74 - Expanding mesenchymal stem cells with platelet lysate vs FBS increases bioreactor efficiency but alters cytokine prolife

Abstract

Bone-marrow derived mesenchymal stem cells (BM-MSCs) are a leading cell product candidate for the treatment of multiple inflammatory, degenerative, and chronic diseases. However, conventional culture methods (with T175 cm2 culture flasks) lack practicable scalability. The production of a lot containing 500 million clinical-grade BM-MSCs is a labor-intensive process that requires a highly-coordinated effort of four well-trained technical staff members. Therefore, bioreactors have been developed as a cost effective and scalable method of producing BM-MSCs. We have validated a Quantum bioreactor (Terumo) method using passage 1, human BM-MSCs isolated from 4 different donors to efficiently produce potent clinical-grade products using both 5% human Platelet Lysate (hPL) and 20% Fetal Bovine Serum (FBS) containing medias. Our harvested products were characterized by proliferation analysis, daily cell lactate production, colony forming unit (CFU) analysis, cell surface protein expression, and cytokine profile. Head to head comparison of hPL and FBS cultured products showed a 5.1 ± 1.46-fold, (P < 0.05) increased efficiency of bioreactor expansion of BM-MSC in hPL media, producing 7.68 × 108 ± 1.25 vs 2.21 × 108 ± 0.92 cells with a seeding of 1.0–2.5 × 107 cells, respectively (P < 0.05). Furthermore, BM-MSCs formed CFUs and had positive expression of CD105, CD90, CD44, and CD75 and negative expression of CD45, CD31, and CD34. Interestingly, cytokine analysis of 80 cytokine targets revealed differences in the BM-MSC secretomes produced in hPL vs FBS media. Expansion in hPL upregulated RANTES (+8.1 ± 1.3-fold, P < 0.05) and downregulated IL-6 (−2.13 ± 0.72-fold, P < 0.05), SDF-1 (−1.62 ± 0.18-fold, P < 0.001), VEGF (−1.57 ± 0.24-fold, P < 0.01), and Angiogenin (−1.6 ± 0.11-fold, P < 0.001), significantly. Additionally, consistent upregulations of IL-8 (+2.29 ± 0.69-fold) and MCP-3 (+6.76 ± 3.6-fold) was documented in hPL-produced BM-MSCs. Together, these results confirm the superiority of hPL expansion of BM-MSCs in the Quantum bioreactor but reveals an alteration to the cytokine prolife. To date, many clinical trials have been done with BM-MSC produced in FBS containing media. However, due to concerns involving the use of animal-derived products, GMP-facilities are beginning to switch to hPL containing media. Our results show that this switch is feasible, however changes in BM-MSC cytokine profile should be taken into consideration depending upon the clinical application.