Processes for making and using a mesenchymal stem cell derived secretome

Abstract

The present application provides methods and processes for making and using a mesenchymal stem cell secretome, as well as methods for treating ocular conditions and/disorders with the mesenchymal stem cell secretome described herein.

1 .- 141 . (canceled) 
     
     
         142 . A composition that induces ocular wound healing comprising a mesenchymal stem cell (MSC) secretome and a tonicity modifying agent, wherein the ability of the composition to induce ocular wound healing is indicated by a migration assay comprising:
 a) adding corneal cells to a chamber comprising a porous membrane, wherein the corneal cells are supplemented with basal media in the absence of the composition;   b) placing the chamber into a container comprising the basal media in the presence of the composition, wherein the corneal cells in the chamber are separated from the composition in the container by the porous membrane;   c) incubating the corneal cells; and   d) measuring the corneal cells that have migrated and/or proliferated through the porous membrane as indicative of the ability of the composition to induce migration and/or proliferation of the corneal cells.   
     
     
         143 . The composition according to  claim 142 , wherein step c) of the migration assay comprise incubating the corneal cells for about 12-72 hours. 
     
     
         144 . The composition according to  claim 142 , wherein step c) of the migration assay comprise incubating the corneal cells for about 18-24 hours. 
     
     
         145 . The composition according to  claim 142 , wherein the migration assay further comprises, prior to step b) of placing the chamber into the container, a step of concentrating the composition, and optionally a step of buffer exchanging for the composition. 
     
     
         146 . The composition according to  claim 142 , wherein the migration assay further comprises, prior to step b) of placing the chamber into the container, a step of diluting the composition, and optionally a step of buffer exchanging for the composition. 
     
     
         147 . The composition according to  claim 142 , wherein the concentrated composition comprises at least 45 μg/ml proteins. 
     
     
         148 . A method of screening for or determining the ability of a composition to induce migration and/or proliferation of corneal cells, the method comprising:
 a) adding corneal cells to a chamber comprising a porous membrane, wherein the corneal cells are supplemented with basal media in the absence of the composition;
 b) placing the chamber into a container comprising the basal media in the presence of the composition, wherein the corneal cells in the chamber are separated from the composition in the container by the porous membrane; 
 c) incubating the corneal cells; and 
 d) measuring the corneal cells that have migrated and/or proliferated through the porous membrane as indicative of the ability of the composition to induce migration and/or proliferation of the corneal cells. 
   
     
     
         149 . A stable mesenchymal stem cell (MSC) secretome formulation comprising an MSC secretome, wherein the formulation further comprises:
 i. 1 mg-3 mg monobasic sodium phosphate per mL;   ii. 5 mg-12 mg dibasic sodium phosphate per mL;   iii. 11.5 mg-13 mg mannitol per mL;   iv. 23 mg-25 mg trehalose dihydrate per mL;   wherein the pH is about 4.7 to about 7.5.   
     
     
         150 . The stable mesenchymal stem cell (MSC) secretome formulation according to  claim 149 , wherein the formulation comprises 2-400 μg of the MSC secretome per mL. 
     
     
         151 . The stable mesenchymal stem cell (MSC) secretome formulation according to  claim 149 , wherein the MSC is a bone marrow-derived MSC. 
     
     
         152 . The stable mesenchymal stem cell (MSC) secretome formulation according to claim  8 , wherein the MSC secretome is a bone marrow-derived secretome comprising: HGF;
 Pentraxin-3 (TSG-14); VEGF; TIMP-1; Serpin E1; <5 ng/mL IL-8, and a tonicity modifying agent.   
     
     
         153 . A stable mesenchymal stem cell (MSC) secretome formulation comprising an MSC secretome, wherein the formulation further comprises:
 i. 4%-5% w/w monobasic sodium phosphate;   ii. 21.5%-23% w/w dibasic sodium phosphate;   iii. 23%-25% w/w mannitol;   iv. 46%-48% w/w trehalose dehydrate;   wherein the pH is about 4.7 to about 7.5.   
     
     
         154 . A method of treating an ocular condition in a subject in need thereof comprising administering to the subject a stable mesenchymal stem cell (MSC) secretome formulation comprising an MSC secretome, wherein the formulation further comprises:
 i. 1 mg-3 mg monobasic sodium phosphate per mL;   ii. 5 mg-12 mg dibasic sodium phosphate anhydrous per mL;   iii. 11.5 mg-13 mg mannitol per mL;   iv. 23 mg-25 mg trehalose dihydrate per mL;   wherein the pH is about 4.7 to about 7.5.   
     
     
         155 . The method of  claim 154 , wherein the condition is selected from the group consisting of: ocular wound, ocular scarring, ocular neovascularization, increased intraocular pressure, dry eye disease, damaged corneal surface, damaged ocular nerve tissue, retina condition, persistent corneal epithelial defects (PCED), Graft v. Host Disease (GvHD), and Stevens-Johnson Syndrome. 
     
     
         156 . A method of treating an ocular condition in a subject in need thereof comprising administering to the subject a stable mesenchymal stem cell (MSC) secretome formulation comprising an MSC secretome, wherein the formulation further comprises:
 i. 4%-5% w/w monobasic sodium phosphate;   ii. 21.5%-23% w/w dibasic sodium phosphate;   iii. 23%-25% w/w mannitol;   iv. 46%-48% w/w trehalose dehydrate;   wherein the pH is about 4.7 to about 7.5.   
     
     
         157 . A method of making a mesenchymal stem cell (MSC) secretome composition comprising:
 i. culturing mesenchymal stem cells (MSCs) in a first culture media;   ii. removing the first culture media from step (i) from the MSCs;   iii. washing the MSCs in step (ii);   iv. adding a second culture media and culturing for about 1-5 days;   v. harvesting the second culture media from step (iv) as conditioned media; and   vi. processing the conditioned media in step (v) into the MSC secretome composition.   
     
     
         158 . The method according to  claim 157 , wherein step (vi) processing the conditioned media in step (v) into the secretome composition comprises:
 a) filtering the harvested conditioned media from step (v) to remove cell particulate;   b) concentrating the filtered conditioned media from step (a); and   c) buffer exchanging with the formulation buffer.   
     
     
         159 . A bone marrow-derived mesenchymal stem cell (MSC) secretome composition comprising: HGF; Pentraxin-3 (TSG-14); VEGF; TIMP-1; Serpin E1; <5 ng/mL IL-8, and a tonicity modifying agent. 
     
     
         160 . A method of treatment for an ocular condition in a subject in need thereof comprising administering to the subject a bone marrow-derived mesenchymal stem cell (MSC) secretome composition, wherein the MSC secretome composition comprises: HGF; Pentraxin-3 (TSG-14); VEGF; TIMP-1; Serpin E1; and <5 ng/mL IL-8. 
     
     
         161 . A composition that induces ocular wound healing comprising a mesenchymal stem cell (MSC) secretome and a tonicity modifying agent, wherein the ability of the composition to promote ocular wound healing is indicated by a wound healing assay comprising:
 a) providing a layer of corneal cells;   b) introducing a wound gap to the layer of corneal cells; and   c) determining whether the wound gap heals in the presence of the composition, wherein the composition is administered to the corneal cells either before or after step b);   wherein closure of the wound gap is indicative of the ability of the composition to induce ocular wound healing.