KR20070019638A - Biodegradable polymer-ligand conjugates and their uses in isolation of cellular subpopulations and in cryopreservation, culture and transplantation of cells - Google Patents

Abstract

The present invention relates to a biodegradable particle-cell composition having at least one biodegradable particle, at least one receptor covalently bound to the particle, and cells immobilized on the receptor. The particles are polylactide, polylactide-lysine copolymer, polylactide-lysine-polyethylene glycol copolymer, starch, or collagen. The receptor may be an antibody, fragment of an antibody, avidin, streptavidin, or biotin moiety. In addition, the particles may have an extracellular matrix other than collagen. The particle-cell composition can be used for selection of cells from a population, cell culture of fixed-dependent cells, cryopreservation of fixed-dependent cells, and transplantation as cell therapy.

none

Description

Biodegradable POLYMER-LIGAND CONJUGATES AND THEIR USES IN ISOLATION OF CELLULAR SUBPOPULATIONS AND IN CRYOPRESERVATION, CULTURE AND TRANSPLANTATION OF CELLS

The present invention relates generally to medical devices used in vivo and in vitro for the production and transportation of medically useful materials. More specifically, the present invention relates to biodegradable natural or synthetic resin compositions conjugated with reactive ligands. The present invention also relates to methods of using the compositions for specific enrichment of cells, cell cryopreservation, ex vivo maintenance of cells, and cell therapy.

Eukaryotic cells in isolated cell culture are characteristically of two types. One type is capable of survival and proliferation in suspension culture. Among the cells, cells derived from cancer and lymphoma and cells transformed with chemical or viral agents are suitable for this type of survival. On the other hand, the second type of cell needs to be anchored to the base for survival and proliferation of the cell. Among the cells belonging to the latter category are adherent cells, such as cells derived from solid tissue and not transformed, adherent cell types, such as cells derived from liver, lung, brain, and the like, and especially progenitor cell populations from solid tissue. Often, such cells require attachment to extracellular matrix components and maintenance of serum free hormonal defined medium for growth and / or survival. The matrix component (s) can be a protein such as collagen or laminin or a proteoglycan such as heparan sulfate proteoglycan. The composition of the hormonal confinement medium depends on each cell type and the stage of maturation or lineage of the cell type; Thus, the progenitor cells of a given lineage have requirements that overlap with the mature cells of the lineage, but also have some other needs. Even in cases where definition is not easy, these in vitro requirements for various adherent cell types could be defined; That is, they can be established in conventional cell cultures but have not been readily used in bioreactors that can be used for medical treatment, mass cell culture, or medical or industrial use. In addition, a working environment for the preservation of adherent cell types such as cryopreservation is impractical when cells need to be regenerated after thawing or used in a variety of ways. Thus, adherent cells require unique methods for long-term preservation of cells, separation of one cell type from other types of cells, and handling of the cells in predetermined medical uses.

Biodegradable polymers have been used in tissue engineering. Among the biocompatible and biodegradable polymers used in tissue engineering, the most widely studied, are poly- (alpha-hydroxy acid) based polymers and related copolymers. Some of these polymers have been licensed from F.D.A. for medical use. Therefore, they are used as the starting polymer material which is the easiest to implement in the present invention. However, cell adhesion to the polymer is problematic.

The compositions and methods disclosed herein relate to issues associated with anchorage-dependent cells, thereby satisfying the need for sorting, cell preservation, cell proliferation, and medical use of cells.

The present invention provides a biodegradable polymer particle-cell composition comprising at least one biodegradable particle, at least one receptor covalently bonded to the particle, and cells anchored to the at least one receptor. Receptors can be any suitable group, including but not limited to antibodies, antibody fragments, avidin, streptavidin, or biotin moiety, carbohydrates, synthetic ligands, protein A, protein G, or combinations thereof. . Furthermore, the receptor may itself be a ligand capable of ligand-receptor interaction.

In another aspect, the present invention provides a cryopreservation method for fixed-dependent cells comprising immobilizing the cells in a composition comprising at least one biodegradable particle and freezing the mixture in the presence of a suitable cryopreservative. The cells may be provided for interaction with the particles substantially as in a single cell suspension.

In another aspect, the present invention provides at least one biodegradable polymer, at least one receptor covalently bound to the polymer, at least one cell anchored to the at least one receptor, and at least one anchored to the at least one receptor It provides a composition comprising cells of, and provides a cell separation method comprising removing at least one cell that is not immobilized on the polymer. The polymer can also be typed into macroparticles, microparticles or nano-particles with functional receptor groups.

In another aspect, the present invention provides a composition having at least one biodegradable polymer, at least one covalently linked receptor, and at least one cell attached to the at least one receptor; Provided is a cell culture method of fixed-dependent cells comprising contacting the composition with a cell culture medium.

In another embodiment, the present invention provides a composition having at least one biodegradable polymer, at least one covalently linked receptor, and at least one cell attached to the at least one receptor; Contacting said composition with cell culture medium, said cells comprising at least one hepatic precursor, mesenchymal cell, mesenchymal precursor, muscle cell, which are heart cells, neuronal cells, glial cells, fibroblasts, stem cells, epithelium A method of cell culture of fixed-dependent cells comprising cells, endothelial cells, or combinations thereof is provided.

In another aspect, the invention relates to administering to a subject an effective amount of a composition comprising at least one biodegradable polymer, at least one receptor covalently bound to the polymer, and at least one cell immobilized on the at least one receptor A method of treating a subject in need of cell therapy is provided. Polymers for cell therapy can be typed into macroparticles, microparticles or nanoparticles.

The present invention relates to a composition having a biodegradable polymer covalently bound to a receptor or ligand. The present invention further relates to the above compositions which are further combined with the cells. The cell may be immobilized to a receptor ligand or receptor. The receptor ligand or receptor may be an antibody against a cell surface antigen or receptor, an avidin, streptavidin, or a biotin moiety, or an antibody fragment. The composition may further comprise one or more extracellular matrix components such as collagen, fibronectin, laminin, or a combination thereof. The invention also relates to methods of using the compositions for selection and isolation of cell populations, cryopreservation of cell particle combinations, and cell culture of fixed-dependent cells.

Justice

Serum-free, hormonal confinement medium for diploid cells (HDM-drainage cells). This medium has been found to induce colony expansion, colony formation, or complete cell separation of the diploid subpopulations of liver tissue cells. This medium does not contain copper, contains small amounts of calcium (<0.5 mM), insulin (1-5 μg / ml), transferrin / Fe (1-10 μg / ml), and lipid mixtures (high purity fatty acid-free Free fatty acid mixture bound to albumin; optional but useful additive may consist of any nutritive basal medium (eg, RPMI 1640, HAM's F12) further supplemented with 10 μg / ml of high density lipoprotein. Details regarding fatty acid production are appended here as in Appendix A.

Embryonic stromal feeders as defined herein are mesenchymal stromal feeder cells derived from embryonic tissue. Ideal for liver cells are stromal cells derived from embryonic liver; Although there is some vague evidence, there is some evidence of tissue specificity. We have defined age limitations for non-human rats (for example, embryo stroma is ideally obtained from embryonic rat livers of E13-E17 in the conception period). For humans, we can only speculate on the corresponding duration of conception, such as the human embryonic liver of 12-18 weeks of conception. There is no data in the laboratory to confirm that conjecture. However, most importantly, these feeder cells are age-specific and the most active form is obtained from embryonic tissue. Embryonic stromal cell lines, “STO” cells, obtained from mouse embryos and commonly used for the preservation of embryonic stem cells (ES cells) can be used. STO cells do not give much the same effect as embryonic liver strom, but the researchers It is sufficient to use them to eliminate the need for primary culture preparation of embryonic tissue.

Colony forming expansion as defined herein refers to cells that can be repeatedly passaged and expanded at very low seeding densities (approximately 1 cell / dish).

Colony formation includes cell colony formation from inoculated cells but includes a limited number of divisions (typically 5-7 cell divisions) in a relatively short period (1-2 weeks). The cells cannot be passaged easily. Unlike colony expansion, colony formation can include differentiation steps that prevent infinite cell division and passaging.

Primitive liver stem cells as defined herein have the ability to colonize expansion and co-expansion of cytokeratin (CK 19) and albumin (ie bile and liver cell markers respectively) but without alpha-fetoprotein expression. It is a cell. In human liver lineages obtained from fetal livers, these cells also co-express N-CAM, epithelial CAM (EP-CAM) and CD 133 and expand colony forming in tissue culture plastic and HDM-ploploid cells.

Proximal liver stem cells (or hepatoblasts) as defined herein are pluripotent cells with colony-forming expansion capacity and coexpression of cytokeratin 19 (CK 19), albumin, and alpha-fetoprotein. to be. In human liver lineages obtained from fetal livers, these cells may co-express I-CAM, epithelial CAM (Ep-CAM) and CD 133 and in embryonic stromal feeders (eg STO cells) and HDM-doubled cells Colony formation will swell.

The committed precursor cells as defined herein are unipotent precursor cells capable of growing into liver cells (committed liver precursor cells) or bile epithelial cells (committed bile precursor cells). These cells will form colonies in embryonic stromal feeder and HDM-ploploid cells. It is still unclear whether they can expand colony formation under these or other conditions.

Biploid adult liver cells (called “small liver cells”) as defined herein range in size from 15-20 μm and express a variety of adult specific functions (eg, PEPCK, glycogen), EP-CAM, Diploid liver cells that form colonies under various conditions without expressing CD 133 or N-CAM but are ideal if embryonic stromal feeder and HDM diploid cells are supplemented with 10-50 ng / ml epidermal growth factor (EGF) .

Drainage liver cells as defined herein are diploid liver cells (which can reach tetraploid or 4N-32N in mammalian species). These are mature cells of the liver and have been found to undergo cytoplasmic division under DNA synthesis and limited but regenerative conditions.

Progenitor cells as defined herein is a broad term encompassing all subpopulations of stem cells and committed progenitor cells.

Precursor as defined herein is a functional term indicating that a particular subset of cells is a precursor to another subset of cells. For example, primordial liver stem cells are precursors of hepatoblast; Hepatoblast is a precursor of a committed precursor; Diploid adult liver cells are precursors of diploid liver cells.

As used herein, the term “frozen preservation” relates to freezing cells and / or tissues in an environment that maintains the viability of the cells for continuous thawing. General techniques for cryopreservation of cells are well known in the art: see, for example, Doyle et al., (1995), Cell & Tissue Culture: Laboratory Procedures, Jhon Wiley & Sons, Chichester; and Ho and Wang (eds.), 1991, Animal Cell Bioreactors, Cutteworth-Heinemann, Boston, which are incorporated herein by reference.

The biodegradable polymer-ligand conjugates of the present invention are terms for cell-receptive particles or more simply particles. These terms, as used in all embodiments of the biodegradable polymer-ligand conjugates, include, but are not limited to, direct antibody conjugates, conjugates to fragments of antibodies, avidin conjugates, biotin conjugates, fibronectin conjugates, biodegradable particles, and PEG linkers. Antibody conjugates with long spacer linkers such as and anti-antibody conjugates.

6.1. Polymer  Produce

Several types of biocompatible and biodegradable polymers include, but are not limited to, polyacted, polyacted-lysine copolymers, polyacted-lysine-polyethylene glycol copolymers, starches, alginates and proteins. Suitable for use in the invention. Suitable proteins are collagen, gelatin, poly-lysine, laminin, fibronectin, or combinations thereof. One embodiment of the invention uses poly- (alpha-hydroxy acid) -lysine copolymers, and / or poly (lactide-co-glycolide, PLGA) copolymers. PLGA can be activated by a coupling agent, such as but not limited to glutaraldehyde, before coupling with amino, including ligands or proteins (Seifert, Romaniuk and Groth, 1997 Biomaterials 18: 1495-1502). In addition, the biodegradable PLGA polymers also contain protein A or other proteins by bifunctional linkers such as (3 [(2-aminoethyl) dithiol] propionic acid, AEDP), which are amino groups or commercial linkers of protein G. May be coupled with the receptor. In the present invention, polymers and copolymers of the poly- (alpha-hydroxy acid) series are used to prepare biocompatible and biodegradable beads without surface reactors, providing a core structure of degradable polymer particles.

As used herein, a polymer or polymer matrix is used if the polymer and any degradation products of the polymer are almost nontoxic to the recipient and there are no noticeable detrimental effects on the subject's body, such as a noticeable immunological response at the site of injection. "Biocompatibility".

As used herein, “biodegradable” means that the composition degrades in vivo to form smaller chemical species. Degradation can occur by, for example, enzymatic, chemical and / or physical processes. Suitable biocompatible, biodegradable polymers are, for example, poly (lactide), poly (glycolide), poly (lactide-co-glycolide), poly (lactic acid), poly (glycolic acid), poly (lactic acid) Co-glycolic acid), polycaprolactone, polycarbonate, poly (amino acid), polyorthoesters, polyetheresters, copolymers of polyethylene glycol and polyorthoesters, mixtures and copolymers thereof, including but not limited to Do not.

For example, biocompatible, non-biodegradable polymers suitable for use in the present invention include polyacrylates, polymers of ethylene-vinyl acetate and other acyl substituted cellulose acetates, non-degradable polyurethanes, polystyrene, polyvinyl chloride, polyvinyl Non-biodegradable polymers selected from the group consisting of fluoride, poly (vinyl imidazole), chlorosulfonate polyolefins, polyethylene oxides, and combinations and copolymers thereof.

The terminal functionality of the polymer can also be modified. For example, the polyester can be a barrier, non-blocking or a mixture of barrier and non-blocking polyester. Blocked polyesters are as defined typically in the art, especially with blocked carboxy end groups. In general, the blocking group is derived from the initiator of the polymerization and is typically an alkyl group. Non-blocking polyesters are as defined typically in the art, especially with carboxyl end groups.

Acceptable molecular weights for the polymers used in the present invention can be determined by those skilled in the art in view of factors such as the desired rate of polymer degradation, physical properties such as mechanical forces, and rate of degradation of the polymer in solvent. Typically, the acceptable range of molecular weight is from about 2,000 Daltons to about 2,000,000 Daltons. In a preferred embodiment, the polymer is a biodegradable polymer or copolymer. In a more preferred embodiment, the polymer is a poly (lactide-co-glycolide) (hereinafter, “1” having a molecular weight of about 5,000 Daltons to 70,000 Daltons, although the ratio of lactide to glycolide is not limited thereto, “ PLGA ") or a derivative. More preferably, the molecular weight of PLGA used in the present invention is 5,000 Daltons to 42,000 Daltons.

In one embodiment, copolymers comprising amino acids with reactive side chains such as lysine are copolymerized with lactic acid containing monomers, glycolic acid containing monomers, or other monomers having similar polymerization mechanisms. By way of example, the lactic acid containing monomer can be lactide and the glycolic acid containing monomer can be glycolide. The reaction region of amino acids is protected with standard protecting groups. Similarly, polymers having side groups to be protected can be deprotected to make reactive amino groups. The deprotected poly (lactic) acid-lysine copolymer is used to make poly (lactic) acid-lysine copolymers with the desired porous particles and then join the epsilon amino groups of lysine residues to form a tethered conjugate directly. Thereby covalently bound with the receiving formulation. In some embodiments the reactor contains a small group of ligand groups, including but not limited to antibodies, antibody fragments, collagen, laminin, fibronectin, avidin or streptavidin, or biotin and RGD containing peptides, Protein A or Protein G However, the protein may be, but is not limited to.

As used herein, antibodies contemplated for use in the present invention are polyclonal antibodies, monoclonal antibodies (mAbs), human-compatible or chimeric antibodies, single chain antibodies, Fab fragments, F (ab '). Sub. 2 fragments, fragments prepared by Fab expression libraries, anti-idiotypic (anti-Id) antibodies, and any epitope-binding fragments thereof, including but not limited to.

As used herein, small molecular ligand groups have a molecular weight of 10,000 Daltons or less, more preferably 5,000 Daltons or less. For example, combination techniques can be used to design libraries of combinations of small organic molecules or small peptides. Generally, for example, Kenan et al., Trends Biochem. Sc., 19: 57-64 (1994); Gallop et al., J. Med. Chem., 37: 1233-1251 (1994); Gordon et al., J. Med. Chem., 37: 1385-1401 (1994); Ecker et al., Biotechnology, 13: 351-360 (1995). Such combinatorial libraries of compounds can be used as receptors in the present invention. Random peptides include, for example, recombinantly expressed libraries (eg, face display libraries), or in vitro translation based libraries (eg, mRNA display libraries, such as Wilson et al., Proc Natl Acad Sci 98: 3750-3755 (2001).) Small molecular ligands also include carbohydrates and compounds such as those described in US Pat. No. 5,792,783, wherein the small molecular ligands have a molecular weight of about 1000 Daltons or less, and are vascular targets or vessels. Defined as an organic molecule that acts as a ligand for cell labeling), a peptide selected by the face-display technique described in US Pat. No. 5,403,484, a peptide designed de novo complementary to a tumor expression receptor; Or other receptor target groups.

As used herein, the term “RGD” refers not only to the peptide sequence Arg-Gly-Asp, but also generally refers to a hierarchy of microscopic or core peptide sequences that mediate specific interactions with integrins. Thus, "RDG target sequence" encompasses the entire class of integrin binding domains. Inducing molecules to the surface of the cell is known to promote molecular uptake through endocetic means. See, eg, Hart et al., J. Biol. Chem. 269: 12468-74 (1994) (internalisation of phage bearing RGD); Goldman et al, Gene Ther. 3: 811-18 (1996) (RGD-mediatedadenoviral infection) and Hart et al., Gene Ther. 4: 1225-30 (1997) (RGD-mediated transfection). Thus, in many cases the target domain will work well as an internalization domain. Many such target signals are known in the art. One kind of target signal that specifically binds to integrin (point of extracellular matrix attachment) bears a peptide signal sequence based on Arg-Gly-Asp (RGD). However, other classes include peptides having the center of Ile-Lys-Val-Ala-Val (IKVAV). Weeks et al. , Cell Inmunol. 153: 94-104 (1994).

1 refers to the hydrophilic properties of lysine bonds that allow for coupling reactions to proceed in aqueous media.

As shown in FIG. 2, to further expand the copolymer's ability in tethering proteins (including, for example, antibodies), polyethylene glycol ("PEG") bonds can be activated by sulfonyl chloride and analogues. And may be coupled to primary amine groups such as, but not limited to, lysyl residues or epsilon-amino groups of proteins, thus forming extended bonds with three-dimensional dispersion and structural properties. Commercially available linker structures of various lengths and linearities are suitable for the present invention, and various surface dispersions can be obtained. While not intending to be limiting, various linkers sold by Pierce Chemical Co. are suitable for use in the methods of the present invention. Alternatively, such linker structures can be synthesized using conventional organic synthetic chemistries available in the art. Surface dispersion of the receiving region is an important characteristic that affects the density and dispersion of cell-target receiving molecules on the surface of the new polymer. In some cases, the surface dispersion of the accepting cluster region to be applied should be sufficient to enable cellular contact that is important for cell growth and differentiation, migration and morphology. (Eg, Cima, L.G 1994, J. Cellular Biochemistry 56: 155-161). The surface dispersion of the receiving area can be determined routinely on a case-by-case basis using special methods available to those skilled in the art. Such features determine, but are not limited to, the binding of receptors labeled by radial or fluorescent materials targeted by ligands on the polymer surface (eg, Rolwey JA, Madlambayan, G., Mooney, DJ 1999, Biomaterials 20 45-53; Massia, SP, Hubbell, JA 1991, J. Cell Biology 114: 1089-1100), X-ray and neuroreflex analysis (e.g. Russell, TP 1990 Material Science Reports 5: 171-271), And binding assays of immunofluorescent labeled antibodies of surface receptors (eg, Massia, SP, Hubbell, JA 1991, J. Cell Biology 114: 1089-1100). As shown in FIG. 2, depending on the linker structure, the terminal copolymer can have straight or branched linkers with single or multiple reactive groups. The linkers are preferably hydrophilic and can be exposed to an aqueous medium, making the incoming coupling agent accessible.

6.2. Scaffold ( scaffold ) or Bead  new Polymer  Produce

Another important aspect of the present invention relates to the manufacture of biodegradable polymers from particles, beads, fibers, or scaffolds. Porous particles having a size of 1000 micrometers (microns) or less can be prepared by the process according to the invention. The present invention also discloses a method for modifying surface porosity, internal porosity of particles, degradability and dispersion of surface receptors. Polymer particles larger than about 500 microns in diameter, called macroparticles, are prepared by rapid freezing at low temperatures of polymer droplets with NaCl or similar crystal particles of defined size. The polymer particles include, but are not limited to, about 500 microns, about 550 microns, about 600 microns, about 650 microns, about 700 microns, about 750 microns, about 800 microns, about 850 microns, about 900 microns, about 950 microns, It may have a size of about 1000 microns, about 1050 microns, about 1,100 microns, or larger if necessary. This method forms a porous structure while leaching the crystals embedded by a solvent selected for dissolution of the non-polymer crystals.

To produce particles having a size of about 200 to about 500 microns, called microparticles, a polymer emulsion of the defined formula is dispersed in small droplets in an aqueous medium in the presence of a surfactant. Continued dispersion of the droplets allows extraction and evaporation of the solvent, leaving solidified polymer particles. Polymer microparticles may be of a size such as, but not limited to, about 200 microns, about 250 microns, about 300 microns, about 350 microns, about 400 microns, about 450 microns, about 500 microns, and the like. Small polymer particles, up to about 200 microns in diameter, called nanoparticles, are prepared by quickly dispersing the polymer solution into small droplets using ultrasonic shear forces typically delivered by ultrasonic nebulizers.

Small particles of polymer are solidified at low temperatures, and the solvent of the polymer is removed by a second or third solvent. Polymer microparticles have a size such as, but not limited to, about 25 microns, about 50 microns, about 75 microns, about 100 microns, about 125 microns, about 150 microns, about 175 microns, about 200 microns, and the like. Thus, the particles can be macroparticles, microparticles, nanoparticles or combinations thereof. The polymer may be formed from fibers including tubular fibers.

6.3. Polylactic acid (-Lee Sin Copolymer ) And other on Protein  Direct coupling

Interesting proteins can be conjugated to biodegradable polymer particles or scaffolds using crosslinking reagents. Preferred proteins are, but are not limited to, antibodies, avidin, streptavidin, extracellular matrix proteins, peptides comprising RGD sequences, protein A / G.

Antibodies and other proteins that target cell surface labels can be directly conjugated with epsilon amino groups of the lysyl residues of the copolymer present on the polymer bead surface, thereby forming a tethered antibody or other protein on the surface. Although not limited thereto, various coupling reagents such as, for example, commercial glutaraldehyde (eg, Pierce Chemical Co) can be used to couple antibodies or other proteins to biodegradable polymers. For example, antibodies or other proteins, and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride in the presence of the particles can be reacted with a buffer in the pH 4-6 range. Tethering may also occur in a two-step process, generally using 6- (4-azido-2-nitrophenylamino) hexanoic acid N-hydroxy succinimide ester. In this method, the particles initially react with the succinimide reagent in the dark at pH 6.5 to 8.5. Thereafter, the antibody or other protein is added and the coupling is initiated by 250-350 nanometer irradiation to produce reactive nirene. The nitrene is inserted into adjacent molecules containing the antibody. Unreacted reagent can then be removed by washing with an aqueous medium.

Many other reagents that crosslink primary amine groups are equally suitable for tethering antibodies or other proteins to biodegradable particles, and include the following reagents: S-acetylmercaptosuccinic anhydride; S-acetylthioglycolic acid N-hydroxy-succinimide ester; 4-azidobenzoic acid N-hydroxy succinimide ester; N- (5-azido-2-nitrobenzoyloxy) succinimide; Bromoacetic acid N-hydroxysuccinimide esters; Dimethyl 3,3'-dithio-bis (propionimate) dihydrochloride; Dimethyl pimelimate dihydrochloride; Dimethyl suverimidate dihydrochloride; 4,4 ', dithio-bis (phenyl azide); 3,3 ', dithio-bis (propionic acid) N- (hydroxysuccinimide ester); Ethylene glycol-bis (succinic acid N-hydroxy succinimide ester); 6- (iodoacetamido) caproic acid N-hydroxysuccinimide ester; Iodoacetic acid N-hydroxy succinimide ester; 3-maleimidobenzoic acid N-hydroxysuccinimide ester; Gamma-maleimidobutyric acid N-hydroxy succinimide ester; Epsilon maleimidocaproic acid N-hydroxysuccinimide ester; 4- (N-maleimidomethyl) cyclohexane-1-carboxylic acid N-hydroxy succinimide ester; 4- (N-maleimidomethyl) cyclohexane-1-carboxylic acid 3-sulfo-N-succinimide ester sodium salt; Beta maleimidopropionic acid N-hydroxysuccinimide ester; Bis (polyoxyethylenebis [imidazoyl carbonyl]); 3- (2-pyridyldithio) propionic acid N-hydroxysuccinimide ester; Suveric acid bis (N-hydroxy succinimide ester); And bis (sulfosuccinimidyl) suverate.

Coupling of the biodegradable particles with the antibody or other protein can occur at various crosslinker concentrations (about 10 ^-9 to about 10 ^-3 M). In one embodiment, about ^10-5 M concentration is used.

The antibody concentration may be between about 20 ng / ml and about 20 mg / ml. Other protein concentrations may be between about 5 mg / ml and about 50 mg / ml. In one embodiment, the antibody or other protein concentration against the coupling agent is about 2 mg / ml. The concentration of the particles is between about 10 ^-10 and about 10 ^-2 M lysine equivalents. In one embodiment, the particle concentration is about 10 ⁻³ M lysine equivalents.

Optimization of the surface distribution, the length of the tether, and the interaction between the antibody or other protein and the cell surface label is modified by those skilled in the art, for example by using polyethylene glycol (PEG) linkers for coupling of biodegradable polymers to the antibody. Can be. One such polyethylene glycol linker is one such as bis (poly-oxyethylene bis [imidazoyl carbonyl]) described above. The specificity of the tethered antibody preferentially determines the cell selectivity of the antibody-polymer conjugate. Antibody fragments comprising F _{ab ′} , for example F _ab or F _ab fragments, are suitable for tethering of this biodegradable polymer.

The monoclonal antibodies used in the present invention can be obtained by any technique that provides for the production of antibody molecules by sustained cell lines in culture. Such techniques include Kohler and Milstein's hybridoma technology (Nature, 256: 495-497,1975; and US Pat. No. 4,376,110), human B-cell hybridoma technology (Kosbor et al., Immunology Today, 4: 72, 1983; Cole et al., Proc. Natl. Acad. Sci. USA, 80: 2026-2030,1983, and BV-Hybridoma technology (Cole et al., Monoclonal Antibodies and Cancer Therapy (Alan R. Liss, Inc. 1985), pp. 77-96). Such antibodies may be of any immunoglobulin class, including IgG, IgM, IgE, IgA, IgD and subclasses thereof. Hybridomas making the mAbs of the invention can be cultured in vitro or in vivo. High titer production of mAbs in vivo may provide a preferred method of preparation.

In addition to using monoclonal antibodies in the method according to the invention, chimeric antibodies and single chain antibodies can also be used. Chimeric antibodies are molecules that differ in different parts from other animal species, such as having a variable region derived from a murine mAb and a constant region derived from human immunoglobulins. A "chimeric antibody" can be prepared by splicing a gene derived from a human antibody molecule with appropriate biological activity with a gene derived from a mouse antibody molecule with appropriate antigen specificity (Morrison et al., Proc. Natl. Acad. Sci., 81: 6851-6855, 1984; Neuberger et al., Nature, 312: 604-608, 1984; Takeda et al., Nature, 314: 452-454, 1985; and US Pat. No. 4,816,567).

Alternatively, preparation of single chain antibodies (eg, US Pat. No. 4,946,778; Bird, Science, 242: 423-426, 1988; Huston et al., Proc. Natl. Acad. Sci. USA, 85: 5879-5883, 1988; and Ward et al., Nature, 334: 544-546, 1989), and techniques for the preparation of humanized monoclonal antibodies (US Pat. No. 5,225,539) are useful for the preparation of single chain antibodies useful in the methods of the present invention. Can be used.

In one embodiment, the particles are coated with a growth-permissive, natural extracellular matrix (“ECM”) and crosslinked to form a matrix surface for cell anchoring to the matrix. Thus, such ECM-coated particles provide an attachment support for fixed-dependent cells. The crosslinker is used to attach EMC to the particles using standard methods in the art. The ECM may comprise a variant of collagen, fibronectin, laminin, or a combination thereof.

In another embodiment, avidin or streptavidin can be conjugated by crosslinking to biodegradable particles using standard methods and crosslinking agents in the art.

The polymer molecules can be crosslinked with proteins according to any suitable method to form the active conjugates according to the invention. For example, biodegradable polymers can be crosslinked to proteins using bi (2)-or multi-functional crosslinkers covalently attached to two or more polymers and protein molecules. Preferred difunctional crosslinkers are aldehydes, epoxies, succinimides, carbodiimides, maleimides, azides, carbonates, isocyanates, divinyl sulfones, alcohols, amines, imidates, anhydrides, halides, silanes, diazoacetates, And derivatives such as aziridine. Alternatively, crosslinking may be performed using oxidants and other reagents such as periodicates that activate side chains or moieties on the polymer to react with other side chains or moieties to form crosslinks. Additional methods for crosslinking include exposing the polymer and protein to radiation such as gamma rays to activate the side polymer to allow crosslinking reactions.

Conjugates can be formed between biodegradable particles and proteins, which proteins are polyclonal antibodies, monoclonal antibodies, chimeric antibodies or fragments thereof, collagen I, collagen III, collagen IV, laminin, fibronectin, avidin, and strepbs. Includes but is not limited to tavidin.

6.4. Polymer Bead  Of reactive groups at the surface Biotinylation

To produce a robust, chemically flexible surface for coupling with antibodies, the present invention uses biotin-avidin complex or biotin-streptavidin as a means for tethering antibodies to biodegradable particle surfaces. do. Referring to FIG. 3, the epsilon-NH ₂ group of lysyl of the copolymer is biotinylated by conventional or commercial biotinizing agents. A preferred commercial reagent kit is Sigma's BK-101 product using a sulfo-NHS biotinizing agent. In some cases, cleavable biotinizing agents such as, for example, commercial kit BK-200 (Sigma) can be used. Biotin can be introduced into the biodegradable polymer and the conjugate of the separately prepared antibody with avidin or streptavidin can be reacted with the biotinylated polymer. Avidin-antibody conjugates or separate streptavidin antibody conjugates can be prepared, for example, by standard methods using the crosslinking agents described above.

In other embodiments, the biodegradable polymer can be covalently linked to avidin or streptavidin using carbodiimide, or other reagents described above. The avidin or streptavidin-linked biodegradable polymer then reacts with the biotinylated antibody to produce a tethered antibody although noncovalent to the biodegradable polymer particles. With reference to FIG. 4, this method allows the use of any biotinylated antibody associated with the streptavidin surface and produces a tethered antibody on the surface that targets the cell surface label.

6.5. Coupling of Antibodies by Antibody-Antibody Conjugation

Another embodiment of the invention for antibody tethering is shown in FIG. 5. 5 shows the use of species-specific antibodies directed against the F _c portion of cell targeting antibodies in animal species different from those used to elicit targeted antibodies to cell surface labels. For example, antibodies to cell surface labels in mice bind to anti-F _c monoclonal antibodies raised against F _c labels in mice. Anti-F _c antibodies can be directly conjugated with poly (lactic acid) -lysine copolymers or activated PEG linkages of the copolymers, creating an antibody surface that targets each cell surface label. Alternatively, the species-specific antibody can be biotinylated and then conjugated with the avidin or streptavidin surface on the polymer particles as shown in FIG. 5. Thus, the present invention creates antibody surfaces that recognize groups of antibodies that share a common F _c domain. An advantage of this method is that antibodies to cell surface labels can be tethered to the polymer particle surface without the need for preliminary chemical modification.

6.6. Cell surface markers Targeted  Screening of Antibodies

In the present invention, a wide range of antibodies against the surface label of liver cells and non-liver cells can be used. Such antibodies include commercial antibodies, antibodies made by the inventors, and antibodies made by others. Such antibodies include antibodies against ICAM-1, anti-rat RT1A ^{a, b, 1} or human equivalents thereof, anti-MHC I antibodies, antibodies against integrins, antibodies against growth factor receptors, and antibodies against glycoproteins. It may include.

1 shows conjugation by direct coupling of lysine with ε-amino groups in the protein receptor.

2 shows conjugation with polyethylene glycol residues.

3 depicts conjugation using biotin-streptavidin or biotin-avidin coupling.

4 shows conjugation using biotinylated polyethylene glycol bonds.

5 depicts species-specific, conjugation using second antibody binding.

Examples of Compositions and Uses of the Invention

The following specific examples are provided to provide a better understanding of the various aspects for practicing the present invention. These specific embodiments are for illustrative purposes only, and the following description should not be construed as limiting the invention in any way. Such limitations are, of course, only set by the claims.

7.1. Use of biodegradable polymer-antibody conjugates for cell binding and cell population separation

Antibody and tethered polymer particles that target cell surface labels were incubated with suspensions of mixed cell populations under conditions similar to physiological conditions. Thus, the temperature was between 0 and 40 ° C., the pH was between about 6 and about 7.5, and an isotonic solution was used. In one embodiment, the cells are incubated with the particle-antibody conjugates at Hank's BSS at about 25 ° C., pH 7.0, for at least about 30 minutes. Antibody-surface receptor interaction facilitated binding of targeted cells to polymer beads. The present invention represents the interaction of multiple cells with each biodegradable polymer particle, or the interaction of multiple microparticle beads with a single cell, or any ratio between them. One skilled in the art can adjust the surface density of the antibody and the length of the tether to optimize cell-particle interaction for several purposes. This means a particular cell population recognized by an antibody attached to said particle-antibody conjugate. Thus, the particles allow for easy separation of one cell population from the mixed population. In other words, the present invention constitutes a positive sort method and enrichment of selected cell populations. The particle-antibody conjugates can also be used well in negative sorting or depletion procedures, i.e., in removing cell populations of interest using antibodies selected for a particular population.

In one embodiment, the particle-antibody conjugates can be used to separate mesenchymal cells, to separate them from other cells, including hepatic progenitor cells. The particle-antibody conjugates prepared with antibodies against mesenchymal cells were incubated with a mixed cell population containing mesenchymal cells. After incubation, the particles with adherent cells were separated and seeded in a cell culture chamber with separate compartments. Thereafter, other progenitor cells, such as liver progenitor cells, were seeded in different chambers. In this embodiment, the compartment is for example observed when the adjacent contact with the medium (contiguous media connection), liver progenitor cells and remote interaction with mesenchymal stem cells, as in the trans-well dish ^®.

The particles can be used to enhance cells in a cell population by cells immobilized on the particles. The cells fixed to the particles can be liver cells, hepatic precursors, fibroblasts, endocrine cells, endothelial cells, or any fixed-dependent cells.

The cells not immobilized on the biodegradable particles can be any non-fixed dependent cells, including hematopoietic cells, hematopoietic precursors, erythrocytes, leukemia cells, and lymphoma cells, and cells with surface receptors not targeted by the antibody-polymer surface. have..

7.2. Of particle-cell conjugates Extracellular  Use of biodegradable polymer-antibody conjugates for culture and its use in three-dimensional bioreactors

Biodegradable particles conjugated with the extracellular matrix as described above were incubated with fixed-dependent cells. The use of extracellular matrix provided a desirable growth environment for fixed-dependent cells and facilitated the transfer of cell suspension from one vessel to another. This method also facilitates the expansion of the cell population and the sampling of the cell population.

A large number of various fixed-dependent cells suitable for use with biodegradable particle extracellular matrix conjugates include hepatic precursors, mesenchymal cells, mesenchymal precursors, muscle cells, which are heart cells, neuronal cells, glial cells, fibroblasts , Stem cells, epithelial cells, and endothelial cells. In addition, endocrine cells are suitable for growth on particle-extracellular matrix conjugates.

In addition, the particle-cell combination is suitable for growth in three-dimensional culture in a bioreactor. This use provides a flow of nutrient medium and nutrient gas to the adherent cell population and facilitates the exchange of metabolites and metabolic wastes as needed.

7.3. Biodegradability for Cryopreservation of Fixed-Dependent Cells Polymer - Protein  Use of the conjugate

By attaching enhanced cells to the biodegradable polymer support, the compositions according to the invention can also improve the survival and recovery of cryopreserved cells. Early cryopreservation methods for hematopoietic cells normally in suspension and cell lines adapted to cell culture have been successful, but not for fixed-dependent cell types. Cryopreservation of fixed-dependent liver cells by conventional methods of resuspension with trypsin or other scavengers resulted in a very large loss of cell activity. In addition, cells lost their differentiation properties and the ability to adhere to solid surfaces. The present invention applies derivatized biodegradable particles for cell fixation. The particle-extracellular matrix conjugate was provided for cell attachment and then exposed to vitrification solution to block ice crystal formation. Preferred cryo-preservation or vitrification solutions comprise 5-15%, typically 10% of dimethyl sulfoxide (v / v) in the serum-reinforced medium. Another vitrification solution contains 10% (v / v) of dimethyl sulfoxide in defined medium (ie, no serum or plasma). In addition, the particle-bound cells are not removed from the particles after thawing. This improvement is important because cells embedded in a separate material, such as an extracellular matrix, or alginate, must be resuspended after thawing for practical use according to most research or clinical needs. However, enzymatic treatment of cells immediately after thawing causes loss of survival in many cells in most cases. Cells are particularly vulnerable to handling and enzymatic treatment immediately after conventional cryopreservation and thawing. By avoiding enzymatic treatment after thawing, the cells on the beads are more robust. Cells on the particles can simply be washed with cell culture medium and used immediately without further handling. This process improves the survival and function of cryopreserved fixed-dependent cells and simplifies cell-banking and cell-typing operations.

7.4. Biodegradability for Cell Transplantation Polymer - Protein  Use of the conjugate

In another embodiment of the present invention, the method according to the present invention provides a powerful means for producing enhanced fixed-dependent cells for transplantation. Conjugates of biodegradable polymer-protein-cells can be directly implanted into blood vessels or the recipient organ. Polymers are designed to degrade into constituent molecules naturally present in vivo in cooperation with enhanced progenitor cell growth and maturation and formation of natural extracellular matrix and tissue structures. In addition, decomposition and removal of the polymer material minimizes the problem of rejection.

7.5. Cell strengthening by negative sorting

If the desired cell type does not exhibit a unique recognizable cell surface label, negative sorts, any repeat negative sorts, may be used to enhance the desired cells in the population. Preferred cases are as follows.

Biodegradable particle-antibody conjugates for glycophorin A (particle-Ab (GA)) are prepared according to the methods described above. An actual single cell suspension (10 ⁶ cells / ml concentration) of 10 ⁷ embryonic liver cells was mixed with 0.5 g wet weight particle-Ab (GA) conjugates. By "substantially" in the specification is meant that at least about 70% of the cells are not associated with other cells. In one embodiment, the substantially single cell suspension has at least about 90% of the cells not associated with other cells. The mixture consisted of Dubelco's Modified Eagle Medium and Ham's F12 (DMEM / F12, GIBCO / BRL, Grand Island, NY) for 1 hour at 24 ° C., 20 ng / ml EGF (Collaborative Biomedical Products ), 5 μg / ml insulin (Sigma), 10 ⁻⁷ M dexamethasone (Sigma), 10 μg / ml iron-saturated transferrin (Sigma), 4.4 × 10 ⁻³ M nicotinamide (Sigma), 0.2% (w / v) bovine serum albumin (Sigma), 5 x 10 ^-5 M 2-mercaptoethanol (Sigma), 7.6 μeq / l free fatty acid, 2 x 10 ^-3 M glutamine (GIBCO / BRL), 1 x 10 ^-6 Incubated in defined medium (HDM) with M CuSO ₄ , 3 × 10 ⁻⁸ MH ₂ SeO ₃ and antibiotics. Cells remaining in the supernatant and not attached to the beads were incubated in fresh medium or used for subsequent sorting.

7.6. Cell enrichment by positive sorting

If the desired cell exhibits at least one unique recognizable cell surface label, positive sorts, any repeat positive sorts or a combination of positive and negative sorts, may be used to enhance the desired cells in the population. Preferred cases are as follows.

Biodegradable particle-antibody conjugates for ICAM-1 (particle-Ab (ICAM-1)) are prepared according to the methods described above. An actual single cell suspension (10 ⁶ cells / ml concentration) of 10 ⁷ embryonic liver cells was mixed with 0.5 g wet weight Particle-Ab (ICAM-1) conjugates. The mixture consisted of Dubelco's Modified Eagle Medium and Ham's F12 (DMEM / F12, GIBCO / BRL, Grand Island, NY) for 1 hour at 24 ° C., 20 ng / ml EGF (Collaborative Biomedical Products ), 5 μg / ml insulin (Sigma), 10 ⁻⁷ M dexamethasone (Sigma), 10 μg / ml iron-saturated transferrin (Sigma), 4.4 × 10 ⁻³ M nicotinamide (Sigma), 0.2% (w / v) bovine serum albumin (Sigma), 5 x 10 ^-5 M 2-mercaptoethanol (Sigma), 7.6 μeq / l free fatty acid, 2 x 10 ^-3 M glutamine (GIBCO / BRL), 1 x 10 ^-6 Incubated in defined medium (HDM) with M CuSO ₄ , 3 × 10 ⁻⁸ MH ₂ SeO ₃ and antibiotics. Cells attached to the particles were incubated in fresh medium.

In another example, biodegradable particle-antibody against EpCAM-1 (particle-Ab (EpCAM-1)) / NCAM-1 (particle-Ab (NCAM-1)) was prepared according to the method described above. In another embodiment, biodegradable particle-antibodies against EpCAM-1 (particle-Ab (EpCAM-1)) / ICAM-1 (particle-Ab (ICAM-1)) were prepared according to the methods described above. Biodegradable particle-antibodies having at least one unique recognizable cell surface label can be used to enhance the desired cells in the population.

7.7. particle- ECM  Cell Culture on Conjugates

Populations of hepatic progenitor cells enriched by any method were incubated in HDM with biodegradable particles conjugated with collagen IV. Collagen IV-particles were prepared according to the method described above, and the diameter of the particles was 500 microns and the ratio of collagen IV to particles was 0.02 (w / w). A total wet weight of 10 g of collagen IV-particles was suspended in 500 ml of HDM at 37 ° C., 95% (v / v) air / 5% (v / v) CO ₂ atmosphere. Collagen IV-particles were seeded with 10 ⁶ hepatic progenitors and medium was changed every two days. The particles were gently stirred to maintain a suspended state. Cell metabolism was monitored by pH and glucose concentration changes and cell growth was monitored by measuring DNA content. The addition of new particles to the culture mixture provided a new growth surface for the growth culture.

In another embodiment, the hepatic progenitor cell population enriched by any method is combined with other suitable specialized matrix chemistries, hyaluronic acid, and heparin glycan sulfate generally present in the fetal form of laminin known to those skilled in the art. Incubated with conjugated biodegradable particles.

7.8. Cell cryopreservation using particle-adhered cells

As in Example 6.4, fixed-dependent cells growing on biodegradable particles resuspend the particles with attached cells in a solution of 10% (v / v) dimethyl sulfoxide in HDM, and 1 × 10 ⁶ cells. It is cryopreserved by transferring a portion containing sterile ampoules or vials. The ampoule or vial is properly sealed and the temperature is gradually lowered by 1 ° C. per minute between about −80 ° C. and about −160 ° C. Store cells at about -160 ° C without limitation until needed. If necessary, the ampoule or vial is thawed quickly, for example in a lukewarm bath. The contents are then transferred to a culture vessel with culture medium (HDM) aseptically.

7.9. Transplantation of Liver Progenitors in Liver Disorder Models

Liver disorder rat model was used to evaluate xenograft cell transplantation therapy. Liver disorders were modeled by surgical removal of about 70% of the liver and / or biliary duct ligation in an experimental group of 10 male rats (125-160 g body weight). Ten sham control groups of matched age and gender were similarly anesthetized and livers were manipulated with an open midline, but no bile duct ligation and liver removal.

Enhanced populations of hepatic precursors immobilized on biodegradable beads were prepared according to the method described above. In summary, the livers of 12 fetal (14 day embryonic) rat pups were aseptically removed, diced, washed with 1 mM EDTA in Hank's BSS without calcium or magnesium and having a pH of 7.0. Suspensions in proximity to single cells were prepared by incubating for 20 minutes in Hank's BSS containing 0.5 mg / ml collagenase.

Sterile biodegradable particles conjugated with antibodies to ICAM-1 were prepared according to the method described above. Single intercellular suspensions from 12 pups were incubated with ICAM-1-microparticles with a packing volume of 1.5 ml at 25 ° C. for 1 hour. The particles were then diluted with 10-fold volume of HDM and allowed to stand at 1 × g for 5 minutes before decanting the supernatant. Thereafter, the method was repeated. The particles were gently resuspended in fresh HDM and incubated at 37 ° C. under 95% air, 5% CO ₂ (v / v) for 5 days.

Three days after liver incision or mock operation, rats (both experimental and mock controls) were incised 5 mm intraperitoneally to expose the spleen. Half of each experimental and mock control group animal was randomly selected and 0.1 ml of biodegradable-particle-ICAM-I-embryonic liver cell composition, respectively, was injected directly into the spleen. All incisions were sealed with a surgical stapler. Cyclosporin A, 1 mg / kg body weight, was administered intraperitoneally daily as an immunosuppressive agent.

Blood levels of bilirubin, gamma glutamyl transferase and alanine aminotransferase activity were monitored 2 days before hepatic or mock hepatic incision operation and 3, 7, 14, and 28 days after the operation. Body observations of body weight, water consumption, and drowsiness were recorded on the same day. On day 28 after liver incision, all live animals were killed for spleen and liver tissue evaluation.

All publications, patents, and patent documents referenced herein are incorporated by reference in their entirety.

The present invention has been described with reference to the above specific and preferred embodiments and methods. However, it should be understood that many modifications may be made while retaining the spirit and scope of the invention. Therefore, it is not to be limited by the foregoing embodiments, but the scope of the present invention is limited only by the claims.

Appendix A

Table 1. Preparation of Free Fatty Acid (FFA) Mixtures

Source of Purified Fatty Acids: See Table 2

The preparation of stock free fatty acids was made by dissolving each component in 100% ethanol. The ingredients are as follows:

Palmitic acid (solid) 1 M stock; Soluble in hot alcohol

Palmitoleic acid 1 M stock; Easily soluble in alcohol

Oleic acid 1 M stock; Easily soluble in alcohol

Linoleic acid 1 M stock; Easily soluble in alcohol

Linoleic acid 1 M stock; Easily soluble in alcohol

Stearic acid (solid) 151 mM stock, 1 g dissolved in 21 ml of alcohol,

Need to heat up.

These stocks can be stabilized by bubbling each with nitrogen and then storing at -20 ° C.

Free fatty acid mixture stock solution:

Palmitic Acid 31.0 mM

Palmitoleic Acid 2.8 mM

Oleic Acid 13.4 mM

Linoleic Acid 35.6 mM

Linoleic Acid 5.6 mM

Stearic acid 11.6 mM

In total, a total of 100 mM free fatty acids were obtained. Stocks with all free fatty acids can also be stabilized by bubbling with nitrogen and storing at -20 ° C.

Final solution:

76 μl of free fatty acid mixture stock per liter of culture medium was added to a final concentration of 7.6 μEq. Free fatty acids are purified and toxic unless provided with fatty acid-free, endotoxin-free serum albumin (eg, Pentex type V albumin). Albumin was formulated at a typical concentration of 0.1-0.2% in the basal medium or PBS used.

Table 2. Sources of basal medium, growth factors, matrix components and other culture components

factor Source Growth factor / hormone Prolactin (Luteotropic Hormone) Sigma-Aldrich US Biological Cortex Biochemical Inc. ICN Biomedicals Epithelial growth factor (EGF) mice; Receptor Grade Human Recombinant Mouse Recombination  Collaborative Biomedicals Sigma-Aldrich Pepro Tech Upstate Biologicals Accurate Chemicals Clonetics Products Antigenix American Inc. Accurate Chemicals Antigenix American Inc. Transferrin: Holo-iron saturated bovine, human Sigma-Aldrich Clonetics Somatotropin: Growth Hormone Human Peitutari Human Recombination  Sigma-Aldrich Accurate Chemicals ICN Biomedicals Hydrocortisone Sigma-Aldrich Clonetics Calbiochem Alfa Aesar Bishop Canada ICN Biomedicals Dexamethasone Sigma-Aldrich Clonetics American Pharmacia Biotech Accurate Chemicals Calbiochem ICN Biomedicals

Glucagon Forcin Pancreas Sigma-Aldrich BIOTREND Chemikalien Other Reinforcement HDL: High Density Lipoprotein Human Plasma  Sigma-Aldrich Chemicon International Biodesign International Per Immune BioResource Technology Academy Biomedical Co. Biodesign international Free fatty acids Linoleic Sigma-Aldrich Altech Associates Inc., ICN Biomedicals Linolenic Sigma-Aldrich Altech Associates Inc., Oleic Sigma-Aldrich Altech Associates Inc., ICN Biomedicals Palmitic Sigma-Aldrich Altech Associates Inc., ICN Biomedicals Stearic Sigma-Aldrich Altech Associates Inc., ICN Biomedicals

Bovine Serum Albumin V Free Fatty Acid Free Sigma-Aldrich Genmini Bio-Products Nicotinamide (niacinamide) Sigma-Aldrich Calbiochem ICN Biomedicals Spectrum Laboratory Products TCI America Putrescine Sigma-Aldrich Advanced ChemTech Inc. Crescent Chemicals ICN Biomedicals Spectrum Laboratory Products 3 ', 3', 5'-triiodo-L-tyronine (T3) Sigma-Aldrich Toronto Research Chemicals ICN Biomedicals Novabiochem TCI America microelement Copper pentahydrate Sigma-Aldrich Chem Services Inc. Crescent Chemicals Gallade Chemical, Inc. ICN Biomedicals MV Laboratories, Inc. Spectrum Laboratory Products Strem Chemicals, Inc. Zinc Sulfate Heptahydrate Sigma-Aldrich Crescent Chemicals ICN Biomedicals MV Laboratories, Inc. Selenium Acid: Sigma-Aldrich Crescent Chemicals ICN Biomedicals MV Laboratories, Inc.

Foundation DMEM / F12 Gibco BRL BioWhittaker Mediatech Inc. Specialty Media-Division of Cell & Molecular Technologies RPMI 1640 Gibco BRL Biologos Inc. BioSource International ICN Biomedicals BioWhittaker Liver cell medium Sigma, Clonetics Keratinocyte basal medium Clones Extracellular matrix components Fibronectin bobbin human bobbin, human, rat, mouse human bobbin, chicken, hose, human, mouse bobbin, human, mouse salmon , rat  Sigma-Aldrich Collaborative Biomedicals Accurate Chemicals Biosource International BIOTREND Chemikalien Chemicon International Calbiochem Laminin mouse human  Sigma-Aldrich Collaborative Biomedicals EY Laboratories Alexis Corp. Biosource International Alexis Corp. Chemicon International BIOTREND Chemikalien Collagen Type I Collaborative Biomedicals Sigma-Aldrich BioShop Canada BIOTREND Chemikalien Collagen Type II Sigma-Aldrich Chemicon International Accurate Chemicals

Collagen Type III Chemicon International Accurate Chemicals BIOTREND Chemikalien Collagen Type IV Collaborative Biomedicals Sigma-Aldrich BIOTREND Chemikalien Matrigel Collaborative Biomedicals Clonetics Unbleached Heparin Sigma BioChemika Clonetics CarboMer, Inc. Alfa Aesar PolySciences, Inc Heparan sulfate Sigma-Aldrich BioChemika CarboMer, Inc. US Biologicals Seikagaku USA Calbiochem ICN Biomedicals Carrageenan (heparin-like reagent purified from seaweed. Three forms are available: lambda, kappa and iota, which vary in solubility) Sigma-Aldrich BioChemika CarboMer, Inc. ICN Biomedicals TCI America Suramin (heparin-like molecule found to have potent anti-microbial and anti-tumor activity) Sigma-Aldrich BioChemika Calbiochem Alexis Corp. BIOMOL Research Laboratories, Inc. ICN Biomedicals A.G. Scientifics American Qualex International Inc. Heparan Sulfate Proteoglycan (HS-PG) from EHS Tumors Collaborative Biomedicals Sigma-Aldrich Chemicon International

Claims (26)

A composition comprising at least one biodegradable particle, at least one receptor covalently bound to the particle, and at least one cell anchored to the at least one receptor. The composition of claim 1, wherein the receptor comprises an antibody, fragment of the antibody, avidin, streptavidin, biotin moiety, or a combination thereof. The composition of claim 1 wherein the particles comprise polylactide, polylactide-lysine copolymer, polylactide-lysine-polyethylene glycol copolymer, starch, or protein. The composition of claim 1 further comprising an extracellular matrix. The composition of claim 4, wherein the extracellular matrix comprises collagen, fibronectin, laminin, or a combination thereof. The composition of claim 1 wherein the particles are macroparticles, microparticles, or nanoparticles. The composition of claim 1 wherein the cells are selected from the group consisting of liver cells, hepatic precursors, and hematopoietic precursors. The composition of claim 1, wherein the particles are biocompatible. The composition of claim 1 wherein the receiver is stable in at least one aqueous or organic solvent. (a) immobilizing cells to a composition comprising at least one biodegradable particle to form a mixture, (b) freezing the mixture, (c) A method of cryopreservation of anchorage-dependent cells comprising thawing and recovering cells from the cell-polymer particle conjugate. The method of claim 10, wherein the biodegradable particles further comprise a receptor covalently bonded to the particles. The method of claim 11, wherein the receptor comprises an antibody, fragment of the antibody, avidin, streptavidin, biotin moiety, or a combination thereof. The method of claim 10, further comprising an extracellular matrix. The method of claim 10 further comprising a cryopreservation solution. The method of claim 14, wherein the cryopreservation solution comprises 10% (v / v) dimethyl sulfoxide. (a) providing a composition comprising at least one biodegradable particle, at least one receptor covalently bound to the particle, at least one cell immobilized on at least one receptor, and at least one cell unfixed to the receptor and, (b) removing the cells comprising at least one cell unfixed to the biodegradable particles. The method of claim 16, wherein the receptor comprises an antibody, fragment of the antibody, avidin, streptavidin, biotin moiety, or a combination thereof. The method of claim 16, wherein the cells immobilized on the biodegradable particles comprise liver cells, or liver precursors. The composition of claim 16 wherein the cells not immobilized on the biodegradable particles comprise hematopoietic precursors. (a) providing a composition comprising at least one biodegradable particle, at least one receptor covalently bound to the particle and at least one cell immobilized on the at least one receptor; (b) A method of cell culturing fixed-dependent cells comprising contacting the composition with a cell culture medium. The method of claim 20, wherein the composition further comprises an extracellular matrix. The method of claim 20, wherein the cells comprise at least one of hepatic precursors, hematopoietic precursors, fibroblasts, mesenchymal cells, heart cells, endothelial cells, epithelial cells, neuronal cells, glial cells, endocrine cells, or a combination thereof. Cell therapy, comprising administering to a subject an effective amount of a composition comprising at least one biodegradable particle, at least one receptor covalently bound to the particle, and at least one cell immobilized on the at least one receptor How to treat a subject. The method of claim 23, wherein the cells comprise liver precursors. The method of claim 23, wherein the composition is administered by intravenous, intraarterial, intramuscular, parenteral or a combination thereof. The method of claim 23, wherein the effective amount ranges from about 10 ² to about 10 ¹¹ cells.

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