Method for obtaining an enriched population of functional mesenchymal stem cells, cells obtained thereof compositions comprising the same

Abstract

A method to obtain a composition comprising an enriched population of functional mesenchymal stem cells for hypothermic transport and local administration of said enriched population of functional mesenchymal stem cells in therapy. Finally also described is the use of said enriched population of functional mesenchymal stem cells, and compositions comprising them, obtained by the described method, in autologous or allogeneic treatment of diseases susceptible to mesenchymal stem cell therapy, either by local or systemic treatments, and more particularly in the treatment of osteoarticular diseases such as degenerative disc disease, osteoarthritis, and bone repair; in lupus erythematosus, graft-versus-host disease, and other autoimmune diseases; in peripheral vascular insufficiency and other cardiovascular diseases.

1 . A method for obtaining a composition comprising functional mesenchymal stem cells suitable for administration in therapy, said method comprising the steps of:
 a. suspending an ex vivo sample of bone marrow mesenchymal stem cells in a cryoprotecting medium comprising 5% to 10% dimethyl sulfoxide at a concentration of 5×10 6  to 10×10 6 cells/ml;   b. cryopreserving the sample of bone marrow mesenchymal stem cells, cooling them first from −70° C. to −90° C. for at least 24 hours prior to storing the sample in liquid nitrogen;   c. restoring the sample of bone marrow mesenchymal stem cells, by performing the following steps:
 c1. thawing the sample of the bone marrow mesenchymal stem cells by progressively increasing the temperature up to 35-39° C. during 1 to 5 minutes; 
 c2. diluting the sample 10 to 30 times the initial sample volume with suitable culture medium; 
 c3. centrifuging the sample, discarding the supernatant and resuspending the pellet of mesenchymal stem cells in suitable culture medium; 
 c4. selecting the mesenchymal stem cells with a viability of at least 70%; 
 c5. seeding the mesenchymal stem cells selected in step (c4) on a plastic support and incubating said mesenchymal stem cells with a suitable culture medium comprising 7.5% to 10% CO 2  and at least 5% fetal bovine serum at a cell concentration between 1000 to 5000 cells/cm 2 , with adequate culture conditions at 35-39° C., 
 c6. replacing with fresh suitable culture medium comprising 7.5% to 10% CO 2  and at least 5% fetal bovine serum, at regular time intervals and isolating the mesenchymal stem cells from the support when the cells occupy 80 to 100% of the support's surface; 
 c7. selecting the mesenchymal stem cells that: 
 show adherence to plastic; and 
 present a viability of at least 70%; and 
 present an expression ≥90% of CD90, CD 166, CD73 and CD 105; and 
 present an expression >10% of CD 14, CD34, CD45 and HLA-DR>10%; and 
 do not feature chromosomal aberrations; and 
 present capacity to differentiate into osteoblasts, adipocytes and chondrocytes; and 
   d. suspending the mesenchymal stem cells isolated in step (c7) in an adequate medium for transport and storage at 2-8° C. to obtain a composition comprising functional mesenchymal stem cells, wherein said adequate medium for transport and storage at 2-8° C. is an isotonic medium comprising 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid 0.25 to 1 mM.   
     
     
         2 . A method according to  claim 1 , wherein the cryoprotecting medium of step (a) comprises fetal bovine serum and 5% to 10% DMSO. 
     
     
         3 . A method according to  claim 1 , wherein the cryoprotecting medium of step (a) is an animal component-free, serum-free and protein-free medium, comprising 5% or 10% DMSO. 
     
     
         4 . A method according to  claim 1 , wherein step (b) comprises cryopreserving the sample of bone marrow mesenchymal stem cells at a speed of 1° C./min from −70° C. to −90° C. for at least 25 hours prior to storing the sample in liquid nitrogen. 
     
     
         5 . (canceled) 
     
     
         6 . A method according to  claim 1 , wherein the isotonic medium comprising 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid 0.25 mM to 1 mM, is an animal component-free serum-free protein-free medium. 
     
     
         7 . A method according to  claim 1 , wherein the isotonic medium comprising 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid 0.25 mM to 1 mM comprises a 1:9 to 9:1 mixture (v/v) of a first composition comprising Na +  130 mM, K +  4 mM, Ca 2+  1.35 mM, Chloride 109 mM and lactate 16 mM, with a second composition comprising Na +  159 mM, K +  5 mM, Mg 2+  0.8 mM, Chloride 77 mM, dihydrogen phosphate 28 mM, citrate 10 mM and acetate 32 mM, supplemented with glucose 5 mM, and human serum albumin 0.1% to 0.5%. 
     
     
         8 . A composition comprising a population of at least 0.5×10 6  mesenchymal stem cells and 6-hydroxy-2.5.7.8-tetramethylchroman-2-carboxylic acid 0.25 mM to 1 mM obtainable according to the method of  claim 1 , wherein said mesenchymal stem cells retain for at least 72 hours during hypothermic transport at 2-8° C. the functionality of fresh mesenchymal stem cells, including:
 showing adherence to plastic; and 
 presenting a viability of at least 70%; and 
 presenting an expression >90% of CD90, CD 166, CD73 and CD 105; and 
 presenting an expression ≥10% of CD14, CD34, CD45 and HLA-DR ≥10%; and 
 not featuring chromosomal aberrations; and 
 presenting capacity to differentiate into osteoblasts, adipocytes and chondrocytes. 
 
     
     
         9 . A composition according to  claim 8  for use as a medicament. 
     
     
         10 . A composition according to  claim 8 , for use in autologous or allogeneic treatment of osteoarticular diseases, autoimmune diseases and cardiovascular diseases. 
     
     
         11 . The composition for use according to  claim 9 , wherein said composition is to be administered at a cell density of 1×10 6  to 10×10 6  cells/ml and at 0.5 to 90 million cells. 
     
     
         12 . The composition for use according to  claim 10 , wherein the osteoarticular diseases are selected from the group consisting of degenerative disc disease, osteoarthritis, meniscus injuries, and rheumatoid arthritis, 
     
     
         13 . The composition for use according to  claim 10 , wherein the autoimmune diseases are selected from the group consisting of lupus erythematosus, graft-versus-host disease, and systemic sclerosis. 
     
     
         14 . The composition for use according to any of  claim 10 , wherein the cardiovascular diseases are selected from the group consisting of peripheral vascular insufficiency, myocardial infarction, stroke, and ischemia.