Expansion of Human Mesenchymal Stromal/Stem Cells Using Standardized Xeno-Free, Serum-Free Culture Condition

Aims: mesenchymal stromal/stem cells (MSCs) is one of the most promising cell therapies to treat immune related diseases such as GvHD, Crohn's disease, osteoarthritis as well as to support function of heart, liver, and central nervous system. Many advanced clinical trials has been taking place to investigate the safety and efficacy of MSC therapy, however, it remains challenging to compare the results of these trials due to variations in culture conditions and tissue origins. These variations pose the urgent need of a standardized large-scale production and quality control of these cells become more urgent, as the use of MSCs are spreading around the globe and disease backgrounds. Numerous culture protocols for MSCs are available using FBS-containing, human platelet lysate/plasma supplementing, or serum-free xeno-free condition, either commercial or self-prepared media. In this study, we have established a standardized expansion protocol that can be used for MSCs derived from bone marrow (BM), adipose tissue (AD), and umbilical cord (UC). Methods: we tested commercial xeno-free, serum-free media (MesenCult™-ACF Plus Medium/Stem Cell Technologies, MSC NutriStem® XF Medium/BI, Stempro® MSC SFM Medium/ThermoFisher, PowerStem MSC1 Medium/PAN, and StemMACS™ MSC Expansion Media/Miltenyi) in order to identify a common culture condition for all of these MSC sources. The cells were analysed for their population doubling time from P2 to P6, expression of surface markers including the positive markers CD73, CD90, and CD105, and the negative markers CD34, CD45, CD11b, CD19, and HLA-DR. Karyotype was conducted at early and later passage (P3 and P6, respectively). MSCs were tested for their differentiation capacity into osteoblast/osteocyte, adipocyte, and chondrocyte lineages. Bacterial and fungal sterility and mycoplasma test were performed and endotoxin concentration was determined before the cells were transfused into patients. Results: using UC for the primary screening, we identified two medium candidates that supported the isolation and culture of UC-MSCs. MSCs derived from BM and AD are capable of expansion in these media as well. However, one candidate induced an increased HLA-DR expression so that it failed to fulfill the minimal criteria of MSCs (Dominici et al., 2006). The last one showed the best results for MSCs from all tested tissues. Under our established protocol, MSCs grew exponentially until P6, which was the highest analyzed passage. The cells expressed CD73, CD90, CD105 in more than 95% cells and showed less than 2% of negative markers (CD34, CD45, CD11b, CD19, and HLA-DR). Karyotype analysis confirmed no chromosomal aberrations of the cultured cells. The cells were able to differentiate into osteoblast/osteocyte, adipocyte, and chondrocyte lineages. We are now conducting phase 1 clinical trial to address the safety and feasibility of UC-MSCs in the treatment of bronchopulmonary dysplasia and chronic obstructive pulmonary disease. Conclusion: we have established a standardized protocol for the xeno-free, serum-free culture of MSCs from different sources. BM-, AD-, and UC-derived MSCs expand in large-scale and maintain their cellular characteristics, karyotype integrity, and can be applied for cell therapy. Disclosures No relevant conflicts of interest to declare.