Normal cells, such as fibroblasts or other tissue or organ cell types, are genetically engineered to express biologically active, therapeutic agents, such as proteins that are normally produced in small amounts, for example, MIS, or other members of the TGF-beta family Herceptin.TM., interferons, and anti-angiogenic factors. These cells are seeded into a matrix for implantation into the patient to be treated. Cells may also be engineered to include a lethal gene, so that implanted cells can be destroyed once treatment is completed. Cells can be implanted in a variety of different matrices. In a preferred embodiment, these matrices are implantable and biodegradable over a period of time equal to or less than the expected period of treatment, when cells engraft to form a functional tissue producing the desired biologically active agent. Implantation may be ectopic or in some cases orthotopic. Representative cell types include tissue specific cells, progenitor cells, and stem cells. Matrices can be formed of synthetic or natural materials, by chemical coupling at the time of implantation, using standard techniques for formation of fibrous matrices from polymeric fibers, and using micromachining or microfabrication techniques. These devices and strategies are used as delivery systems via standard or minimally invasive implantation techniques for any number of parenterally deliverable recombinant proteins, particularly those that are difficult to produce in large amounts and/or active forms using conventional methods of purification, for the treatment of a variety of conditions.

DETAILED DESCRIPTION OF THE INVENTION

A strength of biological modifiers is that they impart specificity to treatment paradigms to allow for prolonged parenteral therapy, and eliminate many of the side effects and inconveniences associated with conventional therapies. Problems which are often encountered with these molecules include purification and enrichment. Most are manufactured in the laboratory using recombinant technology. A small number are selected for scale up by the pharmaceutical industry. As an important step for their purification, they undergo rigorous purification schemes using separation methods that may alter their chemical characteristics. As is often the case, the end product is a small percentage of the starting material, and is frequently less potent. To obviate the loss of quantity and potency, a polymer scaffolding or matrix has been used to proliferate cells producing the biological modifiers. When this scaffold or matrix is implanted into an organism, it becomes vascularized or otherwise connected to the vasculature, the seeded cells grow to fill the scaffold, the biological modifiers are secreted directly into the bloodstream or adjacent cells, and, in a preferred embodiment, the scaffold is resorbed, leaving a new secretory tissue. Elimination of the purification steps enhances yield and avoids the problems with contamination, cost and loss of biological activity.

I. Materials for Production of Secretory Tissues

The materials required for production in vivo of biologically active molecules include cells which produce the biologically active molecules and matrices for proliferation of the engineered cells which can be implanted in vivo to form new secretory tissues. In one embodiment, cells are obtained which already produce the desired biological modifiers. In another embodiment, cells are genetically engineered to produce the biological modifiers. In this embodiment, it is also necessary to provide the appropriate genes, means for transfection of the cells, and means for expression of the genes.

A. Cells to be Engineered

As a proof of principle, CHO cells permanently transfected by calcium phosphate precipitation with the MIS gene on a CMV promotor and clonally selected for the highest MIS producers were used in preparations and implantations. The devices were seeded with cells for 4 7 days prior to implantation and MIS levels measured in the serum by a sensitive MIS ELISA. The next step was to implant non-tumor cells, such as fibroblasts, both cell lines and then the patient's own fibroblasts to avoid rejection. These cells likewise can be engineered to express, and secrete the desired biological molecule(s). Other representative cell types include other patient specific differentiated cells, progenitor or embryonic or pluripotential stem cells.

Cells to be engineered can be obtained from established cell culture lines, by biopsy or from the patient or other individuals of compatible tissue types. The preferred cells are those obtained from the patient to be treated. In those cases where the patient's own cells are not used, the patient will also be treated with appropriate immunosuppressants such as cyclosporine to avoid destruction of the implanted cells during therapy.

In the preferred embodiments, cells are obtained directly from the donor, washed, and cultured using techniques known to those skilled in the art of tissue culture. Cells are then transfected with the gene of interest and seeded at various cell counts onto a matrix such as a polymeric mesh to achieve optimal production of a biological such as MIS.

Cell attachment and viability can be assessed using scanning electron microscopy, histology, and quantitative assessment, for example, by ELISA, fluorescent labelled or radioactive labelled antibodies. The function of the implanted cells can be determined using a combination of the above-techniques and functional assays. Studies using protein assays can be performed to quantitate cell mass on the polymer scaffolds. These studies of cell mass can then be correlated with cell functional studies to determine the appropriate cell mass.

B. Biologically Active Molecules

Any biologically active molecule which has been cloned or for which a cellular source is available can be used. Representative molecules are those having a known activity which selectively reduces the symptoms of the disorder to be treated, such as MIS, Herceptin, interferons, endostatin, and growth factors such as tumor necrosis factor. For example, for the treatment of malignant or benign hyperplasia, the biologically active molecules include anti-angiogenic compounds, MIS and other hormones which selectively or preferentially bind to the cells to be killed or inactivated. Alternatively, the cells can be engineered to correct the defect in the cells which results in overproliferation.

The goal is not to form a permanent new tissue, but to provide an implanted "bioreactor" to produce therapeutic biologicals for a defined period effective to cause cessation of cell proliferation, regression of abnormal tissue, or cell death. Various devices and strategies are used as delivery systems which can be transplanted by standard or by minimally invasive implantation techniques for any number of parenterally deliverable recombinant proteins, particularly those that are difficult to produce in large amounts and/or active forms using conventional methods of purification, for the treatment of a variety of conditions that produce abnormal growth, including treatment of malignant and benign neoplasias, vascular malformations (hemangiomas), inflammatory conditions, keloid formation, endometriosis, congenital or endocrine abnormalities, and other conditions that can produce abnormal growth such as infection.

C. Vectors for Engineering Cells

Examples of recombinant DNA techniques include cloning, mutagenesis, and transformation. Recombinant DNA techniques are disclosed in Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y. (1982). Vectors, including adeno-associated viruses, adenoviruses, retroviruses, and tissue specific vectors, are commercially available. Vectors can include secretory sequences, so that the biological modifier will diffuse out of the cell in which it is expressed and into the vascular supply or interstitial spaces in order to expose the cells of interest to concentrations of the protein that are effective to treat the patient. The vector or expression vehicle, and in particular the sites chosen therein for insertion of the selected DNA fragment and the expression control sequence, are determined by a variety of factors, e.g., number of sites susceptible to cleavage by a particular restriction enzyme, size of the protein to be expressed, expression characteristics such as start and stop codons relative to the vector sequences, and other factors recognized by those of skill in the art. The choice of a vector, expression control sequence, and insertion site for DNA sequence encoding the biological modifier is determined by a balance of these factors.

It should be understood that the DNA sequences coding for the biological modifier that are inserted at the selected site of a cloning or expression vehicle may include nucleotides which are not part of the actual gene coding for the biological modifier or may include only a fragment of the actual gene. It is only required that whatever DNA sequence is employed, a transformed host cell will produce the biological modifier. For example, MIS DNA sequences may be fused in the same reading frame in an expression vector with at least a portion of a DNA sequence coding for at least one eukaryotic or prokaryotic signal sequence, or combinations thereof. Such constructions enable the production of, for example, a methionyl or other peptidyl-MIS polypeptide. This N-terminal methionine or peptide may either then be cleaved intra- or extra-cellularly by a variety of known processes or the MIS polypeptide with the methionine or peptide attached may be used, uncleaved.

The complete nucleotide and amino acid sequence for human and bovine MIS, and cloning and expression vehicles, are provided in U.S. Pat. No. 5,047,336 to Cate, et al. and Cate et al., Cell 45:685 698 (1986), and are also available using publicly available gene data bases and commercial suppliers.

D. Matrices

There are three basic types of matrices that can be used: devices formed by micromachining, micromolding or other microfabrication techniques, fibrous polymeric scaffolds, and hydrogels.

1. Microfabricated Device Design and Manufacture

Preferred materials for making devices to be seeded with cells are biodegradable polymers, although in some embodiments non-degradable materials may be preferred or may be used as structural support or as components of a device formed of biodegradable polymer. The polymer composition can be selected both to determine the rate of degradation as well as to optimize proliferation. Many biodegradable, biocompatible polymeric materials can be used to form the device, or guide channels within the device, including both natural and synthetic polymers, and combinations thereof. Examples of natural polymers include proteins such as collagen, collagen-glycosaminoglycan copolymers, polysaccharides such as the celluloses (including derivatized celluloses such as methylcelluloses), extracellular basement membrane matrices such as Biomatrix, and polyhydroxyalkanoates such as polyhydroxybutyrate (PHB) and polyhydroxybutyrate-co-valerate (PHBV) which are produced by bacterial fermentation processes. Synthetic polymers include polyesters such as polyhydroxyacids like polylactic acid (PLA), polyglycolic acid (PGA) and compolymers thereof (PLGA), some polyamides and poly(meth)acrylates, and polyanhdyrides. Examples of non-degradable polymers include ethylenevinylacetate (EVA), polycarbonates, and some polyamides.

The surface morphology of the devices can affect cell growth. Bioactive materials may also be incorporated into the device or a sustained release matrix within the device to promote cell viability or proliferation. These materials can be incorporated into the polymer at a loading designed to release by diffusion and/or degradation of the polymer forming the device over a desired time period, ranging from days to weeks. Alternatively, the bioactive substance may be incorporated into a matrix loaded into or adjacent to the device. These matrices may be formed of the same materials as the device or may consist of polymeric materials incorporated within the tracts or channels, for example, hydrogel matrices of the types described in the literature (for example, Wells, et al., Exp. Neurol. (1997) 146(2):395 402; Chamberlain, et al., Biomaterials 1998 19(15):1393 1403; and Woerly, et al., (1999) J. Tissue Engineering 5(5):467 488) for use in promoting nerve growth. Examples of such materials include polyamide, methylcellulose, polyethyleneoxide block compolymers such as the Pluronics, especially F127 (BASF), collagen, and extracellular matrix (ECM) of the type sold as Biomatrix. Other useful materials include the polymer foams reported by Hadlock, et al., Laryngoscope (1999) 109(9):1412 1416.

Microfabrication techniques include micromachining, solid free form (SFF) techniques, and micromolding techniques, as well as other techniques based on well-established methods used to make integrated circuits, electronic packages and other microelectronic devices, having dimensions as small as a few nanometers and which can be mass produced at low per-unit costs.

Micromachining Techniques

Micromachining techniques are described in the literature, for example, by Rai-Choudhury, ed. Handbook of Microlithography, Micromaching & Microfabrication (SPIE Optical Engineering Press, Bellingham, Wash. 1997), the teachings of which are incorporated herein. The techniques can be used to form the device directly, or as discussed below, to form molds which are then used to form the devices.

Other microfabrication processes that may be used include lithography; etching techniques, such as wet chemical, dry, and photoresist removal; thermal oxidation; film deposition, such as evaporation (filament, electron beam, flash, and shadowing and step coverage), sputtering, chemical vapor deposition (CVD), epitaxy (vapor phase, liquid phase, and molecular beam), electroplating, screen printing, lamination, laser machining, and laser ablation (including projection ablation). See generally Jaeger, Introduction to Microelectronic Fabrication (Addison-Wesley Publishing Co., Reading Mass. 1988); Runyan, et al., Semiconductor Integrated Circuit Processing Technology (Addison-Wesley Publishing Co., Reading Mass. 1990); Proceedings of the IEEE Micro Electro Mechanical Systems Conference 1987 1998; Rai-Choudhury, ed., Handbook of Microlithography, Micromachining & Microfabrication (SPIE Optical Engineering Press, Bellingham, Wash. 1997).

Deep plasma etching can be used to create structures with diameters on the order of 0.1 .mu.m or larger. In this process, an appropriate masking material is deposited onto a substrate and patterned into dots having the diameter of the desired tracts or channels. The wafer is then subjected to a carefully controlled plasma. Those regions protected by the metal mask remain and form the tracts.

Another method for forming devices including tracts or channels is to use microfabrication techniques such as photolithography, plasma etching, or laser ablation to make a mold form, transferring that mold form to other materials using standard mold transfer techniques, such as embossing or injection molding, and reproducing the shape of the original mold form using the newly-created mold to yield the final device. Alternatively, the creation of the mold form could be skipped and the mold could be microfabricated directly, which could then be used to create the final device.

Micromolding Techniques

Another method of fabricating tracts or channels utilizes micromold plating techniques. A photo-defined mold first is first produced, for example, by spin casting a thick layer, typically 150 .mu.m, of an epoxy onto a substrate that has been coated with a thin sacrificial layer, typically about 10 to 50 nm. Arrays of cylindrical holes are then photolithographically defined through the epoxy layer, which typically is about 150 .mu.m thick. (Despont, et al., "High-Aspect-Ratio, Ultrathick, Negative-Tone Near-UV Photoresist for MEMS," Proc. of IEEE 10.sup.th Annual International Workshop on MEMS, Nagoya, Japan, pp. 518 522 (Jan. 26 30, 1997)). The diameter of these cylindrical holes defines the outer diameter of the tracts. The upper surface of the substrate, the sacrificial layer, is then partially removed at the bottom of the cylindrical holes in the photoresist. The exact method chosen depends on the choice of substrate. For example, the process has been successfully performed on silicon and glass substrates (in which the upper surface is etched using isotropic wet or dry etching techniques) and copper-clad printed wiring board substrates. In the latter case, the copper laminate is selectively removed using wet etching. Then a seed layer, such as Ti/Cu/Ti (e.g., 30 nm/200 nm/30 nm), is conformally DC sputter-deposited onto the upper surface of the epoxy mold and onto the sidewalls of the cylindrical holes. The seed layer should be electrically isolated from the substrate. Subsequently, one or more electroplatable metals or alloys, such as Ni, NiFe, Au, Cu, or Ti, are electroplated onto the seed layer. The surrounding epoxy is then removed, leaving molds which each have an interior annular hole that extends through the base metal supporting the tracts. The rate and duration of electroplating is controlled in order to define the wall thickness and inner diameter of the tracts.

The molds made as described above and injection molding techniques can be applied to form the tracts or channels in the molds (Weber, et al., "Micromolding--a powerful tool for the large scale production of precise microstructures", Proc. SPIE--International Soc. Optical Engineer. 2879, 156 167 (1996); Schift, et al., "Fabrication of replicated high precision insert elements for micro-optical bench arrangements" Proc. SPIE--International Soc. Optical Engineer. 3513, 122 134 (1998). These micromolding techniques can provide relatively less expensive replication, i.e. lower cost of mass production.

Solid Free Form Manufacturing Techniques

As defined herein, SFF refers to any manufacturing technique that builds a complex three dimensional object as a series of two dimensional layers. The SFF methods can be adapted for use with a variety of polymeric, inorganic, and composite materials to create structures with defined compositions, strengths, and densities, using computer aided design (CAD).

Examples of SFF methods include stereo-lithography (SLA), selective laser sintering (SLS), ballistic particle manufacturing (BPM), fusion deposition modeling (FDM), and three dimensional printing (3DP). In a preferred embodiment, 3DP is used to precisely create channels and pores within a matrix to control subsequent cell growth and proliferation in the matrix of one or more cell types having a defined function, such as nerve cells.

The macrostructure and porous parameters can be manipulated by controlling printing parameters, the type of polymer and particle size, as well as the solvent and/or binder. Porosity of the matrix walls, as well as the matrix per se, can be manipulated using SFF methods, especially 3DP. Structural elements that maintain the integrity of the devices during erosion can also be incorporated. For example, to provide support, the walls of the device can be filled with resorbable inorganic material, which can further provide a source of mineral for the regenerating tissue. Most importantly, these features can be designed and tailored using computer assisted design (CAD) for individual patients to individualize the fit of the device.

Three Dimensional Printing (3DP).

3DP is described by Sachs, et al., "CAD-Casting: Direct Fabrication of Ceramic Shells and Cores by Three Dimensional Printing" Manufacturing Review 5(2), 117 126 (1992) and U.S. Pat. No. 5,204,055 to Sachs, et al., the teachings of which are incorporated herein. Suitable devices include both those with a continuous jet stream print head and a drop-on-demand stream print head. A high speed printer of the continuous type, for example, is the Dijit printer made and sold by Diconix, Inc., of Dayton, Ohio, which has a line printing bar containing approximately 1,500 jets which can deliver up to 60 million droplets per second in a continuous fashion and can print at speeds up to 900 feet per minute. Both raster and vector apparatuses can be used. A raster apparatus is where the printhead goes back and forth across the bed with the jet turning on and off. This can have problems when the material is likely to clog the jet upon settling. A vector apparatus is similar to an x-y printer. Although potentially slower, the vector printer may yield a more uniform finish.

3DP is used to create a solid object by ink-jet printing a binder into selected areas of sequentially deposited layers of powder. Each layer is created by spreading a thin layer of powder over the surface of a powder bed. The powder bed is supported by a piston which descends upon powder spreading and printing of each layer (or, conversely, the ink jets and spreader are raised after printing of each layer and the bed remains stationary). Instructions for each layer are derived directly from a computer-aided design (CAD) representation of the component. The area to be printed is obtained by computing the area of intersection between the desired plane and the CAD representation of the object. The individual sliced segments or layers are joined to form the three dimensional structure. The unbound powder supports temporarily unconnected portions of the component as the structure is built but is removed after completion of printing.

As shown in U.S. Pat. No. 5,204,055, the 3DP apparatus includes a powder dispersion head which is driven reciprocally in a shuttle motion along the length of the powder bed. A linear stepping motor assembly is used to move the powder distribution head and the binder deposition head. The powdered material is dispensed in a confined region as the dispensing head is moved in discrete steps along the mold length to form a relatively loose layer having a typical thickness of about 100 to 200 microns, for example. An ink-jet print head having a plurality of ink-jet dispensers is also driven by the stepping motor assembly in the same reciprocal manner so as to follow the motion of the powder head and to selectively produce jets of a liquid binder material at selected regions which represent the walls of each cavity, thereby causing the powdered material at such regions to become bonded. The binder jets are dispensed along a line of the printhead which is moved in substantially the same manner as the dispensing head. Typical binder droplet sizes are between about 15 to 50 microns in diameter. The powder/binder layer forming process is repeated so as to build up the device layer by layer. While the layers become hardened or at least partially hardened as each of the layers is laid down, once the desired final part configuration is achieved and the layering process is complete, in some applications it may be desirable that the form and its contents be heated or cured at a suitably selected temperature to further promote binding of the powder particles. In either case, whether or not further curing is required, the loose, unbonded powder particles are removed using a suitable technique, such as ultrasonic cleaning, to leave a finished device. Finer feature size is also achieved by printing polymer solutions rather than pure solvents.

Stereo-lithography (SLA) and selective laser sintering (SLS).

SFF methods are particularly useful for their ability to control composition and microstructure on a small scale for the construction of these medical devices. The SFF methods, in addition to 3DP, that can be utilized to some degree as described herein are stereo-lithography (SLA), selective laser sintering (SLS), ballistic particle manufacturing (BPM), and fusion deposition modeling (FDM).

Stereolithography is based on the use of a focused ultra-violet (UV) laser which is vector scanned over the top of a bath of a photopolymerizable liquid polymer material. The UV laser causes the bath to polymerize where the laser beam strikes the surface of the bath, resulting in the creation of a first solid plastic layer at and just below the surface. The solid layer is then lowered into the bath and the laser generated polymerization process is repeated for the generation of the next layer, and so on, until a plurality of superimposed layers forming the desired device is obtained. The most recently created layer in each case is always lowered to a position for the creation of the next layer slightly below the surface of the liquid bath. A system for stereolithography is made and sold by 3D Systems, Inc., of Valencia, Calif., which is readily adaptable for use with biocompatible polymeric materials.

SLS also uses a focused laser beam, but to sinter areas of a loosely compacted plastic powder, the powder being applied layer by layer. In this method, a thin layer of powder is spread evenly onto a flat surface with a roller mechanism. The powder is then raster-scanned with a high-power laser beam. The powder material that is struck by the laser beam is fused, while the other areas of powder remain dissociated. Successive layers of powder are deposited and raster-scanned, one on top of another, until an entire part is complete. Each layer is sintered deeply enough to bond it to the preceding layer. A suitable system adaptable for use in making medical devices is available from DTM Corporation of Austin, Tex.

Ballistic Particle Manufacturing (BPM) and Fusion Deposition Modeling (FDM)

BPM uses an ink-jet printing apparatus wherein an ink-jet stream of liquid polymer or polymer composite material is used to create three-dimensional objects under computer control, similar to the way an ink-jet printer produces two-dimensional graphic printing. The device is formed by printing successive cross-sections, one layer after another, to a target using a cold welding or rapid solidification technique, which causes bonding between the particles and the successive layers. This approach as applied to metal or metal composites has been proposed by Automated Dynamic Corporation of Troy, N.Y.

FDM employs an x-y plotter with a z motion to position an extrudable filament formed of a polymeric material, rendered fluid by heat or the presence of a solvent. A suitable system is available from Stratasys, Incorporated of Minneapolis, Minn.

Polymer Materials, Binders and Solvents for Use in SSF Techniques

Depending on the processing method, the material forming the matrix may be in solution, as in the case of SLA, or in particle form, as in the case of SLS, BPM, FDM, and 3DP. In the preferred embodiment, the material is a polymer. In SLS, the polymer must be photopolymerizable. In the other methods, the material is preferably in particulate form and is solidified by application of heat, solvent, or binder (adhesive). In the case of SLS and FDM, it is preferable to select polymers having relatively low melting points, to avoid exposing incorporated bioactive agent to elevated temperatures.

A number of materials are commonly used to form a matrix. Unless otherwise specified, the term "polymer" will be used to include any of the materials used to form the matrix, including polymers and monomers which can be polymerized or adhered to form an integral unit, as well as inorganic and organic materials, as discussed below. In a preferred embodiment the particles are formed of a polymer which can be dissolved in an organic solvent and solidified by removal of the solvent, such as a synthetic thermoplastic polymer, for example, ethylene vinyl acetate, poly(anhydrides), polyorthoesters, polymers of lactic acid and glycolic acid and other .alpha. hydroxy acids, polyhydroxyalkanoates, and polyphosphazenes, a protein polymer, for example, albumin or collagen, or a polysaccharide. The polymer can be non-biodegradable or biodegradable, typically via hydrolysis or enzymatic cleavage. Examples of non-polymeric materials which can be used to form a part of the device or matrix for drug delivery include organic and inorganic materials such as hydoxyapatite, calcium carbonate, buffering agents, and lactose, as well as other common excipients used in drugs, which are solidified by application of adhesive or binder rather than solvent. In the case of polymers for use in making devices for cell attachment and growth, polymers are selected based on the ability of the polymer to elicit the appropriate biological response from cells, for example, attachment, migration, proliferation and gene expression.

Photopolymerizable, biocompatible water-soluble polymers include polyethylene glycol tetraacrylate (Mw 18,500) which can be photopolymerized with an argon laser under biologically compatible conditions using an initiator such as triethanolamine, N-vinylpyrrolidone, and eosin Y. Similar photopolymerizable macromers having a poly(ethylene glycol) central block, extended with hydrolyzable oligomers such as oligo(d,l-lactic acid) or oligo(glycolic acid) and terminated with acrylate groups, may be used.

Examples of biocompatible polymers with low melting temperatures include polyethyleneglycol 400 (PEG) which melts at 4 8.degree. C., PEG 600 which melts at 20 25.degree. C., and PEG 1500 which melts at 44 48.degree. C. Another low melting material is stearic acid, which melts at 70.degree. C.

Other suitable polymers can be obtained by reference to The Polymer Handbook, 3rd edition (Wiley, N.Y., 1989), the teachings of which are incorporated herein.

A preferred material is a polyester in the polylactide/polyglycolide family. These polymers have received a great deal of attention in the drug delivery and tissue regeneration areas for a number of reasons. They have been in use for over 20 years in surgical sutures, are Food and Drug Administration (FDA)-approved and have a long and favorable clinical record. A wide range of physical properties and degradation times can be achieved by varying the monomer ratios in lactide/glycolide copolymers: poly-L-lactic acid (PLLA) and poly-glycolic acid (PGA) exhibit a high degree of crystallinity and degrade relatively slowly, while copolymers of PLLA and PGA, PLGAs, are amorphous and rapidly degraded.

Solvents and/or binder are used in the preferred method, 3DP, as well as SLA and BPM. The binder can be a solvent for the polymer and/or bioactive agent or an adhesive which binds the polymer particles. Solvents for most of the thermoplastic polymers are known, for example, methylene chloride or other organic solvents. Organic and aqueous solvents for the protein and polysaccharide polymers are also known, although an aqueous solution, for example, containing a crosslinking agent such as carbodiimide or glutaraldehyde, is preferred if denaturation of the protein is to be avoided. In some cases, however, binding is best achieved by denaturation of the protein.

The binder can be the same material as is used in conventional powder processing methods or may be designed to ultimately yield the same binder through chemical or physical changes that take place in the powder bed after printing, for example, as a result of heating, photopolymerization, or catalysis.

These methods and materials are further described in PCT/US96/09344 "Vascularized TissueRegeneration Matrices Formed by Solid Free-Form Fabrication Methods" Massachusetts Institute of Technology and Children's Medical Center Corporation.

2. Fibrous Scaffolds for Implantation

Fibrous scaffolding can be used to implant the cells, for example, as described in U.S. Pat. No. 5,759,830 to Vacanti, et al. The design and construction of the scaffolding is of primary importance. The matrix should be a pliable, non-toxic, porous template for vascular ingrowth. The pores should allow vascular ingrowth and the injection of cells into the scaffold without damage to the cells or patient. The scaffolds are generally characterized by interstitial spacing or interconnected pores in the range of at least between approximately 100 and 300 microns in diameter. The matrix should be shaped to maximize surface area, to allow adequate diffusion of nutrients and growth factors to the cells and to allow the ingrowth of new blood vessels and connective tissue.

The same type of polymers can be used as in the Solid Free Form Manufacturing techniques described above. In the preferred embodiment, the matrix is formed of a bioabsorbable, or biodegradable, synthetic polymer such as a polyanhydride, polyorthoester, polyhydroxy acid such as polylactic acid, polyglycolic acid, or a natural polymer like polyalkanoates such as polyhydroxybutyrate and copolymers or blends thereof. Proteins such as collagen can be used, but is not as controllable and is not preferred. These materials are all commercially available. Non-biodegradable polymers, including polymethacrylate and silicon polymers, can be used, depending on the ultimate disposition of the growing cells.

In some embodiments, attachment of the cells to the polymer is enhanced by coating the polymers with compounds such as basement membrane components, agar, agarose, gelatin, gum arabic, collagens types I, II, III, IV, and V, fibronectin, laminin, glycosaminoglycans, mixtures thereof, and other materials, especially attachment peptides and polymers having attachment peptides or other cell surface ligands bound thereto, known to those skilled in the art of cell culture. Vitrogen--100 collagen (PCO 701) has been used in these experiments.

3. Hydrogel Matrices for Implantation

Polymeric materials which are capable of forming a hydrogel can be utilized. The polymer is mixed with cells for implantation into the body and is permitted to crosslink to form a hydrogel matrix containing the cells either before or after implantation in the body. In one embodiment, the polymer forms a hydrogel within the body upon contact with a crosslinking agent. A hydrogel is defined as a substance formed when an organic polymer (natural or synthetic) is crosslinked via covalent, ionic, or hydrogen bonds to create a three-dimensional open-lattice structure which entraps water molecules to form a gel. Naturally occurring and synthetic hydrogel forming polymers, polymer mixtures and copolymers may be utilized as hydrogel precursors. See for example, U.S. Pat. No. 5,709,854 and WO 94/25080 by Reprogenesis.

In one embodiment, calcium alginate and certain other polymers that can form ionic hydrogels which are malleable. For example, a hydrogel can be produced by cross-linking the anionic salt of alginic acid, a carbohydrate polymer isolated from seaweed, with calcium cations, whose strength increases with either increasing concentrations of calcium ions or alginate. The alginate solution is mixed with the cells to be implanted to form an alginate suspension which is injected directly into a patient prior to hardening of the suspension. The suspension then hardens over a short period of time due to the presence in vivo of physiological concentrations of calcium ions. Modified alginate derivatives, for example, more rapidly degradable or which are derivatized with hydrophobic, water-labile chains, e.g., oligomers of .epsilon.-caprolactone, may be synthesized which have an improved ability to form hydrogels. Additionally, polysaccharides which gel by exposure to monovalent cations, including bacterial polysaccharides, such as gellan gum, and plant polysaccharides, such as carrageenans, may be crosslinked to form a hydrogel using methods analogous to those available for the crosslinking of alginates described above. Additional examples of materials which can be used to form a hydrogel include polyphosphazines and polyacrylates, which are crosslinked ionically, or block copolymers such as Pluronics.TM. or Tetronics.TM., polyethylene oxide-polypropylene glycol block copolymers which are crosslinked by temperature or pH, respectively. Other materials include proteins such as fibrin (although this is not preferred since thrombin may stimulate tumor growth via a pathway that MIS may have to overcome, such as EGF-stimulated proliferation), polymers such as polyvinylpyrrolidone, hyaluronic acid and collagen. Polymers such as polysaccharides that are very viscous liquids or are thixotropic, and form a gel over time by the slow evolution of structure, are also useful. For example, hyaluronic acid, which forms an injectable gel with a consistency like a hair gel, may be utilized. Modified hyaluronic acid derivatives are particularly useful. Polymer mixtures also may be utilized. For example, a mixture of polyethylene oxide and polyacrylic acid which gels by hydrogen bonding upon mixing may be utilized. In one embodiment, a mixture of a 5% w/w solution of polyacrylic acid with a 5% w/w polyethylene oxide (polyethylene glycol, polyoxyethylene) 100,000 can be combined to form a gel over the course of time, e.g., as quickly as within a few seconds.

Covalently crosslinkable hydrogel precursors also are useful. For example, a water soluble polyamine, such as chitosan, can be cross-linked with a water soluble diisothiocyanate, such as polyethylene glycol diisothiocyanate. The isothiocyanates will react with the amines to form a chemically crosslinked gel. Aldehyde reactions with amines, e.g., with polyethylene glycol dialdehyde also may be utilized. A hydroxylated water soluble polymer also may be utilized.

Alternatively, polymers may be utilized which include substituents which are crosslinked by a radical reaction upon contact with a radical initiator. For example, polymers including ethylenically unsaturated groups which can be photochemically crosslinked may be utilized, as disclosed in WO 93/17669. Additionally, water soluble polymers which include cinnamoyl groups which may be photochemically crosslinked may be utilized, as disclosed in Matsuda et al., ASAID Trans., 38:154 157 (1992).

II. Methods for Engineering and Implantation of Cells

A. Disorders to be Treated

A variety of conditions that produce abnormal growth, including treatment of malignant and benign neoplasias, vascular malformations (hemangiomas), inflammatory conditions including those resulting from infection, especially chronic or recalcitrant conditions such as those in the sinuses or which are cystic, keloid formation, endometriosis, congenital or endocrine abnormalities such as testotoxicosis (Teixeira et al, PNAS 1999) and other conditions that produce abnormal growth, can be treated.

Examples of tumor cells that can be treated with MIS include primary and metastatic growth of the following: ovarian adenocarcinomas, endometrial adenocarcinomas, cervical carcinomas, vulvar epidermoid carcinomas, ocular melanomas, prostate, breast, and germ cell tumors. As initially demonstrated with MIS transfected cells, this methodology can be used for delivery of a large number of proteins to control abnormal tissue growth, particularly other members of the TGF.beta. family. Coupled with minimally invasive delivery systems, the biodegradable implants producing the therapeutic proteins from transfected autologous cells can be introduced into a variety of sites to deliver therapeutics, particularly where a local effect is advantageous. This allows use of a variety of recombinant proteins without the need for complex purification protocols.

B. Engineering of Cells

In the preferred embodiment, patient cells are transfected with the gene to be expressed, for example, rhMIS cDNA, to produce cells having stably incorporated therein the DNA encoding the molecules to be expressed. Methods yielding transient expression, such as most adenoviral vectors, are not preferred. Stable transfectants are obtained by culturing and selection for expression of the encoded molecule(s). Those cells that exhibit stable expression are seeded onto/into the appropriate matrix and then implanted using techniques such as those described in the following examples.

C. Seeding of Matrices

The level of expression of the bioactive molecules is measured prior to implantation to insure that an adequate number of cells is implanted. In general, the higher the number of cells implanted, the better. Cells are preferably cultured initially in vitro, then implanted before the matrix degrades but when the level of bioactive molecules is highest. An example of a suitable seeding density is between 1 and 10.times.10.sup.6 cells on a matrix with a surface area of 0.25 cm.sup.2.

D. Implantation of Matrices

The devices are implanted into the patient at the site in need of treatment using standard surgical techniques. In one embodiment, the device is constructed, seeded with cells, and cultured in vitro prior to implantation. The cells are cultured in the device, tested for high MIS production by ELISA, then implanted.

The technique described herein can be used for delivery of many different cell types for different purposes. Other endocrine producing transfectant cells can also be implanted. The matrix may be implanted in one or more different areas of the body to suit a particular application. Matrices with hepatocytes or other high oxygen organ cells may be implanted into the mesentery to insure a good blood supply. Sites other than the mesentery for injection or implantation of cells include the ovarian pedicle, subcutaneous tissue, retroperitoneum, properitoneal space, and intramuscular space. The use of ovarian pedicle for MIS producing implants cause ovary and fallopian tubes to adhere to implants (Kristjansen, et al., 1994).
 

Claim 1 of 7 Claims

1. A cell-matrix structure for implantation into a patient comprising a polymeric matrix and cells attached thereto in an amount sufficient to stop or regress abnormal cell or tissue growth in the patient, wherein the cells in the cell-matrix structure are genetically engineered to stably express Mullerian Inhibiting Substance (MIS).

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