Accelerated and safe expansion of human mesenchymal stromal cells in animal serum-free medium for transplantation and regenerative medicine
ABSTRACT 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.
- SourceAvailable from: Tomoyuki Kawase[Show abstract] [Hide abstract]
ABSTRACT: 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.Cytotherapy 10/2014; 17(1). DOI:10.1016/j.jcyt.2014.08.005 · 3.10 Impact Factor
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ABSTRACT: Bone marrow-derived mesenchymal stem cells have become an attractive cell source for periodontal ligament regeneration treatment because of their potential to engraft to several tissue types after injury. Most researchers have focused on the transplantation process, but few have paid attention to cell safety concerns and rapid proliferation before transplantation. Using serum-free medium to culture stem cells may be an effective method to avoid problems associated with exogenous serum and the addition of growth factors to promote cell proliferation. Here, we randomly divided our serum-free cultures and treated them with different levels of epidermal growth factor (EGF). We then evaluated changes in rates of cell adhesion, proliferation, apoptosis, and cell cycle ratio as well as their differentiation potential. The data showed that all of these parameters were significantly different when comparing serum-free cultures with and without 10 nM/L EGF (p < 0.05/0.01); however, cells with 10 nM/L EGF did not respond differently than cells grown in standard serum-containing media without EGF (p > 0.05). In summary, our results demonstrate that 10 nM/L EGF was the optimal dose for serum-free culture, which can replace traditional standard serum medium for in vitro expansion of miniature pig bone marrow-derived mesenchymal stem cells.In Vitro Cellular & Developmental Biology - Animal 09/2013; 49(10). DOI:10.1007/s11626-013-9665-6 · 1.00 Impact Factor
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ABSTRACT: One of the major challenges in tissue engineering is to supply larger three-dimensional (3D) bioengineered tissue transplants with sufficient amounts of nutrients and oxygen and to allow metabolite removal. Consequently, artificial vascularization strategies of such transplants are desired. One strategy focuses on endothelial cells capable of initiating new vessel formation, which are settled on scaffolds commonly used in tissue engineering. A bottleneck in this strategy is to obtain sufficient amounts of endothelial cells, as they can be harvested only in small quantities directly from human tissues. Thus, protocols are required to expand appropriate cells in sufficient amounts without interfering with their capability to settle on scaffold materials and to initiate vessel formation. Here, we analysed whether umbilical cord blood (CB)-derived endothelial colony-forming cells (ECFCs) fulfil these requirements. In a first set of experiments, we showed that marginally expanded ECFCs settle and survive on different scaffold biomaterials. Next, we improved ECFC culture conditions and developed a protocol for ECFC expansion compatible with 'Good Manufacturing Practice' (GMP) standards. We replaced animal sera with human platelet lysates and used a novel type of tissue-culture ware. ECFCs cultured under the new conditions revealed significantly lower apoptosis and increased proliferation rates. Simultaneously, their viability was increased. Since extensively expanded ECFCs could still settle on scaffold biomaterials and were able to form tubular structures in Matrigel assays, we conclude that these ex vivo-expanded ECFCs are a novel, very potent cell source for scaffold-based tissue engineering. Copyright © 2013 John Wiley & Sons, Ltd.Journal of Tissue Engineering and Regenerative Medicine 02/2013; DOI:10.1002/term.1673 · 4.43 Impact Factor