A process for the production of a composition for the treatment of a subject suffering from osteoarthritis characterised in that it comprises the following:
- a) isolating stromal vascular fraction (SVF) from the adipose tissue of at least two healthy donors that are different from the subject to be treated but belong to the same species;
- b) providing the mesenchymal stem cells, that is, the MSCs obtained from each isolated fraction separately, preferably by digestion with collagenase;
- c) sedimenting the obtained MSCs separately and resuspending preferably in balanced saline buffer, for example Hank's buffer;
- d) propagating the resuspended MSCs separately, preferably at least until 70%, 80%, or 90% confluency, then freezing them separately to generate a P1 cell bank in a way that selecting and keeping those propagated MSCs prior freezing that are verified pathogen free, morphologically healthy, and based on the analysis of their cell surface markers, they are CD 105, CD73 positive but do not carry surface markers characteristic of hematopoietic stem cells and progenitor cells and preferably do not carry surface markers characteristic of blood cell development lineages, that is, the MSCs are preferably CD45, CD34, CD14, CD11b, CD31, CD79α, and CD19 negative, and their PD value during culturing is preferably in the range of 0.5 to 5, more preferably in the range of 1 to 4, most preferably about 3;
- e) thawing a part of the separate P1 cell banks and further propagating MSCs to at least until 70%, 80%, or 90% confluency, then freezing them separately to generate a P2cell bank in a way that selecting and keeping those cultured MSCs prior freezing that are verified pathogen free, morphologically healthy, and based on the analysis of their cell surface markers, they are CD105, CD73 positive but do not carry surface markers characteristic of hematopoietic stem cells and progenitor cells and preferably do not carry surface markers characteristic of blood cell development lineages, that is, the MSCs are preferably CD45, CD34, CD14, CD11b, CD31, CD79α and CD19 negative, and their PD value during culturing is preferably in the range of 0.5 to 5, more preferably in the range of 1 to 4, and most preferably about 3, and the level of kynurenine metabolite production of the MSCs is in the range of 30 to 50 ng/ml, preferably about 30 ng/ml, and the level of THBS1 cytokine production of the MSCs is in the range of 50 to 80 ng/ml, preferably about 50 ng/ml;
- f) repeating step e) until the number of MSCs reaches the value of 105 to 108, preferably 105 to 5×107 separately, and the CPD value of the MSCs is preferably in the range of 3 to 14, more preferably in the range of 2 to 12, most preferably about 8;
- g) optionally mixing the MSCs thus obtained, propagated from each donor separately, wherein the mixture contains isolated MSCs producing kynurenine at about 50 ng/ml and THBS1 cytokine at about 80 ng/ml in an amount less than 50% of the mixture and isolated MSCs producing kynurenine at about 30 ng/ml and THBS1 cytokine at about 50 ng/ml in an amount of higher then 50% of the mixture and;
- h) adding 5% DMSO Hank's (HBSS) solution to the balanced composition.