CELL SUPPORTING SERUM

Abstract

Failing and degenerating organs may be induced to recover by administering a serum augmenting natural paracrine to extend replenishment. The serum may be collected from a stressed culture of cells and administered to an organ and/or tissue. The stressed serum may also be combined with cells to be implanted to increase recovery, viability and/or establishment of cells.

Claims

1. Cryopreserved cells, comprising: frozen cells; a cryopreservation solution; and a sufficient amount of a stressed serum.

2. The cryopreserved cells of claim 1, wherein the stressed serum is obtained by: seeding a first surface with a first culture of cells; growing the first culture to a monolayer of less than 100% confluence in the presence of a first growth medium as to provide at least one cell free area on the first surface, the first grown medium comprising nutrients and at least one growth factor, wherein the first growth medium promotes proliferation of the first culture; after growing the first culture to less than 100% confluence, replacing the growth medium with a collection medium, the collection medium lacking at least one of the growth factors of the growth medium; maintaining the first culture for a period of time in the presence of the collection medium to produce a conditioned medium; and collecting at least a portion of the conditioned medium after the period of time, said conditioned medium comprising the stressed serum.

3. The cryopreserved cells of claim 2, wherein obtaining the stressed serum further comprises: seeding a second culture onto the at least one cell free area of the first surface, the second culture comprising cells different from the first culture; growing the first and second culture to less than 100% confluence in the presence the second growth medium, the second growth medium comprising of nutrients and at least one growth factor, wherein the second growth medium promotes proliferation of the second culture; after growing the first and second cultures to less than 100% confluence, replacing the second growth medium with a collection medium, the collection medium lacking at least one of the growth factors of the second growth medium; maintaining the first and second cultures for a period of time in the presence of the collection medium to produce a conditioned medium; and collecting at least a portion of the conditioned medium after the period of time, said conditioned medium comprising the stressed serum.

4. The cryopreserved cells of claim 1, wherein the stressed serum comprises a sufficient amount of at least one of amount of an angio-modifying formulation, a morphogenesis formulation, an extracellular matrix formulation, an extracellular matrix modification formulation, an immune promoting formulation and a cytoskeleton formulation.

5. A graft comprising: a biocompatible matrix; cells within biocompatible matrix; and a sufficient amount of stressed serum within the biocompatible matrix.

6. The graft of claim 5, wherein the stressed serum is obtained by: seeding a first surface with a first culture of cells; growing the first culture to a monolayer of less than 100% confluence in the presence of a first growth medium as to provide at least one cell free area on the first surface, the first grown medium comprising nutrients and at least one growth factor, wherein the first growth medium promotes proliferation of the first culture; after growing the first culture to less than 100% confluence, replacing the growth medium with a collection medium, the collection medium lacking at least one of the growth factors of the growth medium; maintaining the first culture for a period of time in the presence of the collection medium to produce a conditioned medium; and collecting at least a portion of the conditioned medium after the period of time, said conditioned medium comprising the stressed serum.

7. The graft of claim 5, wherein obtaining the stressed serum further comprises: seeding a second culture onto the at least one cell free area of the first surface, the second culture comprising cells different from the first culture; growing the first and second culture to less than 100% confluence in the presence the second growth medium, the second growth medium comprising of nutrients and at least one growth factor, wherein the second growth medium promotes proliferation of the second culture; after growing the first and second cultures to less than 100% confluence, replacing the second growth medium with a collection medium, the collection medium lacking at least one of the growth factors of the second growth medium; maintaining the first and second cultures for a period of time in the presence of the collection medium to produce a conditioned medium; and collecting at least a portion of the conditioned medium after the period of time, said conditioned medium comprising the stressed serum.

8. The graft of claim 5, wherein the stressed serum comprises a sufficient amount of at least one of amount of an angio-modifying formulation, a morphogenesis formulation, an extracellular matrix formulation, an extracellular matrix modification formulation, an immune promoting formulation and a cytoskeleton formulation.

9. A method of recovering cryopreserved cells, comprising thawing the cells in a stressed serum solution.

10. The method of claim 9, wherein the stressed serum is obtained by: seeding a first surface with a first culture of cells; growing the first culture to a monolayer of less than 100% confluence in the presence of a first growth medium as to provide at least one cell free area on the first surface, the first grown medium comprising nutrients and at least one growth factor, wherein the first growth medium promotes proliferation of the first culture; after growing the first culture to less than 100% confluence, replacing the growth medium with a collection medium, the collection medium lacking at least one of the growth factors of the growth medium; maintaining the first culture for a period of time in the presence of the collection medium to produce a conditioned medium; and collecting at least a portion of the conditioned medium after the period of time, said conditioned medium comprising the stressed serum.

11. The method of claim 9, wherein obtaining the stressed serum further comprises: seeding a second culture onto the at least one cell free area of the first surface, the second culture comprising cells different from the first culture; growing the first and second culture to less than 100% confluence in the presence the second growth medium, the second growth medium comprising of nutrients and at least one growth factor, wherein the second growth medium promotes proliferation of the second culture; after growing the first and second cultures to less than 100% confluence, replacing the second growth medium with a collection medium, the collection medium lacking at least one of the growth factors of the second growth medium; maintaining the first and second cultures for a period of time in the presence of the collection medium to produce a conditioned medium; and collecting at least a portion of the conditioned medium after the period of time, said conditioned medium comprising the stressed serum.

12. The method of claim 9, wherein the stressed serum comprises a sufficient amount of at least one of amount of an angio-modifying formulation, a morphogenesis formulation, an extracellular matrix formulation, an extracellular matrix modification formulation, an immune promoting formulation and a cytoskeleton formulation.

Description

DETAILED DESCRIPTION

[0073] A method for obtaining a stressed serum suitable for administration to organs and/or tissue with or without cells to be implanted will be described more fully with reference to specific examples. The serum, however, may be obtained in different manners, and thus should not be construed as limited to the specific examples provided. Accordingly, the serum may be obtained by a different ordering and/or sequence of the various steps and/or procedures detailed in the provided examples. For example, two or more steps may be performed concurrently or with partial concurrence. Also, some steps that are performed as discrete steps in the following examples may be combined, and steps being performed as a combined step may be separated into discrete steps, the sequence of certain steps may be reversed or otherwise varied, and the nature or number of discrete steps may be altered or varied. Accordingly, the provided examples are not intended to exclude any of such means of obtaining a stressed serum suitable for in combination with implanted stem cells.

[0074] Likewise, different reagents, techniques, materials and/or equipment other than those specifically mentioned may be utilized to provide the stressed serum.

[0075] A stressed serum may be produced by stressing a culture including proliferative cells. The culture of cells may be obtained by first establishing a monolayer of a first cell culture on a surface. After a monolayer of a first culture is established, a second culture may then be seeded onto cell free areas within the monolayer and established. Additional cultures may then be seeded and established until a monolayer the having the desired cellular composition is obtained. The monolayer of the final culture is then stressed to obtain a serum by conditioning a collection medium. The obtained stressed serum may then be administered to an organ and/or tissue to be treated and/or combined with cells to be implanted before cryopreservation, during thawing, prior to implantation and/or after implantation. When combined prior to preservation, the stressed serum may be incorporated into the cryopreservation solution. When combined during thawing, cryopreserved cells may be thawed in a solution containing the stressed serum. When combined prior to implantation, the stressed serum may be incorporated into a solution, matrix and/or graft containing the cells. When combined after implantation, the stressed serum may be incorporated into a matrix placed over, under and/or adjacent the implanted cells and/or a solution sprayed onto the implanted cells.

[0076] A stressed serum may be obtained from a proliferative monolayer comprising a culture of one or more cell types, such as, but not limited to, a proliferative monolayer of keratinocytes and fibroblasts. The co-cultured monolayer may be established by first partially submerging a vial of frozen keratinocytes (obtained from LifeLine Cell Technologies) in a 37 C. water bath, without submerging the top of the vial. The vial is allowed to thaw in the water bath until a small piece of ice remains. The vial is then removed and sprayed with an ethanol solution. In a hood, keratinocytes are seeded from the vial at 2,500 to 5,000 cells per cm.sup.2 onto a culture treated surface. The surface should be provided with an appropriate volume of a suitable growth medium, such as a medium including Basal DermaLife Media (LifeLine Cell Technologies) and growth factors comprising bovine pituitary extract, L-glutamine, hydrocortisone hemisuccinate, transforming growth factor, insulin, epinephrine and/or ApoTransferrin. The seeded surface is then placed in an incubator and grown at 37 C. in the presence of humidified air comprising 5% CO.sub.2. As to remove any residue DMSO and/or other solvents that may be present in the cryogenic solution, the growth medium may be changed every 24 to 48 hours following initiation of the monoculture. After which time, the growth medium may be changed every 48 to 72 hours.

[0077] Other means of obtaining the initial keratinocytes may also be employed. For instance, keratinocytes may be isolated from neonatal foreskin retrieved from circumcision using the techniques detailed in U.S. application Ser. No. 14/597,796, filed Jan. 15, 2015, the teachings of which are hereby incorporated by reference in their entirety.

[0078] The keratinocytes are allowed to grow in the growth medium until 80-90% confluence is achieved. Voids are then created within the established monolayer by removing the growth medium and washing twice with an appropriate volume of a buffer solution, such as phosphate buffer solution without calcium or magnesium. After washing with buffer solution, a sufficient volume of an enzymatic cell detachment solution to promote detachment of the cells from the surface is added. The cell detachment solution may comprise proteolytic and/or collagenolytic enzymes. For instance, detachment of the cells may be promoted by adding 1 ml of Accutase Solution, available for Innovative Cell Technologies, Inc, per 25 cm.sup.2 of growth area. The enzymatically treated monolayer may then be incubated at 37 C. until the keratinocytes start balling. The surface is then tilted to collect the enzyme solution with a pipette. The collected solution is sprayed at focused points onto the monolayer to create voids in about 50% of the monolayer. The enzyme solution and detached keratinocytes are then removed. The remaining monolayer is provided with a sufficient volume of the growth medium and returned to the incubator. For example, an amount of medium providing 10 ml of medium per 55 cm.sup.2 of growth area may be sufficient.

[0079] Simultaneously, a monolayer of fibroblasts is cultured on a second surface by submerging a vial of frozen fibroblasts (obtained from LifeLine Cell Technologies) in a 37 C. water bath, without submerging the top of the vial. The vial is allowed to thaw in the water bath until a small piece of ice remains. The vial is then removed and sprayed with an ethanol solution. In a hood, fibroblasts are seeded from the vial at 2,500 to 5,000 cells per cm.sup.2 on to a culture treated surface. The surface should be provided with an appropriate volume of a suitable growth medium, such as a medium including Basal DermaLife Media (LifeLine Cell Technologies) and growth factors comprising L-glutamine, hydrocortisone hemisuccinate, lineolic acid, licithin, human serum albumin, basic fibroblasts growth factor, epidermal growth factor, transforming growth factor, insulin and/or vitamin C. The seeded surface is then placed in an incubator and grown at 37 C. in the presence of humidified air comprising 5% CO.sub.2. As to remove any residue DMSO and/or other solvents that may be present in the cryogenic solution, the growth medium may be changed every 24 to 48 hours following initiation of the monoculture. After which time, the growth medium may be changed every 48 to 72 hours.

[0080] Other means of obtaining the initial fibroblasts may also be employed. For instance, fibroblasts may be isolated from neonatal foreskin retrieved from circumcision using the techniques detailed in co-pending U.S. application Ser. No. 14/597,796.

[0081] When the fibroblast monolayer reaches approximately 80 to 90% confluence, the surface is transferred to a hood and the growth medium removed. The fibroblasts monolayer is then washed with a buffer solution, such as phosphate buffer solution without calcium or magnesium. A sufficient volume of a cell detachment solution to promote detachment of the fibroblasts from the second surface is then added. For instance, detachment of the fibroblasts may be promoted by adding 1 ml of Accutase Cell Detachment Solution, manufactured by Innovative Cell Technologies, Inc, per 25 cm.sup.2 of growth area. The fibroblast cells are then incubated in the cell detachment solution at 37 C. until all the cells have detached. A homogenous suspension of cells is then obtained by mixing and the fibroblasts suspension is seeded onto cell free areas within the keratinocyte monolayer. The seeded culture is then returned to the incubator. The co-culture is then grown in the keratinocyte growth medium until 80 to 95% confluence is achieved.

[0082] A monolayer of co-cultured cells may also be achieved by culturing keratinocytes in the keratinocyte growth medium until approximately 50% confluence is achieved. Cell free areas on the surface may then be seeded with the cultured fibroblasts suspension. For example, a co-culture in a T175 cm.sup.2 flask would be overlayed with 1.5 ml of fibroblast suspension generated from a confluent T75 cm.sup.2 flask of fibroblasts dissociated using 3 ml of Accutase. The co-culture may then be grown in the incubator until approximately 80 to 95% confluence is achieved.

[0083] The co-culture of cells may be stressed to provide a therapeutic serum suitable for use in a cosmetic preparation. Stressing the co-culture may be achieved by selectively removing nutrients, growth factors and/or other favorable conditions. The stress need not be severe. Accordingly, sufficient stress may be induced by removing all or a portion of the growth factors while maintaining nutrient levels. Growth factors may be removed by extracting the keratinocyte growth medium from the surface and rinsing the co-culture monolayer twice with a sufficient volume of a buffer solution, such as phosphate buffer solution lacking calcium and magnesium. As to ensure all growth factors are removed, the co-culture may be incubated for a period of time in a collection medium that is added to the surface and then discarded prior to serum collection. For instance, growth factors may be removed prior to serum collection by adding approximately 5.0 ml of a collection medium per 55 cm.sup.2 of growth area and incubating for approximately six hours.

[0084] The collection medium may comprise a minimum essential medium with Earl's salt and have the nutrients of the keratinocyte growth medium.

[0085] After removal of the growth factors, a sufficient volume of fresh collection medium is added, and the surface returned to the incubator for a sufficient period of time to produce a conditioned medium form the collection medium. For example, incubating the co-culture in approximately 10.0 ml of fresh collection medium per 55 cm.sup.2 of growth area for approximately 48 hours may be sufficient to produce a conditioned medium from the collection medium. After incubating for a sufficient period of time, approximately 50% of the collection medium is removed and replaced with an approximately equal amount of fresh collection medium. The co-culture is then incubated for approximately 48 hours to produce more conditioned medium. After which time, all of the conditioned medium is removed.

[0086] The co-culture is then allowed to recover by removing the stress and incubating for a period of time. For instance, incubating in the presence of approximately 10.0 ml per 55 cm.sup.2 of growth area of the keratinocyte growth medium for approximately 24 to 72 hours may provide sufficient recovery. During recovery, the co-culture may be refreshed by seeding fresh cells of one or more of the cultures onto the monolayer.

[0087] After recovering, serum collection is repeated.

[0088] The process of the serum collection and recovery may be repeated until the co-cultures no longer produce serum of the desired quality. For instance, three passes may be utilized. The quality of serum may begin to degrade when one or more of the cultures used to initially establish the co-culture reach 80% of their life expectancy as defined by the maximum number of population doublings.

[0089] The conditioned medium collected may be filtered using a suitable filter, such as a 0.45 m Millipore filter. The serum collected from filtering the conditioned medium may be tested for sterility, virology and/or stability factors. Depending on the intended use of the serum, such testing may not be necessary.

[0090] The stressed serum may also be synthetically generated by combining formulations of proteins with a suitable solvent, such as water. The proteins of each formulation may be obtained from various suppliers and/or harvested from various organisms. The proteins may also be isolated from serums created by cell cultures.

[0091] The stressed serum may comprise a sufficient amount of an angio-modifying formulation comprising agrin isoform 6 and calpastatin isoform 6. The amount of agrin peptides may be less than the amount of calpastatin peptides within the formulation, with a preferred ratio of 8:13.

[0092] In addition to agrin isoform 6 and calpastatin isoform 6, the angio-modifying formulation may further comprise at least one of: [0093] Protein Jagged-1, with the amount of protein-jagged-1 peptides being less than the amount of calpastatin peptides and less than the amount of agrin peptides, a preferred ratio of 5:13 with respect to calpastatin peptides; [0094] Isoform 2 of Growth arrest-specific protein 6, with the amount of growth arrest-specific protein 6 peptides being less than the amount of calpastatin peptides and less than the amount of agrin peptides, a preferred ratio of 7:13 with respect to calpastatin peptides; [0095] Vascular endothelial growth factor C, with the amount of vascular endothelial growth factor peptides being less than the amount of calpastatin peptides and more than the amount of agrin peptides, a preferred ratio of 10:13 with respect to calpastatin peptides; [0096] 72 kDa type IV collagenase, with the amount of collagenase peptides being greater than the amount of calpastatin peptides and more than the amount of agrin peptides, a preferred ratio of 22:13 with respect to calpastatin peptides; and [0097] Desmoglein-2, with the amount of desmoglein-2 peptides being less than the amount of calpastatin peptides and less than the amount of agrin peptides, a preferred ratio of 3:13 with respect to calpastatin peptides.

[0098] The stressed serum may comprise a sufficient amount of morphogenesis formulation comprising tenascin isoform 4 and fibronectin isoform 3. The amount of tenascin peptides may be less than the amount of fibronectin peptides, with a preferred ratio of 8:41.

[0099] In addition to tenascin isoform 4 and fibronectin isoform 3, the morphogenesis formulation may further comprise at least one of: [0100] Transforming growth factor beta induced protein ig-h3, with the amount of ig-h3 peptides being less than the amount of fibronectin peptides and more than the amount of tenascin peptides, a preferred ratio of 31:41 with respect to fibronectin peptides; [0101] Plasminogen activator inhibitor 1, with the amount of plasminogen activator inhibitor 1 peptides being less than the amount of fibronectin peptides and more than the amount of tenascin peptides, a preferred ratio of 38:41 with respect to fibronectin peptides; [0102] Amyloid beta A4 protein, with the amount of amyloid beta A4 peptides being than the amount of fibronectin peptides and more than the amount of tenascin peptides, a preferred ratio of 18:41 with respect to fibronectin peptides; [0103] Glucose-6-phosphate isomerase, with the amount of isomerase peptides being less than the amount of fibronectin peptides and more than the amount of tenascin peptides, a preferred ratio of 19:41 with respect to fibronectin peptides; [0104] Long isoform of serine protease inhibitor Kazal-type 5, with the amount of serine protease inhibitor being less than the amount of fibronectin peptides and more than the amount of tenascin peptides, a preferred ratio of 9:41 with respect to fibronectin peptides; [0105] Cadherin-3, with the amount of cadherin-3 peptides being less than the amount of fibronectin peptides and more than the amount of tenascin peptides, a preferred ratio of 9:41 with respect to fibronectin peptides; [0106] Pappalysin-1, with the amount of pappalysin-1 peptides being less than the amount of fibronectin peptides and less than the amount of tenascin peptides, a preferred ratio of 4:41 with respect to fibronectin peptides; [0107] Insulin-like growth factor-binding protein 7, with the amount insulin-like growth factor-binding protein 7 peptides being less than the amount of fibronectin peptides and more than the amount of tenascin peptides, a preferred ratio of 24:41 with respect to fibronectin peptides; [0108] Kallikrein-10, with the amount of kallikrein-10 peptides being less than the amount of fibronectin peptides and more than the amount of tenascin peptides, a preferred ratio of 22:41 with respect to fibronectin peptides; [0109] Protocadherin fat 1, with the amount of protocadherin fat 1 peptides being less than the amount of fibronectin peptides and less than the amount of tenascin peptides, a preferred ratio of 1:41 with respect to fibronectin peptides; [0110] Syntenin-1, with the amount of syntenin-1 peptides being less than the amount of fibronectin peptides and more than the amount of tenascin peptides, a preferred ratio of 14:41 with respect to fibronectin proteins; [0111] Proliferation-associated protein 2G4, with the amount of proliferation-associated protein peptides being less than the amount of fibronectin peptides and more than the amount of tenascin peptides, a preferred ratio of 10:41 with respect to fibronectin peptides; [0112] Protein CYR61, with the amount of protein CYR61 peptides being less than the amount of fibronectin peptides and more than the amount of tenascin peptides, a preferred ratio of 10:41 with respect to fibronectin peptides; [0113] Keratinocyte proline-rich protein, with the amount of proline-rich protein peptides being less than the amount of fibronectin peptides and less than the amount of tenascin peptides, a preferred ratio of 6:41 with respect to fibronectin peptides; [0114] Brain-specific serein protease 4, with the amount of serine protease peptides being less than the amount of fibronectin peptides, a preferred ratio of 8:41 with respect to fibronectin peptides; [0115] Cadherin 13 isoform 4, with the amount of cadherin 13 peptides being less than the amount of fibronectin peptides and less than the amount of tenascin peptides, a preferred ratio of 4:41 with respect to fibronectin peptides; [0116] Integrin alpha-2, with the amount of integrin alpha 2 peptides being less than the amount of fibronectin peptides and less than the amount of tenascin peptides, a preferred ratio of 2:41 with respect to fibronectin peptides; [0117] Integrin beta-1, with the amount of beta-1 peptides being less than the amount of fibronectin peptides and less than the amount of tenascin peptides, a preferred ratio of 3:41 with respect to fibronectin peptides; and [0118] Neural cell adhesion molecule L1 isoform 2, with the amount of adhesion molecule peptides being less than the amount of fibronectin peptides and less than the amount of tenascin peptides, a preferred ratio of 2:41 with respect to fibronectin peptides.

[0119] The stressed serum may comprise a sufficient amount of an extracellular matrix formulation comprising laminin subunit alpha 3.

[0120] In addition to laminin alpha subunit 3, the extracellular matrix formulation may further comprise at least one of: [0121] Laminin subunit gamma 2, with the amount of gamma 2 peptides being greater than the amount of alpha 3 peptides, a preferred ratio of 42:30; [0122] Laminin subunit beta 3, with the amount of beta 3 peptides being greater than the amount of alpha 3 peptides, a preferred ratio of 36:30; [0123] Collagen alpha-1(I), with the amount of alpha-1(I) peptides being less than the amount of alpha 3 peptides, a preferred ratio of 11:30; [0124] Collagen alpha-1(III), with the amount of alpha-1(III) peptides being less than the amount of alpha 3 peptides, a preferred ratio of 4:30; [0125] Collagen alpha-1(VI), with the amount of alpha-(VI) peptides being less than the amount of alpha 3 peptides, a preferred ratio of 28:30; [0126] Collagen alpha-2(I), with the amount of alpha-2(I) peptides being less than the amount of alpha 3 peptides, a preferred ratio of 15:30; [0127] Collagen alpha-2(VI), with the amount of alpha-2(VI) peptides being less than the amount of alpha 3 peptides, a preferred ratio of 11:30; [0128] Laminin subunit alpha 4, with the amount of alpha 4 peptides being less than the amount of alpha 3 peptides, a preferred ratio of 7:30; [0129] Laminin subunit beta 1, with the amount of beta 1 peptides being less than the amount of alpha 3 peptides, a preferred ratio of 8:30; [0130] Laminin subunit gamma 1, with the amount of gamma 1 peptides being less than the amount of alpha 3 peptides, a preferred ratio of 12:30; [0131] Laminin 332; and [0132] Laminin 411.

[0133] The stressed serum may comprise a sufficient amount of an immune promoting formulation comprising serapin B7, complement component C1s and complement component C3. The amount of C1s peptides may be less than the amount of complement C3 peptides and the amount of serapin B7 peptides may be more than the amount of the C3 peptides, with a preferred ratio of C1s to C3 peptides 12:14, and a preferred ratio of serapin peptides to C3 peptides of 18:14.

[0134] In addition to serapin B7, complement component C1s and complement component C3, the immune promoting formulation may further comprise at least one of: [0135] Elafin, with the amount of elafin peptides being greater than the amount of serpin, C3 or C1s peptides, a preferred ratio of 74:14 with respect to C3 peptides; [0136] Matrix metalloproteinase-9, with the amount of metalloproteinase peptides being less the amount of serpin, C3 or C1s peptides, a preferred ratio of 9:14 with respect to C3 peptides; [0137] Stromelysin-2, with the amount of stromelysin-2 peptides being less the amount of serpin, C3 or C1s peptides, a preferred ratio of 9:14 with respect to C3 peptides; [0138] HLA class I histocompatibility antigen, Cw-6 alpha chain, with the amount Cw-6 alpha chain peptides being less the amount of serpin, C3 or C1s peptides, a preferred ratio of 6:14 with respect to C3 peptides; [0139] Quinone oxidoreductase PIG3, with the amount PIG3 peptides being less the amount of serpin, C3 or C1s peptides, a preferred ratio of 10:14 with respect to C3 peptides; [0140] Superoxide dismutase, with the amount of dismutase peptides being greater than the amount of C1s peptides and less than the serapin peptides, a preferred ratio of 1:1 with respect to C3 peptides; [0141] Metallothionein-2, with the amount of metallothionein peptides being more than the amount of C1s, C3 or serapin peptides, a preferred ratio of 42:14 with respect to C3 peptides; [0142] Alpha-1 antichymotrypsin, with the amount of anitchymotrypsin peptides being less the amount of serpin, C3 or C1s peptides, a preferred ratio of 6:14 with respect to C3 peptides; [0143] Interleukin-1 receptor-like 1, with the amount of interleukin-1 receptor-like peptides being less the amount of serpin, C3 or C1s peptides, a preferred ratio of 4:14 with respect to C3 peptides; and Neutrophil chemotactic agents.

[0144] The Neutrophil chemotactic agents may comprise at least one of: [0145] Interleukin-6, with the amount of interleukin-6 peptides being more the amount of serpin, C3 or C1s peptides, a preferred ratio of 23:14 with respect to C3 peptides; [0146] Growth regulated alpha protein, with the amount of alpha protein peptides being more the amount of serpin, C3 or C1s peptides, a preferred ratio of 32:14 with respect to C3 peptides; [0147] Protein S100-A8, with the amount S100-A8 peptides being more the amount of serpin, C3 or C1s peptides, a preferred ratio of 30:14 with respect to C3 peptides; [0148] Interleukin-8, with the amount of interleukin-8 peptides being more the amount of serpin, C3 or C1s peptides, a preferred ratio of 26:14 with respect to C3 peptides; and [0149] CXC motif chemokine 5, the amount of chemokine peptides being more the amount of serpin, C3 or C1s peptides, a preferred ratio of 19:14 with respect to C3 peptides.

[0150] The stressed serum may comprise a sufficient amount of an extracellular matrix modification formulation comprising interstitial collagenase and stomelysin-1. The amount of stomelysin-1 peptides may be less than the amount of collagenase peptides, with a preferred ratio of 46:72.

[0151] In addition to interstitial collagenase and stomelysin-1, the extracellular matrix modification formulation may further comprise at least one of: [0152] Cathepsin L2, with that amount of cathepsin peptides less than the amount of stomelysin peptides and less than the amount of collagenase peptides, a preferred ratio of 37:72 with respect to collagenase peptides; [0153] Latent transforming growth factor beta-binding protein 2, with the amount of latent transforming growth factor beta-binding protein peptides less than the amount of stomelysin peptides and less than the amount of collagenase peptides, a preferred ratio of 6:72 with respect to collagenase peptides; [0154] Aminopeptidase N, with the amount of aminopeptidase peptides peptides less than the amount of stomelysin peptides and less than the amount of collagenase peptides, a preferred ratio of 6:72 with respect to collagenase peptides; [0155] Decorin, with the amount of decorin peptides less than the amount of stomelysin peptides and less than the amount of collagenase peptides, a preferred ratio of 27:72 with respect to collagenase peptides; [0156] Urokinase-type plasminogen activator, with the amount of plasminogen activators peptides less than the amount of stomelysin peptides and less than the amount of collagenase peptides, a preferred ratio of 20:72 with respect to collagenase peptides; [0157] Lumican, with the amount lumican peptides less than the amount of stomelysin peptides and less than the amount of collagenase peptides, a preferred ratio of 21:72 with respect to collagenase peptides; [0158] Cystatin-M, with the amount of cystatin peptides less than the amount of stomelysin peptides and less than the amount of collagenase peptides, a preferred ratio of 30:72 with respect to collagenase peptides; [0159] Lysyl oxidase homolog 2, with the amount of homolog peptides less than the amount of stomelysin peptides and less than the amount of collagenase peptides, a preferred ratio of 5:72 with respect to collagenase; [0160] Cystatin-C, the amount of cystatin peptides less than the amount of stomelysin peptides and less than the amount of collagenase peptides, a preferred ratio of 25:72 with respect to collagenase peptides; [0161] Protein-lysine 6-oxidase, the amount of oxidase peptides less than the amount of stomelysin peptides and less than the amount of collagenase peptides, a preferred ratio of 8:72 with respect to collagenase peptides; [0162] Tissue factor pathway inhibitor 2, the amount of inhibitor peptides less than the amount of stomelysin peptides and less than the amount of collagenase peptides, a preferred ratio of 14:72 with respect to colleganase; and [0163] Procollagen-lysine, 2-oxoglutarate 5-dioxgenase isoform 2, the amount of dioxgenase peptides less than the amount of stomelysin peptides and less than the amount of collagenase peptides, a preferred ratio of 3:72 with respect to colleganase.

[0164] The stressed serum may comprise a sufficient amount of cytoskeleton formulation comprising translationally-controlled tumor protein.

[0165] In addition to translationally-controlled tumor protein, the cytoskeleton formulation may further comprise at least one of: [0166] Filamin A, with the amount of filamin peptides being less than amount translationally-controlled tumor protein peptides, a preferred ratio of 13:44; [0167] Alpha-actin-1, with the amount of alpha-actin peptides being less than amount translationally-controlled tumor protein peptides, a preferred ratio of 31:44; [0168] Microtubule associated protein 4, with the amount of microtubule associated protein peptides being less than amount translationally-controlled tumor protein peptides, a preferred ratio of 19:44; [0169] Moesin, with the amount of moesin peptides being less than amount translationally-controlled tumor protein peptides, a preferred ratio of 35:44; [0170] Vinculin, with the amount of vinculin peptides being less than amount translationally-controlled tumor protein peptides, a preferred ratio of 11:44; [0171] Involucrin, with the amount involucrin peptides being less than amount translationally-controlled tumor protein peptides, a preferred ratio of 18:44; [0172] Gelsolin isoform 2, with the amount gelsolin peptides being less than amount translationally-controlled tumor protein peptides, a preferred ratio of 10:44; [0173] PDZ and LIM domain protein 1, with amount of PDZ and LIM domain protein peptides being less than amount translationally-controlled tumor protein peptides, a preferred ratio of 20:44; [0174] Caldesmon isoform 5, with the amount of caldesmon peptides being less than amount translationally-controlled tumor protein peptides, a preferred ratio of 13:44; [0175] LIM domain and actin-binding protein 1, with the amount of LIM domain and actin-binding protein peptides being less than amount translationally-controlled tumor protein peptides, a preferred ratio of 7:44; [0176] Myosin regulatory light chain 12B, with the amount light chain peptides being less than amount translationally-controlled tumor protein peptides, a preferred ratio of 21:44; [0177] Small proline rich protein 3, with the amount protein 3 peptides being less than amount translationally-controlled tumor protein peptides, a preferred ratio of 22:44; [0178] Smooth muscle isoform of myosin light polypeptide 6, with the amount of myosin peptides being less than amount translationally-controlled tumor protein peptides, a preferred ratio of 23:44; [0179] Jupiter microtubule associated homolog 1, with the amount of Jupiter peptides being less than amount translationally-controlled tumor protein peptides, a preferred ratio of 21:44; [0180] Small proline rich protein 2A, with the amount of protein 2A peptides being less than amount translationally-controlled tumor protein peptides, a preferred ratio of 45:44; and [0181] Myotrophin, with the amount of myotrophin peptides being less than amount translationally-controlled tumor protein peptides, a preferred ratio of 22:44.

[0182] The stressed serum may further comprise at least one of: [0183] A chaperone formulation comprising at least one of 78 kDa glucose-regulated protein and heat shock 70 kDA protein to assist protein folding and/or assembly; [0184] Nidogen-1 to maintain the basement membrane; [0185] Serum albumin; and [0186] A metabolism formulation comprising at least one of pyruvate kinase, phospholipid transfer protein,

[0187] The stressed serum collected may be combined with cells to be implanted before cryopreservation, during thawing, prior to implantation and/or after implantation. For instance, the stressed serum may be incorporated into a cryopreservation solution. The stressed serum may also be combined with cells by thawing cryopreserved cells in a solution comprising a sufficient amount of the stressed. The stressed serum may also be combined with the cells by incorporating a sufficient amount of the stressed serum into a graft and/or matrix containing the cells. The stressed serum may also be combined with the cells by including a sufficient amount of the stressed serum within an injection solution, spray and/or other delivery vehicle utilized to implant the stem cells. Cells may also be combined with the stressed serum by placing a matrix, such as, but not limited to, a hydrogel and/or bandage, containing a sufficient amount of the stressed serum over, under, adjacent and/or in proximity to implanted cells. Applying a solution, such as, but limited to a spray or injection, containing the stressed serum onto and/or adjacent the implanted cells may also combine the stem cells with the stressed serum.

[0188] It is thus understood from the description herein that aspects of the invention provide new cryopreserved cells comprising: frozen cells; a cryopreservation solution; and a sufficient amount of a stressed serum. In embodiments, the stressed serum is obtained by: seeding a first surface with a first culture of cells; growing the first culture to a monolayer of less than 100% confluence in the presence of a first growth medium as to provide at least one cell free area on the first surface, the first grown medium comprising nutrients and at least one growth factor, wherein the first growth medium promotes proliferation of the first culture; after growing the first culture to less than 100% confluence, replacing the growth medium with a collection medium, the collection medium lacking at least one of the growth factors of the growth medium; maintaining the first culture for a period of time in the presence of the collection medium to produce a conditioned medium; and collecting at least a portion of the conditioned medium after the period of time, said conditioned medium comprising the stressed serum. In embodiments, the obtaining the stressed serum further comprises: seeding a second culture onto the at least one cell free area of the first surface, the second culture comprising cells different from the first culture; growing the first and second culture to less than 100% confluence in the presence the second growth medium, the second growth medium comprising of nutrients and at least one growth factor, wherein the second growth medium promotes proliferation of the second culture; after growing the first and second cultures to less than 100% confluence, replacing the second growth medium with a collection medium, the collection medium lacking at least one of the growth factors of the second growth medium; maintaining the first and second cultures for a period of time in the presence of the collection medium to produce a conditioned medium; and collecting at least a portion of the conditioned medium after the period of time, said conditioned medium comprising the stressed serum. In embodiments, the stressed serum comprises a sufficient amount of at least one of amount of an angio-modifying formulation, a morphogenesis formulation, an extracellular matrix formulation, an extracellular matrix modification formulation, an immune promoting formulation and a cytoskeleton formulation.

[0189] It is thus understood from the description herein that aspects of the invention provide a new graft comprising: a biocompatible matrix; cells within biocompatible matrix; and a sufficient amount of stressed serum within the biocompatible matrix. In embodiments, the stressed serum is obtained by: seeding a first surface with a first culture of cells; growing the first culture to a monolayer of less than 100% confluence in the presence of a first growth medium as to provide at least one cell free area on the first surface, the first grown medium comprising nutrients and at least one growth factor, wherein the first growth medium promotes proliferation of the first culture; after growing the first culture to less than 100% confluence, replacing the growth medium with a collection medium, the collection medium lacking at least one of the growth factors of the growth medium; maintaining the first culture for a period of time in the presence of the collection medium to produce a conditioned medium; and collecting at least a portion of the conditioned medium after the period of time, said conditioned medium comprising the stressed serum. In embodiments, the obtaining the stressed serum further comprises: seeding a second culture onto the at least one cell free area of the first surface, the second culture comprising cells different from the first culture; growing the first and second culture to less than 100% confluence in the presence the second growth medium, the second growth medium comprising of nutrients and at least one growth factor, wherein the second growth medium promotes proliferation of the second culture; after growing the first and second cultures to less than 100% confluence, replacing the second growth medium with a collection medium, the collection medium lacking at least one of the growth factors of the second growth medium; maintaining the first and second cultures for a period of time in the presence of the collection medium to produce a conditioned medium; and collecting at least a portion of the conditioned medium after the period of time, said conditioned medium comprising the stressed serum. In embodiments, the stressed serum comprises a sufficient amount of at least one of amount of an angio-modifying formulation, a morphogenesis formulation, an extracellular matrix formulation, an extracellular matrix modification formulation, an immune promoting formulation and a cytoskeleton formulation.

[0190] It is thus understood from the description herein that aspects of the invention provide a new method of recovering cryopreserved cells, comprising thawing the cells in a stressed serum solution. In embodiments, the stressed serum is obtained by: seeding a first surface with a first culture of cells; growing the first culture to a monolayer of less than 100% confluence in the presence of a first growth medium as to provide at least one cell free area on the first surface, the first grown medium comprising nutrients and at least one growth factor, wherein the first growth medium promotes proliferation of the first culture; after growing the first culture to less than 100% confluence, replacing the growth medium with a collection medium, the collection medium lacking at least one of the growth factors of the growth medium; maintaining the first culture for a period of time in the presence of the collection medium to produce a conditioned medium; and collecting at least a portion of the conditioned medium after the period of time, said conditioned medium comprising the stressed serum. In embodiments, the obtaining the stressed serum further comprises: seeding a second culture onto the at least one cell free area of the first surface, the second culture comprising cells different from the first culture; growing the first and second culture to less than 100% confluence in the presence the second growth medium, the second growth medium comprising of nutrients and at least one growth factor, wherein the second growth medium promotes proliferation of the second culture; after growing the first and second cultures to less than 100% confluence, replacing the second growth medium with a collection medium, the collection medium lacking at least one of the growth factors of the second growth medium; maintaining the first and second cultures for a period of time in the presence of the collection medium to produce a conditioned medium; and collecting at least a portion of the conditioned medium after the period of time, said conditioned medium comprising the stressed serum. In embodiments, the stressed serum comprises a sufficient amount of at least one of amount of an angio-modifying formulation, a morphogenesis formulation, an extracellular matrix formulation, an extracellular matrix modification formulation, an immune promoting formulation and a cytoskeleton formulation.

[0191] It is thus understood from the description herein that aspects of the invention provide a new method of producing a stressed serum improving replenishment of cells, comprising: seeding a first surface with a first culture of cells; growing the first culture to a monolayer of less than 100% confluence in the presence of a first growth medium as to provide at least one cell free area on the first surface, the first grown medium comprising nutrients and at least one growth factor, wherein the first growth medium promotes proliferation of the first culture; after growing the first culture to less than 100% confluence, replacing the growth medium with a collection medium, the collection medium lacking at least one of the growth factors of the growth medium; maintaining the first culture for a period of time in the presence of the collection medium to produce a conditioned medium; and collecting at least a portion of the conditioned medium after the period of time, said conditioned medium comprising the stressed serum. In embodiments, the first culture of cells comprises at least one of stem cells, cardiac cells, fibroblasts, keratinocytes and hepatic cells. In embodiments, the method further comprising: seeding a second culture onto the at least one cell free area of the first surface, the second culture comprising cells different from the first culture; growing the first and second culture to less than 100% confluence in the presence the second growth medium, the second growth medium comprising of nutrients and at least one growth factor, wherein the second growth medium promotes proliferation of the second culture; after growing the first and second cultures to less than 100% confluence, replacing the second growth medium with a collection medium, the collection medium lacking at least one of the growth factors of the second growth medium; maintaining the first and second cultures for a period of time in the presence of the collection medium to produce a conditioned medium; and collecting at least a portion of the conditioned medium after the period of time, said conditioned medium comprising the stressed serum, wherein at least one of the first culture and second cultures comprise stem cells.

[0192] It is thus understood from the description herein that aspects of the invention provide a new method of treating replenishing cells within a tissue, comprising administering to a tissue to be treated stressed serum comprising a sufficient amount of at least one of amount of an angio-modifying formulation, a morphogenesis formulation, an extracellular matrix formulation, an extracellular matrix modification formulation, an immune promoting formulation and a cytoskeleton formulation.

[0193] It is thus understood from the description herein that aspects of the invention provide a new method of implanting cells, comprising: combining cells with a stressed serum, the stressed serum comprising a sufficient amount of at least one of amount of an angio-modifying formulation, a morphogenesis formulation, an extracellular matrix formulation, an extracellular matrix modification formulation, an immune promoting formulation and a cytoskeleton formulation; and implanting the cells into a patient. In embodiments, the stems are frozen and the stressed serum is combined during thawing. In embodiments, the method further comprising combining the cells and stressed serum with a cryopreservation solution; and freezing the combination of cells, stressed serum and cryopreservation solution.

[0194] It is thus understood from the description herein that aspects of the invention provide a new therapeutic matrix, comprising: a biocompatible matrix; and a sufficient amount of a stressed serum within the biocompatible matrix. In embodiments, the stressed serum is obtained by: seeding a first surface with a first culture of cells; growing the first culture to a monolayer of less than 100% confluence in the presence of a first growth medium as to provide at least one cell free area on the first surface, the first grown medium comprising nutrients and at least one growth factor, wherein the first growth medium promotes proliferation of the first culture; after growing the first culture to less than 100% confluence, replacing the growth medium with a collection medium, the collection medium lacking at least one of the growth factors of the growth medium; maintaining the first culture for a period of time in the presence of the collection medium to produce a conditioned medium; and collecting at least a portion of the conditioned medium after the period of time, said conditioned medium comprising the stressed serum. In embodiments, the obtaining the stressed serum further comprises: seeding a second culture onto the at least one cell free area of the first surface, the second culture comprising cells different from the first culture; growing the first and second culture to less than 100% confluence in the presence the second growth medium, the second growth medium comprising of nutrients and at least one growth factor, wherein the second growth medium promotes proliferation of the second culture; after growing the first and second cultures to less than 100% confluence, replacing the second growth medium with a collection medium, the collection medium lacking at least one of the growth factors of the second growth medium; maintaining the first and second cultures for a period of time in the presence of the collection medium to produce a conditioned medium; and collecting at least a portion of the conditioned medium after the period of time, said conditioned medium comprising the stressed serum. In embodiments, the stressed serum comprises a sufficient amount of at least one of amount of an angio-modifying formulation, a morphogenesis formulation, an extracellular matrix formulation, an extracellular matrix modification formulation, an immune promoting formulation and a cytoskeleton formulation.

[0195] While the present invention has been described herein with respect to the exemplary embodiments, it will become apparent to one of ordinary skill in the art that many modifications, improvements and sub-combinations of the various embodiments, adaptations and variations can be made to the invention without departing from the spirit and scope thereof. Accordingly, the presented embodiment should not be construed as limiting the scope of this disclosure or the accompanying claims.

[0196] Furthermore, it should be appreciated that first and second as used in claims is merely to reference that one (first) precedes another (second) and/or to distinguish similar components from one another. It should also be appreciated that though examples presented above may have included two cultures, this was solely for purposes of illustration and in no way intended to limit the scope of this disclosure or the claims. As such, a first culture may be a first, second, third, etc. culture. Likewise, the second culture may be any culture added subsequent to the first culture. Accordingly, if the first culture is the third culture added, then the second culture may be a fourth, fifth, sixth, etc. culture.