MXPA96004587A - Geles cellula - Google Patents

Abstract

Cell gels composed of cells contained within a cross-linked matrix of cross-linked collagen with bifunctional polyethylene glycol are made by cross-linking the collagen in the presence of the cells. The gels are useful to increase the tissue vi

Description

CELLULAR GELS DESCRIPTION Technical field This invention relates to new formulations and compositions of "cell gels", suitable for augmenting new tissue, comprising a plurality of living cells contained within a cross-linked matrix material, such as cross-linked collagen.

BACKGROUND OF THE INVENTION A method number for increasing or replacing tissue is known in the art. The above methods employed biocompatible implants that, after implantation, are colonized by host cells via cell entry, inward growth, and migration. For example, Daniels et al. (U.S. Patent No. 3,949,073) described, inter alia, the preparation and injection of soluble collagen in appropriate locations of a subject with a REF: 23272 promoter of fibril formation (described as a patent polymerization promoter) to form fibrous collagen implants, in situ, to augment hard or soft tissue. These implants are rapidly colonized by the host cells and are formed with vascular tissue. Now, this material is commercially available from Collagen Corporation (Palo Alto, CA) under the trademark Zyder ® Collagen Implant. The colonization by cells of similar materials has been examined. For example, a wide variety of microcarriers have been developed based on gelatin, dextran, cellulose, acrylamide, fluorocarbon-polylysine and polystyrene (see, for example, Reuveny, Advances in Cell Culture, 1995, Vol. 4, pp. 213-247 , Issemann et al., In Vitro Cellular and Developmental Biology, 1985, Vol. 21, No. 7, pp. 391-401 and references therein). An important application of these microcarriers is in the culture of cells dependent on the anchorage or firm support, where these cells join and proliferate on the surfaces of the preformed microcarriers. Cell binding and proliferation can be limited to the outer surface of the microcarrier, or for porous microcarriers, inward growth and proliferation can occur within the interior pore spaces. For example, Nilsson et al. (Biotechnology, 1986, Vol. 4, pp. 989-990) describe the preparation of porous microcarriers from gelatin (denatured collagen) which allows the cells to grow inside the porous gelatin pellet. Similarly, Adema et al.

(BioPharm, 1990, Vol. 3, No. 7, pp. 20-23) describe the preparation of fortified porous microcarriers from a cross-linked glutaraldehyde-collagen-glycosaminoglycan (GAG) copolymer. Similar materials have been used in the preparation of tissue replacements. For example, Eisenberg (U.S. Patent No. 5,282,859) discloses an equivalent living skin comprising, inter alia, a dermal layer of fibroblast cells cultured in a cross-linked, porous collagen sponge prepared by inoculating cross-linked bovine collagen sponge membranes commercially available. Saintingny et al. (Acta. Derm. Venereol. (Stockholm), 1993, Vol. 73, pp. 175-180) describe epidermal reconstruction by seeding or inoculating fibroblast cells in a porous, porous substrate comprising a chitosan-collagen copolymer. -glycosaminoglycan. The introduction of living cells directly into tissue replacement reduces or removes the need for cell migration or inward growth before colonization (see, for example, review by Nanchahal et al., British Journal of Plastic Surgery, 1993, Vol. 45, pp. 354-363 and references therein). Yannas and collaborators (Patent North American No. 4,418,691, United States Patent No. 4,458,678, Proc. Nati Acad. Sci., 1989, Vol. 86, No. 3, pp. 933-937) describe, inter alia, the introduction of viable cells into a fibrous lattice by surgical techniques, using force, or another of manipulation (all referred to as "seeding") in order to promote the growth of cells or the generation of tissue in a wound. In particular, Yannas et al. Describe a fibrous lattice comprising collagen that is crosslinked with glycosaminoglycan (a polysaccharide component found in, inter alia, the collective tissue) in which cells are fixed by centrifugal force. Bell et al. (Proc. Nati, Acad. Sci., 1979, Vol. 76, pp. 1274-1278, Plastic and Reconstructive Surgery, 1981, Vol. 67, pp. 386-392, British Journal of Dermatology, 1986, Vol. 114, pp. 91-101) describe a method with which the collagen in solution is mixed with a suspension of cells and the pH is adjusted subsequently to cause the collagen to exit the solution in the form of fibrils, producing a gel or lattice in which the cells are distributed in a more or less uniform way. Over a period of days, the mold gel subsequently undergoes compaction by the mobile activity of the cells to produce a tissue of firm consistency. Rowling and collaborators (Biomaterials, 1990, Vol. 11, pp. 181-185) found that these dermal equivalents exhibited improved resistance to enzymatic degradation after 20-30 days in culture. Weinberg et al. (U.S. Patent No. 4,837,379) describe fibrin-containing tissue equivalents comprising collagen, fibrin, and fixed cells (as "contractile agents"). In addition, tissue equivalents may additionally include an agent that can cross-link fibrin and collagen, e.g., factor XIII, (a natural blood coagulation factor), to improve strength and stability. Weinberg also describes a method for preparing tissue equivalents including, inter alia, the step of simultaneously mixing collagen, fibrin (obtained in situ from the reaction of fibrinogen with thrombin), and cells to form a gel. Freeman (Methods in Enzymology, 1987, Volume 135, pp. 216-22) describes, inter alia, cell immobilization by gel entrapment of whole cells in prepolymerized, crosslinked polyacrylamide hydrazide gels. This entrapment procedure is based on the dispersion of the cells in an aqueous solution of a synthetic polymer, soluble in water, linear, which is then crosslinked, in the presence of cells and under moderate physiological conditions, by the addition of a dialdehyde such as glyoxal. Rhee et al. (U.S. Patent No. 5,162,430, U.S. Patent No. 5,264,214, Bovine Collagen Modified by PEG, in Poly (Ethylene Glycol) Chemistry: Biotechnical and Biomedical Applications, ed J. Milton Harris, Plenum Press, New York, 1992, pp. 183-198) describe collagen-polymer conjugates in which the collagen, preferably reconstituted atelopeptide collagen, is chemically bound to a synthetic hydrophilic polymer, preferably polyethylene glycol. By using polyfunctional polymers, cross-linked collagen is obtained.

Description of the invention One aspect of the present invention relates to a composition of cellular gels comprising a plurality of cells contained within a matrix material cross-linked with a polymeric, synthetic crosslinking agent. Another aspect of the invention relates to a method for making or manufacturing a cell gel composition comprising: (a) providing a cell mixture, a matrix forming material, and a polymeric, synthetic crosslinking agent; and (b) subjecting the mixture under conditions that cause the matrix-forming material to be crosslinked by the crosslinking agent. Yet another aspect of the invention relates to a method for augmenting tissue at a site within a living mammal comprising placing the cellular gel composition, described above at that site.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 (a) is a graph of the relative absorbance data recorded for the collagen-SPEG cellular gels of Example 1. Figure 1 (b) is a graph of the relative absorbance data, normalized for cell gels of collagen-SPEG of Example 1.

Modes for carrying out the invention A. Composition and Formulations of Cellular Gels.

The term "cell gel" as used herein refers to composition and formulations of cell gels comprising a plurality of living cells contained within a matrix material that has been cross-linked with a cross-linking agent, which are useful for increase the living tissue. The term "increase", as used herein, refers to the repair, prevention, or relief of defects, particularly defects due to the loss or absence of hard or soft tissue, by providing, augmenting or replacing this tissue. The term "matrix forming material" as used herein refers to crosslinkable polymers. That is, polymers that have functional groups that allow the polymers to crosslink. Examples of matrix-forming materials include collagen, fibrin, fibrinogen, chitin, chitosan, its derivatives and analogs, and mixtures thereof, if obtained from natural sources or in a synthetic manner. Preferably, the matrix-forming material comprises collagen or collagen derivatives. Collagen is the main protein component of bone, cartilage, skin, and connective tissue in animals. Collagen is typically isolated from human placentas, bovine leather, cartilage, or bones; usually, the bones are dried, descened, crushed, and demineralized to extract the collagen, while the leather and cartilage are usually crumbled and digested with enzymes proteolytic (different from collagenase). Since collagen is resistant to most proteolytic enzymes, this procedure conveniently serves to remove most of the contaminating protein found with collagen. The collagen can be denatured by boiling, which produces the familiar gelatin product. The term "collagen", as used herein, refers to all forms of collagen, including native collagens that have been processed or otherwise modified, and collagens that have been produced by genetic engineering (ie, recombinant collagen) . Suitable collagens include all types, preferably types I, II and III. Collagens can be soluble (for example, Vitrogen® 100, collagen in solution, commercially available), and may have or omit the telopeptide regions. Preferably, the collagen will be fibrillar, reconstituted atelopeptide collagen, e.g., Zyderm® collagen implant (ZCI) or atelopeptide collagen in solution (CIS, for its acronym in English). Various forms of collagen are commercially available or can be prepared by the processes described in for example, U.S. Patent Nos. 3,949,073; 4,488,911; 4,424,208; 4,582,640; 4,642,117; 4,557,764; and 4,689,399, all incorporated herein by reference.

The term "crosslinked matrix material" as used herein refers to a matrix material in which one or more crosslinkable polymers have been crosslinked by chemical reaction with one or more crosslinking agents to form covalent linkages therewith. The term "crosslinking agent" as used herein refers to compounds that (i) have functional groups that are capable of chemically reacting with functional groups of the matrix-forming material to form covalent bonds, and (ii) are nominally not cytotoxic The term "functional groups that are capable of reacting chemically," as used herein, includes functional groups that can be activated or derivatized to be capable of chemically reacting with functional groups of the matrix-forming material to form covalent bonds. The term "synthetic crosslinking agent" as used herein refers to crosslinking agents that are non-natural. In one embodiment, the synthetic crosslinking agent is derived from a polymeric compound. Examples of polymeric, synthetic crosslinking agents include polyethylene glycols, polyfunctional, activated. For example, the difunctional polyethylene glycol (dPEG) is first reacted with a dicarboxylic acid anhydride, such as glutaric anhydride, and subsequently reacted with an activating agent, such as N-hydroxysuccinimide, to form succinimidyl poly (ethylene glycol) glutarate. (denoted in the present SPEG), a polymeric, synthetic crosslinking agent. In the present, PEGs of molecular weight are preferred from about 400 to about 20,000, most preferably from about 1,000 to about 7,000 Examples of dicarboxylic acid anhydride include oxalic anhydride, malonic anhydride, succinic anhydride, glutaric anhydride, adipic anhydride, 1,8-naphthalene dicarboxylic anhydride, 1,4,5,8-naphthaletracarboxylic dianhydride, and the like. Activating agents are those compounds that react with carboxylic acids to produce activated esters, -COOR, wherein R is a good leaving group. Examples of activation groups include N-hydroxysuccinimide, N, N'-disuccinimidyl oxalate, N, N'-disuccinimidyl carbonate, and the like. Also see US Patent No. 4,179,337 issued by Davis for additional link groups. The crosslinking agents react chemically with a matrix forming material to form crosslinks. Any known method can be used to derivatize and subsequently react the crosslinking agent and the matrix forming material to form crosslinks. For example, collagen molecules, which possess a number of available usin groups with free amino groups (-NH2), can be cross-linked with active ester portions, such as those of succimidyl-poly (ethylene glycol) glutarate, for form amide bonds (-CONH-). The degree of crosslinking can be expressed as the number of functional groups per molecule (initial) of the matrix-forming material that are included in the crosslinking. For example, for collagen, the number of available lysins comprised in the crosslinking can vary from a single residue to 100% of the lysines, preferably 10% -50%, and more preferably 20% -30%. The number of reactive plant residues can be determined by normal methods, for example, with the reaction with TNBS (2, 3, 4-trinitrobenzenesulfonic acid). The term "nominally non-cytotoxic" as used herein refers to crosslinking agents, which, when added to the cellular environment (ie, added to a cell suspension) at concentrations useful for crosslinking, do not reduce substantially cell viability, for example, do not reduce cell viability by more than 50% for 7 days, and are not physiologically harmful. The term "cells" as used in the The present invention relates to living cells, preferably mammalian cells, including, for example, human cells. The cells can be autogeneic, isogeneic, allogeneic, or xenogeneic, more preferably autogeneic or halogenic. Cells that have been genetically engineered are included. Cell gels can contain different cell types, which can be chosen to act synergistically, for example, in tissue formation. Examples of cell types include muscle cells, nerve cells, epithelial cells, connective tissue cells, and organ cells. Examples of cells include fibroblast cells, smooth muscle cells, striated muscle cells, heart muscle cells, nerve cells, epithelial cells, endothelial cells, bone cells, bone progenitor cells, bone marrow cells, cells blood, brain cells, kidney cells, liver cells, lung cells, pancreatic cells, spleen cells, breast cells, foreskin cells, ovarian cells, testis cells and prostate cells. Other mammalian cells are useful in the practice of the invention and do not They exclude from consideration in the present. Alternatively, cell gels can be prepared using eukaryotic cells, prokaryotic non-mammalian cells, or viruses. The cell gel compositions and formulations of the present invention further have properties that are (i) nominally non-immunogenic and (ii) bioerodible. The term "nominally non-immunogenic" as used herein refers to materials that do not elicit substantial immune response, inflammation, or foreign body reaction when administered. The term "bioerodible" as used herein refers to the potential of a material to be eroded or degraded by the action of enzymes (including, for example, proteinases, such as collagenases), or other biological processes, to produce substances Non-toxic or by-products that are compatible with bodily processes. The degree to which a material is bioerodible can be indicated by its period of bioerosion. The term "bioerosion period" as used herein refers to the period of time after which substantial bioerosion of the crosslinked matrix material has occurred. The period of bioerosion can vary for the particular indication, and will reflect the needs to place, support and initially house the trapped cells, to allow the growth and proliferation of those cells, and to finally allow the complete or almost complete erosion of the matrix material. reticulated, original. Examples of erosion periods for cell gels include 20-45 days, for cell gels associated with the dermal, 30-90 days for cell gels associated with bone, 10-30 days for gels associated with the nerve.

The term "administer" as used herein is generic for the methods of applying, attaching, implanting, injecting and the like. If the cell gel material is a suspension, injection is the preferred method of administration.

B. Preparation and use of cell gels.

The compositions and formulations of cell gels of the invention can be prepared by a one-step method comprising simultaneously mixing the matrix-forming material, 'l the crosslinking agent, and the cell suspension. Alternatively, the compositions and formulations of cell gels can be prepared by a two-step number, such as pre-mixing the cell suspension and the matrix-forming material, followed by the addition of the cross-linking agent; premixing the cell suspension and the cross-linking agent, followed by the addition of the matrix-forming material; and premixing the matrix-forming material and the cross-linking agent, followed by the addition of a cell suspension.

The physical properties of the resulting cell gel compositions and formulations, such as viscosity, consistency, and texture, can be adjusted by varying the concentrations of the reactants, the reaction conditions, the reaction time, and other factors. The term "physical properties" as used herein includes, for example, viscosity, consistency, texture, modulus of elasticity, surface properties, surface roughness, pore size, pore shape, pore interconnection, and the like. For example, the viscosity of the cell gels can be increased by increasing the concentration of the matrix-forming material in the reaction mixture. For example, for collagen-SPEG cellular gels, preferred concentrations of collagen are 5-10 mg / mL, most preferably about 10-75 mg / mL, most preferably 30-60 mg / mL. The degree of cross-linking present in the cell gels can be varied according to the molar ratio of the matrix-forming material to the cross-linking agent. The increase in the concentrations of the crosslinking agent relative to the concentrations of the matrix-forming material produces cell gels with high viscosities which can be characterized as gel-like, plastics, semi-solid or solid. Conversely, the decrease in the relative concentration of the cross-linking agent produces cell gels with lower viscosities that can be characterized as fluids or liquids. You can get a continuum of viscosities, textures and consistencies. For example, for cellular collagen-SPEG gels, collagen molar ratios: SPEG of about 200: 1 to about 5: 1 produce cell gels that are fluid and can be injected, while ratios of about 5: 1 to about 1 : 10 produce semisolid cell gels, and ratios greater than about 1:10 to about 1:75 produce heavier, more rigid, cross-linked cell gels. Ratios of about 1:50 typically lead to cell gels wherein all available collagen lysines are comprised in crosslinking. The degree of crosslinking present in the cell gels can be further controlled by adjusting the reaction temperature and the reaction time. The increased reaction temperature (but not higher than 37 ° C) will increase the rate of formation of the crosslinks. Conversely, reducing the reaction temperature will decrease the rate of formation of the crosslinks. For example, the reaction of collagen and SPEG to form crosslinks is rapid at room temperature, but substantially slower at 5 ° C. During the reaction, the degree of crosslinking increases with the increase in reaction times; by adjusting other conditions, such as concentrations and temperature, adequate reaction rates (and therefore reaction time) can be obtained. Other factors, such as pH, can be adjusted to vary the properties of the cell gel. For example, for cell gels obtained using collagen in solution and activated PEG, a wide range of fibrillar collagen content can be obtained by varying the pH of the reaction mixture. In addition, a microgel material in the form of particles can be obtained by stirring a cell gel reaction mixture comprising, for example, collagen in solution in activated PEG, during cross-linking (for example, by stirring or passing between syringes). The salt concentration of the reaction mixture can also be adjusted to control the properties of the cell gels. Reagents, such as the matrix-forming material, the cross-linking agent, and the cell suspension, can be dispersed in a pharmaceutically acceptable carrier prior to mixing. For example, since SPEG is subjected to hydrolysis, it is typically stored dried at -2 ° C before use, and prepared as an aqueous mixture immediately before use. Alternatively, the SPEG can be prepared as a non-aqueous suspension (using, for example, glycerol, PEG, triglycerides, DMSO and the like) or as a suspension with reduced concentrations of water. These reduced aqueous or non-aqueous suspensions can be used to further control the rates and reaction times. Similarly, reduced aqueous or non-aqueous cell suspensions can be prepared to control the properties of cell gels.

The term "cell suspension" as used herein refers to living cells dispersed in a liquid, preferably an aqueous medium. Examples of suitable liquids include, for example, salt solutions, physiologically quenched and cell culture media, and may include components such as glycerol, DMSO, triglycerides, and the like, and may additionally contain supplements of media known in the art, including for example, serum, growth factors, hormones, sugars, amino acids, vitamins, metalloproteins, lipoproteins, and the like. Methods for the preparation of cell suspensions by dissociation from an aggregate of living cells are well established and known to those skilled in the art (see for example, RI Freshney, Culture of Animal Cells - A Manual of Basic techniques, 2nd Edition, Alan Rl Liss, Inc. New York). For example, a common method comprises treating a cellular aggregate with EDTA, or an enzyme, such as trypsin, collagenase, and the like, which causes the cells to disengage from other cells or solid surfaces.

The concentrations of the cell suspension can be chosen to optimize the texture and viscosity of the cell gels, the rate of subsequent colonization of the gel, and / or the viability of the cells within the gel. In the present, cell suspension concentrations resulting in concentrations of the reaction mixture from about 1 x 10 4 to 1 x 10 b cells / mL are preferred, and concentrations of about 1 x 10 5 cells / mL are more preferred. The cell gels of the invention may additionally include biologically active factors to aid in the healing or regrowth of normal tissue. For example, factors such as heparin, epidermal growth factor (EGF), transforming growth factor (TGF) alpha, TGF-β (including any combination of TGF-β) TGF- can be incorporated. ßl, TGF-ß2, platelet-derived growth factor (PDGF-AA, PDGF-AB, PDGF-BB), acid fibroblast growth factor (FGF), basic FGF, connective tissue activation peptides (CTAP), factors Growth hormone-like insulin ß-thromboglobulin, tumor necrosis factor (TNF), interleukins, colony stimulation factors (CsFs), erythropoietin (EPO), nerve growth factors (NGF), interferons (IFN), osteogenic factors, and the like. The incorporation of these factors, and appropriate combinations of the factors, can facilitate the transformation of cellular gels or can be used in the treatment of wounds. The cell gels of the invention containing growth factors are particularly suitable for the sustained administration of factors, as in the case of the promotion of wound healing. Osteoinductive factors and cofactors (including TGF-β) can be advantageously incorporated into compositions intended for the replacement, augmentation and / or repair of bone defects. Cell gels containing biological growth factors such as EGF and TGF-β are prepared by mixing an appropriate amount of the factor in the composition, or by incorporating the factor into the matrix-forming material before treatment with the cross-linking agent.

The cell gels of the invention containing biological growth factors such as EGF and TGF-β are prepared by mixing an appropriate amount of the factor in the composition. The factors can be chemically linked to the matrix-forming material, or to the cross-linked matrix material, for example, by employing a suitable amount of cross-linking agent during the preparation of the cell gel. For example, the factors can be covalently attached to the matrix-forming material in the same manner as the matrix-forming material is cross-linked. By binding the factor molecules to the reticulated matrix material, the effective amount of the factor is substantially reduced. The term "effective amount" refers to the amount of composition required in order to obtain the desired effect. In this way, a "tissue growth promoting amount" of a composition containing a growth factor refers to the amount of the factor necessary in order to stimulate tissue growth to a detectable degree. Tissue, in this context, includes connective tissue, bone, cartilage, epidermis, dermis, blood, and other tissues. In addition, the factors covalently bound to the matrix-forming material serve as effective matrices for delivery of controlled-release drugs. For example, the factors can be chemically linked to the collagen using an activated PEG: the factor is first reacted with a molar excess of activated PEG in a diluted solution for a period of about 5 min to about 1 hour. The factor is preferably provided at a concentration - from about 1 μg / mL to about 5 mg / mL, while the activated PEG is added It is preferably at a final concentration that provides a molar excess of 30 to 80 times. The resulting conjugate factor is then added to a collagen, aqueous mixture (from about 1 to about 60 mg / mL) at pH 7-8 and allowed to react further. The resulting composition is allowed to stand overnight at room temperature. The pellet is collected by centrifugation, and washed with PBS by forming a vigorous vortex in order to remove the unbound factor. The resulting collagen-factor material is then used in the matrix-forming material in the preparation of a cellular gel.

Particulate materials may additionally be included in the cell gel, eg, hydrogel or collagen-dPEG beads, hydroxyapatite / tricalcium phosphate particles, particulate material of polylactic acid / polyglycolic acid (PLA / PGA), or Teflon beads, to provide a more bulky or stiffer cell gel after crosslinking. Suitable formulations for the repair of bone defects or non-unions can be prepared by providing cellular gels with a high concentration of the matrix-forming material, high concentrations of the cross-linking agent, or by mixing with suitable particulate materials. . The term "suitable particulate material" as used herein refers to a particulate material that is substantially insoluble in water, that is biocompatible, and that is immiscible with the matrix-forming material or the material of reticulated matrix. The particles of the particulate material may be fibrillar or may vary in size from about 1 to 500 μm in diameter and are in a manner similar to pellets or beads or irregularly shaped. For example, for injectable cell gels, such as those useful for soft tissue augmentation, the preferred particle sizes are less than about 150 μm. For cell gels useful for bone-related enhancement, preferred particle sizes greater than about 100 μm. Particulate materials, for example, include without limitation, fibrillated cross-linked collagen, gelatin beads or pellets, cross-linked PEG-collagen particles, polytetrafluoroethylene beads, beads or silicone rubber pellets, hydrogel beads, carbide beads of silicon, glass beads, carbon fibers, PLA / PGA fibers, and polyethylene terephthalate (PET) fibers. The presently preferred particulate materials are hydroxyapatite and tricalcium phosphate. Injectable plastic, malleable, cell gel compositions can be prepared by adjusting the reaction parameters as indicated above, or by the addition of a sufficient amount of a pharmaceutically acceptable carrier, such as water or glycerol. The term "sufficient amount" as used herein is applied to the amount of carrier used in combination with the cellular gels of the invention. A sufficient amount is that amount which when mixed with the cell gel returns it in a desired physical form, for example, injectable solution, injectable suspension, plastic or malleable implant, rigid implant, and so on. The term "injectable" as used herein refers to materials that have a texture and viscosity that allows their flow through a suitable surgical needle and i using typical pressures and injection. For example, an injectable material can be forced through a 32 gauge needle under normal pressure. The mixture is injected directly into the site in need of augmentation, such as the tendon or cartilage, and essentially inflammation or reaction to the foreign body is not caused. Injectable cell gel formulations are useful for dermal augmentation, for example for filling in skin wrinkles, and the provision of support for skin surfaces, increase of the sphincter, (for example, for the restoration of continence), revascularization of the tissue, distribution of deposit cells, blockage of blood vessels of tumors, therapy, and contraceptive / infertility treatments. Alternatively, the cell gels can be administered by injection before the crosslinking is complete. For example, an aqueous mixture of matrix-forming material and cell suspension is combined with a low-concentration solution containing the cross-linking agent, mixed, and the reaction mixture is injected or applied before the viscosity is sufficiently increased. to return to the difficult injection (usually around 10 minutes). Mixing can be achieved by passing the mixture between two syringes equipped with Luer fixation cubes, or through an individual syringe that has dual compartments (eg double barrel). The reaction mixture reacts to form crosslinks in situ (ie, at the site in need of augmentation), and can be further crosslinked to the endogenous tissue, anchoring the cell gel in place. Similarly, a mixture comprising cells and the matrix-forming material can be injected at the site in need of augmentation, and subsequently injected with a mixture comprising a cross-linking agent at the same site. Alternatively, a mixture comprising cells and a crosslinking agent can be injected at the site in need of augmentation, and subsequently injected with a matrix-forming material. If desired, the more viscous, denser formulations can be formed or molded into any desired shape, for example, in sheets or membranes, in meshes, in tubes or cylinders, in hooks, cords or ropes, and the like. Flexible sheets or membranous forms of cell gels can be prepared by methods analogous to those known in the art for the preparation of gel membranes (see for example US Pat. Nos. 4,600,533; 4,412,947; and 4,242,291). For example, a mixture of a fibrillar collagen or CIS, preferably fibrillar alopeptide collagen such as ZCI) of high concentration (10-100 mg / mL), activated PEG (having a molecular weight of about 3,400), and the cell suspension is mold in a flat sheet container, and allow to react for 2-3 hours at 37 ° C. The resultant collagen cell gel is removed from the excess reaction solution using a sterile spatula or the like, and can be washed with PBS to remove the excess unreacted crosslinking agent. More flexible membranous forms are achieved by using lower collagen concentrations and higher concentrations of the crosslinking agent as starting materials. Similarly, sheaths or tubular shapes of cell gel compositions can be prepared which are useful to replace or augment vascular structures, such as blood vessels, or as a sheath of nerve tissue. The compositions of the invention can be prepared in a form that is dense and rigid enough to replace the cartilage or bones that are weightless, for example, finger bones. These compositions are useful for repairing and supporting tissue that requires some degree of structure, for example in the reconstruction of the nose, ear, knee, larynx, tracheal rings, and joint surfaces. Tendon, ligament, and blood vessel tissue can also be replaced using appropriately formed cartilaginous material. In these applications, the cellular gel is formed or molded in general in one form; In the case of tendons and ligaments, it may be preferable to form filaments to weave them into cords or ropes. All publications, patents, and patent applications cited in this specification are incorporated herein by reference as if each publication or individual patent application was specifically and individually indicated to be incorporated by reference.

C. Examples The invention will be further understood with reference to the following examples, which are of a purely exemplary nature, and are not intended to be used to limit the scope of the invention.

Example 1 Preparation of Collagen Cell Gels-SPEG Fibroblast, dermal, human cells were used in the 5th passage. Dermal, human fibroblast cells were subcultured from those in the reservoir with the American Type Culture Collection, 12301, Parklawn Drive, Rockville, MD 20852, USA, under the accession number of ATCC, CRL 1885. On day 0, the cells were treated with trypsin using 25 mg / mL trypsin in EDTA (2 mM) and the cells were pelleted by centrifuging at 150-200 g for 10 minutes at room temperature. The solid was discarded, and the pellet was redispersed in 5 ml of the DME medium (Eagle's medium, modified Dulbecco's, with 4.00 mm L-glutamine, 1000 mg / L glucose, 100 mg / L pyruvate sodium). Cell concentration was determined using a hemocytometer. The cell suspension was diluted with DME medium at a concentration of 1 x 10 5 cells / mL. The controls were prepared using aliquots of 1 μl, 10 2 μl and 100 μl of the stock solution of the cell suspension were used to obtain concentrations of 100 cells / well (1A1, 1A2, 2A1, 2A2), 1,000 cells / well (1A3, 1A4, 2A3, 2A4), and 10,000 cells / well (1A5, 1A6, 2A5, 2A6) in DME medium in the culture studies (indices such as 1A3 indicate plate 1, row A, column 3). A combination of Zyderm I collagen, (Collagen Corporation, Palo Alto CA), was prepared by combining a total of 12 L of different samples of Zyderm I and mixing through a sterile bridge (such as a tap) to ensure uniformity.

Zyderm I is an aqueous mixture of fibrillar collagen (300,000 g / mol) prepared with a concentration of 35 mg / mL or 1.17 x 10 ~ 4 mol / 1. A stock or concentrate of succinimidyl-poly (ethylene glycol) glutarate (SPEG, 3,400 g / mol) was prepared by dissolving 45.0 mg of the activated PEG in 10 ml of PBS (phosphate buffered exit solution), to give a concentration of 4.5 mg / mL or approximately 1.32 x 10"J mol / 1 Zyderm I controls (1B4, 1B5, 1B6) were prepared using aliquots of 0.5 ml of a combination of Zyderm I. Cultures of Zyderm I / cells were prepared by mixing 1.5 ml of Zyderm I with 30 μl of the concentrated cell suspension Aliquots of 0.5 mL were placed in each well (1B1, 1B2, 1B3), to give 1,000 cells / well A cellular collagen / SPEG gel was prepared (10: 1) by mixing 2 ml of Zyderm I combination, 10 μl of activated PEG solution, and 40 μl of concentrated cell suspension, 0.5 mL aliquots were placed in each well (1C1, 1C2, 1C3) to give 1,000 cavity cells. prepared collagen / PEG controls (10: 1) when mixing 2 mL of combination of Zyderm I with 10 > , μl of concentrated PEG solution. Aliquots of 0.5 L were placed in each cavity (1C4, 1C5, 1C6). A cellular gel of collagen / SPEG (100: 1) was prepared by mixing 2 ml of concentrated collagen solution, 1 μl of activated PEG solution, and 40 μl of concentrated cell suspension. Aliquots of 0.5 mL were placed in each well (1D1, 1D2, 1D3) to give 1,000 cells / well. Collagen / PEG controls were prepared (100: 1) by mixing 2 mL of concentrated collagen solution with 10 μL of concentrated PEG solution. Aliquots of 0.5 mL were placed in each well (1D4, 1D5, 1D6).

The culture plates were centrifuged at 4 ° C at 150-200 g for 2 minutes. The feeding, by the addition of 1 mL of DME-complete (DME medium that also comprises 10% Fetal Bovine Serum and 1% penicillin or streptomycin) to each cavity, was performed on day 0, day 1, day 3, and day 5. On day 3 and day 7, an assay was performed with Alamar Blue: 100 μL of Alamar Blue per 1 mL of medium was added to each well and the absorbance was recorded at 590 nm subsequently using an EX fluorometer 550 EM 590, Cambrigde, 3 and 6 hours after incubation. After the measurements were recorded at 6 hours, the medium was removed and 1 mL of fresh medium was added. The data are summarized in Table 1. The analyzed data are presented in Table 2. The "Normalized" intensity presented for the samples containing cells reflects the difference between the average absorbance recorded for the sample containing cells minus the average absorbance recorded for the analogous samples that lack cells. The normalized cell proliferation data are basically shown in Figure 1. Proliferation data show that cell viability is not adversely affected by growth within the crosslinked collagen-PEG cell gel. After the final assay with Alamar Blue on day 7, the cell culture controls (from 1A1 to 1A6, from 2A1 to 2A6) were treated with trypsin and counted using a hemocytometer. The control of 10 cells / cavity had too few cells to count. The control of 10J cells / cavity had an average count of 9.1 x 10J cells / cavity. The 104 cells / cavity had an average count of 3.9 x 104 cells / cavity. ^ After performing the tests with Alamar Blue on day 7, all the cavities containing collagen were fixed with 10% buffered formalin, overnight. Preliminary results from histology indicated that the cells were present throughout the materials of the collagen / cell and collagen-PEG cellular gels, and were not adversely affected by the cross-linked collagen matrix.

Table 1 Alamar Blue Test Results Table 2 Results of the Alamar Blue Analyzed Test It is noted that in relation to this date, the best method known by the applicant to carry out the present invention is that which is clear from the present description of the invention. Having described the invention as above, the content of the following is claimed as property:

Claims (11)

1. A cellular gel composition, characterized in that it comprises a plurality of cells contained within a matrix material crosslinked with a crosslinking agent, polymeric, synthetic.

2. The composition according to claim 1, characterized in that the composition can be injected.

3. The composition according to claim 1, characterized in that the matrix material is collagen.

4. The composition according to claim 1, characterized in that the crosslinking agent is a polyfunctional polyethylene glycol.

5. The composition according to claim 1, characterized in that the crosslinking agent is polysuccinimidyl-poly-ethylene glycol glutarate.

6. The composition according to claim 4, characterized in that the molecular weight of the polyfunctional polyethylene glycol is about 400 to about 20,000.

7. The composition according to claim 1, characterized in that the molar ratio of the matrix-forming material to the cross-linking agent is in the range of about 200: 1 to about 5: 1.

8. The composition according to claim 3, characterized in that the cross-linking agent binds about 10% to about 50% of the available collagen-containing waste.

9. The composition according to claim 2, characterized in that the matrix material is collagen, the crosslinking agent is a polyfunctional polyethylene glycol having an average molecular weight of about 400, up to about 20,000 and the molar ratio of collagen to polyfunctional polyethylene glycol is in the range from about 1: 1 to about 1:20.

10. A method for manufacturing a cellular gel composition, characterized in that it comprises: (a) providing a mixture of cells, a matrix-forming material, and a polymeric, synthetic cross-linking agent; and (b) subjecting the mixture to conditions that cause the matrix-forming material to be cross-linked by the cross-linking agent.

11. A method for enhancing tissue at a site within a living mammal, characterized in that it comprises placing the cellular gel composition of claim 1 at that site.