OPTIMIZATION of the Cultivation of Mesenchymal STEM CELLS for CLINICAL USE

Studies with mesenchymal stem cells (MSCs) have shown its benefits in hematology, mainly for Graft-versus-host disease (Lancet 371:1579–86, 2008), with three unsettled matters: (1)MSCs expansion in medium supplemented with Fetal Calf Serum (FCS) and its risk of xenoreaction (Blood 89:776–9, 1997); (2)The optimal number of cells needed for therapy is not yet defined, but there is an empirical indication for 2×10⁶células/Kg with the need to optimize expansion, number and time wise; and (3)the utilization of third party donors. This study was designed to determine the superiority or no-inferiority of the Platelet Lysates (PL) over FCS on the expansion of MSC, the optimal cell plating density and days between each pass, and to investigate if in our conditions total nucleated cells (TNC) obtained from the washouts of HSCT explants can expand to be used at clinical grade.

TNC were removed from the filters and bags used in the HSCT (Cytotherapy 11:403–13, 2009) and after the first passage were plated in different concentrations (2000/cm², 3000/cm², 4000/cm², 5000/cm², 6000/cm² and 7000/cm²) with 10% FBS or 10% PL, and the number of days reach 80% of confluence was observe (Transfusion, 49:2680–5, 2009). Next, cultures with the same plating density were fed either with 10% PL or 10% FCS and were trypsinized after seven days and counted to analyze how much they have grown in that time period. And finally, cultures were allowed to growth up to Passage 3 (P3) to test the ability to obtain clinical grade number of cells. The proliferation of mesenchymal stem cells in the presence of PL and SFB was averaged 11.88 and 2.5 times, respectively, in a period of 7 days (p = 0.005). The highest concentration of plating cells using PL, took less time (6 days) to reach confluence as compared with the three lower (7.55 to 8.55 days) (p = 0.005), and at P3 with PL we obtained from 10×10⁹ up to 10 × 10¹¹ cells.

This study suggests that the PL is the best choice as a supplement to expand MSC, and allow the proliferation of a sufficient number of MSC at P3 for clinical use obtained from the washouts of HSCT explants.

No relevant conflicts of interest to declare.