WO2008121422A2

WO2008121422A2 - Implantable cell matrix composition for delivery of bioactive compounds

Info

Publication number: WO2008121422A2
Application number: PCT/US2008/004311
Authority: WO
Inventors: Shai Yehoshua Schubert
Original assignee: Moma Therapeutics
Priority date: 2007-04-01
Filing date: 2008-04-01
Publication date: 2008-10-09
Also published as: WO2008121422A3

Abstract

This invention relates to methods and compositions for cell therapy which employ monocytic cells (e.g., monocytes) placed in a matrix, which can then be implanted at a selected site in the body and deliver localized, controlled doses of therapeutic products produced and secreted by the monocytes. The invention further relates to kits and methods for creating and using the matrix containing the monocytic cells.

Description

IMPLANTABLE CELL MATRIX COMPOSITION FOR DELIVERY OF

BIOACTIVE COMPOUNDS

RELATED APPLICATIONS This application claims the benefit of prior-filed provisional patent application

U.S. Serial No. 60/909,485 (filed April 1, 2007) entitled "Induction of Angiogenesis in Ischemic Tissue with Implanted Matrix containing Monocytes", U.S. Serial No. 60/957,169 (filed August 22, 2007) entitled "Methods and Compositions for Localized Cell Therapy with Implanted Matrix containing Monocytes" and U.S. Serial No. 61/019,594 (filed January 7, 2008) entitled "Methods and Compositions for Delivery of Monocyte Secreted Factors using an Implanted Matrix Containing Monocytes". The entire content of the above-referenced applications is incorporated herein by reference.

BACKGROUND OF THE INVENTION Monocytes are a type of leukocyte, or white blood cell, which have an integral role in the innate immune system. Following the appearance of signals delivered from a specific site in the body, monocytes are mobilized by chemotactic signals and adhere to the activated endothelium through interactions mediated by adhesion molecules like P- CAM, V-CAM and I-CAM on endothelial cells and CDl 8 and CDl IB on monocytes. Following their adhesion to the endothelium, monocytes transmigrate into the tissue and differentiate into macrophages.

Together with neutrophils, eosinophils and natural killer cells, monocytes function as a first-line defense to detect, eliminate or contain invading microbes and toxic macromolecules. Monocytes responses towards these targets are rapid and triggered by structures, commonly referred to as Pathogen- Associated Molecular Patterns (PAMP). Whenever the innate immunity is unable to handle an invading microorganism, monocytes function as effector cells of the adaptive immune system, after receiving the appropriate activation and information from antigen-specific T and B- lymphocytes. Monocytes have also essential functions in wound healing and resolution of inflammation, coordinating cell migration, extra-cellular matrix remodeling and angiogenesis, all of which are required for tissue repair. Consequently, the ultimate goal of monocytes is the maintenance of tissue homeostasis and integrity. This is achieved by various monocyte functions, such as, secretion of specific proteins, scavenging, elimination of pathogen and tumor cells, clearance of senescent cells, control of tissue cell growth and modulation of the extra- cellular milieu.

To accomplish these tasks, monocytes exhibit a highly flexible gene expression program that allows them to adapt and respond to changes in their surrounding micro- environment, as well as to recruit, engage and coordinate other cell types in restoring normal tissue structure and function. These various monocyte activities are not displayed concomitantly and, in fact, some of these activities are clearly contradictory (e.g., degradation versus synthesis of extracellular matrix). Thus, tissue monocytes are functionally heterogeneous under basal conditions, and exhibit a large degree of variability upon activation by endogenous factors or exogenous stimuli {see Vega, MA et al, Inmunologia 2006, 25(4): 248-272). Monocyte implantation at site of ischemic tissue has been attempted as a therapeutic approach for the treatment of various conditions such as cancer, heart disease, ischemia, nerve injury, wound healing and diabetes. In the treatment of myocardial infarction, monocyte therapy has been used to promote vascular growth and regain heart functionality in the ischemic heart. Monocytes secrete vascular growth factors which induce and support angiogenesis. Various studies and clinical trials have demonstrated the beneficial effect of monocyte therapy (e.g., naked monocyte therapy) to the ischemic heart by reducing the damage of tissue ischemia, increasing angiogenesis and tissue perfusion, and therefore recovering the heart's functionality after an ischemic event (Arras, M et al., J Clin Invest, 1998. 101 :40-50; Hattori, R and Matsubara, H, MoI CeU Biochem, 2004. 264: 151-5; Higashi, Y et al., Circulation, 2004. 109: 1215-8; Ishida, A et al., Circ J, 2005.69:1260-5; Iwase, T, et al., Cardiovasc Res, 2005. 66:543- 51; Li, TS, et al., Am J Physiol Heart Circ Physiol, 2003. 285:H931-7; Saigawa, T, et al., Circ J, 2004. 68:1189-93; Strauer, BE, et al., Circulation, 2002. 106: 1913-8; Perin, E.C., Geng, YJ. Geng, and Willerson, J.T., Circulation, 2003. 107:935-8; Sherman, W., et al. , Nat Clin Pract Cardiovasc Med, 2006. 3 : S57-64).

Similarly, monocyte therapy has been used for the delivery of therapeutic proteins by genetic manipulation, activation or transformation of the monocytes (Muhlebach, M.D., et al., MoI Ther, 2005. 12:1206-16; Lu, Y., et al., Cell MoI Biol, 2003. 49: 1151-6; Spiekeπnann, K., et al., Eur J Haematol, 2001. 67:63-71; US 2006/0257359). The use of monocytes has also been described for nerve repair and spinal cord injury treatment (Lazarov-Spiegler, O., Solomon, A.S., and Schwartz, M. Glia, 1998. 24:329-37; Rapalino, O., et al., Nat Med, 1998. 4:814-21; Schwartz, M., et al., Neurosurgery, 1999. 44:1041-6; U.S. Patent No. 6,267,955).

While some progress has been made in the field of cellular therapy and therapeutic protein delivery for tissue repair, there exists a need to improve the delivery of cells and cellular factors in vivo. In particular, it would be highly advantageous to keep the monocytes confined to the target tissue and away from host cells and tissues that can alter the behavior, phenotype, differentiation and/or activation state of the monocytes.

SUMMARY OF THE INVENTION Compositions and methods for delivery of compounds comprising a matrix comprising monocytic cells has been developed. Embodiments of the invention feature liquid, semi-solid and solid matrices with embedded cells and methods for their formation and use. The matrices may be injectable or implantable. In some embodiments, monocytes are introduced to a matrix and are embedded in the matrix; the monocytes may be polarized either before or after embedding in the matrix. In some embodiments featuring polarized monocytic cells, cells are polarized to a Ml phenotype. In some embodiments featuring polarized monocytic cells, cells are polarized to a M2 phenotype and may be characterized as belonging to a M2a, M2b or M2c phenotype. Monocytic cells may be incubated with polarizing agents before being contacted with a matrix of the invention in some embodiments. In some embodiments, a matrix of the invention is formulated to incorporate agents and compounds that exert one or more effects upon cells embedded in the matrix. Embodiments of the invention include matrices with embedded cells in which the cells produce, release and/or secrete compounds. In additional embodiments, the matrix is permeable to the compounds produced. Compounds produced or secreted by cells within a matrix may diffuse out of the matrix and create effects in the surrounding environment in some embodiments. Additional embodiments of the invention include kits comprising a matrix of the invention and instructions for practicing the invention. In some embodiments, a kit of the invention includes cells to be used with the matrix.

One embodiment of the invention features an implantable, semi-solid matrix comprising a hydrogel material and monocytic cells embedded therein. In particular embodiments, monocytic cells are capable of producing a secreted product and in particular embodiments, the matrix is permeable to a product secreted by the monocytic cells.

Additional embodiments of the invention feature an injectable composition comprising a hydrogel material and monocytic cells that are added to the injectable composition, where the monocytic cells are capable of producing a secreted product. The injectable composition in convertible to a semi-solid state in some embodiments and the injectable composition can be permeable to the product secreted by the monocytic cells. Some embodiments feature injectable compositions that can be converted to a semi-solid state by exposing the composition to heat, ionizing radiation or ultraviolet radiation. In some embodiments, the monocytic cells are capable of producing more than one product. In some embodiments, a product secreted by a monocytic cell is an angiogenic factor. Vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), platelet derived growth factor (PDGF), hepatocyte growth factor/scatter factor (HGF/SF), epidermal growth factor (EGF), and/or Interleukin-8 (IL-8) are secreted by a monocytic cell in some embodiments. A product secreted by a monocytic cell may be an immunosuppressive factor in some embodiments. A monocytic cell in some embodiments may secreted one or more immunosuppressive factors such as IL-4, IL-IO and/or TGF-β. In some embodiments, a monocytic cell may be a monocyte precursor cell and may be a bone marrow cell and/or a monocyte progenitor cell in some embodiments.

Monocytic cells may be activated monocytes in particular embodiments. Monocytic cells may be activated and/or polarized towards a M2 phenotype in some embodiments. Monocytic cells may produce one or more M2 phenotypic markers in some embodiments. In some embodiments, monocytic cells producing a M2 phenotypic marker produces the market at a level that is increased as compared to a monocytic cell having a Ml phenotype. In particular embodiments, monocytic cells are activated before they come in contact with a matrix and is some embodiments, cells are not activated before they come in contact with a matrix. In some embodiments, monocytic cells are pre-activated towards a M2 phenotype. In some embodiments, a matrix comprises one or more agents that activate a monocytic cell towards a M2 phenotype.

One aspect of the invention features a matrix with monocytic cells where the monocytic cells have a M2 phenotypic profile. In additional aspects of the invention, a matrix with monocytic cells where the monocytic cells have a Ml phenotype profile. In some embodiments, a monocytic cell produces at least one marker of a Ml phenotype profile. A monocytic cell producing at least one marker of a Ml phenotype may produce the marker at a level that is increased in comparison to monocytic cells having a M2 phenotype. In some embodiments, monocytic cells are pre-activated towards the Ml phenotype. A matrix in particular embodiments may comprise an activating agent that activates towards a Ml phenotype.

An additional aspect of the invention includes a matrix comprising a macrophage. In some embodiments, a cell comprised by a matrix that is injected or implanted into an organism comes from an autologous source. In some embodiments, a cell comprised by a matrix that is injected or implanted into an organism comes from an allogenic source. In some embodiments of the invention, a cell comprised by a matrix that is injected or implanted into an organism comes from a xenogenic source. In some embodiments, a cell comprised by a matrix is a genetically engineered cell.

Aspects of the invention include a matrix comprising a hydrogel which may comprise one or more polymers. In some embodiments, the hydrogel comprises polylactic acid, polyglycolic acid, other polyhydroxy acids, copolymers of two or more polyhydroxy acids, polyorthoesters, polyanhydrides, gelatin, collagen, cellulose, derivatized cellulose, chitosan, alginate, thiol-modified hyaluronan, and/or combinations or copolymers thereof. Cross-linkers are comprised by a matrix of the invention in some embodiments.

In particular embodiments, a matrix of the invention comprises one or more of glutaraldehyde, diphenylphosphoryl azide, transglutaminase, dimethyl suberimidate, DMS-treated collagen, dimethyl 3,3'-dithiobispropionimidate, multivalent ions, calcium ions, N,N methylene-bisacrylamide (MBA), acrylamide, allyl methacrylate, ethylene glycol dimethacrylate, tripolyphosphate, and combinations thereof.

Additional aspects of the invention include methods for delivering a secreted product to a localized site in a subject. In some embodiments, a method of the invention includes delivering a matrix to a localized site and maintaining the matrix in the localized site for a period of time sufficient for the secreted product to interact with the localized site. In some embodiments of the invention, a subject that receives a matrix of the invention has a disease, has a condition and/or is at risk for a disease and/or a condition. In some embodiments, the disease and/or condition that a subject has or has a risk for is coronary artery disease, peripheral artery disease, limb ischemia, ischemic wound, ischemic ulcer, ischemic bowel disease, atherosclerotic ischemic disease, muscle flaps, skin flaps, organ transplant, nasolabial folds, wrinkles, conditions which result in scar formation, conditions requiring plastic surgery and/or conditions requiring a cosmetic procedure. Some aspects of the invention feature a method for treating a subject that has or is at risk of having ischemia. In some embodiments, a matrix or composition of the invention is administered at a site of ischemia. In some embodiments, the administration of a matrix or composition produces a secretion of an angiogenic factor at the site for a time sufficient to prevent or lessen the effects of a perfusion injury associated with the ischemia. In some embodiments, a method for treating a subject that has or is at risk for cardiac ischemia is featured. In some embodiments, the method for administering the matrix of composition to a site is injection.

Additional aspects of the invention include methods for reducing the nasolabial folds or wrinkles in a subject, comprising administering at the site of the nasolabial folds or wrinkles a matrix or composition of the invention, such that secretion of an angiogenic factor is effected for a time sufficient to detectably reduce the number and/or depth of the nasolabial folds or wrinkles. In some embodiments, a method for reducing the nasolabial folds or wrinkles in a subject comprises injecting a matrix or composition of the invention. In some aspects of the invention, a method for reducing an immune response at a localized site in a subject is featured, wherein a matrix or composition of the invention is administered at a site and an immunosuppressive factor is secreted for a time sufficient to detectable reduce the immune response at the localized site. In some embodiments, a matrix or composition of the invention administered to reduce an immune response at a localized site in a subject is injected.

Further embodiments of the invention include a kit comprising a hydrogel material and instructions for the use of the hydrogel material to form a semi-solid matrix comprising monocytic cells embedded within. In some embodiments, the monocytic cells embedded within produce a secreted product to which the matrix is permeable. In some embodiments, a kit will comprise a monocytic cell having a M2 phenotype which produces a secreted product at a level that is increased as compared to a monocytic cell having an Ml phenotype. In some embodiments, a kit comprises a cross-linking agent for cross-linking the hydrogel to form a semi-solid matrix. In some embodiments, a kit includes instructions for combining the hydrogel material, monocytic cells and, in particular embodiments, a cross-linking agents, such that an injectable liquid is formed that transitions to a semi-solid matrix following injection into a subject. In some embodiments, instructions are provided in a kit of the invention for pre-activating monocytic cells towards the M2 phenotype.

Aspects of the invention include kits that comprise one or more hydrogels, and may comprise polylactic acid, polyglycolic acid, other polyhydroxy acids, copolymers of two or more polyhydroxy acids, polyorthoesters, polyanhydrides, gelatin, collagen, cellulose, derivatized cellulose, chitosan, alginate, thiol-modified hyaluronan, and/or combinations thereof. In some embodiments, a cross-linking agent is comprised by a kit of the invention and may comprise glutaraldehyde, diphenylphosphoryl azide, transglutaminase, dimethyl suberimidate, DMS-treated collagen, dimethyl 3,3'- dithiobispropionimidate, multivalent ions, calcium ions, N,N methylene-bisacrylamide (MBA), acrylamide, allyl methacrylate, ethylene glycol dimethacrylate, and tripolyphosphate.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows projected data for the survival of monocytes under in vitro cell culture conditions, comparing the survival of monocytes embedded in a matrix (top line) to those grown in ordinary tissue culture well plates.

Figure 2 shows projected data for the release over time of VEGF from gelfoam collagen matrix cultures embedded with VEGF transduced monocytes.

Figure 3 shows VEGF- 165 production by matrix-free monocytes incubated in media with the TLR agonist LPS, with or without the adenosine receptor A2 agonist NECA. Figure 4 shows VEGF- 165 production by matrix-free monocytes incubated in media with the TLR agonist CL097, with or without the adenosine receptor A2 agonist NECA.

Figure 5 shows VEGF- 165 production by matrix-free monocytes incubated in media with the adenosine receptor A2 agonist NECA alone. Figure 6 shows the effects of LPS and NECA on VEGF- 165 secretion kinetics from a polarizing collagen matrix embedded with positively selected monocytes that were not pre-polarized.

Figure 7 shows the effects of CL097 and NECA on VEGF- 165 secretion kinetics from a polarizing collagen matrix embedded with positively selected monocytes that were not pre-polarized.

Figure 8 shows the effects of NECA alone on VEGF-165 secretion kinetics from a polarizing collagen matrix embedded with positively selected monocytes that were not pre-polarized. Figure 9 shows the effects of LPS and NECA on VEGF-165 secretion kinetics from a polarizing collagen matrix embedded with negatively selected monocytes that were not pre-polarized.

Figure 10 shows the effects of CL097 and NECA on VEGF-165 secretion kinetics from a polarizing collagen matrix embedded with negatively selected monocytes that were not pre-polarized..

Figure 11 shows the effects of NECA alone on VEGF-165 secretion kinetics from a polarizing collagen matrix embedded with negatively selected monocytes that were not pre-polarized.

Figure 12 shows the effects of LPS and NECA on VEGF-165 secretion kinetics from a polarizing collagen matrix embedded with negatively selected, pre-polarized monocytes.

Figure 13 shows the effects of CL097 and NECA on VEGF-165 secretion kinetics from a polarizing collagen matrix embedded with negatively selected, pre-polarized monocytes.

Figure 14 shows the effects of NECA alone on VEGF-165 secretion kinetics from a polarizing collagen matrix embedded with negatively selected, pre-polarized monocytes.

DETAILED DESCRIPTION OF THE INVENTION

The present invention features methods and compositions for cell therapy which employ the preparation of monocytic cells, in particular monocytes, activation and/or genetic manipulation of cells, the placement of cells in a matrix and the placement of a cell-bearing matrix in vivo as an implantable device that delivers localized, controlled doses of secreted therapeutic cell products to selected tissue areas for a defined period of time.

Exemplary aspects of the invention provide for implantation of monocytic cells (e.g., monocytes) by placing a defined number of cells in a defined size matrix that confines the cells within the matrix and delivering the matrix to a target tissue. In this way, the cells (e.g., monocytes) remain in the intended placement location independently of their receptor mediated binding capacity and the dosage of therapeutic proteins secreted and defused to the target tissue can be controlled. In preferred embodiments, monocytes engrafted in a matrix of the invention form an implantable device to be used in a controlled, defined and predictable manner, making it highly advantageous over naked monocytes.

The plasticity of monocyte differentiation and the underlying gene expression and product secretion can be manipulated by activating monocytes towards a specific Ml or M2 phenotype. This will result in the secretion of specific sets of proteins associated with a desired phenotype. When the secretion of those proteins is controlled and localized, a therapeutic benefit may be obtained. This can be achieved by a) polarizing before and during containment: directing the monocytes to a specific phenotype where desired therapeutic factors are secreted by the cells; b) injection and protection: delivering the monocytes in a way that will result in local delivery of the monocyte derived therapeutic factors while preventing direct interaction of the cells with the target tissue; c) localizing: keeping the cells in place and preventing their migration (monocytes as well as other cells tend to migrate rapidly from site of delivery); and d) maintenance: maintaining the cells in the desired phenotype for the duration of the treatment (avoid their premature differentiation due to exposure to local cytokines, contact with local tissue and local conditions).

One embodiment of the present invention provides a method for preparing monocytes (e.g., by separating monocytes from the blood), differentiating monocytes into a particular phenotype, and placing monocytes in a hydrogel matrix under conditions which allow for the delivery of the cells to a specific location in the body for therapeutic purposes. The monocytes are prevented from direct contact with the cells of the tissue to be treated. This allows therapeutically effective, monocyte-produced factors to diffuse through the matrix and reach the tissue, while preventing the rapid migration of the monocytes from the site of delivery. This provides an effective use of monocytes as producers of therapeutic agents for the treatment of different indications. It also provides a means to keep a physical separation between the delivered cells and the organism being treated, in order to avoid host versus graft reactions by the cells producing the therapeutic agents.

In a preferred embodiment, the invention provides a semisolid matrix comprising a hydrogel material, with monocytes embedded within the matrix and producing a secreted product, wherein the matrix is permeable to the secreted product.

I. Definitions

In order that the present invention may be more readily understood, certain terms are first defined.

Monocytes are mononuclear phagocytic leukocytes formed in the bone marrow that transport to tissues where they develop into a wide variety of cells, including macrophages. The term "monocytic cell" as used herein refers both to monocytes and any cell terminally differentiated from monocytes (e.g., macrophages), as well as cells capable of differentiating into monocytes. Thus, the term "monocytic cell" includes not only differentiated monocytes, but also pluripotent stem cell and committed progenitor cells which differentiate into monocytes, as well as other effector cells which terminally differentiate from monocytes (e.g., macrophages and/or mononuclear phagocytes, and the like). These other cells are well-known and have been previously described (Zhao, et al., Proc Natl Acad Sci USA, 2003, 100:2426-31, Strauss-Ayali, et al, J Leukoc Biol, 2007, 82:244-52, Tacke and Randolph, Immunobiology, 2006, 211:609-18, Vega, MA et al, Inmunologia 2006, 25(4): 248-272). The term "monocytic cell" also includes monocyte-derived multipotential cells (MOMC), which can display morphological and phenotypic features of endothelial and mesenchymal cells (Seta and Kuwana, Keio J Med, 2007, 56:41-47).

The terms "Ml monocyte" and "monocytes having a Ml phenotype" are used interchangeably and each refers to exhibiting an inflammatory and/or phagocytic phenotype. Ml monocytes are generally recognized by increased expression and/or secretion of one or more inflammatory markers or cytokines and/or by decreased expression and/or secretion of one or more immunoregulatory markers and/or cytokines. Patterns of expression or secretion of Ml -specific markers and/or cytokines are also referred to as Ml monocyte profiles. Ml monocytes are generally characterized as being TNF-α^high, IL-I ^Uφ, IFN-γ¹¹¹⁸¹¹, and/or VEGF ^low In some embodiments, Ml monocytes can further be characterized as being IL-12^hieh, IL-10^low , and/or IL-23^high (Alberto Mantovani, Blood, 2006, Vol. 108, No. 2, 408-409). Ml monocytes can also be characterized by the type of response generated, for example, generation of a Th type 1 (ThI) response, a cytotoxic response (direct or indirect) and/ an inflammatory response (e.g., a type I inflammatory response).

The terms "M2 monocyte" and "monocytes having a M2 phenotype" are used interchangeably and each refers to monocytes exhibiting an immunoregulatory (e.g., immunosuppressive) phenotype and/or an angiogenic phenotype. M2 monocytes are generally recognized by increased expression and/or secretion of one or more immunoregulatory markers and/or cytokines and/or by decreased expression and/or secretion of one or more inflammatory markers or cytokines. M2 monocytes are generally characterized as being TNF-α ^low, IL- 1 ^low, IFN-γ ^low and/or VEGF "*. M2 monocytes can further be characterized as being IL-IO¹"⁸¹¹ and/or TGF-β¹"⁸*¹. In some embodiments, M2 monocytes can optionally (or further) be characterized as being IL- 12^low, IL-IO¹"⁸¹¹ and/or IL-23^low. In some embodiments, M2 monocytes can optionally (or further) be characterized as being IL-12^low, IL-23^low, IL-IO*⁸*¹, TNF-α^low, IL-I receptor antagonist (IL-lra)^ωgh, decoy IL- 1 type II receptor** IL-lb^low, caspasel^low, scavenger receptor¹"⁸¹¹, mannose receptor¹"⁸¹¹, or galactose-type receptor¹"⁸¹¹. Orientation of arginine metabolism to ornithine and polyamine can also signify the M2 monocytic phenotype. M2 monocytes also typically promote a Th type 2 response. Immunoregulatory activity and/or the inflammatory phenotype (e.g., type II inflammation) are also characteristic of M2 monocytic cells.

The terms "high" or "low", when used to characterize marker or factor secretion or expression refers to an increase or decrease, respectively in the marker or factor as compared to an appropriate control. For example, the term "high" can refer to a 50% increase in a level of expression or secretion of marker or factor as compared to an appropriate control. The term "high" can refer to a 100% (i.e., 2-fold) increase in a level of expression or secretion of marker or factor as compared to an appropriate control. The term "high" can also refer to 3-, 4-, 5-, 10-, 50-, 100- or greater fold increase in a level of expression or secretion of marker or factor as compared to an appropriate control. The term "low" can refer to a 50% decrease in a level of expression or secretion of marker or factor as compared to an appropriate control. The term "low" can refer to a 100% (i.e., 2-fold) decrease in a level of expression or secretion of marker or factor as compared to an appropriate control. The term "low" can also refer to 3-, 4-, 5-, 10-, 50-, 100- or greater fold decrease in a level of expression or secretion of marker or factor as compared to an appropriate control. Appropriate controls include, but are not limited to monocytic cells (e.g., monocytes of a different cellular phenotype (e.g., Ml versus M2 monocytes), monocytes of a different sub-phenotype, monocytes of a certain cellular phenotype versus a specific sub-phenotype, activated versus unactivated monocytes, cells of a different bioactive phenotype (see below), as well as predetermines values and/or levels.

Certain of the above-described factors (e.g., cytokines) or markers can also be determinative or characteristic of a particular bioactive phenotype, for example, an angiogenic and/or immunosuppressive phenotype. In exemplary embodiments, VEGF¹"⁸¹¹, optionally in addition to other M2 markers, is indicative of an angiogenic phenotype. In other embodiments, TNFα^low, optionally in addition to other M2 markers, is indicative of an angiogenic phenotype. Inverse levels or profiles are generally indicative of an inflammatory phenotype, including, for example, TNFα^hlgh and/or VEGF^low. Preferred markers of an immunosuppressive phenotype include, but are not limited to IL- 10"* TGF-β¹^.

The term "profile", as used herein, refers to a set of characteristics of a monocytic cell, the detection of which provides information as to the activity of that monocytic cell. A characteristics of the set may be the secretion of one or more compounds by the cell; the absence of detectable secretion of one or more compounds by a cell; a change in the amount or concentration of the secretion of one or more compounds by the cell; a change in the level of secretion of one or more compounds by a cell relative to the secretion of one or more other compounds by that cell or by another cell; or any effects or conditions induced, created, reduced or ended in cells or tissues adjacent to or in the vicinity of the monocytic cell. As used herein, the term "angiogenic" means relating to angiogenesis. As used herein, the term "angiogenesis", used interchangeably with "vascularization", refers to the process of growth or formation of new blood vessels from pre-existing vessels. Angiogenesis or vascularization provides tissue with, for example, blood supply and nutrients.

As used herein, the term "immunosuppressive" means relating to immunosuppression. As used herein, the term "immunosuppression" refers to the process of suppressing an organism's immune response, for example, an immune response to an allogenic implant, etc.

As used herein, the term "inflammatory" means relating to inflammation. The term "inflammation" refers to the process by which vascular tissues responds to harmful stimuli, such as pathogens, damaged cells, or irritants. "Inflammation includes, but is not limited to secretion of and response to inflammatory factors, e.g., inflammatory cytokines.

The term "polymer" as used herein refers to any monomer or polymer molecular species that can be polymerized to form a matrix of the invention. As such, a solution containing one or more polymers may contain a chemical species comprising one subunit of a polymer compound (e.g., a monomer) or two or more subunits covalently linked with each other. In some embodiments, a polymer for use in creating matrix of the invention will have one or more reactive groups per molecule. In some embodiments, a polymer may have an essentially linear structure. In some embodiments, a polymer may have a branched structure, comprising at least one branch point from which two or more portions of the polymer molecule originate.

The term "linker" as used herein refers to connections between molecules of the polymer network wherein one or molecules is bound or physically associated with two or more other molecules of the polymer network simultaneously.

The term "matrix" as used herein refers to a liquid, semi-solid or solid polymer substance (e.g., cross-linked polymeric substance) that has the capacity to comprise cells. In preferred embodiments, the term "matrix" refers to a biodegradable hydrogel that may be in any polymerization state.

The term "hydrogel" as used herein refers to a polymeric substance that absorbs at least 90% of its weight in water. The term "semisolid matrix" as used herein refers to a composition of matter that has a rigidity and viscosity intermediate between a solid and a liquid e.g., a gel. Biocompatible materials are generally considered to be materials that perform with an appropriate host response in a specific application, with the additional quality of not having toxic or injurious effects on biological systems. The term "biocompatible" as used herein refers to the ability of a hydrogel to perform with an appropriate host response when delivered as described in the present invention.

The term "activation" as used herein refers to the induction of monocytic cells, for example, monocytes (e.g., unactivated monocytes, previously activated monocytes, and the like) towards a differentiated phenotype as determined by a change in the secretion of one or more proteins characteristic of said phenotype. Activation may be caused by the addition of exogenous agents to the monocytes or the matrix containing the monocytes, as well as by conditions in the matrix. The term "activation" also includes spontaneous differentiation caused by placing untreated monocytes in a matrix of this invention.

The term "polarization" as used herein refers to a phenotypic shift in the behavior of a monocyte that can be initiated by introduction of one or more compounds to the monocyte and/or by a change in the local micro environment of the monocyte. As used in particular embodiments, the polarization of a monocyte will lead to a transient state exhibited by the upregulation of production and secretion of one or more particular compounds and the downregulation of the production and secretion of one or more other compounds. Polarization can lead to a transient Ml or M2 state for monocytes. In exemplary embodiments of the invention, polarization may be actively induced before incorporation of cells into a matrix of the invention. In other embodiments, a matrix of the invention can be designed to induce polarization of cells after the cells have been incorporated into a matrix of the invention. The term "delivery" as used herein refers to the introduction or transport of cells into an organism. In exemplary embodiments of the invention, cells are contacted with a polymer-based matrix before delivery. Delivery may be achieved by any means of introduction of a substance to an organism, including by injection; by application to a surface or membrane of an organism, organ system, organ or tissue; by oral gavage; by insertion; by ingestion; by inhalation; and by implantation. The term "delivery" as used herein also refers to the introduction or transport of bioactive compounds into a cell or organism. In exemplary embodiments of the invention, delivery of bioactive compounds is via cells and/or matrices of the invention described herein. The term "protection" as used herein refers to the differential effects of delivery on monocytes encompassed by a matrix as opposed to naked monocytes delivered to the same location in an organism. Protection may refer to the effects, or lack thereof, on monocytes that occur due to exposure to host compounds, host cells, host signals or aspects of the local microenvironment created within the host in a normal state or a diseased state. Protection may also refer to the differences in the microenvironment of a monocyte encompassed by a matrix as compared to a monocyte under the same circumstances that is not encompassed by a matrix. Protection can refer to positive effects that increase the activity of a cell encompassed by a matrix and to negative effects that decrease the activity of a cell encompassed by a matrix. Protection can refer to a promotion, a prolonging or a shortening of a phase, activation, phenotype or other state of activity or behavior of a cell in a matrix.

The term "microenvironment" refers to the physical conditions and chemical composition of the area immediately surrounding a cell or a group of cells. Aspects of the microenvironment may include: the amount of physical stress on a cell; other aspects of the physical environment, including temperature and physical state; and the identity, concentration and rate of change in concentration over time of nutrients, metabolites, hormones, growth factors, adhesion molecules and other molecules contacting cells, drugs and any other compound or species. The microenvironment of a cell can be impacted by activity of that cell, by the activity or presence of adjacent cells (either within or outside of a matrix), by the activity or presence of cells and tissues outside of a matrix and by any organs, tissues or cells that are present or active in an organism. The microenvironment of a cell can be impacted by treatment of the cell before incorporation into a matrix, by an interaction between the cell and a matrix, by the formula, content and method of creating of a matrix, and by the method of polymerization, introduction of cells into and delivery of a matrix.

The term "introduction" as used herein refers to the mixing or contacting of two or more elements. The term "embedded" refers to the result of the introduction of cells and polymer matrix wherein the majority of the cells are contained within the mass of the polymer matrix.

Some embodiments of the invention feature an implantable, semisolid matrix that includes both hydrogel material and embedded monocytic cells. The monocytic cells within the matrix are able to produce a secreted product. The matrix can be permeable to the product, so that the product will diffuse through the gel and can move out of the matrix.

Additional embodiments of the invention include an injectable composition that includes a hydrogel and monocytic cells that are able to produce a secreted product. The injectable composition is able to be converted to a semi-solid state. When in its semisolid state, the injectable composition is permeable to the product of the monocytic cells. The product can diffuse through the semi-solid injectable composition and into the surrounding area. In some embodiments, the semi-solid state is induced by a change in the surrounding environment, such as a change in temperature or pressure or some form of physical stimulus. In some embodiments, the semi-solid state is induced by ionizing radiation, ultraviolet radiation and/or some other form of electromagnetic or radiated energy. In some embodiments, the semi-solid state may emerge over time. The emergence of a semi-solid state may be due to a manipulation of the injectable composition after the composition is formulated or may be due to the formulation steps themselves.

In some embodiments of the invention, a monocytic cell of the invention is capable of producing one secreted product. In some embodiments, a monocytic cell of the invention is capable of producing two or more secreted products.

In some embodiments, one or more of the secreted products that a monocytic cell of the invention can produce is an angiogenic factor. Some angiogenic factors that a monocytic cell of the invention may produce in some embodiments are vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), platelet derived growth factor (PDGF), hepatocyte growth factor/scatter factor (HGF/SF), epidermal growth factor (EGF) and Interleukin-8 (IL-8). Some embodiments of the invention feature a monocytic cell that can produce one or more immunosuppressive factors, such as IL-4, IL-IO and/or TGFβ.

Embodiments of the invention may comprise any one or more of a number of different monocytic cells. For example, in some embodiments, a matrix of the invention comprises a monocyte precursor cell, such as a bone marrow cell and/or a monocyte progenitor cell. Some embodiments feature matrices with activated monocyte. In some embodiments, monocytic cells are activated and/or treated before introduction of the cells to a matrix of the invention. In some embodiments, monocytic cells are not activated nor treated before introduction of the cells to a matrix of the invention. Monocytic cells of some embodiments are activated towards a M2 phenotype. In some embodiments, monocytic cells activated towards a M2 phenotype produce one or more marker of a M2 phenotype. Monocytic cells of the invention may produce markers of a M2 phenotype that comprise a profile of a M2 phenotype. In some embodiments, a marker or a profile that indicates the presence of a monocytic cell of an M2 phenotype may also indicate the presence of a monocytic cell of a particular M2 subphenotype, such as M2a, M2b or M2c.

Monocytic cells of the invention may be activated or polarized towards a phenotype and/or subphenotype before introduction of a monocytic cell to a matrix of the invention. In some embodiments, a matrix of the invention includes a polarizing or activating agent before a monocytic cell is introduced to the matrix. In some embodiments, a monocytic cell becomes activated or polarized after introduction to a matrix of the invention by virtue of activating or polarizing compounds in the matrix to which the cell is exposed after introduction. In some embodiments, the activating or polarizing compounds in a matrix of the invention will induce a M2 phenotype or subphenotype in a monocytic cell. In some embodiments, the M2 phenotype or subphenotype will be angiogenic. In some embodiments, the M2 phenotype or subphenotype will by immunosuppressive.

In some embodiments, a monocytic cell will be activated or polarized towards a Ml phenotype. Monocytic cells of the invention may display one or more markers of a Ml phenotype and may produce a marker of a Ml phenotype at a level that is increased in comparison to one or more markers of a M2 phenotype. In some embodiments, matrices of the invention feature the inclusion of agents and/or compounds that activate or polarize a monocytic cell towards a Ml phenotype. A monocytic cell that is pre- activated or pre-polarized towards a Ml phenotype at the time of introduction to a matrix of the invention is a feature of some embodiments.

In some embodiments, a monocytic cell is a macrophage. Cells in some embodiments may come from the same organism to which the matrix is later delivered, i.e., autologous cells. Cells in some embodiments may come from a different organism of the same species as the organism to which the matrix is later delivered, i.e., allogenous cells. Cells in some embodiments may come from an organism of a different species than the organism to which the matrix is later delivered, i.e., xenogenous cells. Cells in some embodiments, coming from an autologous, allogenous or xenogenous source, may be genetically manipulated or modified before, during or after introduction of the cells to a matrix of the invention.

Some embodiments of the invention feature hydrogels that comprise one or more ingredients, such as polymers, cross-linkers, compounds that can affect the physical or chemical properties of a matrix of the invention and compounds that can affect the cells embedded within, adjacent or in proximity to a matrix of the invention. Some embodiments feature one or more polymers, including polylactic acid, polyglycolic acid, other polyhydroxy acids, copolymers of two or more polyhydroxy acids, polyorthoesters, polyanhydrides, gelatin, collagen, cellulose, derivatized cellulose, chitosan, alginate, thiol-modified hyaluronan, and combinations or copolymers thereof. Some embodiments that feature a polyhydroxy acid comprise a polyhydroxy acid that is a polylactic acid or a polyglycolic acid. Some embodiments of the invention include cross-linkers, including glutaraldehyde, diphenylphosphoryl azide, transglutaminase,, dimethyl suberimidate, DMS-treated collagen, dimethyl 3,3'-dithiobispropionimidate, multivalent ions, calcium ions, N,N methylene-bisacrylamide (MBA), acrylamide, allyl methacrylate, ethylene glycol dimethacrylate, tripolyphosphate, and combinations thereof.

Embodiments of the invention include methods for delivering a secreted product to a site, location or localized site. These methods include the delivery of a matrix or a composition of the invention to a localized site and maintaining the matrix or composition at that site for a sufficient period of time to allow the secreted product to interact with the localized site. In exemplary embodiments, the methods feature delivery of a matrix or composition of the invention under conditions that promote angiogenesis and/or immunosuppression. In particular embodiments of the invention, a number of different diseases or conditions may be addressed or treated by a composition of the invention, including coronary artery disease, peripheral artery disease, limb ischemia, ischemic wound, ischemic ulcer, ischemic bowel disease, atherosclerotic ischemic disease, muscle flaps, skin flaps, organ transplant, nasolabial folds, wrinkles, conditions which result in scar formation, conditions requiring plastic surgery and conditions requiring a cosmetic procedure. In some methods of the invention wherein an ischemic condition or disease is addressed or treated, a angiogenic secreted product is delivered for a sufficient time to prevent or lessen the damage, harm or effects of a perfusion injury associated with the ischemic condition or disease. Some methods of the invention are useful to address or treat a cardiac ischemic condition or disease. In some embodiments of methods of the invention for treating or addressing an ischemic condition or disease, the composition of the invention is injectable.

Methods of the invention include methods for the treatment of folds or wrinkles in the outer layers or epidermis of an organism, including methods for reducing nasolabial folds and/or wrinkles. In some embodiments, the method of the injection comprises the injecting of a composition into the nasolabial fold or wrinkle or into an adjacent area.

Some methods of the invention feature the reduction of an immune response at a localized site in a subject by the introduction, insertion or injection of a composition to the site and allowing the composition to deliver a product that is able to reduce the immune response in the area.

Kits that have a hydrogel material of the invention and instructions for using the hydrogel material in order to form a semisolid matrix embedded with monocytic cells are also features of some embodiments of the invention. The monocytic cells may have a M2 phenotype in some embodiments. The monocytic cells may also secrete a product at a level that is higher than the secretion usually seen in monocytic cells that have a Ml phenotype.

Kits of the invention may also have a cross-linking agent. In some embodiments, kits include a hydrogel material or ingredient, monocytic cells, one or more cross-linkers and instructions for combining kit materials to produce an injectable liquid. In some embodiments, the injectable liquid will, at some time post-mixing, transition into a semisolid or solid matrix . In some embodiments, the injectable liquid can be injected into an organism and will form a semi-solid matrix after injection. In some embodiments, the instructions have information on how to pre-activate the monocytic cells towards an M2 phenotype and some embodiments may include such an agent as a component of the kit.

Embodiments of the invention can include hydrogels that are formed from one or more ingredients, including polylactic acid, polyglycolic acid, other polyhydroxy acids, copolymers of two or more polyhydroxy acids, polyorthoesters, polyanhydrides, gelatin, collagen, cellulose, derivatized cellulose, chitosan, alginate, thiol-modified hyaluronan, and combinations thereof. In some embodiments, a hydrogel of the invention includes one or more cross-linkers, which may be glutaraldehyde, diphenylphosphoryl azide, transglutaminase, dimethyl suberimidate, DMS-treated collagen, dimethyl 3,3'- dithiobispropionimidate, multivalent ions, calcium ions, N,N methylene-bisacrylamide (MBA), acrylamide, allyl methacrylate, ethylene glycol dimethacrylate, and tripolyphosphate.

π. Selection and Isolation of Cells

The present invention features monocytic cells, e.g., monocytes, for use in delivery of bioactive compounds, e.g., therapeutic factors, to subjects in need thereof. Monocytes are white blood cells originating from pluripotent stem cells in bone marrow. These pluripotent cells must first differentiate into committed progenitor cells, then into monocytes. Monocytes can also further differentiate into other effector cells, such as macrophages. As used herein, the term "monocytic cell" includes not only differentiated monocytes, but also pluripotent stem cell and committed progenitor cells which differentiate into monocytes or any other cell type, as well as other effector cells which terminally differentiate from monocytes, progenitor cells derived from circulating blood and bone marrow cells. As used herein, the term "monocyte" refers to a monocytic cell committed to differentiated, or at least partially differentiated, into the monocytic phenotype, for example, secreting or expressing markers (or marker profiles) characteristic of monocyte differentiation. Monocytes can be fully differentiated into the monocytic phenotype and, in certain instances, can be further differentiated into cells derived therefrom.

Monocytic cells of the invention can be isolated, purified, partially purified, or found enriched in unfractionated bone marrow cells (BMC), fractionated BMC, bone marrow-derived stem cells and bone marrow derived progenitor cells. Monocytes useful in the present invention can also be isolated from the whole blood of an individual of either the same species as the patient to be treated with the matrix ("donor"), or a compatible xenogenic species. In some embodiments, the monocytes are autologous and the donor is the patient to be treated with the matrix. In other embodiments, the monocytes are allogenic and may be obtained from a blood bank or another donor. Using allogenic monocytes allows for greater availability, as such monocytes may be obtained from blood banks and the like. Non-autologous monocytes can be used in the matrices and methods of this invention because the monocytes are embedded in the matrix and therefore not exposed to the patient's immune system. Additionally, factors such as IL-IO secreted by the non-autologous monocytes suppress a potential immune response by the patient. Both allogenic and autologous monocytes may be stored for a period of time prior to use under standard blood bank storage conditions that preserve the ability of the cells to survive when incubated under appropriate conditions. In some embodiments, monocytes are obtained from the patient by separation from whole blood drawn from the patient in an amount sufficient to produce the number of monocytes desired for the treatment. Alternatively, monocytes can be taken from donor blood (allograft). Patients or blood donors can be treated to increase circulating monocyte number by treatment with granulocyte-colony stimulating factor (G-CSF), AMD3100 or other agents which simulate cell mobilization from the bone marrow to the peripheral blood for several days prior to blood drawing. Monocytes can be separated from whole blood by different separation techniques which are well known in the art. These may include, but are not limited to, positive selection by specific antibodies targeting monocyte specific surface markers such as CD14 and CDl Ib; negative selection by specific antibodies targeting specific surface markers of blood cells other then monocytes; gradient centrifugation and matrix adherence. In particular embodiments of the present invention, monocytes can be used alone or in combination with other cells with in the matrix. The use of monocytes with other cells includes but is not limited to non specific separation methods such as "buffy coat" taken from gradient centrifugation separation techniques such as used for the enrichment of BMCs or peripheral blood monocytes

Cells for use with embodiments of the invention include host cells (autologous) and cells obtained or isolated from allogenic or xenogenic biological samples. Particular embodiments include leukocytes and other cells that interact with a variety of different leukocyte adhesion molecules. In particular embodiments, cells can be selected and/or isolated by virtue of their expression of or interaction with one or more leukocyte adhesion molecules or the counter-receptors or ligands of these molecules, including selectins (such as L-selectin (CD62L), P-selectin (CD62P) and E-selectin (ELAM-I and CD62E)), members of the immunoglobulin adhesion molecule superfamily (such as integrins and VLA proteins) and intercellular adhesion molecules (such as ICAM-I, ICAM-2 and ICAM-3), for example. In certain embodiments, cells are isolated from an organism at a time immediately prior to embedding in the matrix. In certain embodiments of the invention, cells are isolated from an organism and then undergo culturing and/or treatment prior to embedding in a matrix.

DI. Characterization and Treatment of Cells

The present invention features the use of cells, in particular, monocytic cells (e.g., monocyte precursor cells, monocytes, and/or cells differentiated therefrom) for the delivery of bioactive compounds (e.g., angiogenic factors, immunosuppressive factors, and the like) to tissues of a subject.

Monocytic cells, in particular monocytes, can be directed to exhibit unique functional properties by changes in their cellular environment. The ability of monocytes to exhibit different functional phenotypes is usually described as "activation". These phenotypes have been categorized into four functional phenotypes (Ml, M2a, M2b and M2c) (Mantovani, A et al, Trends Immunol 2004, 25: 677-686; Mantovani, A et al, Immunity 2005, 23:344-346; Mosser, DM, J Leukoc Biol 2003, 73:209-212). Ml polarization produces monocytes with potentiated cytotoxic properties capable of producing large amounts of pro-inflammatory cytokines, expressing high levels of major histocompatibility complex (MHC) molecules, and is implicated in the killing of pathogens and tumor cells. M2 polarized monocytes are more prominently involved in immunoregulation, immunosuppression, encapsulation and containment of parasites, tissue repair, tissue remodeling and angiogenesis (Mantovani, A. et al, Trends Immunol 2004, 25: 677-686; Mantovani, A et al, Immunity 2005, 23:344-346; Mantovani, A et al, Eur J Immunol 2007, 37:14-16; Pinhal-Enfield, G et al, Am J Pathol 2003,163:711-721; Zhao, Y et al., Proc Natl Acad Sci USA 2003, 100:2426-2431 ; Mantovani, A, Blood 2006, 108(2):408-409).

The M2 phenotype includes various forms of monocyte activation. The M2 phenotype includes monocytes exposed to IL-4 or IL- 13, immune complexes, IL-10, and glucocorticoid hormones. The M2 phenotype generally shows a secretion profile of IL-I ^low, IFN-γ^l0W and/or VEGF*⁸¹¹. M2 monocytes may also have an JL-\O^iύφ and/or TGF-β ^Wgh profile (optionally combined with the above profile in whole or in part). Various versions of M2 monocytes can also share an IL-12^low, IL-23 ^low, JL-l⁽f^φ, TNF-α^low phenotype or profile (in whole, in part or combined in whole or in part with an above profile). They can also have high levels of scavenger, mannose, and galactose-type receptors, for example, as part of a profile. M2 monocytes can also orient arginine metabolism to ornithine and polyamine, which is involved in growth promotion. M2 monocytes can also be IL-I receptor antagonist (IL-lra)¹"⁸*¹, decoy IL-I type II receptor¹"⁸¹¹, EL-lb^low and caspase l^low . M2 monocytes also typically promote a Th type 2 response. Immunoregulatory activity and/or the inflammatory phenotype {e.g., type II inflammation) are also characteristic of M2 monocytic cells (Mantovani, A et al., Trends Immunol, 2004, 25:677-86; Mantovani, A et al., Immunity, 2005, 23:344-6; Mantovani, A et al., Eur J Immunol, 2007, 37: 14-6; Pinhal-Enfield, G et al., Am J Pathol, 2003, 163:711-21; Zhao, Y et al., Proc Natl Acad Sci USA, 2003, 100:2426-31), for example, as part of a profile.

The various versions of the M2 phenotype may be sub-divided into three subphenotypes, all of whom share the common characteristic of lacking features of the inflammatory Ml phenotype, such as a high capacity for presenting antigen, characteristic inflammatory cytokine production (e.g., TL-U^, IL-23^high, IL-10^low ) and high production of toxic reactive compounds such as nitric oxide and reactive oxygen species. High levels of IL-10 secretion are also a common characteristic of the M2 subphenotypes. Although they share common characteristics, each of the M2 subphenotypes are distinguishable by particular subphenotype inducers, particular secretion profiles and particular activities and purposes for the subphenotype. The M2a subphenotype is particularly induced by exposure to IL-4 and IL- 13 and is involved in allergic and anti-parasitic responses of the immune system. The M2b subphenotype is particularly induced by exposure to immune complexes and contributes to immune system regulation and suppression of immune system activity. Exposure to IL-10 particularly induces the M2c phenotype, which is involved in tissue repair and remodeling, including angiogenesis, as well as modulation of immune reactions. The M2 subphenotypes also differ in their secretion profiles. For example, monocytic cells of a M2a phenotype will secrete prominent levels of IL- Ira, whereas EL-I and EL-6 secretion is a feature of the M2b phenotype and secretion of high levels of TGFβ is a marker of the M2c phenotype (Mantovani, A et al., Trends Immunol, 2004, 25:677-86).

Monocytic cells for use in the present invention can be activated by employing biological or chemical inducers. In some embodiments, inducers of a polarized state, phenotype and/or subphenotype may be included in the matrix. Monocytic cells for use in the present invention can be genetically modified to express specific proteins such as monocyte chemoattractants, growth factors and therapeutic proteins. The treatment of monocytes can be done before or after embedding them in the matrix by adding biological or chemical inducers or by incorporating the inducers into the matrix.

A. Polarization

In some embodiments, the monocytes in the matrices of this invention are polarized towards an M2 phenotype. In one embodiment, the monocytes have an M2 phenotype at the time they are combined with one or more of the matrix components to form the matrix. This may be achieved by in vitro exposure of monocytes to appropriate M2 polarizing conditions prior to transferring them into a hydrogel matrix and before placing the matrix containing the monocytes at the location in the body were the therapeutic effect is needed. In some embodiments, monocytes are unpolarized before introduction to a matrix. In some embodiments, monocytes are polarized before introduction into a matrix. In some embodiments, monocytes that are unpolarized before introduction to a matrix become polarized as a result of becoming embedded in the matrix. In some embodiments, previously unpolarized monocytes that become polarized upon matrix embedding do so due to the micro environment of the monocytes within the matrix. In some embodiments, previously polarized monocytes are further polarized as a result of becoming embedded in the matrix (e.g., due to the microenvironment in the matrix). The microenvironment in the matrix may be due to inherent properties of the polymer itself and may be due to compounds incorporated into the matrix that are not required for its structural properties. The microenvironment in a matrix may be due to influence on the matrix by the organism that manifests upon delivery or at some time after delivery. The microenvironment may be due to the activity of cells in the matrix and may be the result of the production of homologous or heterologous compounds by cells in the matrix or exterior to the matrix.

In some embodiments it may be beneficial to block the activation of monocytes towards the Ml phenotype. This can be achieved, for example, by blocking the CD40 receptor, which is involved in Ml activation through interaction with cytokines like TNFα and BL-2. This can be achieved by adding CD40 inhibitors such as Trapidil, specific CD40 blocking antibodies, peptides or specific CD40 siRNA (Pluvinet, et al, Blood, 2004, 104:3642-6) into the matrix and/or during the polarization stage. The phenotype of monocytes for inclusion in a matrix may be evaluated by detection or measurement of compounds produced by the monocytes. Monocytes of a particular phenotype or subclass of a phenotype will exhibit a particular profile of secreted compounds. Monocytes with a Ml phenotype typically exhibit a profile featuring the upregulation of production of TNFα, IL-I, and/or IFNγ and a downregulation of production of VEGF. Monocytes with a M2 phenotype typically exhibit a profile featuring the upregulation of EL-IO, TGFβ and/or other antiinflammatory cytokines, in addition to growth factors such as VEGF, and a downregulation of TNFα, IL-I and/or IFNγ. As used herein, the term "VEGF" refers to "vascular endothelial growth factor". VEGF stimulates vascular endothelial cell growth, survival, and proliferation. This protein plays an important role in angiogenesis. The generic term "VEGF" commonly refers to VEGF-A, although there are at least six known VEGF family members, known as VEGFs A-E and placental growth factor (PlGF). VEGF A is of primary importance in the present invention due to its established role in angiogenesis and vascular maintenance. VEGF-A binds the receptors VEGFR-I, VEGFR-2 and neuropilin-1. VEGF-E and PlGF have also been implicated in angiogenesis. There are 4 major iso forms of VEGF-A (VEGF), each coded for by a different portion of the VEGF gene. These isoforms are VEGF121, VEGF 165, VEGFl 89, and VEGF206. Although these isoforms behave identically in solution, they differ in their ability to bind heparin and the extracellular matrix.

Monocyte phenotype may be evaluated in some embodiments by a measurement of the production of one, some or all of the factors that are characteristic of a monocyte of a particular phenotype. In some embodiments, the achievement of an angiogenic M2 phenotype is evidenced by an upregulation of VEGF. In some embodiments, the achievement of an immunosuppressive M2 phenotype is determined by the detection of an upregulation of BL-IO production that is greater in magnitude than a concurrent upregulation of TGFβ. In some embodiments, the achievement of an inflammatory Ml phenotype is evidenced by an upregulation of TNFα production.

Polarization of monocytes may take place before and/or after introduction of the cells to a matrix, in some embodiments. The polarization of monocytes before and/or after introduction to a matrix can be achieved by exposure of the cells to one or more polarizing agents, such as agonists and activators of adenosine receptors and/or To 11- like receptors. Polarization in the matrix can be achieved, for example, by inclusion of one or more polarizing factors in the matrix, or by other means of controlling the cellular microenvironment in the matrix.

Adenosine is a purine nucleoside released from hypoxic and ischemic tissues, where it acts via 4 subtypes (Al, A2A, A2B, A3) of G protein-coupled cell surface receptors to restore homeostasis by increasing blood supply and decreasing energy demand. Adenosine receptor activation was demonstrated to induce angiogenesis in vivo (Adam N. Clark, 2007, Circulation Research; 101 ;1130-1138). Adenosine receptors activation can be used in order to activate monocytes towards the angiogenic phenotype and are useful in reducing the inflammatory characteristics such as of TNFα expression when administrated together with inflammation inducers such as toll like receptor agonists to benefit an angiogenic effect (Grace Pinhal-Enfield, 2003, American Journal of Pathology; 163:711-721). In the present invention any of the adenosine receptors Al, A2A, A2B, A3 or their combinations can be targeted by specific agonists in combination with any of IL-10, IL-4, IL-13, an IL-I receptor ligand, PGE2, TGF-β, TNFα, lactic acid and lactic acid analogues, lipoteichoic acid, NADH dehydrogenase subunit 1, poly (adenosine diphosphate-ribose) polymerase, pyruvate, hydrogen ions, Colony Stimulating Factor-1, TLRl, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLRlO, pyruvate and pyruvate analogues, hypoxic conditions, acidic conditions, and combination thereof to increase the angiogenic potency of monocytes and/or to reduce the level of inflammatory cytokines such as TNFα.

In some embodiments, a combination of the adenosine receptor agonist NECA and the Toll-like receptor activator LPS is used to polarize monocytes towards a M2 phenotype. In some embodiments, a combination of the adenosine receptor agonist NECA and the Toll-like receptor activator CLO97 is used to polarize monocytes towards a M2 phenotype. In some embodiments, lactic acid may be used to polarize monocytes towards a M2 phenotype. In some embodiments, a change in physical conditions in the microenvironments is used to polarize monocytes towards a phenotype, such as the reduction in molecular oxygen tension to polarize monocytes towards a M2 phenotype.

In some embodiments, the polarized phenotype of a monocyte will effectively continue for at least up to 10 days to 2 weeks. In some embodiments, the polarized phenotype will effectively continue for at least up to 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 weeks. In a preferred embodiments, the polarized phenotype of a monocyte embedded in a matrix will continue at least or up to 100 days or more.

In some embodiments of the invention, any number of a range of polarizing agents may be used, including but not limited to: IL-10, DL-4, IL-13, an IL-I receptor ligand, PGE2, TGF-β, TNFα, lactic acid, lipoteichoic acid, NADH dehydrogenase subunit 1, poly (adenosine diphosphate-ribose) polymerase, pyruvate, hydrogen ions, Colony Stimulating Factor- 1, adenosine, an adenosine analogue, NECA, LPS, Pam3CSK4, E. coli LPS, R848, imiquimod, non-methylated CpGDNA, ODN2006, thioredoxin peroxidase, a CD-40 blocking compound, Trapidil, a CD40 blocking antibody, a CD40 blocking peptide, a CD40 siRNA, an IL-2 antagonist, a TNFα antagonist, a TLR2 agonist (e.g., Paπi3CysSerLys₄, peptidoglycan (Ppg), PamCys), a TLR3 agonist (e.g., IPH 31XX), a TLR4 agonist (e.g., Aminoalkyl glucosaminide phosphates [AGPs], E6020, CRX-675, 5D24.D4, RC-527), a TLR7 agonist (e.g., Imiquimod, 3M-003, Aldara, 852A, R850, R848, CL097), a TLR8 agonist (3M-002), a TLR9 agonist (Flagellin, Vaxlmmune, CpG ODN (AVE0675, HYB2093), CYT005- AUQbGlO, dSLIM), adenosine Al agonists (R-PIA, CPA TCPA, CVT-3146, CVT-510, GR 79236, WAG 994), adenosine A2 agonists (CGS 21680, APEC, 2HE-NECA), and/or adenosine A3 agonists (e.g., IB-MECA, CI-IB-MECA, 3'-Aminoadenosine-5'- uronamides). In some embodiments, any number of polarizing conditions may be used, including but not limited to: exposure to P. gingivalis, activation by an acidic pH (e.g., wherein the acidic pH is less than pH 7.4) and/or activation by hypoxic conditions.

B. Activity of Polarized Cells

Monocytes polarized in a M2 phenotype can secrete a number of different compounds which can affect the behavior and activity of cells in a matrix and in an organism. In exemplary aspects of the invention, the secreted compounds have a desired in vivo and/or therapeutic effect (e.g., an angiogenic and/or immunosuppressive effect) on cells or tissues surrounding the matrix (e.g., cells or tissue at an injection or implantation site). In certain aspects of the invention, delivery of compounds (e.g., angiogenic and/or immunosuppressive compounds) is performed under conditions sufficient to promote angiogenesis (providing blood supply, nutrients, etc.) to tissue, in particular, damaged tissues targeted by the methods of the invention. In preferred aspects, promotion of angiogenesis is without significant activation of an inflammatory or immune response. Successful promotion of angiogenesis (and/or immunosuppression) can be determined utilizing any one of a variety of assays described herein or in the art (e.g., histochemical assays, using light or electron microscopy; monitoring of blood flow using a laser Doppler perfusion imaging system (PeriScan PIM II, Lisca AB, Sweden); etc.). Activities can be assayed either in vivo (e.g., using labeled assay components and/or imaging techniques) or in vitro (e.g., using samples or specimens derived from a subject). Activities can be assayed either directly or indirectly.

In certain preferred embodiments, angiogenic endpoints (e.g., microvessel density post-treatment, changes in indicators of a hypoxic state, changes in VEGF expression, changes in eNOS and 5-HT₂A receptor expression, changes in levels of apoptosis, necrosis and mitotic scores, etc.) are assayed. Such endpoints can be assayed in living subjects (e.g., in animal models of angiogenesis or in human subjects, for example, undergoing therapies of the invention) using non-invasive detection methodologies. Alternatively, such endpoints can be assayed in subjects post mortem. Assaying such endpoints in animal models and/or in human subjects post mortem is useful in assessing the effectiveness of various agents (e.g., cell-embedded matrices) to be utilized in further or improved applications with other subjects. In other embodiments, certain clinical endpoints or parameters can be assessed as indicators of the above angiogenic and/or immunosuppressive activities or endpoints. For example, decrease in pain due to muscle ischemia, healing of ischemic ulcer, decrease in levels of circulating inflammatory or immune response factors, reduction of number and/or depth of wrinkles or folds, and other art-recognized endpoints can be assessed.

Compounds secreted by monocytes in a M2 phenotype may be naturally occurring compounds, which may be secreted in naturally occurring amounts. In some embodiments, monocytes are genetically engineered to produce compounds in the M2 phenotype that are not naturally occurring and/or produced in not naturally occurring amounts.

In one embodiment, the secreted product is a naturally occurring ("homologous") molecule characteristically produced by M2 monocytes. The term "characteristically produced by M2 monocytes" as used herein means a molecule that is secreted in greater quantity by a M2 monocyte as compared to an Ml monocyte. In another embodiment, the secreted product is a cloned ("heterologous¹) molecule that is produced by the M2 monocyte as a result of genetic manipulation of the monocyte (e.g., a DNA cloned into monocytes by standard molecular cloning techniques that directs transcription and/or expression of the secreted product). In certain embodiments, the secreted product is produced under the control of a promoter that is upregulated in an M2 monocyte as compared to a Ml monocyte. Examples of promoters that are upregulated in an M2 monocyte include, but are not limited to, promoters that control the expression of VEGF (e.g., VEGF 165), platelet derived growth factor (PDGF), IL-IO or TGF-β. When the secreted product is a homologous molecule, its production and secretion may be controlled by other naturally occurring factors produced by M2 monocytes, by heterologous factors produced by the M2 monocytes as a result of genetic manipulation, by pre-incubation of the monocytes with an exogenous factor that causes production of the secreted molecule, by inclusion in the matrix of an exogenous factor that causes production of the secreted molecule, or by any combination of the foregoing. In some embodiments, the exogenous factor will also be responsible for polarizing the monocytes to the M2 phenotype.

In a more specific embodiment, the injectable composition comprises monocytes having an M2 phenotype and producing at least one secreted product at a level that is increased as compared to monocytes having an Ml phenotype.

In certain embodiments, it is desirable for the monocytes to have a specific subtype of M2 phenotype. As with conversion to the M2 phenotype, subtype-specific polarization is achieved by incubation of the monocytes with specific agents and/or under specific conditions before embedding the monocytes in the matrix. Alternatively, subtype-specific polarization is achieved by inclusion of specific agents in the matrix containing the monocyte or by incubation of the matrix containing the monocytes under specific conditions.

In one embodiment, the M2 monocytes have an angiogenic phenotype. An angiogenic phenotype is characterized by the secretion of at least one angiogenic growth factor, such as vascular endothelial growth factor (VEGF) and/or platelet derived growth factor (PDGF). The angiogenic phenotype may be achieved by using conditions, reagents and combinations thereof known in the art. In a specific embodiment, an angiogenic phenotype is achieved by treating monocytes with a combination of Toll-like receptor 4 (TLR-4) agonist and adenosine receptor - A2AR agonist (Pinhal-Enfield, et al., Am J Pathol, 2003, 163:711-21); or by incubation in hypoxic conditions (e.g., oxygen concentration lower than normal (less than 20%), in more specific embodiments less than 10%, and in more specific embodiments ranging between 0.0001% and 5%).

In other embodiments the angiogenic phenotype is achieved by treatment of monocytes with one or more of the following agents: IL-10, IL-4, IL-13, IL-I receptor ligands, PGE2, TGF-β, TNFα, lactic acid, lipoteichoic acid, NADl (NADH dehydrogenase subunit 1), poly (adenosine diphosphate-ribose) polymerase, pyruvate, hydrogen ions, CSF-I (Colony Stimulating Factor- 1), adenosine, adenosine analogues, adenosine receptor agonists such as NECA, TLR2 agonist such as P. gingivalis, LPS or Pam3CSK4, TLR4 agonist such as E. coli LPS, TLR7 and TLR8 agonist such as R848 (resiquimod) or a water-soluble derivative thereof (e.g., CLO97), or imiquimod, TLR9 agonist such as non-methylated CpG DNA or ODN2006, or thioredoxin peroxidase.

In still other embodiments, the angiogenic phenotype is achieved by incubating the monocytes or the matrix containing the monocytes in medium having a pH lower then 7.4. In exemplary embodiments, the pH ranges between 3 and 7.3. In specific embodiments, the pH ranges between 3 and 6, between 3 and 5, or between 6 and 4. The successful differentiation towards the angiogenic M2 phenotype is determined, for example, by the secretion by the monocytes of one or more of VEGF (e.g., VEGF 165) and/or PDGF (e.g., PDGF-AA, PDGF-AB and/or PDGF-BB). In another embodiment, the M2 monocytes have an immunosuppressive phenotype. The immunosuppressive phenotype is achieved, for example, by contacting the monocytes or the matrix containing the monocytes with EL-IO.

In some embodiments the immunosuppressive phenotype is achieved by treatment of monocytes with one or more of the following agents: IL-10, IL-4, IL-13, PGE2, TGF-β , lactic acid, lipoteichoic acid, NADl (NADH dehydrogenase subunit 1), poly (adenosine diphosphate-ribose) polymerase, pyruvate, hydrogen ions, CSF-I (Colony Stimulating Factor-1), adenosine, adenosine analogues, adenosine receptor agonists such as NECA. In still other embodiments, the immunosuppressive phenotype is achieved by incubating the monocytes or the matrix containing the monocytes in medium having a pH lower then 7.4. In a more specific embodiment, the pH ranges between 3 and 7.3. In specific embodiments, the pH ranges between 3 and 6, between 3 and 5, or between 6 and 4.

Each of the above agents and conditions can be modified to achieve the angiogenic phenotype by those of skill in the art. hi exemplary embodiments, the successful differentiation towards the M2 phenotype is measured by the secretion of EL- 10 and/or TGF-β. In another embodiment, the M2 monocytes with an immunosuppressive phenotype are myeloid suppressor cells, a distinct population of cells derived from monocytes. These cells have been identified in humans as CDH⁺HLA-DR^"710 cells, and have been shown to actively secrete the immune inhibitory factor TGF-β. Myeloid suppressor cells seem to regulate adaptive immunity to cancer, by suppressing immune effector cells.

As described above, exemplary aspects of the invention feature monocytic cells secreting compounds that have a desired in vivo and/or therapeutic effect (e.g., an angiogenic and/or immunosuppressive effect) on cells or tissues surrounding the matrix (e.g., cells or tissue at an injection or implantation site). Such compounds are preferably secreted at biologically or therapeutically effective levels, for example at pM or nM levels (or in pg or ng quantities). Exemplary matrices are capable, for example, of providing from about 10 to about 1000 pg of factor per injection (e.g., from about 10 to about 1000 pg of VEGF per injection). Other matrices are capable, for example, of providing from about 10 to about 1000 ng of factor per injection. Matrices capable of providing, for example, about 100 to about 500, 600, 700, 800 or 900 pg per injection are preferred. Multiple injections are clearly intended to be within the scope of the invention. Ranges and values between and intermediate to all ranges recited herein are intended to also be encompassed by the instant invention.

IV. Matrix

The present invention features monocytic cells embedded in certain matrices for the delivery of compounds to target sites surrounding the matrices (e.g., at desired sites of injection, implantation, and the like). A matrix of the invention can include cells pre- activated or polarized towards a particular phenotype and can further include activating and/or polarizing agents as described herein for the purpose of activating, further activating, polarizing, further polarizing and/or maintaining the activation or polarization state of embedded monocytic cells. The matrix used with embodiments of the invention can be a biocompatible matrix suitable for implanting in contact adjacent to, or at the site of the target tissue or at site were localized delivery of monocytes is therapeutically desired. Preferably, the matrix is a biodegradable material, such as a synthetic polymer degrading by hydrolysis, for example, polyhydroxy acids like polylactic acid, polyglycolic acid and copolymers thereof, polyorthoesters, polyanhydrides, proteins such as gelatin and collagen, or carbohydrates or polysaccharides such as cellulose and derivatized celluloses, chitosan, alginate, thiol-modified hyaluronan or combinations thereof, so that over the course of several days or weeks after implantation of the matrix material, the matrix gradually disappears. In a preferred embodiment, the matrix is a hydrogel, defined as a matrix wherein typically approximately 90% by weight of the matrix is absorbed with water. Other hydrogels for use with embodiments of the invention can be formed by ionic or covalent cross linking of a variety of water soluble polymers such as polyphosphazenes, polysaccharides such as alginate, and proteins such as gelatin.

In certain embodiments, the hydrogel material will be capable of forming a semi- solid matrix on its own. One example of such a hydrogel material is liquid collagen in physiologic pH which converts to a semisolid state upon exposure to body temperature. In other embodiments, when the hydrogel material cannot on its own form a semisolid matrix or produce the desired physical properties, the matrix will further comprise a cross-linking agent which will form the semisolid matrix with the hydrogel material. Examples of cross-linking agents useful in the matrix of this invention include, but are not limited to, glutaraldehyde, diphenylphosphoryl azide, transglutaminase, dimethyl suberimidate, DMS-treated collagen, dimethyl 3,3'-dithiobispropionimidate, multivalent ions, calcium ions, N,N methylene-bisacrylamide (MBA), acrylamide, allyl methacrylate, ethylene glycol dimethacrylate, and tripolyphosphate. In certain embodiments, the matrix is biodegradable through hydrolysis of the hydrogel polymer. The stability of the matrix to degradation can be altered through the use of different hydrogel materials, different cross-linking agents and combinations thereof. The desired stability of the matrices of this invention ranges from several days to several weeks, depending upon the disease or condition to be treated by the matrix. In some embodiments, the hydrogel material and/or the matrix will initially be in a liquid state and then converted to a semisolid state through one or more of the following: change in pH, the addition of copolymer(s), irradiation, temperature change, the addition of a catalyzer, or the addition of a cross linker. Such liquid compositions are also part of the present invention.

In both a liquid state and in the final semi-solid state, the matrix of this invention is injectable into a patient at the site of desired treatment. Thus, according to one embodiment, the invention provides an injectable composition comprising: a. a hydrogel; and b. monocytes producing a secreted product, wherein the composition is in or is convertible to a semisolid state, and when in the semisolid state, the composition permeable to the secreted product. Polymer consistency in the matrix can be manipulated to produce soft or hard matrices for different delivery methods such as injection or implantation.

According to some embodiments, the matrix may additionally comprise an insoluble, hydratable biocompatible polymer scaffold, such as Gelfoam®. In these embodiments, the monocytes are typically adhered to the scaffold and then the scaffold containing the adhered monocytes is surrounded by the semisolid hydrogel (and optionally a cross-linker).

A matrix of the invention may be formulated with a variety of different polymer ingredients and polymers in different states in order to achieve desired attributes in the matrix. For example, in some embodiments of the invention, solid collagen or gelatin particles or granules are mixed with liquid collagen before introduction of cells and delivery to an organism. This is done to create a matrix with a reduced tendency for dispersion than a matrix formed with liquid collagen alone. In some embodiments, a matrix can be formulated with a combination of polymers of different types and/or different physical states to create a matrix with particular dimensions or attributes upon delivery. Matrices of the invention may be formulated with a particular polymer or a particular combination of polymers (of different molecular formulas and/or physical states) to enhance cohesion of the matrix upon delivery and/or to reduce immersion of the matrix into tissues, fibers, internal spaces or other structures or voids within the organism. In some embodiments, use of a particular polymer or a particular combination of polymers used to formulate a matrix creates a matrix upon delivery that has a reduced surface area in relation to volume as compared to a matrix formulated with one kind of polymer alone. In certain embodiments, a matrix with a reduced surface area to volume ratio can have a more spherical shape.

The internal microenvironment for a cell embedded in a matrix can be affected by a number of physical attributes of the matrix, including the combination of polymers used to formulate the matrix and the physical states of those polymers; the reaction of polymers used to formulate the matrix to changes in pressure, temperature, pH and other physical indices, upon formulation, introduction of cells, incubation before delivery or delivery to an organism, or after a time period following delivery of the matrix to an organism. In particular embodiments, for a matrix of a particular volume, the depth of the matrix will vary according to the shape of the matrix, the depth being equal to the minimum distance from the surface of the matrix to the point within the matrix that is farther from the surface than any other point. In particular embodiments, a matrix can be designed to have a reduced surface area to volume ratio in order to create a matrix within an organism of increased depth. In some embodiments, a matrix of increased depth will have a center that experiences a difference in microenvironment from the edge of the matrix. In preferred embodiments, the difference in microenvironment between the center of a matrix and locations more edgeward in a matrix of reduced surface area to volume can be exploited to induce a desired phenotype in a cell encompassed by the matrix. In some preferred embodiments, the difference in microenvironment is a reduced molecular oxygen concentration or tension in the center of the matrix. In some embodiments, the difference in microenvironment is a reduction of nutrients, metabolites and/or other factors, including growth factors, below optimal conditions for growth and/or maintenance of a cell. In some embodiments, the difference in microenvironment is an increase in compounds secreted from a cell, including compounds that are deleterious to the growth and/or maintenance of a cell and compounds that are the by-products of cellular processes, including compounds produced as a result of a lack of a compound (below normal optimal levels), including a reduction in the tension or concentration of molecular oxygen. V. Use of Matrix with Incorporated Cells

Monocytes can be placed in a matrix by placing them over a hydrated biocompatible polymer scaffold and letting the cells bind to the matrix by attachment. Alternatively cells can be incorporated in the matrix during hydrogel gelation process to produce cells embedded within the matrix. This can be achieved by merging the cells into a polymer solution to be polymerized later on by one or more of the following: changing PH, adding copolymer(s), irradiation, temperature change or adding a catalyzer. Polymer consistency can be manipulated to produce soft or hard polymer for different delivery methods such as injection or implantation. A combination of solid scaffold polymer and a polymer gel can be used in combination to embed and immobilize the cells. Monocyte density in the matrix can vary from about 1 cell or about 1000 cells per square centimeter to about 1000 X lO⁶ per square centimeter. In some embodiments, the density of monocytes in the matrix can vary from about 10³ cells/cm³to about 10⁹ cells/cm³, depending upon the condition to be treated and the desired magnitude of therapeutic effect. Matrix pH during gelation process with cells can range between pH=2 and pH=14. In particular embodiments, the monocytes are maintained at a pH of between 6.0 and 8.5 once they are combined with one or more matrix components.

The temperature of the matrix component(s) to which the monocytes are added and the ultimate temperature of the matrix may be any temperature between 2 and 85°C. In some embodiments, the monocytes are maintained at a temperature of between about 25° and 42°C once they are combined with one or more matrix components. Preferred temperatures for forming cell-embedded matrices are about 35-40⁰C (e.g., about 37°C) but can be varied, based on preferred temperatures for matrix polymerization as needed. Cells, matrices, and cell-embedded matrices can also be maintained at different conditions, for example, at less than 4°C, about 4-10⁰C, about 25⁰C, etc., at various stages of preparation or storage.

In particular embodiments, cells are mixed with the polymer under aseptic conditions. This may be achieved by adding a pre-counted number of monocytic cells directly to the polymer in a container with a pipette in a biosafety cabinet. Mixing the cells with the matrix can also be done by placing the matrix and the cells in two syringes. The first and second syringes are connected by a three way stopcock set to allow flow between the syringes. Monocytes are mixed with the matrix solution by injecting and withdrawing the solution into the syringe containing the cells several times until a uniform collagen/cell suspension is achieved. Is some embodiments the mixing of monocytes with the matrix is performed at 4⁰C, such as when the matrix is liquid collagen; in other embodiments, the cells are mixed with the matrix at room temperature, such as when the matrix is alginate

The viscosity of the matrix containing the cells should be in the range of between 10 and 900,000 centiPoise (cP). In a specific embodiment the viscosity of the matrix is between 200 to 2000 cP.

The matrices of this invention preferably contain monocytes that are embedded within the matrix, but not present on the matrix surface. In preferred embodiments, the effective use of the matrix is enhanced when monocytes are not allowed to come into direct physical contact with the site of therapy. Such contact will potentially alter the monocytes and reduce their therapeutic potential. Embedding the monocytes in the matrix can be achieved in various ways. Monocytes can be incubated with an insoluble hydrated biocompatible polymer scaffold, such as Gelfoam® and allowed to bind to the scaffold by attachment. The scaffold is then combined with the hydrogel and optionally the cross-linker to form the matrix containing the embedded monocytes. Alternatively monocytes can be mixed with one or more other matrix components prior to or during the formation of the semisolid state to produce a matrix with monocytes embedded therein (e.g., when the matrix component(s) is in a liquid state). Alternatively, matrix in the form of semisolid gel can be mixed with monocytes to form a mixture of gel particles with monocytes embedded therein.

In a more specific embodiment, the injectable liquid composition converts to a semisolid state immediately following injection into a patient at a desired site of treatment. This avoids the need for invasive surgery to deliver the semisolid matrix to the patient.

The matrix containing the monocytes can be placed adjacent to the target tissue by implanting the matrix during surgery under septic conditions. If needed, the matrix can be confined to its target location by using surgical tissue adhesive and/or sawing or other confining methods well known in the art. When the matrix used is suitable for injection such as when the matrix is a liquid gel or made of small particles, the matrix containing cells can be injected to the target location using a syringe/needle or catheter. In one embodiment, the invention provides a semisolid matrix comprising: a. a hydrogel material; and b. monocytes embedded within the matrix and producing a secreted product, wherein the matrix is permeable to the secreted product. In a more specific embodiment, the injectable composition comprises monocytes having an M2 phenotype and producing a secreted product at a level that is increased as compared to monocytes having an Ml phenotype.

In another embodiment, the monocytes do not have an M2 phenotype when placed in the matrix, but are converted to the M2 phenotype after a period of time within the matrix. This may be achieved in a number of different ways including, but not limited to, placing the appropriate monocyte activating agent(s) in the matrix, for example IL-IO; incubating the matrix containing the monocytes in a solution containing one or more activating agents which will diffuse into the matrix; and placing the matrix containing the monocytes in hypoxia conditions with low oxygen concentrations. Activation to the M2 phenotype may also occur spontaneously within the matrix without the addition of any activating agents or incubation under activating conditions.

Regardless of the phenotype of the monocytes in the matrix, the matrix once prepared and containing the monocytes is placed in the patient at a site where the therapeutic effect is needed.

A matrix of the invention may comprise one or more compounds or elements that induce a polarization or activation state in a cell encompassed by the matrix or prolongs such a state. In some embodiments, a compound or factor present in a matrix may decline or increase in concentration over time, which may lead to a shift in the activity, behavior or phenotype of a cell in the matrix. In a preferred embodiment, a compound incorporated into a matrix to induce or prolong a M2 phenotype is lactic acid. In embodiments where M2 monocytes are present in the matrix of this invention, the M2 monocytes produce a secreted product at a level that is increased as compared to monocytes having an Ml phenotype. The secreted product is typically a protein, but may under certain circumstances be a RNA molecule, hormone, proteoglycan or any other secreted molecule. The secreted product is transported outside the monocyte cell membrane and into the matrix. It then diffuses through and ultimately outside of the matrix so that it may interact with the patient's cells and organs at the location in which the matrix is placed. Matrices of the invention comprising M2 monocytes having an angiogenic phenotype are useful for the treatment of ischemic conditions including but not limited to ischemic heart disease (coronary artery disease), peripheral artery disease (PAD), limb ischemia, would healing, ischemic bowel disease, and atherosclerotic ischemia disease; the healing of muscle flaps and skin flaps; for organ transplant; for the cosmetic treatment of nasolabial folds and wrinkles, prevention of scar formation, and as an adjunct to plastic surgery and cosmetic procedures.

Matrices of the invention comprising M2 monocytes having an immunosuppressive phenotype are useful in organ transplant, medical device transplant, wound healing, treating autoimmune diseases, treating acute inflammation, treating chronic inflammation, and in inhibiting cell proliferation.

It will be apparent that conditions for producing the angiogenic phenotype and the immunosuppressive phenotype overlap in some embodiments. This is because the two phenotypes are distinguished by the factors that the monocytes secrete, rather than by the factors that induce the particular phenotype. In yet another embodiment the matrix of this invention comprises monocytes having an Ml phenotype and producing a secreted product at a level that is increased as compared to monocytes having an M2 phenotype.

In certain embodiments, a matrix of the invention is injected. In other embodiments, the matrix of the invention is implanted, for example, as a presolidifϊed matrix containing cells into a target tissue, under the skin or otherwise.

The matrix may be injected to the treated tissue in a single or multiple injections based on the condition and the area treated. When the application is during open surgery then the matrix can be applied locally with an applicator. For example, if an ischemic muscle is treated then matrix injection will be within the ischemic muscle. If a surgery involving muscle flap of muscle transplant is performed then delivery may be within the implanted muscle by injection or at the implantation interface by topical applicator. When an immunosuppressive effect is desired using immunosuppressive monocytes in matrix, the delivery may be surrounding the source of immune response such as implanted organ or device. In such case the matrix can be applied locally with an applicator during surgery or injected adjacent to the source of immune response.

The matrix of the present invention is useful for providing a monocyte-secreted product to a localized site in a patient. This is achieved by contacting the matrix with the localized site in the patient for a period of time sufficient for the secreted product to interact with the localized site. The matrix may be injected into the localized site in the patient as a liquid form with a syringe, catheter or any injection device and then converted to the semisolid form upon contact with the localized site. Alternatively, the semisolid form can be prepared and then implanted at the localized site through surgery, injection, subcutaneous insertion, topical application, or other techniques that would allow access to the localized site. In certain embodiments, the matrix will be affixed to the localized site through the use of a bandage or other device to hold the matrix in place. When injected in either a liquid or a gel form, the matrix will be confined by the tissue into which it is injected. In exemplary embodiments, injection volumes may range from 1 μL to 1 mL. In other exemplary embodiments, injection volumes may range from 10 μL to 100 μL. In yet other exemplary embodiments, injection volumes may range from 20 μL to 50 μL. In some embodiments, more than one injection may be performed over a range of time, location or both. For example, one or more injections (e.g., 2-5, 5-10, 10-20 or more injections) can be performed. Single or multiple injections can be performed, for example, over a range of time with some injections being performed in a first treatment, further injections being performed in a second treatment, etc. Single or multiple injections can also be performed, for example, over a range of locations with some injections being performed at a first location, further injections being performed at a second location, etc. Subject tolerance and/or desired therapeutic efficacy may guide a routine physician in determining injection regimes.

In one embodiment, the invention provides a method of treating a patient suffering from or susceptible to ischemia comprising the step of contacting a site of ischemia in the patient with a matrix of this invention comprising M2 monocytes that secrete a pro-angiogenic factor at a level that is increased as compared to Ml monocytes, and wherein the secretion of the pro-angiogenic product for a time sufficient to prevent or lessen the effects of a perfusion injury associated with the ischemia. In a more specific embodiment, the ischemia is cardiac ischemia.

In another embodiment, the invention provides a method of reducing nasolabial folds or wrinkles in a patient comprising the step of contacting the site of the nasolabial folds or wrinkles with a semisolid biocompatible matrix comprising M2 monocytes that secrete a pro-angiogenic factor at a level that is increased as compared to Ml monocytes, and wherein the secretion of the pro-angiogenic factor is for a time sufficient to detectably reduce the number or depth of the nasolabial folds or wrinkles.

Standard treatment of nasolabial folds or wrinkles typically involves the administration of biocompatible gel matrix as a filling agent in the wrinkled skin. A key product in the market is the Zyderm® treatment derived from highly purified bovine collagen. Other such products include Cosmoplast®, Hylaform® and Radiance®. The method of the present invention provides an active therapy that regenerates skin functions by inducing tissue remodeling and/or angiogenesis. The use of monocytes will result in active rather than passive (when using gel only) therapy with a more profound and longer term effect on the structure of the tissue and its visual properties.

In still another embodiment, the invention provides a method of reducing an immune response at a localized site in a patient comprising the step of contacting the localized site with a semisolid biocompatible matrix of the invention comprising M2 monocytes that secrete an immunosuppression factor at a level that is increased as compared to Ml monocytes, and wherein the secretion of the immunosuppression factor is for a time sufficient to detectably reduce the immune response at the localized site. The use of monocytes contained in a matrix, as opposed to tissue engineered monocytes, as described in embodiments of the present invention, provides a significant improvement compared to the use of cell suspensions or naked cells for the treatment of tissue ischemia and for the delivery of therapeutic products (not just proteins). For example, in some embodiments the monocyte-containing matrix can be localized to selected tissue(s) and can deliver therapeutic products and substances with a higher and more predictable yield, and for a longer duration. The use of a matrix, in particular embodiments, leads to improved cell function and/or cell survival. The use of a matrix in some embodiments can reduce the rate of cell migration into surrounding tissues, the circulatory system and/or the lymphatic system. In some embodiments, the matrix may create a microenvironment for the monocytes and/or other cells contained within the matrix or in the local area. In some embodiments, the matrix creates a microenvironment for monocytes within the matrix, which allows control over monocyte behavior and performance during treatment. The use of a matrix in particular embodiments allows for the continuation of a cell phenotype for a cell embedded in the matrix and can permit a continuation of a cell treatment that induces or maintains a desired state or phenotype of the cell. In some embodiments, the matrix and/or a compound included in the matrix can serve to induce a phenotype or desired state in a cell contained within the matrix. The use of a matrix in particular embodiments permits the inclusion of particular numbers and concentrations of one or more types of cells within the matrix. In particular embodiments, the matrix microenvironment is one of hypoxia, wherein the concentrations of molecular oxygen within the matrix is significantly and/or substantially below that found in normal tissue under normal physiological conditions. In some of these embodiments, the cells incorporated by the matrix are put into a desired state or have a preexisting desired state maintained by the hypoxic conditions within the matrix. Li particular embodiments, a M2 phenotype of a monocyte is created and/or maintained by the hypoxic conditions within a matrix that comprises the monocyte.

Additional embodiments of the invention feature the ability to manipulate the expression of proteins and other biological factors such as hormones by monocytes through genetic manipulation and/or other biological treatments, such as exposure to cytokines, growth factors or hypoxia. In some embodiments, the manipulations of the expression of proteins and/or other biological factors can provide for the use of various embodiments of the invention as a cell based delivery system of specific therapeutic proteins and other biological factors. In these embodiments, a cell-based delivery system of therapeutic proteins can deliver, to specific targets in the body if desired, a plethora of compounds for treatment of a variety of disorders, conditions or states of damage and/or disrepair, such as ischemia, and other undesirable conditions or states. For example, in a particular embodiment, retroviral transfection of monocytes with an expression vector encoding for MCP-I may be used for local recruitment of systemic monocytes by the genetically modified monocytes seeded in the biocompatible matrix. In some embodiments, the use of particular numbers and/or concentrations of one or more types of cells can be optimized to provide a desired duration of compound delivery, desired concentration of compound delivery, or other optimizations of treatment effects. Some embodiments of the invention feature matrix that will degrade, deteriorate or otherwise undergo declining matrix integrity over time, that leads to a matrix of reduced size or volume or the disappearance of the matrix from the implantation location.

In some embodiments, cells are included in matrices of the invention at concentrations of from about 5,000 to about 2x10⁶ cells per ml or per cc of matrix. In other embodiments, cells are included in matrix at a concentration of from about 5,000 to about 10,000 cells per ml or per cc of matrix. In other embodiments, cells are included in matrix at concentrations of from about 10,000 to about 50,000 cells per ml or per cc of matrix, about 50,000 to about 200,000 cells per ml or per cc of matrix, or about 50,000 to about 1x10⁶ cells per ml or per cc of matrix. In exemplary embodiments, cells are included in matrix at concentrations of from about 200,000 or about 500,000 to about 1 xlO⁶ or 2 xlO⁶ cells per ml or per cc of matrix. In other embodiments, cells are included in matrix at concentrations of from about 2 xlO⁶ to about 10 xlO⁶ cells per mL. In yet other embodiments, cells are included in matrix at concentrations of from about 10 xlO⁶ to. about 50 xlO⁶, or about 100 xlO⁶ or more cells per mL or cc of matrix. Polarizing or activating agents can likewise be include in the matrices of the invention at concentrations of from about 1 pM to about 10 mM. In exemplary embodiments, polarizing or activating agents are included at nM or μM concentrations, for example, concentration of about 1 to about 1OnM, about 1OnM to about 10OnM, about 10OnM to about 50OnM, or about 50OnM to about IuM. In other embodiments, polarizing or activating agents are included at μM or mM concentrations, for example, concentration of about 1 to about 10 μM, about 10 μM to about 100 μM, about 100 μM to about 500 μM, or about 500 μM to about ImM. Concentrations in the mM ranges can be used where necessary to induce or maintain a desired state.

In certain aspect, the invention features formulation of monocytic cell-containing matrices such that the cells are physically separated or distanced from the surrounding tissue (e.g., the tissue at the target injection or implantation site). By embedding the cells in a matrix and creating a physical barrier between the host and graft, it becomes possible to use allogenic (or even xenogenic) cells. In certain embodiments, monocytic cells can be encapsulated in multiple layers of matrix such as encapsulation in alginate followed by embedding in collagen gel or encapsulation in gelatin followed by embedding in collagen, and the like. The use of allogenic (or even xenogenic) cells in the present invention is made possible since the physical contact of the cells with the host tissue is prevented by the use of a matrix for the delivery of the cells as well as by the phenotype of the cells within the matrix which include the secretion of the immunosuppressive cytokines IL-IO and TGF-β.

VT. Articles of Manufacture In another embodiment, the present invention provides a kit comprising: a hydrogel material; and instructions for using the hydrogel material to form a biocompatible semisolid matrix comprising monocytes having an M2 phenotype embedded in the matrix, wherein the embedded monocytes produce a secreted product at a level that is increased as compared to Ml monocytes; and the matrix is permeable to the secreted product.

The kits of the invention may further comprise a cross-linking agent. In some embodiments, the kits may also further comprise any of the following: an insoluble scaffold material, one or more agents for inducing the M2 phenotype, one or more reagents for inducing a specific sub-phenotype in M2 monocytes (e.g., one or more of the agents disclosed above for inducing an angiogenic phenotype or an immunosuppressive phenotype), one or more buffers, one more reagents for isolating M2 monocytes from whole blood, or a syringe for injecting the matrix.

Monocytes in the kit can be separated from the matrix and kept in a delivery medium cryopreserved, cold or at room temperature. Alternatively, monocytes can be embedded in the matrix and delivered cryopreserved, cold or at room temperature. The matrix and/or monocytes can be in the delivery system or in a separate container. The delivery system can be one or more syringes, catheter or other injection device. Preferably the injection device will be set for the delivery of small volumes ranging from 10μL-500μL. Preferably, the kit will include instruction for kit assembly and use.

The foregoing disclosure teaches to those of skill in the art the aspects of the invention including how to make and use the invention. The following examples are meant to provide further elucidation of the invention but are not meant as limitations thereof EXAMPLES

Example 1. Isolation and Preparation of Monocytes

Blood is drawn from the patient in amount sufficient to produce the desired number of monocytes and serum volume for monocyte culturing (80-300 ml). The blood for serum preparation and monocyte separation does not have to be taken at the same time and the serum can be prepared and stored in advanced.

Serum can be prepared in a number of different ways, well known to people familiar with the art. For example by letting the blood to clot at 37°C for 12-24 hours and centrifuging the clot at 500g for 15 min.

Monocyte separation from whole blood is performed using the MidiMACS separation kit with monocyte Isolation Kit II (Miltenyi Biotec LTD) by following the manufacturer instructions.

Monocytes can be cultured in RPMI medium with 10% inactivated human serum or DMEM with 5% inactivated fetal bovine serum.

Example 2. Preparation of Gelatin Matrices with Monocytes

In this example, two kinds of collagen matrices are prepared and combined with monocytes. Monocytes can be prepared by the method detailed in Example 1.

Gelfoam^® paste:

Gelfoam^® paste is formulated by hydrating 1 g of Gelfoam powder (Pharmacia/Upjohn) with 5 ml of gelfoam homogenization buffer (e.g., 0.5% sucrose, 2.5% glycine, 5mM L -glutamic acid, 5 mM NaCl, 0.01% polysorbate 80, pH 4.5). Buffer is added directly to the sterile Gelfoam powder jar via pipet using aseptic technique in a biosafety cabinet. The resulting hydrated powder is then mixed with a sterile spatula for approximately two minutes until a cohesive homogeneous thick doughy paste consistency is achieved. Paste volume after hydration and mixing is approximately 6 ml. The pH of Gelfoam paste is raised with the addition of a basic solution and additional agents may be added, if desired. (For example, the paste can be neutralized with 0.5M NaOH to pH 7.4 and the adenosine receptor A2 agonist 5'-(N- ethyl)Carboxamido-adenosine (NECA) lOμM and TLR 7,8 agonist CL097 10OnM can be added and mixed into the gelfoam paste.) One hundred μL of monocytes in saline buffer, with a concentration of 10,000,000 cells/ml, are added into 0.9 ml of Gelfoam paste and gently folded into the gel using aseptic technique in a biosafety cabinet to produce a 1,000,000 monocytes/ml Gelfoam paste monocyte culture. The Gelfoam paste monocyte culture is immediately covered with 4 ml culture medium containing 10% blood donor serum. Medium is changed after 10 min with fresh medium. Gelfoam paste monocyte culture is incubated in cell culture incubator at 37C, 5% CO₂ under humidified conditions. For Gelfoam paste monocyte culture maintenance, medium is changed every 24 hr.

Similar formulations can be created using unfractionated bone marrow cells (BMC), fractionated BMC, bone marrow-derived stem cells and bone marrow-derived progenitor cells.

Formulations can be created with other materials used for the matrix component. For example, liquid collagen can replace the gelfoam paste in the above example by mixing the cells into liquid collagen at about 4°C and injecting the mixture into an organism with a body temperature of around 37°C. At that temperature, the liquid collagen will rapidly solidify into a semi-solid matrix with embedded cells.

Gelfoam^® sponge:

Gelfoam^® sponge (Pharmacia/Upjohn) are cut into 1 cm X 1 cm squares. Each 1 cm X 1 cm square is hydrated by incubation in 1OmL of monocyte medium (see

Example 1) for 12 to 24 hours at 37°C (5% CO₂, humidified conditions).

The sponges are transferred onto an aseptic surface in a biosafety cabinet.

Excess medium is removed so that the sponge remains moist but not soaked in the medium. To each sponge, 100 μl of monocytes in monocyte medium (10,000,000 cells/ml) are slowly added in four steps of 25 μl, to allow the cells to be absorbed onto the sponge. The monocyte absorbed sponges are placed in cell culture incubator at 37°C

(5% CO₂, under humidified conditions) for 4 hr to allow the cells to attach to the sponge.

Each sponge seeded with monocytes is then transferred, using aseptic technique in a biosafety cabinet, into a 50 ml sterile tube containing 15 ml of monocyte medium. The monocyte-seeded sponges are incubated in a cell culture incubator at 37°C (5% CO₂, under humidified conditions). For monocyte culture maintenance, medium is changed every 24 hours. Similar formulations of cells and Gelfoam^® sponges can be made using unfractionated bone marrow cells (BMC), fractionated BMC, bone marrow-derived stem cells and bone marrow derived progenitor cells.

An example of type of formulation is provided by the following experiment, which examines the survival of matrix monocytes as compared to naked monocytes in cell cultures. Twenty Gelfoam^® sponges (lcm x lcm), seeded with 1 million monocytes per sponge as described above, are incubated in a 24 well tissue culture plate in 2 ml of RPMI medium with 10% inactivated bovine fetal serum. The same number of cells are concurrently seeded without matrix directly in a 24 well tissue culture plate in 2 ml of RPMI medium with 2% inactivated bovine fetal serum.

To examine cell survival at each time point, two sponges are digested with 2 ml collagenase for 10 min, stained for Trypan blue and the total cell population, as well as the number of live/dead cells, are counted using a microscope counting chamber (hemocytometer). Control cells are harvested by incubation for 5 min in 2 ml Trypsin EDTA solution and total cell population, as well as the number of live/dead cells, are counted using a hemocytometer.

While not wishing to be bound by any theory, Figure 1 shows the projected data from the cell survival experiment. The number of live monocytes from sponge cultures does not significantly change over the 20 day course of the experiment, with only a slight drop in the number of live cells seen at day 20 as compared to day 15. But in the standard cell cultures, a precipitous drop in the number of viable cells can be seen after day 5, and by day 20, the number of live cells decreases by nearly 40%.

Formations of cells with Gelfoam^® sponges can also be covered with a additional layer of matrix material. In this way, the cells adsorbed onto the surface of a sponge can be thoroughly covered, so that when the sponge is implanted, a barrier exists between the cells of the sponge and the implanted organism's cells, tissues and/or fluids.

Example 3. Preparation of a Collagen Hydrogel Matrix with Polarized Monocytes

In some embodiments of the invention, matrices can be prepared using cells that have already by polarized towards a M2 phenotype or subphenotype before introduction to the matrix materials, as illustrated by the following example. Monocytes are separated from whole blood as described above in Example 1. Monocytes may be selected positively, e.g. through the use of specific antibodies that target monocyte-specific cell surface markers, such as CD 14. The monocytes can also be selected negatively, by targeting the non-monocytic cells or non-monocytes of the blood, leaving behind only the monocytic cells and/or the monocytes.

The separated monocytes are incubated for 24-48 hours in RPMI medium containing 5% inactivated human serum and 10 ng/ml human IL-IO, an agent that polarizes monocytes towards a M2 phenotype. Following incubation, monocytes are trypsinized and washed twice in PBS by centrifugation at 400 x g. A 2 mg/mL collagen solution (4.5 ml, v/v) at physiologic pH is kept cold (~ 4 °C) in a syringe on ice to prevent gelation. Isolated monocytes (I x 10⁷ cells) are resuspended in saline solution (0.5 ml) and collected into a second syringe. The first and second syringes are connected by a three way stopcock set to allow flow between the syringes. Monocytes are mixed with the collagen solution by injecting and withdrawing the collagen solution into the syringe containing the cells several times until a uniform collagen/cell suspension is achieved.

Example 4. Preparation of a Matrix Using a Collagen Hydrogel and Gelfoam Powder Embodiments of the invention feature matrices comprising one or multiple matrix-forming ingredients. Matrix ingredients compatible for use in a matrix of the invention include both solid, semi-solid and liquid ingredients. In this example, two ingredients are used to create a matrix. In this example, monocytes are polarized towards a M2 phenotype before introduction to the matrix. Monocytes are separated from whole blood using standard techniques. The separated monocytes were incubated for 24-48 hours in RPMI medium containing 5% inactivated human serum and agents to polarize the monocytes towards a M2 phenotype (for example, NECA lOμM and CL097 10OnM). Following incubation, monocytes are trypsinized and washed twice in PBS by centrifugation at 400 x g. Gelfoam powder (0.2 g) is hydrated with 5 ml of saline solution. The hydrated Gelfoam powder suspension is cooled to 4 ⁰C and liquid collagen, buffered to physiological pH, is added to a final concentration of 2 mg/mL. The collagen solution containing the hydrated gelfoam powder (4.5 ml) is placed in a syringe and kept at 4 °C to prevent gelation. 1 x 10⁷ monocytes are resuspended in saline solution (0.5 ml) and collected into a second syringe. The two syringes are connected by a three way stopcock set to allow flow between the two syringes. Monocytes are mixed with the collagen/Gelfoam solution by injecting and withdrawing that solution into the syringe containing the cells several times until a uniform collagen/Gelfoam/cell suspension is achieved.

Example 5. Preparation of a Matrix Containing Alginate Hydrogel.

Monocytes were isolated from whole blood and prepared for embedding in the matrix as in Example 1. A 2.5% sterile alginate solution in saline (4.5 ml) containing 0.1 M calcium chloride and 0. IM calcium sulphate was placed in a syringe and left to form a gel for 1 hour. Isolated monocytes (1 x 10⁷ cells) were resuspended in 0.5 ml saline. Cells were rapidly transferred into a syringe and the syringe containing the cell suspension and the syringe containing the alginate gel were connected by a three way stopcock set to allow flow between the two syringes. Cells were mixed into the alginate gel by injecting and withdrawing the alginate gel into the syringe containing the cells several times until a uniform alginate-cell suspension was achieved.

Example 6. Release kinetics for Matrix VEGF Production

Monocytes are separated as described in Example 1 and transduced with IXlO⁷ pfu of a lentiviral vector carrying a cytomegalovirus promoter driving the expression of secretable human vascular endothelial growth factor 165 (VEGF) gene downstream of an internal ribosomal entry site element. Following the transduction, cells are seeded in a hydrated gelfoam paste as described in Example 2 and placed in a 24 well tissue culture plate in 2 ml of RPMI medium with 5% inactivated bovine fetal serum. 100 μL samples are collected from duplicate samples 6 hours following the first addition of medium and every 24 hours following the first sampling. VEGF concentrations in the samples are measured by Human VEGF Quantikine ELISA Kit (R&D).

While not wishing to be bound by any theory, Figure 2 shows the projected data for the release of VEGF from matrices of the invention. As shown in Figure 2, levels of VEGF secreted into the culture media reach 1 ng/ml in about 2.5 days and secretion is maintained at that level by the monocyte matrix cultures for over one week. Example 7. In-vivo matrix implantation

The following Example illustrates a method of testing the in vivo efficacy of an embodiment of the invention using monocytes in the treatment of an induced ischemic condition in an organism. Monocytes are separated from whole Lewis rats blood as described above. The cells are transduced with IXlO⁷ pfu of a lentiviral vector carrying a cytomegalovirus promoter driving the expression of secretable green fluorescent protein (GFP) gene downstream of an internal ribosomal entry site element. 500,000 monocytes are seeded in lOOμL of hydrated Gelfoam powder in RPMI medium with 10% inactivated rat serum. Forty-eight hours after monocyte seeding, cells are washed 3 X in saline buffer in preparation for in-vivo injection. Fifteen Lewis rats undergo left anterior descending artery (LAD) ligation to induce an anterior wall myocardial infarction. Animals are allowed 72 hr to recover from the surgery.

In a second surgery, the left ventricle is exposed and animals are treated as follows: 5 animals receive 4 injections of 25 μL monocyte matrix, 5 animals receive 4 injections of 25 uL matrix only, and 5 animals receive 4 injections of 25 μL of saline only. Animals receive cyclosporine daily (7.5 mg/kg per day orally) to avoid cell rejection. 4 weeks post cell treatment animals are evaluated for left ventricular function by echocardiogram. After echocardiogram evaluation, heart tissue is examined for the presence of GFP and for repair of the ischemic damage that resulted from the induced myocardial infarction.

Echocardiography:

On day 28 following the second surgery, echocardiography of the heart is performed for all three groups (15.8 MHz, Sequoia 256; Acuson). Rats are anesthetized with isoflurane. Standardized views of the heart are obtained at the papillary muscle level. Fractional shortening is determined from m-mode images. The thicknesses of both left ventricular anterior wall (AWT) and interventricular septum (SWT) are measured. Examinations are evaluated by an independent experienced investigator. Histology and Histochemistry:

At 4 weeks post monocyte cell treatment, animals are anesthetized with 2% isoflurane. Hearts are perfused with saline, explanted, and fixed for 2 hours in paraformaldehyde before overnight cryopreservation in 30% sucrose. The tissue is embedded in a histochemistry mounting medium (OCT) and stored at minus 70⁰C. Immunohistochemical staining is performed on 6 μm sections using the following primary antibodies: polyclonal Alexa-Fluor-488-conjugated anti-GFP antibody (Molecular Probes), rabbit anti a-sarcomeric-actin antibody (Sigma- Aldrich), and rabbit anti-connexin 43 antibody (Sigma- Aldrich). Alexa 546-conjugated or Alexa 647- conjugated secondary antibodies (Molecular Probes) are used. Sections are counterstained with Hoechst (Sigma- Aldrich) and analyzed using a Zeiss Axioplan fluorescent microscope.

Example 8. In-vivo matrix implantation with BMC

The following Example illustrates a method of testing the in vivo efficacy of an embodiment of the invention using bone marrow cells in the treatment of an induced ischemic condition in an organism.

Fifteen New Zealand white rabbits undergo ligation of the left anterior descending artery (LAD) to induce an anterior wall myocardial infarction. Animals are allowed 72 hours to recover from the surgery. Unfractionated bone marrow cells (BMC) are seeded in 100 μL of liquid collagen as described for monocytes in Example 3. In a second surgery, the left ventricle is exposed and animals are separated into three treatment groups, as follows: 5 animals receive 4 injections of 25 μL monocytes in matrix to the ischemic hear tissue, 5 animals receive 4 injections of 25 μL matrix only and 5 animals receive 4 injections of 25 μL of saline only. Animals receive cyclosporine daily (7.5 mg/kg per day orally) to avoid cell rejection. Four weeks after treatment, animals are evaluated for left ventricular function using echocardiography. On day 94 echocardiography of the heart is performed for all three groups (15.8 MHz9 Sequoia 256; Acuson). Rabbits are anesthetized with isoflurane. Standardized views of the heart are obtained at the papillary muscle level. Fractional shortening is determined from m-mode images. The thicknesses of both left ventricular anterior wall (AWT) and interventricular septum (SWT) are measured. Examinations are evaluated by an independent experienced investigator data.

Histology and Histochemistry:

At 12 weeks post monocyte cell treatment, animals are anesthetized with 2% isoflurane. Hearts are perfused with saline, explanted, and fixed for 2 hours in paraformaldehyde before overnight cryopreservation in 30% sucrose. The tissue is embedded in a histochemistry mounting medium (OCT) and stored at minus 70°C. Immunohistochemical staining is performed on 6 μM sections using the following primary antibodies: goat anti rabbit a-sarcomeric-actin antibody (Sigma-Aldrich), and goat anti rabbit connexin 43 antibody Alexa 546-conjugated or Alexa 647-conjugated secondary antibodies (Molecular Probes) are used. Sections are counterstained with Hoechst (Sigma-Aldrich) and analyzed using a Zeiss Axioplan fluorescent microscope.

While not wishing to be bound by any theory, Table 1 contains projected data from this experiment. Only treatment with BMC in matrix provides a significant advantage in restoring left ventricle tissue over the no treatment and matrix only controls.

20

Table 1 : Left ventricular thickness 12 weeks post-treatment

Example 8: VEGF secretion kinetics from monocytes in angiogenic polarizing medium

Controlling human monocyte phenotype is a critical step in the generation of monocyte-based angiogenic growth factors delivery system. In this assay, starting with unactivated monocytes, the ability of human monocytes to differentiate into the angiogenic phenotype by exposing them to a combination of TLR agonist together with adenosine receptor A2 agonist was demonstrated. The differentiation of monocytes into the angiogenic phenotype was measured by the increase in VEGF secretion by the monocytes. Monocytes which were exposed to the combination of TLR agonists and adenosine A2 agonist demonstrated angiogenic activation characteristics whereas treatment with TLR agonist or adenosine receptor A2 agonist alone did not induce the angiogenic phenotype in monocytes.

Experimental Procedure:

Unactivated, negatively separated monocytes (250,000) were incubated in 400μl of DMEM with 5% inactivated fetal bovine serum (FBS) supplemented with a TLR agonist (LPS (100 ng/ml ) or CL097(100nM)), an adenosine receptor A2 agonist (NECA (10μM)), both types of compounds or neither type of compound.

Cultures were incubated in a humidified incubator with 5% CO₂ at 37 ⁰C. Medium was changed every 24 hours. VEGF concentration in the medium was measured and the results are plotted in Figures 3, 4 and 5. As seen in Figures 3, 4 and 5, after an initial burst of VEGF release due to activation of the monocytes, only those cells incubated in media containing both a TLR agonist and a adenosine receptor A2 agonist showed increasing and significant VEGF production, rising to a level of about 1650 pg/mL/day for both LPS + NECA and CL097 + NECA. None of the cell cultures containing only a TLR agonist or NECA alone (without any other polarizing agent) displayed any meaningful VEGF secretion.

In these experiments, VEGF concentration in the medium is a marker for the monocyte activation towards the angiogenic phenotype. These results demonstrate the effect of co-activation of TLR 7,8 with adenosine receptor A2 on the polarization of human monocytes towards the angiogenic phenotype. The VEGF secreted in the first 24 hours is the result of a spontaneous release of cytokines observed in unactivated monocytes immediately following their interaction with a surface or a cytokine. This effect is not observed when monocytes are selected in a positive selection method or following binding to a tissue culture plate {see Figure 6, described below), as the release is transient. Example 9: VEGF secretion kinetics from positively selected monocytes in angiogenic polarizing collagen matrix

In this assay, the polarization of positively selected monocytes (activated) into the angiogenic phenotype while embedded in a 3D collagen matrix supplemented with polarizing agents was studied. The ability of the monocyte embedded in gel matrix to act as a VEGF delivery system was evaluated by measuring the release kinetics of VEGF into the medium surrounding the matrix. The data collected demonstrated that, similar to the effect of a TLR agonist together with an adenosine agonist in polarizing medium, positively selected monocytes can be shifted into an angiogenic phenotype in 3D collagen matrix and can act as a delivery system for VEGF.

Experimental procedure:

Human peripheral blood monocytes were separated from whole blood using standard technique of gradient separation and binding of monocytes on plastic culture plates for 60 minutes in DMEM containing 5% inactivated fetal bovine serum (FBS). Unbound cells were removed by gentle wash with DMEM and bound monocytes were treated in DMEM (5% FBS) for 24 hours. Monocytes were removed from the plate by short trypsin treatment and washed in DMEM with 5% inactivated FBS. Collagen gel was prepared using 1 mg/ml rat tail collagen in DMEM pH 7.4 supplemented with a TLR agonist (LPS (100 ng/ml ) or CL097 (10OnM)), an adenosine receptor A2 agonist (NECA (10μM)), both types of compounds or neither type of compound.

Collagen gels were kept at 4⁰C to prevent solidification. 250,000 monocytes were mixed with 200μl collagen gel containing the various agonist combinations and transferred into wells in 48 wells plates. Collagen gels were allowed to solidify in a humidified incubator at 37⁰C for 45 min before 400μl of medium containing DMEM (5% inactivated FBS) was added over the gelled collagen. Medium was replaced every 24 hr and samples were collected and tested for VEGF 165 using standard elisa assay specific for human VEGF 165. Data are shown in Figures 6, 7 and 8.

As seen in Figures 6, 7 and 8, positively selected/activated monocytes incubated with polarizing agents and embedded in a matrix can produce and secrete substantial amounts of VEGF. However, this secretion was only seen when a TLR agonist and an adenosine receptor A2 receptor were used together, not when either was used alone. Example 10: VEGF secretion kinetics from negatively selected monocytes in angiogenic polarizing collagen matrix

In this assay, the polarization of negatively separated (unactivated) monocytes into the angiogenic phenotype while embedded in a 3D collagen matrix supplemented with polarizing agents was examined. The ability of the monocyte-embedded gel matrix to act as a VEGF delivery system was evaluated by measuring the release kinetics of VEGF into the medium surrounding the matrix. The data collected demonstrated that, similar to the effect of a TLR agonist together with an adenosine agonist in polarizing medium, negatively selected monocytes (unactivated) can be shifted into an angiogenic phenotype in 3D collagen matrix and can act as a delivery system for VEGF.

Experimental Procedure:

Human peripheral blood monocytes were separated from whole blood using standard technique of gradient separation and negative selection using magnetic coated beads with specific antibodies for non-monocyte peripheral blood mononuclear cells surface antigens. Collagen gel was prepared using lmg/ml rat tail collagen in DMEM pH 7.4 supplemented with a TLR agonist (LPS (100 ng/ml ) or CL097 (10OnM)), an adenosine receptor A2 agonist (NECA (10μM)), both types of compounds or neither type of compound. Collagen gels were kept at 4°C to prevent solidification. 250,000 monocytes were mixed with 200μl of each type of supplemented collagen gel and transferred into wells in 48 wells plates. Collagen gels were let to solidify in a humidified incubator at 37⁰C for 45 min before 400μl of medium containing DMEM with 5% inactivated FBS was added over the gelled collagen. Medium was replaced every 24 hours and samples were collected and tested for VEGF 165 using standard elisa assay specific for human VEGF 165. Data are shown in Figures 9, 10 and 11.

Prior polarization before embedding in a matrix is not necessary in order to obtain substantial VEGF production from the matrix. However, as seen in Figures 9, 10 and 11, only those matrices that incorporated both a TLR agonist and an adenosine receptor A2 agonist produced increasing and substantial amounts of VEGF. Those matrices with only one agonist produced much less VEGF, with continually declining amounts after the initial burst following activation. Example 11 : VEGF secretion kinetics from pre activated monocytes embedded in polarizing collagen matrix

In this assay, VEGF release kinetics from a collagen matrix embedded with pre- polarized monocytes were tested. Both the possibility of transferring angiogenically activated monocytes into polarizing collagen gel and the stability of VEGF release kinetics of the pre activated monocytes in the collagen gel matrix were examined. The data demonstrate the potential of creating a continuous delivery system for VEGF. VEGF secretion levels were stable throughout the 6 days experiment duration, indicating that the matrix was supporting the angiogenic phenotype of the monocytes.

Experimental Procedure:

Negatively selected monocytes were incubated for 48 hours in culture in DMED with 5% FBS that was supplemented with various combinations of compounds: a TLR agonist (LPS (100 ng/ml ) or CL097 (10OnM)), an adenosine receptor A2 agonist (NECA ( 1 OμM)), both types of compounds or neither type of compound.

Collagen gel was prepared using 1 mg/ml rat tail collagen in DMEM (pH 7.4) supplemented with polarizing agents in the same combinations and amounts as described for the preconditioning of the monocytes above. Collagen gels were kept at 4°C to prevent solidification. 250,000 pre activated monocytes from each treatment group were mixed with 200μl collagen gel supplemented with the same compounds and transferred into wells in 48 wells plates. Collagen gels were let to solidify in a humidified incubator at 37⁰C for 45 min before 400μl of medium containing DMEM with 5% inactivated FBS was added over the gelled collagen. Medium was replaced every 24 hours and samples were collected and tested for VEGF 165 using standard elisa assay specific for human VEGF165. Data are shown in Figures 12, 13 and 14.

As seen in Figures 12, 13 and 14, use of monocytes that are polarized before embedding in a matrix that also has polarizing agents created initially high levels of VEGF production (nearly 1200 pg/mL/day on day one) that were sustained through day six. However, as seen above, the high levels of VEGF secretion were only seen in the matrices where cells were polarized with, and subsequently exposed to after embedding in the matrix, both a TLR agonist and an adenosine receptor A2 agonist, not with either of those types of compounds alone. Example 12: Effect of angiogenic monocytes in collagen matrix on endothelial tube formation

In this assay, the ability of angiogenic monocytes embedded in collagen gel matrix to induce tube formation in endothelial cells as an indication of their angiogenic effect on vascular endothelial cells was measured. Only monocytes preactivated towards the angiogenic phenotype induced tube formation in endothelial cells. This data is in agreement with the previous experiments demonstrating that angiogenic monocytes, but not untreated monocytes, secrete VEGF while in a matrix.

Experimental Procedure:

Negatively selected monocytes were incubated for 48 hr in DMED with 5% FBS either supplemented with CL097 (10OnM) and NECA (lOμM) or with no supplementation.

Collagen gel was prepared using 1 mg/ml rat tail collagen in DMEM (pH 7.4). 250,000 monocytes from each treatment group were mixed with 200μl collagen gel and transferred into wells in 48 wells plates. Collagen gels were allowed to solidify in a humidified incubator at 37⁰C for 45 min before 400μl of medium containing DMEM with 1% inactivated FBS was added over the gelled collagen. Twenty-four hours following the transfer of cells to the collagen, 20,000 human umbilical vein endothelial cells (HUVECs) were seeded over the gel in DMEM containing 1% FBS and images were taken after 24 hours.

Images taken after 24 hours show distinct grouping of endothelial cells in a network formation in the plates containing gel matrices with polarizing compounds. In the negative control matrices (without polarizing compounds), the endothelial cells displayed a randomized, spotted configuration with no arrangement along lines or in a network.

Although the foregoing invention has been described in detail for purposes of clarity of understanding, it will be obvious that certain modifications may be practiced within the scope of the appended claims. AU publications and patent documents cited herein, as well as text appearing in the figures, are hereby incorporated by reference in their entirety for all purposes to the same extent as if each were so individually denoted.

Claims

What is claimed:

1. An implantable, semisolid matrix comprising a hydrogel material and monocytic cells embedded therein, wherein the monocytic cells are capable of producing a secreted product, and wherein the matrix is permeable to the secreted product when produced.

2. An injectable composition comprising a hydrogel material and monocytic cells added thereto, wherein the monocytic cells are capable of producing a secreted product, and wherein the composition is convertible to a semisolid state that is permeable to the secreted product when produced.

3. The composition of claim 2, wherein composition is converted to the semisolid state is by exposing the composition to heat, ionizing radiation or ultraviolet radiation.

4. The matrix or composition of any one of claims 1 to 3, wherein the monocytic cells are capable of producing more than one secreted product.

5. The matrix or composition of any one of claims 1 to 4, wherein the secreted product is an angiogenic factor.

6. The matrix or composition of claim 5, wherein the angiogenic factor is selected from the group consisting of vascular endothelial growth factor (VEGF)_, fibroblast growth factor (FGF), platelet derived growth factor (PDGF), hepatocyte growth factor/scatter factor (HGF/SF), epidermal growth factor (EGF), and Interleukin-8 (IL-8).

7. The matrix or composition of any one of claims 1 to 4, wherein the secreted product is an immunosuppressive factor.

8. The matrix or composition of claim 7, wherein the immunosuppressive factor is selected from the group consisting of IL-4, IL-10 and TGF-β.

9. The matrix or composition of any one the preceding claims, wherein the monocytic cells are monocyte precursor cells.

10. The matrix or composition of claim 9, wherein the monocyte precursor cells are bone marrow cells or monocyte progenitor cells.

11. The matrix or composition of any one of claims 1-8, wherein the monocytic cells are activated monocytes.

12. The matrix or composition of any one of the preceding claims, wherein the monocytic cells are activated towards the angiogenic phenotype.

13. The matrix or composition of any one of claims 1-6 and 9-12, wherein the monocytes are activated towards the M2 phenotype.

14. The matrix of composition of claim 13, wherein the monocytes produce at least one M2 phenotypic marker.

15. The matrix or composition of claim 14, wherein the M2 phenotypic marker is produced at a level that is increased as compared to monocytes having an Ml phenotype.

16. The matrix or composition of claim 15, wherein the monocytes are pre- activated towards the M2 phenotype.

17. The matrix or composition of claim 16, additionally comprising an agent that activates the monocytes towards the M2 phenotype.

18. The matrix or composition of claim 17, wherein the monocytes have a M2 phenotype profile

19. The matrix or composition of any one of claims 1 -4 and 7- 12, wherein the monocytes are activated towards the Ml phenotype.

20. The matrix or composition of claim 19, wherein the monocytes produce at least one Ml phenotypic marker.

21. The matrix or composition of claim 20, wherein the Ml phenotypic marker is produced at a level that is increased as compared to monocytes having an M2 phenotype.

22. The matrix or composition of claim 21, wherein the monocytes are pre- activated activated towards the Ml phenotype.

23. The matrix or composition of claim 22, additionally comprising an agent that activates monocytes towards the Ml phenotype.

24. The matrix or composition of claim 23, wherein the monocytes have a

Ml phenotype profile

25. The matrix or composition of any one of claims 1-8, wherein the monocytic cells are macrophages.

26. The matrix or composition of any one of the preceding claims, wherein the cells are naturally-occurring cells.

27. The matrix or composition of any one of the preceding claims, wherein the cells are isolated from an allogenic source.

28. The matrix or composition of any one claims 1-26, wherein the cells are isolated from an autologous source.

29. The matrix or composition of any one of claims 1-25 and 27-28, wherein the cells are genetically engineered cells.

30. The matrix or composition of any one of the preceding claims, wherein the hydrogel comprises one or more of copolymers of two or more polyhydroxy acids, polyorthoesters, polyanhydrides, gelatin, collagen, cellulose, derivatized cellulose, chitosan, alginate, thiol-modified hyaluronan, and combinations or copolymers thereof.

31. The matrix or composition of claim 30, wherein the polyhydroxy acid is polylactic acid, polyglycolic acid or other polyhydroxy acid.

32. The matrix or composition of any one of the preceding claims, further comprising a cross-linking agent.

33. The matrix or composition of claim 32, wherein the cross-linking agent is selected from glutaraldehyde, diphenylphosphoryl azide, transglutaminase, dimethyl suberimidate, DMS-treated collagen, dimethyl 3, 3'-dithiobispropionimidate, multivalent ions, calcium ions, N,N methylene-bisacrylamide (MBA), acrylamide, allyl methacrylate, ethylene glycol dimethacrylate, tripolyphosphate, and combinations thereof.

34. A method for delivering a secreted product to a localized site in a subject comprising, providing at the localized site the matrix or composition of any one of the preceding claims; and maintaining the matrix or composition at the localized site for a period of time sufficient for the secreted product to interact with the localized site.

35. The method of claim 34, wherein the subject has or is at risk for a disease or condition selected from the group consisting of coronary artery disease, peripheral artery disease, limb ischemia, ischemic wound, ischemic ulcer, ischemic bowel disease, atherosclerotic ischemic disease, muscle flaps, skin flaps, organ transplant, nasolabial folds, wrinkles, conditions which result in scar formation, conditions requiring plastic surgery and conditions requiring a cosmetic procedure.

36. A method of treating a subject having or at risk for ischemia comprising, the administering at the site of ischemia the matrix or composition of any one of claims 5-6, 9-12, 13-18 and 25-33, wherein secretion of the angiogenic factor is for a time sufficient to prevent or lessen the effects of a perfusion injury associated with the ischemia.

37. The method of claim 36, wherein the ischemia is cardiac ischemia.

38. The method of claim 36 or 37, wherein administering the matrix or composition comprised injecting the matrix or composition.

39. The method of claim 36 or 37, wherein administering the matrix or composition comprises injecting the matrix or composition.

40. A method of reducing nasolabial folds or wrinkles in a subject, comprising administering at the site of the nasolabial folds or wrinkles the matrix or composition of any one of claims 5-6, 9-12, 13-18 and 25-33,, wherein secretion of the angiogenic factor is for a time sufficient to detectably reduce the number or depth of the nasolabial folds or wrinkles.

41. The method of claim 40, wherein administration of the matrix or composition comprises injecting the matrix or composition at the site of the nasolabial folds or wrinkles.

42. A method of reducing an immune response at a localized site in a subject, comprising administering at the localized site the matrix or composition of any one of claims 7-12 and 20-33, wherein secretion of the immunosuppression factor is for a time sufficient to detectably reduce the immune response at the localized site.

43. The method of claim 42, wherein administration of the matrix or composition comprises injection of the matrix or composition.

44. A kit comprising: a. a hydrogel material; b. instructions for using the hydrogel material to form a semisolid matrix comprising monocytes embedded therein, wherein: the embedded monocytes produce a secreted product; and the matrix is permeable to the secreted product.

45. A kit comprising: a. a hydrogel material; b. monocytic cells capable of producing a secreted product; and c. instructions for using the hydrogel material to form a semisolid matrix comprising the monocytic cells embedded therein, wherein: the embedded cells produce a secreted product; and the matrix is permeable to the secreted product.

46. The kit of claim 45, wherein the cells are cryopreserved.

47. The kit of claim 45, wherein the cells are preserved such that they remain viable for prolonged periods at about 4°C to about 8°C.

48. The kit of claim 45, wherein the cells are preserved such that they remain viable for prolonged periods at about 15°C to about 25°C.

49. The kit of any one of claims 46- 48, further comprising a pharmaceutically acceptable wash buffer for treatment of the cells prior to embedding in the matrix.

50. A kit comprising: a. a semisolid matrix comprising hydrogel material; b. monocytic cells capable of producing a secreted product embedded therein; and c. instructions for using the semisolid matrix comprising the monocytic cells embedded therein, wherein the embedded cells produce a secreted product; and the matrix is permeable to the secreted product.

51. The kit of claim 50, wherein the matrix is preserved such that the cells embedded therein remain viable when cryopreserved.

52. The kit of claim 50, wherein the matrix is preserved such that the cells embedded therein remain viable for prolonged periods at about 4°C to about 8°C.

53. The kit of claim 50, wherein the matrix is preserved such that the cells embedded therein remain viable for prolonged periods at about 15°C to about 25°C.

54. The kit of any one of claims 51-53, further comprising a pharmaceutically acceptable wash buffer for treatment of the matrix prior to use.

55. The kit of any one of claims 44-49, wherein the cells or hydrogel material are in separate containers.

56. The kit of any one of claims 44-49, wherein the cells or hydrogel material are in separate delivery devices.

57. The kit of claim 56, wherein the delivery devices are suitable for mixing the cells and hydrogel material.

58. The kit of any one of claims 45-57, wherein the monocytes have an M2 phenotype and produce the secreted product at a level that is increased as compared to monocytes having an Ml phenotype.

59. The kit of any one of claims 45-58, further comprising a cross-Unking agent for cross-linking the hydrogel material to form the semisolid matrix.

60. The kit of claim 59, wherein the cross-linking agent is in a separate container.

61. The kit of claim 59, wherein the cross-linking agent is in a separate delivery device.

62. The kit of any one of claims 45 to 61, further comprising instructions for combining the hydrogel material, the monocytes, and optionally the cross-linking agent to form an injectable liquid that transitions to the semisolid matrix following injection into a patient.

63. The kit of any one of claims 45 to 62, further comprising instructions for pre-activating the monocytes towards the M2 phenotype.

64. The kit of any one of claims 45 to 63, further comprising an agent that activates monocytes towards the M2 phenotype.

65. The kit of any one of claims 45 to 64, wherein the hydrogel is selected from one or more of polylactic acid, polyglycolic acid, other polyhydroxy acids, copolymers of two or more polyhydroxy acids, polyorthoesters, polyanhydrides, gelatin, collagen, cellulose, derivatized cellulose, chitosan, alginate, thiol-modified hyaluronan, and combinations thereof.

66. The kit of any one of claims 45 to 65, wherein the cross-Unking agent is selected from glutaraldehyde, diphenylphosphoryl azide, transglutaminase, dimethyl suberimidate, DMS-treated collagen, dimethyl 3, 3 '-dithiobispropionimidate, multivalent ions, calcium ions, N,N methylene-bisacrylamide (MBA), acrylamide, allyl methacrylate, ethylene glycol dimethacrylate, and tripolyphosphate.

67. The kit of any one of claims 45-67, further comprising a delivery device.

68. The kit of claim 67, wherein the delivering device is a syringe or catheter.