Quality assessment of a serum and xenofree medium for the expansion of human GMP-grade mesenchymal stromal cells.

Background: Cell-based therapies are emerging as a viable modality to treat challenging diseases, resulting in an increasing demand for their large-scale, high-quality production. Production facilities face the issue of batch-to-batch consistency while producing a safe and efficient cell-based product. Controlling culture conditions and particularly media composition is a key factor of success in this challenge. Serum and Xeno-Free Media (SXFM) represent an interesting option to achieve this goal. By reducing batch to batch variability, they increase Good Manufacturing Practices (GMP)-compliance and safety regarding xenogenic transmission, as compared to fetal bovine serum (FBS) supplemented-media or human platelet lysate supplemented medium. Methods: In this study, the isolation, expansion and characteristics including the anti-inflammatory function of human mesenchymal stromal cells (MSC) are compared after culture in MEMα supplemented with human Concentrate Platelet Lysate (hCPL, reference medium) or in MSC-Brew GMP Medium. The latter is a GMP SXFM manufactured in bags under strictly controlled conditions in volumes suitable for expansion to a clinical scale and does not require neither pre-coating of the cell culture units nor the addition of blood derivatives at the isolation step. Results: We showed that MSC derived from human bone-marrow and adipose tissue can be successfully isolated and expanded in this SXFM. Number and size of Colony-Forming Unit fibroblast (CFU-F) is increased compared to cells cultivated in hCPL medium. All cells retained a CD90+, CD73+, CD105+, HLADR-, CD34-, CD45- phenotype. Furthermore, the osteogenic and adipocyte potentials as well as the anti-inflammatory activity were comparable between culture conditions. All cells reached the release criteria established in our production facility to treat inflammatory pathologies. Conclusions: The use of MSC-Brew GMP Medium can therefore be considered for clinical bioprocesses as a safe and efficient substitute for hCPL media.

IL-1ß primed mesenchymal stromal cells moderate hemorrhagic shock-induced organ injuries.

BACKGROUND: Organ damages following hemorrhagic shock (HS) have been partly attributed to an immunological dysfunction. The current challenge in the management of HS patients is to prevent organ injury-induced morbidity and mortality which currently has not etiological treatment available. Mesenchymal stromal cells (MSC) are used in clinical cell therapy for immunomodulation and tissue repair. In vitro priming is often used to improve the immunomodulation efficiency of MSC before administration. OBJECTIVE: Assess the effect of naive MSC (MSCn) or interleukin (IL)-1ß primed (MSCp) treatment in a context of HS-induced organ injury. METHODS: Rats underwent fixed pressure HS and were treated with allogenic MSCn or MSCp. Liver and kidney injuries were evaluated 6h later by histological and biochemical analysis. Whole blood was collected to measure leukocytes phenotypes. Then, in vitro characterization of MSCn or MSCp was carried out. RESULTS: Plasma creatinine, blood urea nitrogen, and cystatin C were decrease by MSCp infusion as well as kidney injury molecule (KIM)-1 on histological kidney sections. Transaminases, GGT, and liver histology were normalized by MSCp. Systemic cytokines (IL-1α, IL-6, and IL-10) as well as CD80, 86, and PD-1/PDL-1 axis were decreased by MSCp on monocytes and granulocytes. In vitro, MSCp showed higher level of secreted immunomodulatory molecules than MSCn. CONCLUSION: An early administration of MSCp moderates HS-induced kidney and liver injury. IL-1ß priming improves MSC efficiency by promoting their immunomodulatory activity. These data provide proof of concept that MSCp could be a therapeutic tool to prevent the appearance of organs injury following HS.

Effect of Preconditioned Mesenchymal Stromal Cells on Early Microvascular Disturbance in a Mouse Sepsis Model.

Septic patients often die in a context of multiple organ dysfunction syndrome (MODS), despite the macro-hemodynamic parameters being normalized and after the onset of antibiotic therapy. Microcirculation injury during sepsis affects capillary permeability and leukocyte-endothelium interactions and is thought to be instrumental in organ injury. Several studies have demonstrated a beneficial effect of mesenchymal stromal cells (MSCs) injection on survival and organ dysfunctions in sepsis models. In vivo activity of MSCs also appears to be very much dependent on the information provided before injection. Indeed preconditioning by interferon γ (IFNγ; MSC-IFNγ) increases immunosuppressive capacity of MSCs in vitro and in vivo. Therefore, the objective was to evaluate the effect of MSC naive or IFNγ preconditioned on leukocyte-endothelium interactions in a polymicrobial sepsis model by intraperitoneal feces injection. Six hours (H6) after this induction, we used intravital microscopy in mice cremaster muscle venules to study the flow behavior of leukocytes. Plasmas were harvested to evaluate inflammation level and endothelial activation. We showed that MSC-IFNγ have a beneficial effect on microcirculation, by increasing the flow of white blood cells (WBCs) and the percentage of venules containing flowing WBCs, by significantly reducing the adhesion of WBCs and by increasing the average red blood cell velocity (VRBC). In conclusion, our results suggest that intravenous injection of preconditioned MSC-IFNγ improves microvascular hemodynamics in early phases of sepsis.