Human bone marrow mesenchymal stromal cells (hMSC) are currently investigated for a variety of therapeutic applications. However, most expansion protocols still use fetal calf serum (FCS) as growth factor supplement which is a potential source of undesired xenogeneic pathogens. We established an expansion protocol for hMSC based on the use of GMP-produced basic medium LP02 supplemented with 5% of platelet lysate (PL) obtained from human thrombocyte concentrates. Compared to FCS-supplemented culture conditions, we found a significant increase in both colony forming unit-fibroblast (CFU-F) as well as cumulative cell numbers after expansion. This accelerated growth is optimized by pooling of at least 10 thrombocyte concentrates. A minimal requirement is the use of 5% of PL with an optimal platelet concentration of 1.5 x 10(9)/ml, and centrifugation of thawed lysate at high speed. Cells expanded by this protocol meet all criteria for mesenchymal stromal cells (MSCs), e.g. plastic adherence, spindle-shaped morphology, surface marker expression, lack of hematopoietic markers, and differentiation capability into three mesenchymal lineages. MSC at passage 6 were cytogenetically normal and retained their immune-privileged potential by suppressing allogeneic reaction of T-cells. Additionally, gene expression profiles show increased mRNA levels of genes involved in cell cycle and DNA replication and downregulation of developmental and differentiation genes, supporting the observation of increased MSC-expansion in PL-supplemented medium. In summary, we have established a GMP-compatible protocol for safe and accelerated expansion of hMSC to be used in cell and tissue therapy.
"Our results are in accordance with these data. Some have reported that PL containing media might also increase the number of CFU-Fs (Lange et al. 2007; Salvade et al. 2010). There are also conflicting results regarding the effect of oxygen concentration on the CFU-F number (Fehrer et al. 2007; Lennon et al. 2001). "
[Show abstract][Hide abstract] ABSTRACT: Efficient xenofree expansion methods to replace fetal bovine serum (FBS)-based culture methods are strongly encouraged by the regulators and are needed to facilitate the adoption of mesenchymal stromal cell (MSC)-based therapies. In the current study we established a clinically-compliant and reproducible animal serum-free culture protocol for bone marrow-(BM-) MSCs based on an optimized platelet-derived supplement. Our study compared two different platelet-derived supplements, platelet lysate PL1 versus PL2, produced by two different methods and lysed with different amounts of freeze-thaw cycles. Our study also explored the effect of a low oxygen concentration on BM-MSCs. FBS-supplemented BM-MSC culture served as control. Growth kinetics, differentiation and immunomodulatory potential, morphology, karyotype and immunophenotype was analysed. Growth kinetics in long-term culture was also studied. Based on the initial results, we chose to further process develop the PL1-supplemented culture protocol at 20 % oxygen. The results from 11 individual BM-MSC batches expanded in the chosen condition were consistent, yielding 6.60 × 10(9) ± 4.74 × 10(9) cells from only 20 ml of bone marrow. The cells suppressed T-cell proliferation, displayed normal karyotype and typical MSC differentiation potential and phenotype. The BM-MSCs were, however, consistently HLA-DR positive when cultured in platelet lysate (7.5-66.1 %). We additionally show that culture media antibiotics and sterile filtration of the platelet lysate can be successfully omitted. We present a robust and reproducible clinically-compliant culture method for BM-MSCs based on platelet lysate, which enables high quantities of HLA-DR positive MSCs at a low passage number (p2) and suitable for clinical use.
"Cytotherapy, 2015; 17: 186e198 (Received 24 June 2014; accepted 21 October 2014) (FCS)-containing media. In comparison to xenogenfree , human platelet lysateebased media, MSCs show reduced proliferation capacity in FCS-based media  . Moreover, FCS poses a regulatory issue due to the additional risk of infection. "
"In contrast, exclusion criteria based on the propensity for mutation, phenotypic plasticity and ability to form neoplasms have been poorly investigated. Only the karyotype test   and animal implantation test  (using immune-deficient mice) have been widely performed to detect abnormal cells within a cell preparation. However, these methodologies are timeconsuming , non-universal and therefore impractical as a required routine test before transplantation. "
[Show abstract][Hide abstract] ABSTRACT: Background aims:
For successful cell transplantation therapy, the quality of cells must be strictly controlled. Unfortunately, to exclude inappropriate cells that possess structurally abnormal chromosomes, currently only karyotyping functions as an assessment. Unfortunately, this methodology is time-consuming and only effective for metaphasic cells. To develop a more efficient, inclusive and sensitive methodology, we examined the phosphorylation of histone H2AX and the p53 levels in normal human periosteal cells exposed to x-rays or other oxidative stressors.
Periosteal cells were obtained from human alveolar bone before being exposed to x-rays, ultraviolet C or hydrogen peroxide. The cell cycle, electric nuclear volume and CD44 expression were evaluated using flow cytometry, and the phosphorylated H2AX (γ-H2AX), p53, p21 and proliferating cell nuclear antigen (PCNA) levels were evaluated by Western blot analyses.
Each oxidative stress dose-dependently arrested cell growth and partially induced premature cellular senescence. In parallel, each oxidative stress rapidly phosphorylated H2AX and stabilized p53, and intense stress sustained these high levels for at least 8 days.
Intensive oxidative stress induces sustained high levels of γ-H2AX and p53, which force cells toward senescence or non-apoptotic cell death. Lower doses of oxidative stress induced more modest and transient increases in γ-H2AX and p53, and these cells eventually survive. However, because DNA is repaired without a template in the majority of these cells, G1 mutations accumulate. Therefore, we recommend that any cell population expressing elevated γ-H2AX and p53 levels be excluded from cell transplantation therapy.
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