| CPC A01N 1/0221 (2013.01) [A01N 1/0226 (2013.01); A61K 35/28 (2013.01); C12N 5/0663 (2013.01)] | 14 Claims |
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1. A method for obtaining a composition comprising functional mesenchymal stem cells that have been cryopreserved, restored, and conditioned for transport and storage and are 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 5×106 to 10×106 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 a volume of the thawed sample from c1 10 to 30 times with suitable culture medium;
c3. centrifuging the diluted sample from c2, 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% CO2 and at least 5% fetal bovine serum at a cell concentration between 1000 to 5000 cells/cm2, with adequate culture conditions at 35-39° C.,
c6. replacing with fresh suitable culture medium comprising 7.5% to 10% CO2 and at least 5% fetal bovine serum maintained at 35-39° C., 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 CD105; and
present an expression ≤10% of CD14, CD34, CD45 and HLA-DR≤10%; and
do not feature chromosomal aberrations; and
present capacity to differentiate into osteoblasts, adipocytes and chondrocytes; and
d. conditioning the mesenchymal stem cells isolated in step (c7) for transport and storage at 2-8° C. by suspending the restored mesenchymal stem cells in an isotonic medium, wherein the conditioned mesenchymal stem cells have a viability of at least 70% at 2-8° C. for at least 72 hours, wherein said isotonic medium comprises 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid 0.25 to 1 mM.
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