shims (41) .
- These outer layers should be less than 100 ⁇ m so that oxygen flux is not unduly impeded.
- a reinforcing fabric or membrane is placed on top of the layer (e.g., of the type illustrated in U.S. Patent No. 5,855,613).
- a cell suspension of physiologically active cells in a substantially uncross-linked second polymer solution is substantially uniformly spread over the exposed layer of the first polymer solution.
- the suspension of cells such as islets or other cells
- the alginate used for suspension of particulates is of a lower viscosity (e.g., at least about 5% less) than the underlying alginate layer to minimize disturbing the bottom layer while sweeping out the particulate suspension.
- a reinforcing fabric or membrane can be placed on top of the spread particulate suspension.
- another layer of a third substantially uncross-linked polymer solution e.g., sodium alginate
- a third substantially uncross-linked polymer solution e.g., sodium alginate
- the over-laying alginate solution can optionally be of a lower viscosity than that of the particulate suspension to minimize disturbance of the suspension during sweeping out of the topmost layer.
- the cross-linking solution can be spread across the surface with a straightedge (43) guided by thick shims or allowed to pool freely.
- the entire laminate can be cross-linked by submerging in a solution of cross-linking ions (e.g., a multivalent ion such as calcium) (44) to cross-link the polymer (alginate) .
- cross-linking ions e.g., a multivalent ion such as calcium
- the entire sheet may be strengthened by immersion in a solution of barium chloride. Barium exchanges with calcium in the sheet, and the stronger barium-alginate bonds result in a sheet that is physically stronger.
- the cell-containing layer of alginate is preferably constrained to a smaller area than the under- and over-lying layers so that the edges of the device will be devoid of cells .
- the reinforcing fabric or membrane is preferably constrained to an area smaller than the under- and over-lying layers and larger than the cell-containing layer so that it may be used as a surgical cuff without any danger of exposing cells in the cell-containing layer during suturing.
- cells includes cells or physiologically active tissue. Such cells are washed in isotonic buffer solution lacking cross-linking agent and resuspended in a soluble polymer solution.
- Suitable apparatus to perform this method includes two flat plastic plates (51) supporting two smooth flat glass plates, e.g., formed of optical glass (52).
- the plastic plates are hinged (53) so that, when the hinge is closed, the flat plates (52) are held apart, preferably spaced equidistant across their facing surfaces by spacers or shims (54) .
- two layers of polymer solutions are formed each having one cross- linked surface adjacent a support surface and an uncross-linked opposed surface.
- This can be performed with a pair of smooth, absorbent membranes (or a single, folded membrane 55) wetted with a solution containing cross-linking agent, such as calcium (see Fig. 10).
- the membranes may be gel films cast from PEG, polyacrylamide, polysulfone, agarose or other polymers.
- the wet membranes are laid out onto rigid flat surfaces, such as glass optical windows (52) . Excess liquid is blotted or displaced from the membrane surfaces.
- the flat surfaces are fitted with spacers (shims or rails) (61) which serve as guides to control thickness of a polymer (e.g., alginate) layer to be spread over the surfaces of the membranes.
- a polymer e.g., alginate
- a liquid solution of polymer, such as sodium alginate, is applied from an impermeable depot resting atop one end of the membranes (61) and spread across the membranes using a straightedge knife (62) guided by the shims (61) .
- the depot and the shims are a single rectangular mask 61.
- a smooth skin of cross-linked gel is rapidly formed at the interface by interaction of the polymer with the cross-linking solution.
- the cross-linking agent diffuses from the membrane into the polymer solution, interacting with the latter and producing a front of gelation (see Fig. 12).
- the degree of cross-linking of the polymer layers decreases in a moving or kinetic gradient from the surface contacting the membranes outward, and is dependent on the composition of the cross-linking solution. Parameters which effect the rate of cross-linking include concentration, counterion, viscosity, and temperature. For example, a dilute solution of a weakly dissociable salt of cross-linker in a viscous solution will result in slower diffusion into the liquid polymer.
- the composition of the polymer solution, composition of the cross-linking solution and the concentration of the cross-linker in the cross-linking solution conditions can be established whereby within a minute or so, the polymer layers are substantially cross-linked to a depth of only several microns.
- This process may be adapted to polymers that gel as a result of a physical process rather than a cross-linking agent, for example, gelation of a liquid that cools to its gel point.
- the next step in the partially pre-cross-linked method is to form a sandwich of an uncross-linked cell suspension layer
- the suspension is deposited onto one of the two partially cross-linked overcoating polymer layers (by syringe 71) .
- the other partially cross-linked layer is lowered on top of the suspension (81) , contacting it, and is pressed down until the spacer shims which control the distance between the two membrane surfaces (Fig. 8) collide.
- the soluble polymer component of said layers is displaced radially (91); see Figs. 13 through 15.
- the gelled and highly viscous, partially gelled polymer components of the layers are unperturbed because of their cross-linked, macromolecular structure, so that the suspended cells are substantially uniformly separated from the membranes by a layer of gelled polymer, the thickness of which is determined by the extent of cross-linking prior to collision of the shims .
- the sandwich consisting of membrane-polymer layer-cell suspension-polymer layer-membrane may then be slid out from between the two rigid, flat surfaces (Fig. 9) .
- This operation can be facilitated by infusing cross-linking solution between the flat surfaces around the sandwich, for instance by using a syringe or suitable device for applying the solution to the space between the surfaces.
- the presence of a solution around the membrane sandwich both continues the cross-linking process and lowers the surface tension between the damp membranes and the flat surfaces .
- the sandwich may then submersed in cross-linking solution (92) and incubated for sufficient time to ensure that polymer is cross-linked entirely throughout its thickness.
- the cross-linking agent can be infused solely between the flat surfaces, or through semipermeable flat, rigid surfaces such as glass frits.
- the membranes are removed from the cell- containing multilayered polymer sheet and the sheet is equilibrated in an appropriate buffered salt solution or nutrient medium, for example, HEPES buffered normal saline with 5 mM CaCl 2 , to prepare it for tissue culture or surgical implantation.
- the entire sheet may be strengthened by immersion in a solution of, e.g., barium chloride. Barium exchanges with calcium in the sheet, and the stronger barium-alginate bonds result in a sheet that is physically stronger.
- a solution of, e.g., barium chloride e.g., barium chloride. Barium exchanges with calcium in the sheet, and the stronger barium-alginate bonds result in a sheet that is physically stronger.
- Figs. 10-16 illustrate formation of a sheet by the partially pre-cross-linked method (the hydrophilic membrane method) .
- Fig- 10 shows a porous membrane (101) prepared by soaking in a solution of a multivalent cation salt cross-linking agent (e.g., calcium gluconate).
- Fig. 11 shows the membrane (101) covered with a layer of liquid sodium alginate (102) at the instant of application before any calcium has diffused out of the membrane.
- Fig. 12 shows calcium diffusing from the membrane (101) into the liquid alginate (102) , gelling the alginate; gelling is complete near the membrane. The amount of calcium in the membrane is chosen so that the alginate remains liquid at a distance from the membrane.
- Fig. 10 shows a porous membrane (101) prepared by soaking in a solution of a multivalent cation salt cross-linking agent (e.g., calcium gluconate).
- Fig. 11 shows the membrane (101) covered with a layer of liquid sodium alginate
- FIG. 13 shows two membranes (101) layered with partially gelled alginate (102) beginning to squeeze a dollop of liquid alginate containing cells or islets (103) .
- Fig. 14 shows the squeezing continues as the cell/alginate suspension is flattened (103) and the liquid alginate from the membrane layer is displaced by the advancing liquid alginate from the cell dollop (arrows) .
- Fig. 15 shows the completion of the squeezing process. The sheet is of uniform thinness and the liquid portion of the membrane alginate has been displaced from view. The spacing of the membrane is determined by shims (54) .
- Fig. 16 shows calcium diffusing in through both membranes and gelling all the liquid alginate into a sheet.
- Proliferating cells entrapped in a sheet of polymer may break out of the sheet during expansion. In order to prevent this eventuality, such cells may be first entrapped within shells which have sufficient mechanical strength to contain them.
- cells which either are free or entrapped within an intraluminal gel matrix can be contained within shells in the form of small, semipermeable hollow fibers, sealed at either end. See, e.g., Sharp et al., DiaJbetes 43 (9) : 1167-70 (1994). These sealed tubes containing cells may be layered onto one of the two partially cross-linked polymer layers of hydrophilic membrane method prior to layering with the apposing layer.
- the cells may be entrapped within microcapsules, such as polylysine stabilized alginate microcapsules.
- microcapsules such as polylysine stabilized alginate microcapsules.
- Such cell-containing microcapsules after stabilization by polylysine treatment and optional dissolution of the core gel matrix, can then be substituted for free cells or tissue in the method for sheet manufacture in both the hydrophilic membrane method and the casting method.
- the polymer for the cell-containing core may have a different composition from that used in preparation of the partially cross-linked coating layers.
- the partially cross-linked layers of the hydrophilic membrane method (or the outer liquid layers of the casting method) which will ultimately separate the cell-containing core of the sheet from the external environment, may be composed of a different alginate composition chosen for desired permeability and biocompatibility properties. In general, the characteristics of the polymer solution are described in U.S.
- Patent No. 5,855,613 except that there is no need for polymers that can be reversibly gelled.
- Polymers other than alginate can be used. These polymers must be liquid in one phase (which allows cell viability) and gelled in another phase (which also allows cell viability) , for instance by cross-linking by addition of a cross-linking agent (which also allows cell viability) .
- a cross-linking agent which also allows cell viability
- chitosan can be substituted for alginate and cross-linked by similar divalent ion diffusion.
- Acrylamide monomer may be cross-linked with ammonium persulfate and TEMED (N,N,N',N' ⁇ tetramethylathylenediamine) .
- Suitable cross-linking agents are multivalent cation salts (e.g., of calcium, barium, strontium, or mixtures thereof). Such salts include as counterions gluconate, lactate, and chloride.
- a free-radical polymer may be made by sweeping the three layers in the dark (or under red light) then exposing the sheet to light.
- polyacrylamide may be cross-linked with TEMED mixed with ammonium persulfate. Agarose, gelled by a change in temperature can be used.
- any method may be used for cross-linking the polymer solution in the casting method or partially cross- linked method so long as it causes a phase transition from liquid to a gel or solid.
- cross- linking takes place by diffusion of cross-linking agent toward the interface of the cell suspension layer from one or more of the sandwiching layers .
- a temperature-induced phase transition may be used.
- 1% agarose in physiological solutions undergoes a liquid-to-gel phase transition at approximately 37 °C.
- Cells for encapsulation can be suspended in 1% liquid agarose thermostated at temperatures slightly higher than this, e.g., 40°C. This liquid suspension is used for the core polymer. After formation of a sandwich with substantially cell-free polymer solutions, cooling below 37 °C would cross-link and thereby gel the suspension, entrapping the cells, without need for diffusible cross-linking agents.
- the substantially uncross-linked polymer solution may be cross-linked by photoactivation using solutions of polymers such as polyethylene glycol derivatized with photolabile cross-linking groups.
- polymers such as polyethylene glycol derivatized with photolabile cross-linking groups.
- the chemistry described in U.S. Patent 5,573,934 can be implemented in the described methods.
- Hubbell describes the use of polyethylene glycol- diacrylates as a soluble "macromer" in which the cells to be entrapped are suspended.
- ethyl eosin and triethanolamine to act as a photon-induced free radical donor.
- the eosin Upon illumination with 415nm light the eosin produces a free radical which can be transferred via the triethanolamine to the acrylate group on the macromer, which becomes activated and cross-links with other acrylate groups forming a larger polymer which will no longer be soluble, but will form a cross- linked gel phase entrapping the cells and thereby forming a cellular implant.
- Other photoactivated cross-linking systems are well-known in the art.
- alginate possesses the best balance of these properties, especially biocompatibility, but this in no way limits the choices available for this or other applications of this invention.
- a mesh or other fabric (13) can be laid down on the first over-coating film together with polymer suspended cells by either the hydrophilic membrane method or the casting method.
- the mesh or fabric can optionally be treated in such a manner as to improve bonding to the polymer. Examples of such treatment are covalent modification of the fabric material to allow coupling of polyaminoacids or other polycations and amination. Wettability of the fabric can also be improved by chemical modification, corona treatment or pre-wetting with dilute alginate or other polymer solution and drying.
- An alternative means of improving mechanical strength of the sheet is to incorporate a cuff of mesh or other fabric by similar means to provide an outer annulus of support, which can be used for suturing the sheet onto a vascularized surface (see Fig. 22) .
- the integral cuff can be placed so as to be distant from the cells within the core and can be imposed in such a way as to ensure its total encapsulation within the coating polymer layers to improve biocompatibility.
- the cuff can be imposed in such a way as to allow presentation to cells of the implant recipient to encourage engraftment of the sheet without stimulation of cellular deposition within the immediate environment of the contained cells .
- the polymer solution of the coat or core can contain microfibrillar collagen, fibrin or other microfibrillar material, which will enhance mechanical strength.
- Treatment of fully cross-linked thin polymer layers or a fully cross-linked cell-containing polymer core with multi-functional reagents such as poly-lysine, poly-asparagine or other poly- cationic polymers provides another means of improving mechanical strength, while simultaneously providing another means of controlling permeability.
- multi-functional reagents such as poly-lysine, poly-asparagine or other poly- cationic polymers
- the presence of unreacted reactive moieties of polymer-bound multi-functional reagent provides a means for bonding between the so treated cross- linked polymer layer and the layer of soluble polymer (coat or core, interchangeably) .
- a number of other optional materials may be co-entrapped with cells in the sheet.
- immobilized enzymes may be desirable for modification of sloughed or secreted cellular substances or immobilized hemoglobin or fluorocarbon fluid may be included to improve oxygen and C0 2 solubility and transport through the gel matrix. It may be desirable to add materials which will be released slowly over time, such as agents to minimize inflammation in response to the initial trauma of surgical implantation. It may also be desirable to co-encapsulate different cell types, where such cells are capable of interacting in some desirable fashion. For example, inclusion of a "feeder" cell may improve viability or survival of a particular cell. Another example is the use of Sertoli cells to induce tolerance (U.S. Patent No. 5,849,285, Selawry, Helena P) .
- hyaluronic acid may be included in the coat.
- a cell line that secretes known vascularization factors may be included in the core and/or coat or materials that encourage vascular growth in adjacent tissues e.g., VEGF.
- Insolublized or entrapped enzymes which catalyze desirable conversion of naturally present substances in the recipient may also be entrapped alone within a sheet.
- the implant may be sutured to the omentum or trapped beneath an omental flap sutured to the surface of a vascularized organ (Fig. 21) .
- a support cuff can optionally be sutured into place exterior to the space between the omental patch and the organ.
- a sheet can be sutured onto any vascularized site including the surface of the liver or other organs or subcutaneously.
- the device may be held in place during engraftment by fibrin glues, preferably autologous plasma concentrates.
- Figs. 17-21 show various views of thin sheets.
- Fig. 17 shows a sheet with a red cell containing core.
- Fig. 18 shows an electron micrograph of the surface of an islet sheet; the islets inside appear as convex areas of the surface.
- Fig. 19 shows an electron micrograph of a sheet reinforced with nonwoven fabric mesh, fractured; the flat surface (11) ; the edge of the broken upper half of the sheet (12); the mesh (13) and the lower half of the sheet (14).
- Fig. 20 shows a sheet retrieved after over 2 months in a dog and
- Fig. 21 shows an islet sheet made by the method of Example 8 being attached to the dog's omentum.
- Fig. 22 shows blood sugars following implant of a canine Islet Sheet into a pancreatectomized diabetic dog of Example 8.
- Fig. 23 shows intravenous glucose tolerance tests (IVGTT) of the same dog 30 days and 60 days after the implant.
- IVGTT intravenous glucose tolerance tests
- Guluronate-rich alginate was purified by a modification of the method of Dorian, et al. (U.S. Patent No. 5,643,594; http://www.isletmedical.com/meth0202.htm (March 31, 1999)). Briefly, 1 gram Protan MVG alginate was dissolved in 1 liter 0.5 M EDTA, 10 mM HEPES, pH 7.0.
- the solution was filtered to 0.45 microns to remove particulates then mixed with 4 grams bleached, activated charcoal for 30 minutes to adsorb organic contaminants (bleaching of charcoal comprised stirring 30 minutes in 100 mL 0.1 M sodium perchlorate then washing by centrifugation 2x times with 100 mL H20, 4 times with 100 L EtOH, 4 times with 100 mL H20) .
- the charcoal adsorbed alginate was filtered sequentially to 0.22 microns then 0.1 microns.
- To the filtered solution was added 10.2 mL 10% MgCl 2 -2H 2 0. While stirring, 3.8 mL of 34% CaCl 2 -2H 2 0 was added to precipitate larger, guluronate-rich chains.
- the precipitate was pelleted and the supernatant was discarded.
- the pellet was dissolved in 150 mL 0.1 M EDTA disodium salt, 10 mM HEPES, pH 7.0. After dilution to 1 liter with water, the solution was filtered three times by concentrating ten fold in a 17 kD hollow fiber cartridge. The retentate was diluted to 250 mL with water and NaCl was added to a final concentration of 100 mM. While stirring vigorously, an equal volume of anhydrous ethanol was added slowly. The precipitated alginate was then pelleted by centrifugation, dissolved in 250 mL 100 mM NaCl, reprecipitated by addition of an equal volume alcohol as above and pelleted.
- the final pellet was resuspended in 40 mL 50 mM NaCl and combined with 160 mL ethanol, pelleted and washed 3 times by centrifugation with 200 mL ethanol to remove water, salts and residual organic contaminants. After the last wash, the pellet was pressed to remove excess alcohol, teased and fluffed with forceps and dried overnight in vacuo at 60 degrees.
- Mannuronate-rich alginate was purified by a modification of the method of Dorian, et al. (U.S. Patent No. 5,429,821; http://www.isletmedical.com/meth0102.htm (March 31, 1999)).
- One gram Kelco HV alginate was dissolved in 1 liter 0.5 mM EDTA, 10 mM HEPES, pH 7.0. The solution was filtered to 0.45 microns then mixed with 4 grams bleached, activated charcoal for 30 minutes (charcoal bleached as in example 1) . The charcoal adsorbed alginate was filtered sequentially to 0.22 microns then 0.1 microns. The filtered solution was then filtered with a hollow-fiber cartridge and alcohol washed as in example 1. After the last wash, the pellet was pressed to remove excess alcohol, teased and fluffed with forceps and dried overnight in vacuo at 60 degrees.
- Purified canine islets were sedimented by gravity from serum free medium and washed twice by gentle centrifugation with a wash buffer comprised of 0.9% NaCl, 3 mM glucose, 0.5 mM sodium citrate, 10 mM HEPES, pH 7.0.
- the washed islets were resuspended in a 3% solution of purified mannuronate-rich alginate (example 2) in 0.9% NaCl, 0.5 mM sodium citrate, 10 mM HEPES, pH 7.0.
- a solution of soluble alginate salt (3% hi-M in normal saline/10 mM HEPES, pH 7.0) is spread as a thin layer on a perfectly smooth surface, such as optical glass (Borofloat Window, Edmund Scientific cat# K45-686.
- a layer was formed by dragging the alginate across the smooth surface with a straightedge (precision stainless steel straightedge, McMaster- Carr cat# 2215A2) while controlling the thickness by means of guide shims fabricated from 75 micron polycarbonate film.
- a reinforcing fabric (Hollytex, Ahlstrom cat# 3251) was placed on top of the alginate layer. An additional 187.5 um shim was stacked atop the 75 um shim.
- the suspension of islets in alginate solution was spread with a straightedge across the surface of the layer..
- a coating layer of alginate (2% hi-M in normal saline/10 mM HEPES, pH 7.0) was spread with a straightedge over the top of the islet-containing layer. The entire laminate was submerged in a solution of cross-linking ions (1.7% CaCl 2 -2H 2 0, 10 mM HEPES. pH 7.0) to cross-link the alginate.
- cross-linking ions (1.7% CaCl 2 -2H 2 0, 10 mM HEPES. pH 7.0
- Partially cross-linked layers of alginate (6 cm wide and 20 cm long) were prepared by spreading to a depth of 125 microns a 5% solution of 0.45 micron sterile filtered purified mannuronate- rich alginate (example 2) in 0.9% NaCl, 0.5 mM sodium citrate, 10 mM HEPES, pH 7.0 onto a 0.2 micron nitrocellulose filter membrane (Micro Filtration Systems) which had been pre-wetted with 4.63% sucrose, 3.2% calcium gluconate, 10 mM HEPES, pH 7.0 and blotted before alginate application.
- a 5% solution of 0.45 micron sterile filtered purified mannuronate- rich alginate (example 2) in 0.9% NaCl, 0.5 mM sodium citrate, 10 mM HEPES, pH 7.0 onto a 0.2 micron nitrocellulose filter membrane (Micro Filtration Systems) which had been pre-wetted with 4.63% sucrose
- the plates were then hinged apart, leaving the membrane/alginate layer/guluronate-rich core/alginate layer/membrane sandwich, overlaid by an optional thin polycarbonate film lying on one of the 2 plates.
- the plastic film provided a dry interface to break the surface tension which otherwise binds the plates together, and was peeled off and the overcoated sheet trapped within the folded membrane was transferred into a dish of 1.7% CaCl 2 -2H 2 0, 10 mM HEPES, pH 7.0 to complete cross-linking of the alginate.
- One fifth volume of 115 mM BaCl 2 was added to the fixative solution and the sheet was incubated for an additional 5 minutes.
- the sheet was rinsed with 5 mM CaCl 2 -2H 2 0, 10 mM HEPES, pH 7.0 and transferred to serum free DMEM culture medium with added 3 mM CaCl 2 -2H 2 0 and 10 mM HEPES, pH 7.0 in normal saline for maintenance during shipment to the surgical facility.
- Purified canine islets were sedimented by gravity from serum free medium and washed twice by gentle centrifugation with a wash buffer comprised of 0.9% NaCl, 3 mM glucose, 0.5 mM sodium citrate, 10 mM HEPES, pH 7.0.
- the washed islets were resuspended in a 5% solution of purified guluronate-rich alginate (example 1) in 0.9% NaCl, 0.5 mM sodium citrate, 10 mM HEPES, pH 7.0.
- Partially cross-linked layers of alginate (6 cm wide and 20 cm long) were prepared by spreading to a depth of 125 microns a 5% solution of purified mannuronate-rich alginate (example 2) in 0.9% NaCl, 0.5 mM sodium citrate, 10 mM HEPES, pH 7.0 onto a 0.2 micron nitrocellulose filter membrane (Micro Filtration Systems) which had been pre-wetted with 4.63% sucrose, 3.2% calcium gluconate, 10 mM HEPES, pH 7.0 and blotted before alginate application.
- mannuronate-rich alginate (example 2) in 0.9% NaCl, 0.5 mM sodium citrate, 10 mM HEPES, pH 7.0 onto a 0.2 micron nitrocellulose filter membrane (Micro Filtration Systems) which had been pre-wetted with 4.63% sucrose, 3.2% calcium gluconate, 10 mM HEPES, pH 7.0 and blotted before
- a 5 cm disk of 25 micron thick polyester non-woven scrim (Ahlstrom) was placed onto the layer in the approximate center of one of the hinged plates.
- Two hundred microliters of the islet suspension in alginate were dispensed from a syringe onto the polyester disk.
- the hinged plates were then pivoted toward each other so that the alginate layer on the membrane filter folded into juxtaposition, flattening the droplet of suspended islets into a disk entrapped within a sandwich of overlying alginate.
- the thickness of the sandwich was determined by spacers which held the two plates at a defined distance from each other.
- the plates were then hinged apart, leaving the membrane/alginate layer/islet suspension/alginate layer/membrane sandwich, overlaid by the thin polycarbonate film lying on one of the 2 plates.
- the plastic film was peeled off and the overcoated sheet trapped within the folded membrane was transferred into a dish of 1.7% CaCl 2 -2H 2 0, 10 mM HEPES, pH 7.0 to complete cross-linking of the alginate.
- One fifth volume of 115 mM BaCl 2 was added to the fixative solution and the sheet was incubated for an additional 5 minutes.
- the sheet was rinsed with 5 mM CaCl 2 -2H 2 0, 10 mM HEPES, pH 7.0 and transferred to serum free DMEM with added 3 mM CaCl 2 *2H 2 0 and 10 mM HEPES, pH 7.0 for maintenance during shipment to the surgical facility.
- Sheets containing Vascular-Endothelial Growth Factor (VEGF) complexed to sucralfate, an ⁇ -D-glucopyranoside, ⁇ -D- fructofuranosyl-, octakis- (hydrogen sulfate) , aluminum complex were constructed by the following method.
- the dry sucralfate/VEGF complex when added to the alginate suspension at 400 mg/ml formed a thick paste which could not be swept easily over the pure alginate layer.
- the paste was applied to a polyester scrim in a smear of uniform 500 ⁇ m thickness.
- a 75 ⁇ m thick layer of liquid 3% alginate was made by sweeping the alginate suspension with a straightedge and shim.
- Agarose 2% (SIGMA Agarose Type I-B Low EEO) was suspended in normal saline/10 mM HEPES, pH 7.0 and heated in a water bath to 60°C to dissolve it. The agarose was cooled to 45 °C (at which temperature it remains liquid) in a water bath and divided into two aliquots . Dextran beads (Sephadex G-50) were suspended in one solution 10% v/v to serve as mock islets and maintained at 45°C.
- Sheets prepared as described in Example 5 were implanted intraperitoneally in mice, subcutaneously in rats, and on the omentum of a dog (Fig. 21) .
- the mice were sacrificed after 3 weeks and the implants removed for histological analysis.
- the rats were sacrificed after 3 weeks and the implants removed for histological analysis.
- the dog was sacrificed after 13 days and the implants removed for histological analysis.
- the explanted sheets were physically intact and minimal foreign body reaction was observed.
- a total of 200 microliters of islets were obtained from 2 mongrel dogs by standard collagenase treatment and Ficoll density gradient purification. Islets were divided into 6 aliquots and encapsulated in polyester reinforced sheets prepared as described above in Example 3. The sheets were implanted into a pancreatectomized beagle on the dog' s omentum (Fig. 21) .
- an intravenous glucose tolerance test was performed according to the standard protocol. Briefly, the dog is injected with 7 ml 50% glucose, and blood sugars are measured at the times shown in the table below.
- the results of these tests are shown in Fig. 23.
- the IVGTT was closer to normal at 60 days compared to 30 days showing an increase in sheet function.
Abstract
Description
Claims
Priority Applications (2)
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CA002421036A CA2421036A1 (en) | 2000-09-20 | 2001-09-19 | Fabrication of thin sheet bio-artificial organs |
AU9118201A AU9118201A (en) | 2000-09-20 | 2001-09-19 | Improved methods for fabrication of thin sheet bio-artificial organs |
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US66548500A | 2000-09-20 | 2000-09-20 | |
US09/665,485 | 2000-09-20 |
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PCT/US2001/029586 WO2002024107A2 (en) | 2000-09-20 | 2001-09-19 | Fabrication of thin sheet bio-artificial organs |
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AU (1) | AU9118201A (en) |
CA (1) | CA2421036A1 (en) |
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Cited By (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP2228035A1 (en) * | 2003-12-23 | 2010-09-15 | FMC Biopolymer AS | Use of alginate matrices to control cell growth |
US8690874B2 (en) | 2000-12-22 | 2014-04-08 | Zimmer Orthobiologics, Inc. | Composition and process for bone growth and repair |
US8801586B2 (en) | 2008-02-29 | 2014-08-12 | Biomet Biologics, Llc | System and process for separating a material |
US9446168B2 (en) | 2010-06-07 | 2016-09-20 | Beta-O2 Technologies Ltd. | Multiple-layer immune barrier for donor cells |
US9540630B2 (en) | 2008-09-17 | 2017-01-10 | Beta O2 Technologies Ltd. | Optimization of alginate encapsulation of islets for transplantation |
US9713810B2 (en) | 2015-03-30 | 2017-07-25 | Biomet Biologics, Llc | Cell washing plunger using centrifugal force |
US9757721B2 (en) | 2015-05-11 | 2017-09-12 | Biomet Biologics, Llc | Cell washing plunger using centrifugal force |
US20170304528A1 (en) * | 2016-04-22 | 2017-10-26 | DeepScience Ltd. | Implantable medical device for delivering cells |
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US5459054A (en) | 1989-12-05 | 1995-10-17 | Neocrin Company | Cells encapsulated in alginate containing a high content of a- l- guluronic acid |
US5529913A (en) | 1991-08-20 | 1996-06-25 | University Of Leicester | Method of removing protein from a water soluble gum and encapsulating cells with the gum |
US5578314A (en) | 1992-05-29 | 1996-11-26 | The Regents Of The University Of California | Multiple layer alginate coatings of biological tissue for transplantation |
US5643594A (en) | 1992-05-29 | 1997-07-01 | The Regents Of The University Of California | Spin encapsulation apparatus and method of use |
US5656468A (en) | 1992-05-29 | 1997-08-12 | The Regents Of The University Of California | Cells or tissue coated with non-fibrogenic alginate less than 200 μm thick |
US5693514A (en) | 1992-05-29 | 1997-12-02 | The Regents Of The Univesity Of California | Non-fibrogenic high mannuronate alginate coated transplants, processes for their manufacture, and methods for their use |
US5843430A (en) | 1994-04-13 | 1998-12-01 | Research Corporation Technologies, Inc. | Methods of treating disease using sertoli cells and allografts or xenografts |
US5855613A (en) | 1995-10-13 | 1999-01-05 | Islet Sheet Medical, Inc. | Retrievable bioartificial implants having dimensions allowing rapid diffusion of oxygen and rapid biological response to physiological change |
Family Cites Families (1)
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US5976780A (en) * | 1996-07-16 | 1999-11-02 | Shah; Kumarpal A. | Encapsulated cell device |
-
2001
- 2001-09-19 WO PCT/US2001/029586 patent/WO2002024107A2/en active Application Filing
- 2001-09-19 CA CA002421036A patent/CA2421036A1/en not_active Abandoned
- 2001-09-19 AU AU9118201A patent/AU9118201A/en active Pending
Patent Citations (8)
Publication number | Priority date | Publication date | Assignee | Title |
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US5459054A (en) | 1989-12-05 | 1995-10-17 | Neocrin Company | Cells encapsulated in alginate containing a high content of a- l- guluronic acid |
US5529913A (en) | 1991-08-20 | 1996-06-25 | University Of Leicester | Method of removing protein from a water soluble gum and encapsulating cells with the gum |
US5578314A (en) | 1992-05-29 | 1996-11-26 | The Regents Of The University Of California | Multiple layer alginate coatings of biological tissue for transplantation |
US5643594A (en) | 1992-05-29 | 1997-07-01 | The Regents Of The University Of California | Spin encapsulation apparatus and method of use |
US5656468A (en) | 1992-05-29 | 1997-08-12 | The Regents Of The University Of California | Cells or tissue coated with non-fibrogenic alginate less than 200 μm thick |
US5693514A (en) | 1992-05-29 | 1997-12-02 | The Regents Of The Univesity Of California | Non-fibrogenic high mannuronate alginate coated transplants, processes for their manufacture, and methods for their use |
US5843430A (en) | 1994-04-13 | 1998-12-01 | Research Corporation Technologies, Inc. | Methods of treating disease using sertoli cells and allografts or xenografts |
US5855613A (en) | 1995-10-13 | 1999-01-05 | Islet Sheet Medical, Inc. | Retrievable bioartificial implants having dimensions allowing rapid diffusion of oxygen and rapid biological response to physiological change |
Cited By (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US8690874B2 (en) | 2000-12-22 | 2014-04-08 | Zimmer Orthobiologics, Inc. | Composition and process for bone growth and repair |
EP2228035A1 (en) * | 2003-12-23 | 2010-09-15 | FMC Biopolymer AS | Use of alginate matrices to control cell growth |
US8541017B2 (en) | 2003-12-23 | 2013-09-24 | Fmc Biopolymer As | Use of alginate matrices to control cell growth |
US8801586B2 (en) | 2008-02-29 | 2014-08-12 | Biomet Biologics, Llc | System and process for separating a material |
US9540630B2 (en) | 2008-09-17 | 2017-01-10 | Beta O2 Technologies Ltd. | Optimization of alginate encapsulation of islets for transplantation |
US9446168B2 (en) | 2010-06-07 | 2016-09-20 | Beta-O2 Technologies Ltd. | Multiple-layer immune barrier for donor cells |
US9713810B2 (en) | 2015-03-30 | 2017-07-25 | Biomet Biologics, Llc | Cell washing plunger using centrifugal force |
US9757721B2 (en) | 2015-05-11 | 2017-09-12 | Biomet Biologics, Llc | Cell washing plunger using centrifugal force |
US20170304528A1 (en) * | 2016-04-22 | 2017-10-26 | DeepScience Ltd. | Implantable medical device for delivering cells |
Also Published As
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AU9118201A (en) | 2002-04-02 |
WO2002024107A3 (en) | 2002-07-04 |
CA2421036A1 (en) | 2002-03-28 |
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