WO1995026761A1

WO1995026761A1 - Cell-gels

Info

Publication number: WO1995026761A1
Application number: PCT/US1995/003991
Authority: WO
Inventors: Trudy D. Estridge; Prema R. Rao
Original assignee: Collagen Corporation
Priority date: 1994-04-04
Filing date: 1995-03-31
Publication date: 1995-10-12
Also published as: AU2202995A; AU682266B2; MX9604587A; EP0754065A1; JPH10501706A; CA2187109A1

Abstract

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

Description

CELL-GELS

Description

Technical Field

This invention relates to novel "cell-gel" formulations and compositions suitable for augmenting living tissue, comprising a plurality of living cells contained within a cross-linked matrix material, such as cross-linked collagen.

Background Art

A number of methods for augmenting or replacing tissue are known in the art. Early methods employed biocompatible implants which, after implantation, are colonized by host cells via cellular ingress, ingrowth, and migration.

For example, Daniels et al . (U.S. Pat. No. 3,949,073) disclose, inter alia , the preparation and injection of soluble collagen into suitable locations of a subject with a fibril-formation promoter (described as a polymerization promoter in the patent) to form fibrous collagen implants in situ , for augmenting hard or soft tissue. Such implants are rapidly colonized by host cells and vascularized. This material is now commercially available from Collagen

Corporation (Palo Alto, CA) under the trademark Zyderm® collagen implant.

The colonization by cells of similar materials has been examined. For example, a wide variety of microcarriers based on gelatin, dextran, cellulose, acrylamide, fluorocarbon-polylysine, and polystyrene have been developed (see, for example, Reuveny, Advances in Cell Culture, 1985, 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 such microcarriers is in the culturing of anchorage dependent cells, wherein such cells attach to and proliferate on the surfaces of pre-formed microcarriers. Cell attachment and proliferation may be limited to the outer surface of the microcarrier, or for porous microcarriers, ingrowth and proliferation within interior pore spaces may occur. For example, Nilsson et al . (Biotechnology. 1986, Vol. 4, pp. 989-990) disclose the preparation of porous microcarriers from gelatin (denatured collagen) which permit cells to grow in the interior of the porous gelatin bead. Similarly, Ade a et al . (BioPharm. 1990, Vol. 3, No. 7, pp.20-23) disclose the preparation of strengthened porous microcarriers from a glutaraldehyde cross-linked collagen-glycosaminoglycan (GAG) copolymer. Similar materials have been used in the preparation of tissue replacements. For example, Eisenberg (U.S. Patent 5,282,859) discloses a living skin equivalent comprising, inter alia , a dermal layer of cultured fibroblast cells in a porous, cross-linked collagen sponge prepared by inoculating commercially available cross-linked, bovine collagen sponge membranes. Saintigny et al . (Acta. Derm. Venereol. (Stockholm). 1993, Vol. 73, pp.175-180) disclose epidermal reconstruction by seeding fibroblast cells on a porous dermal substrate comprising a chitosan-collagen- glycosaminoglycan copolymer.

The introduction of living cells directly into the tissue replacement reduces or removes the need for cellular migration or ingrowth prior to colonization (see, for example, the review by Nanchahal et al . , British Journal of Plastic Surgery. 1992, Vol. 45, pp.354-363 and references therein) . Yannas et al. (U.S. Patent 4,418,691, U.S. Patent 4,458,678, Proc. Natl. Acad. Sci., 1989, Vol. 86, No. 3, pp. 933-937) disclose, inter alia, the introduction of viable cells into a fibrous lattice by surgical, force- utilizing, or other manipulative techniques (all referred to as "seeding") in order to promote the growth of cells or the generation of tissue at a wound. In particular, Yannas et al. discloses a fibrous lattice comprising collagen that is cross-linked with glycosaminoglycan (a polysaccharide component found in, inter alia , connective tissue) into which cells have been embedded by centrifugal force.

Bell et al . (Proc. Natl. 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) disclose a method whereby collagen in solution is mixed with a cell suspension and the pH subsequently adjusted to cause the collagen to come out of solution in the form of fibrils, yielding a gel or lattice in which the cells are more or less uniformly distributed. Over a period of days, the cast gel subsequently undergoes compaction by the motile activity of the cells to yield a tissue of firm consistency. Rowling et al . (Biomaterials_f 1990, Vol. 11, pp.181-185) found that such dermal equivalents exhibited improved resistance to enzymatic degradation after 20-30 days in culture.

Weinberg et al . (U.S. Patent 4,837,379) disclose fibrin-containing tissue equivalents comprising collagen, fibrin, and embedded cells (as "contractile agents") . In addition, the tissue equivalents may further include an agent which can cross-link fibrin and collagen, for example, Factor XIII (a naturally occurring blood coagulation factor) , to enhance strength and stability. Weinberg also discloses a method for preparing tissue equivalents which includes, inter alia , the step of contemporaneously mixing collagen, fibrin (obtained in situ from reaction of fibrinogen with thrombin) , and cells to form a gel.

Freeman (Methods in Enzymology, 1987, Volume 135, pp. 216-222) discloses, inter alia , cell immobilization by gel entrapment of whole cells in cross-linked prepolymerized polyacrylamide-hydrazide gels. This entrapment procedure is based on suspending the cells in an aqueous solution of a linear, water-soluble synthetic polymer, which is then cross-linked, in the presence of cells and under mild physiological conditions, by the addition of a dialdehyde such as glyoxal.

Rhee et al . (U.S. Patent 5,162,430, U.S. Patent 5,264,214, Bovine Collagen Modified by PEG, in Polv(Ethylene Glvcol) Chemistry: Biotechnical and

Biomedical Applications, ed. J. Milton Harris, Plenum Press, New York, 1992, pp. 183-198) disclose collagen- polymer conjugates in which collagen, preferably reconstituted atelopeptide collagen, is chemically bonded to a synthetic hydrophilic polymer, preferably polyethylene glycol. By employing polyfunctional polymers, cross-linked collagen is obtained.

Disclosure of the Invention

One aspect of the present invention relates to a cell- gel composition comprising a plurality of cells contained within a matrix material cross-linked with a synthetic polymeric cross-linking agent.

Another aspect of the invention relates to a method of making a cell-gel composition comprising:

(a) providing a mixture of cells, a matrix forming material, and a synthetic polymeric 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. Still another aspect of the invention relates to a method for augmenting tissue at a site within a living mammal comprising placing the above-described cell-gel composition at said site.

Brief Description of the Drawings

Figure 1(a) is a graph of relative absorbance data recorded for the collagen-SPEG cell-gels of Example 1. Figure 1(b) is a graph of normalized relative absorbance data recorded for the collagen-SPEG cell-gels of Example 1.

Modes for Carrying Out the Invention

A. Cell-Gel Compositions and Formulations

The term "cell-gel" as used herein relates cell-gel compositions and formulations comprising a plurality of living cells contained within a matrix material which has been cross-linked with a cross-linking agent which are useful for augmenting living tissue.

The term "augmenting", as used herein, relates to repairing, preventing, or alleviating defects, particularly defects due to loss or absence of hard or soft tissue, by providing, augmenting, or replacing such tissue.

The term "matrix forming material" as used herein relates to cross-linkable polymers. That is, polymers which have functional groups which permit the polymers to be cross-linked. Examples of matrix forming materials include collagen, fibrin, fibrinogen, chitin, chitosan, their derivatives and analogs, and mixtures thereof, whether obtained from natural sources or synthetically. Preferably, the matrix forming material comprises collagen or collagen derivatives. Collagen is the major protein component of bone, cartilage, skin, and connective tissue in animals. Collagen is typically isolated from natural sources, such as human placentas, bovine hide, cartilage, or bones. Bones are usually dried, defatted, crushed, and demineralized to extract collagen, while hide and cartilage are usually minced and digested with proteolytic enzymes (other than collagenase) . As collagen is resistant to most proteolytic enzymes, this procedure conveniently serves to remove most of the contaminating protein found with collagen. Collagen may be denatured by boiling, which produces the familiar product gelatin.

The term "collagen" as used herein refers to all forms of collagen, including native collagens which 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 may be soluble (for example, commercially available Vitrogen® 100 collagen-in-solution) , and may have or omit the telopeptide regions. Preferably, the collagen will_^ be reconstituted fibrillar atelopeptide collagen, for example Zyderm® I Collagen Implant (ZCI) or atelopeptide collagen in solution (CIS) . Various forms of collagen are available commercially, or may be prepared by the processes described in, for example, U.S. Patents 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 "cross-linked matrix material" as used herein relates to a matrix material in which one or more cross-linkable polymers have been cross-linked by chemical reaction with one or more cross-linking agents to form covalent bonds therewith.

The term "cross-linking agent" as used herein relates to compounds which (i) have functional groups which are able to react chemically with functional groups of the matrix forming material to form covalent bonds, and (ii) are nominally non-cytotoxic. The term "functional groups which are able to react chemically", as used herein, includes functional groups which can be activated or derivatized so as to be able to react chemically with functional groups of the matrix forming material to form covalent bonds. The term "synthetic cross-linking agent" as used herein relates to cross-linking agents which are not naturally occurring. In one embodiment, the synthetic cross-linking agent is derived from a polymeric compound. Examples of synthetic polymeric cross-linking agents include activated polyfunctional polyethylene glycols. For example, 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 succinimidylpoly(ethylene glycol) glutarate (herein denoted SPEG) , a synthetic polymeric cross-linking agent. At present, preferred are PEGs of molecular weight from about

400 to about 20,000, more preferably about 1,000 to about 7,000. Examples of dicarboxylic acid anhydrides include oxalic anhydride, malonic anhydride, succinic anhydride, glutaric anhydride, adipic anhydride, 1,8-naphthalene dicarboxylic anhydride, 1,4,5,8-naphthalenetetracarboxylic dianhydride and the like. Activating agents are those compounds which react with carboxylic acids to yield activated esters, -C00R, wherein R is a good leaving group. Examples of activating groups include N-hydroxysuccinimide, N,N^,-disuccinimidyl oxalate, N,N^,-disuccinimidyl carbonate, and the like. See also U.S. Patent No. 4,179,337 issued to Davis for additional linking groups. Cross-linking agents react chemically with the matrix forming material to form cross-links. Any known method for derivatizing and subsequently reacting the cross-linking agent and matrix forming material to form cross-links may be used. For example, collagen molecules, which possess a number of available lysine groups with free amino (-NH₂) groups, may be cross-linked with active ester moieties, such as those of succinimidylpoly(ethylene glycol) glutarate, to form amide (-CONH-) linkages.

The degree of cross-linking may be expressed as the number of functional groups per (initial) molecule of matrix forming material which are involved in cross-linking. For example, for collagen, the number of available lysines involved in cross-linking may vary from a single residue to 100% of the lysines, preferably 10%-50%, and more preferably 20%-30%. The number of reactive lysine residues may be determined by standard methods, for example by reaction with TNBS (2,3,4-trinitrobenzensulfonic acid). The term "nominally non-cytotoxic" as used herein relates to cross-linking agents which, when added to a cell's environment (i.e. added to a cell suspension) at concentrations useful for effecting cross-linking, do not substantially reduce cell viability, for example, does not reduce cell viability by more than 50% over 7 days, and are not physiologically detrimental.

The term "cells" as used herein relates to living cells, preferably mammalian cells, including, for example, human cells. Cells may be autogeneic, isogeneic, allogeneic, or xenogeneic, more preferably autogeneic or allogeneic. Included are cells which have been genetically engineered. Cell-gels may contain different cell types, which may be chosen to act synergistically, for example, in the formation of tissue. Examples of types of cells 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, blood cells, brain cells, kidney cells, liver cells, lung cells, pancreatic cells, spleen cells, breast cells, foreskin cells, ovary cells, testis cells, and prostate cells. Other mammalian cells are useful in the practice of the invention and are not excluded from consideration here. Alternatively, cell- gels may be prepared using non-mammalian eucaryotic cells, procaryotic cells, or viruses.

The cell-gel compositions and formulations of the present invention further have the properties that they are (i) nominally non-immunogenic and (ii) bioerodable. The term "nominally non-immunogenic" as used herein relates to materials which provoke no substantial immune response, inflammation, or foreign body reaction when administered. The term "bioerodable" as used herein relates to the potential for a material to be eroded or degraded by the action of enzymes (including, for example, proteinases such as collagenase) , or other biological processes, to yield non-toxic substances or byproducts which are compatible with normal bodily processes. The degree to which a material is bioerodable may be indicated by its bioerosion period.

The term "bioerosion period" as used herein relates to the period of time after which substantial bioerosion of the cross-linked matrix material has occurred. The bioerosion period may vary for the particular indication, and will reflect needs to initially locate, support, and house the entrapped cells, to permit the growth and proliferation of those cells, and to ultimately permit the complete or nearly complete erosion of the original cross-linked matrix material. Examples of bioerosion periods for cell-gels include 20-45 days, for dermal- associated cell-gels, 30-90 days for bone-associated cell- gels, 10-30 days for nerve-associated cell gels. The term "administer" as used herein is generic to methods of applying, attaching, implanting, injecting, and the like. If the cell-gel material is a suspension, injection is the preferred method for administration.

B. Preparation and Use of Cell-Gels

The cell-gel compositions and formulations of the invention may be prepared by a one-step method comprising contemporaneous mixing of matrix forming material, cross- linking agent, and cell suspension.

Alternatively, cell-gel compositions and formulations may be prepared by a number of two-step methods, such as premixing of cell suspension and matrix forming material, followed addition of cross-linking agent; premixing of cell suspension and cross-linking agent, followed by addition of matrix forming material; and premixing of matrix forming material and cross-linking agent, followed by addition of a cell suspension.

The physical properties of the resulting cell-gel compositions or formulations, such as viscosity, consistency, and texture, may be adjusted by varying reactant concentrations, reaction conditions, reaction time, or 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 cell-gels may be increased by increasing the concentration of matrix forming material in the reaction mixture. For example, for the collagen-SPEG cell-gels, preferred collagen concentrations are 5-100 mg/mL, more preferably about 10-75 mg/mL, most preferably 30-60 mg/mL.

The degree of cross-linking present in cell-gels may be varied according to the molar ratio of matrix forming material to cross-linking agent. Increasing concentrations of cross-linking agent relative to matrix forming material concentrations yields cell-gels with higher viscosities which may be characterized as gel-like, plastic, semi- solid, or solid. Conversely, decreasing the relative concentration of cross-linking agent yields cell-gels with lower viscosities which may be characterized as fluid or liquid. A continuum of viscosities, textures, and consistencies may be obtained. For example, for collagen- SPEG cell-gels, collagen:SPEG molar ratios of about 200:1 to about 5:1 yield cell-gels which are fluid and injectable, whereas ratios of about 5:1 to about 1:10 yield semi-solid cell-gels, and larger ratios of about 1:10 to about 1:75 yield more rigid, heavily cross-linked cell- gels. Ratios of about 1:50 typically lead to cell-gels wherein all available lysines of collagen are involved in cross-linking.

The degree of cross-linking present in cell-gels may further be controlled by adjusting reaction temperature and reaction time. Increased reaction temperature (up to, but not greatly exceeding 37°C) will increase the rate of formation of cross-links. Conversely, reducing the reaction temperature will decrease the rate of formation of cross-links. For example, reaction of collagen and SPEG to form cross-links is rapid at room temperature, but substantially slower at 5°C. During reaction, the degree of cross-linking increases with increasing reaction times; by adjusting other conditions, such as concentrations and temperature, suitable reaction rates (and therefore reaction times) may be obtained.

Other factors, such as pH, may be adjusted to vary cell-gel properties. For example, for cell-gels obtained using collagen in solution and activated PEG, a wide range of fibrillar collagen content may be obtained by varying the pH of the reaction mixture. Furthermore, a particulate microgel material may be obtained by agitating a cell-gel reaction mixture comprising, for example, collagen in solution and activated PEG, during cross-linking (e.g., by stirring or passing between syringes) . The salt concentration of the reaction mixture may also be adjusted to control cell-gel properties.

Reactants, such as the matrix forming material, the cross-linking agent, and the cell suspension, may be suspended in a pharmaceutically acceptable carrier prior to mixing. For example, since SPEG is subject to hydrolysis, it is typically stored desiccated at -20°C prior to use, and prepared as an aqueous mixture immediately prior to use. Alternatively, SPEG may be prepared as a non-aqueous suspension (using, for example, glycerol, PEG, triglycerides, DMSO, and the like) or as a suspension with reduced water concentrations. Such non-aqueous or reduced- aqueous suspensions may be used to further control reaction rates and times. Similarly, non-aqueous or reduced-aqueous cell suspensions may be prepared to control cell-gel properties.

The term "cell suspension" as used herein relates to living cells suspended in a liquid, preferably aqueous, medium. Examples of appropriate liquids include, for example, physiologically buffered salt solutions and cell culture media, and may include such components as glycerol, DMSO, triglycerides, and the like, and may further contain media supplements known in the art, including for example, serum, growth factors, hormones, sugars, amino acids, vitamins, etalloproteins, lipoproteins, and the like. Methods for the preparation of cell suspensions by dissociation of an aggregate of living cells are well established and known to those of skill in the art (see, for example, R.I. Freshney, Culture of Animal Cells - A Manual of Basic Technioues. 2nd. Edition, Alan R. Liss, Inc., New York). For example, a common method involves treatment of a cellular aggregate with EDTA, or an enzyme, such as trypsin, collagenase, and the like, which causes cells to become detached from other cells or from solid surfaces.

Cell suspension concentrations may be chosen to optimize cell-gel texture and viscosity, the rate of subsequent colonization of the gel, and/or viability of cells within the gel. At present, cell suspension concentrations which result in reaction mixture concentrations of about lxlO⁴ to lxlO⁶ cells/mL are preferred, and concentrations of about 1x10^s cell/mL are more preferred.

Cell-gels of the invention may additionally include biologically active factors to aid in healing or regrowth of normal tissue. For example, one may incorporate factors such as heparin , epidermal growth factor (EGF) , transforming growth factor (TGF) alpha, TGF-/3 (including any combination of TGF-βs) , TGF-01, TGF-/32, platelet derived growth factor (PDGF-AA, PDGF-AB, PDGF-BB) , acidic fibroblast growth factor (FGF) , basic FGF, connective tissue activating peptides (CTAP) , β-thromboglobulin, insulin-like growth factors, tumor necrosis factors (TNF) , interleukins, colony stimulating factors (CSFs) , erythro- poietin (EPO) , nerve growth factor (NGF) , interferons (IFN) , osteogenic factors, and the like. Incorporation of such factors, and appropriate combinations of factors, can facilitate the transformation of the cell-gels, or may be used in the treatment of wounds.

Cell-gels of the invention which contain growth fac¬ tors are particularly suited for sustained administration of factors, as in the case of wound healing promotion.

Osteoinductive factors and cofactors (including TGF-β) may advantageously be incorporated into compositions destined for bone replacement, augmentation, and/or defect repair. Cell-gels of the invention containing biological growth factors such as EGF and TGF-/3 are prepared by mixing an appropriate amount of the factor into the composition, or by incorporating the factor into the matrix forming material prior to treatment with the cross-linking agent. Cell-gels of the invention containing biological growth factors such as EGF and TGF-/3 are prepared by mixing an appropriate amount of the factor into the composition. One may chemically link the factors to the matrix forming material or to the cross-linked matrix material, for example, by employing a suitable amount of cross-linking agent during preparation of the cell-gel. For example, the factors may be covalently attached to the matrix forming material in the same manner that the matrix forming material is cross-linked. By tethering factor molecules to the cross-linked matrix material, the effective amount of factor is substantially reduced. The term "effective amount" refers to the amount of composition required in order to obtain the effect desired. Thus, a "tissue growth promoting amount" of a composition containing a growth factor refers to the amount of factor needed in order to stimulate tissue growth to a detectable degree. Tissue, in this context, includes connective tissue, bone, cartilage, epidermis and dermis, blood, and other tissues. Furthermore, factors covalently tethered to the matrix forming material serve as effective controlled-release drug delivery matrices.

For example,, factors may be chemically linked to collagen using activated PEG: the factor is first reacted with a molar excess of activated PEG in a dilute solution over a period of about 5 min to about 1 hour. The factor is preferably provided at a concentration of about l μg/mL to about 5 mg/mL, while the activated PEG is preferably added to a final concentration providing a 30 to 80-fold molar excess. The resulting conjugated factor is then added to an aqueous collagen mixture (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 ambient temperature. The pellet is collected by centrifugation, and is washed with PBS by vigorous vortexing in order to remove non-bound factor. The resulting collagen-factor material is then used at the matrix forming material in the preparation of a cell-gel.

One may additionally include particulate materials in the cell-gel, for example hydrogel or collagen-dPEG beads, hydroxyapatite/tricalcium phosphate particles, polylactic acid/polyglycolic acid (PLA/PGA) particulates, or teflon beads, to provide a bulkier or more rigid cell-gel after cross-linking.

Formulations suitable for repair of bone defects or nonunions may be prepared by providing cell-gels with high concentration of matrix forming material, high concentrations of cross-linking agent, or by admixture with suitable particulate materials. The term "suitable particulate material" as used herein refers to a particulate material which is substantially insoluble in water, which is biocompatible, and which is immiscible with matrix forming material or cross-linked matrix material. The particles of particulate material may be fibrillar, or may range in size from about 1 to 500 μm in diameter and be bead-like or irregular in shape. For example, for injectable cell-gels, such as those useful for soft tissue augmentation, preferred particles sizes are less than about 150 μm. For cell^gels useful for bone-related augmentation, preferred particles sizes are greater than about 100 μm. Exemplary particulate materials include without limitation fibrillar cross-linked collagen, gelatin beads, cross-linked collagen-PEG particles, poly¬ tetrafluoroethylene beads, silicone rubber beads, hydrogel beads, silicon carbide beads, glass beads, carbon fibers, PLA/PGA fibers, and polyethylene terephthalate (PET) fibers. Presently-preferred particulate materials are hydroxyapatite and tricalcium phosphate.

Malleable, plastic cell-gel compositions which are injectable may be prepared by adjusting 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 cell-gels of the invention. A sufficient amount is that amount which when mixed with the cell-gel renders it in the physical form desired, for example, injectable solution, injectable suspension, plastic or malleable implant, rigid implant, and so forth. The term "injectable" as used herein relates to materials having a texture and viscosity which permits their flow through a suitable surgical needle by employing typical injection pressures. For example, an injectable material may be forced through a 32-gauge needle under normal pressure. The mixture is injected directly into the site in need of augmentation, such as tendon or cartilage and causes essentially no detectable inflammation or foreign body reaction.

Injectable cell-gel formulations are useful for dermal augmentation, for example for filling in dermal creases, and providing support for skin surfaces, sphincter augmentation, (e.g., for restoration of continence), tissue revascularization, depot cell delivery, tumor blood vessel blockage, therapy, and contraception/infertility treatments.

Alternatively, one may administer the cell-gels by injection before cross-linking has completed. For example, an aqueous mixture matrix forming material and cell suspension is combined with a low-concentration solution containing the cross-linking agent, mixed, and the reaction mixture injected or applied before the viscosity increases sufficiently to render injection difficult (usually about 10 minutes) . Mixing may be accomplished by passing the mixture between two syringes equipped with Luer lock hubs, or through a single syringe having dual compartments (e.g., double barrel) . The reaction mixture reacts to form cross- -links in situ (that is, at the site in need of augmentation) , and may additionally cross-link to the endogenous tissue, anchoring the cell-gel in place. Similarly, one may inject a mixture comprising cells and matrix forming material at the site in need of augmentation, and subsequently inject a mixture comprising a cross-linking agent at the same site. Alternatively, one may inject a mixture comprising cells and a cross-linking agent at the site in need of augmentation, and subsequently inject a mixture comprising a matrix forming material.

If desired, denser more viscous formulations may be cast or molded into any desired shape, for example into sheets or membranes, into meshes, into tubes or cylinders, into hooks, cords or ropes, and the like.

Flexible sheets or membranous forms of cell-gels may be prepared by methods analogous to those known in the art for the preparation of gel membranes (see, for example, U.S. Patent Nos. 4,600,533; 4,412,947; and 4,242,291). For example, a mixture of a high concentration (10-100 mg/mL) CIS or fibrillar collagen (preferably atelopeptide fibrillar collagen, such as ZCI) , activated PEG (having a molecular weight of approximately 3,400), and cell suspension is cast into a flat sheet container, and allowed to react for 2-3 hours at 37°C. The resulting collagen- cell-gel is removed from the excess reaction solution using a sterile spatula or the like, and may be washed with PBS to remove excess unreacted cross-linking agent. More flexible membranous forms are achieved by using lower collagen concentrations and higher cross-linking agent concentrations as starting materials.

Similarly, sheaths or tubular forms of cell-gel compositions may be prepared which are useful for replacing or augmenting vascular structures, such as blood vessels, or as a nerve tissue sheath.

Compositions of the invention may be prepared in a form that is dense and rigid enough to substitute for car- tilage or non-weight bearing bones, for example, finger bones. These compositions are useful for repairing and supporting tissue which require some degree of structure, for example in reconstruction of the nose, ear, knee, larynx, tracheal rings, and joint surfaces. One can also replace tendon, ligament and blood vessel tissue using appropriately formed cartilaginoid material. In these applications, the cell-gel is generally cast or molded into a shape; in the case of tendons and ligaments, it may be preferable to form filaments for weaving into cords or ropes.

All publications, patents, and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were 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 purely exemplary in nature, and are not meant to be utilized to limit the scope of the invention.

Example 1

Preparation of collagen-SPEG-cell gels

Human dermal fibroblast cells at the 5th passage were used. The human dermal fibroblast cells were subcultured from those on deposit with the American Type Culture Collection, 12301 Parklawn Drive, Rockville, MD 20852, USA, under ATCC Accession Number CRL 1885. On Day 0, cells were trypsinized using 25 mg/mL trypsin in EDTA (2 mM) and the cells were pelleted by centrifuging at 150-200 g for 10 min at room temperature. The supernatant was discarded, and the pellet resuspended in 5 mL of DME media (Dulbecco's Modified Eagle's Medium with 4.00 mM L-glutamine, 1000 mg/L glucose, 100 mg/L sodium pyruvate) . The cell concentration was determined using a hemocytometer. The cell suspension was diluted with DME media to a concentration of lxlO⁵ cells/mL.

Controls were prepared using aliquots of 1 μL, 10 μL, and 100 μL of stock cell suspension solution 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 media in the culture studies. (Indices such as 1A3 indicate plate 1, row A, column 3) . A pool of Zyderm I collagen (Collagen Corporation, Palo Alto, CA) was prepared by combining a total of 12 L from different Zyderm I samples and mixing through a sterile bridge (such as a stopcock) 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 1.17X10-* mol/L.

A stock solution 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 saline), to give a concentration of 4.5 mg/mL or -1.32X10-³ mol/L.

Zyderm I controls (1B4, 1B5, 1B6) were prepared using 0.5 mL aliquots of Zyderm I pool.

Zyderm I/cell cultures were prepared by mixing 1.5 mL of Zyderm I with 30 μL of stock cell suspension. Aliquots of 0.5 mL were placed in each well (1B1, 1B2, 1B3) , to give 1000 cells/well.

A collagen/SPEG (10:1) cell-gel was prepared by mixing 2 mL of Zyderm I pool, 10 μL of activated PEG solution, and 40 μL of stock cell suspension. Aliquots of 0.5 mL were placed in each well (1C1, 1C2, 1C3) to give 1000 cells/well. Collagen/SPEG (10:1) controls were prepared by mixing 2 mL of Zyderm I pool with 10 μL SPEG stock solution. Aliquots of 0.5 mL were placed in each well (1C4, 1C5, 1C6) .

A collagen/SPEG (100:1) cell-gel was prepared by mixing 2 mL of collagen stock solution, 1 μL of activated PEG solution, and 40 μL of stock cell suspension. Aliquots of 0.5 mL were placed in each well (1D1, 1D2, 1D3) to give 1000 cells/well.

Collagen/SPEG (100:1) controls were prepared by mixing 2 mL of collagen stock solution with 10 μL SPEG stock solution. Aliquots of 0.5 mL were placed in each well (1D4, 1D5, 1D6). Culture plates were centrifuged at 4°C at 150-200 g for

2 min. Feeding, by the addition of 1 mL of DME-complete (DME media further comprising 10% Fetal Bovine Serum and 1% penicillin or streptomycin) to each well, was performed on Day 0, Day 1, Day 3, and Day 5. On Day 3 and Day 7, an Alamar Blue Assay was performed: 100 μL of Alamar Blue per 1 mL of media was added to each well and absorbance at 590 nm subsequently recorded using a Cambridge Fluorometer EX 550 EM 590, both

3 and 6 hours after incubation. After the 6 hour measurements were recorded, the media was removed and 1 mL of fresh media added. The data are summarized in Table 1. The analyzed data are presented in Table 2. "Normalized" intensity presented for cell-containing samples reflect the difference between the average absorbance recorded for the cell-containing sample less the average absorbance recorded for analogous samples lacking cells. Normalized cell proliferation data are shown graphically in Figure l. The proliferation data demonstrate that cell viability is not adversely affected by growth within the cross-linked collagen-SPEG cell-gel.

After the final Alamar Blue Assay on Day 7, the cell culture controls (1A1 though 1A6, 2A1 through 2A6) were trypsinized and counted using a hemocytometer. The lθ² cells/well control had too few cells to count. The 10³ cells/well control had an average count of 9.1X10³ cells/well. The 10⁴ cells/well had an average count of 3.9x10" cells/well.

After performing the Alamar Blue Assays on Day 7, all wells containing collagen were fixed with 10% buffered formalin overnight. Preliminary histology results indicated that cells were present throughout the collagen/cell and collagen-SPEG cell-gel materials, and were not adversely affected by the cross-linked collagen matrix.

Table 1 Alamar Blue Assay Results

Sample Day 3 Day 7

Well Contents Well Init 3 6 3 6 Cell hours hours hours hours Cone

Cells 1A1 10² 1706 1972 2404 3349 1A2 1896 2046 2387 3148 2A1 1675 1644 1679 1936 2A2 1918 2000 2193 2529

Cells 1A3 10³ 3235 6698 9571 16942 1A4 3589 6222 7030 14893 2A3 4197 8549 11167 18070 2A4 6901 13060 12216 19905

Cells 1A5 10⁴ 8184 16366 13707 22437 1A6 9976 17739 12675 22232 2A5 9014 18028 15415 22232 2A6 8529 15701 13930 21378

Zyderm 1B4 — 2162 2108 2371 2786 1B5 2177 2238 2489 2805 1B6 2208 2291 2576 2760

Zyderm & 1B1 10³ 2432 3191 5152 6966 Cells 1B2 2754 3990 6441 9268 1B3 3311 5284 7294 11323

Zyderm & SPEG 1C4 — 2113 2138 2432 2624 (10:1) 1C5 1870 1941 2148 2698 1C6 2118 2162 2306 2698

Zyderm & SPEG 1C1 10³ 2642 3396 6025 7311 (10:1) 1C2 2844 4591 6485 10092 & Cells 1C3 3118 4988 6396 10714

Zyderm & SPEG 1D4 — 2000 2075 2410 2523 (100:1) 1D5 2203 2123 2270 2917 1D6 2233 2244 2377 2851

Zyderm & SPEG 1D1 10³ 2884 5688 7227 12473 (100:1) 1D2 2857 5675 7481 12416 & Cells 1D3 2884 4385 6151 9505 Table 2 Analyzed Alamar Blue Assay Results

Claims

Clai s

1. A cell-gel composition comprising a plurality of cells contained within a matrix material cross-linked with a synthetic polymeric cross-linking agent.

2. The composition of claim 1 wherein the composition is injectable.

3. The composition of claim 1 wherein the matrix material is collagen.

4. The composition of claim 1 wherein the cross-linking agent is a polyfunctional polyethylene glycol.

5. The composition of claim 1 wherein the cross-linking agent is succinimidyl poly(ethylene glycol) glutarate.

6. The composition of claim 4 wherein the average molecular weight of the polyfunctional polyethylene glycol is about 400 to about 20,000.

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

8. The composition of claim 3 wherein the cross-linking agent is bound to about 10% to about 50% of the available lysine residue of the collagen.

9. The composition of claim 2 wherein the matrix material is collagen, the cross-linking agent is a polyfunctional polyethylene glycol having an average molecular weight of about 400 to about 20,000 and the molar ratio of collagen to polyfunctional polyethylene glycol is in the range of about 1:1 to about 1:20.

10. A method of making a cell-gel composition comprising:

(a) providing a mixture of cells, a matrix forming material, and a synthetic polymeric 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 augmenting tissue at a site within a living mammal comprising placing the cell-gel composition of claim 1 at said site.