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.
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ABSTRACT: In addition to osteosynthetic stabilizing techniques and autologous bone transplantations, so-called orthobiologics play an increasing role in the treatment of bone healing disorders. Besides the use of various growth factors, more and more new data suggest that cell-based therapies promote local bone regeneration. For ethical and biological reasons, clinical application of progenitor cells on the musculoskeletal system is limited to autologous, postpartum stem cells. Intraoperative one-step treatment with autologous progenitor cells, in particular, delivered promising results in preliminary clinical studies. This article provides an overview of the rationale for, and characteristics of the clinical application of cell-based therapy to treat osseous defects based on a review of existing literature and our own experience with more than 100 patients. Most clinical trials report successful bone regeneration after the application of mixed cell populations from bone marrow. The autologous application of human bone marrow cells which are not expanded ex vivo has medico-legal advantages. However, there is a lack of prospective randomized studies including controls for cell therapy for bone defects. Autologous bone marrow cell therapy seems to be a promising treatment option which may reduce the amount of bone grafting in future.Orthopedic Reviews 09/2010; 2(2):e20.
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ABSTRACT: Mesenchymal stromal cells (MSCs) as multipotent cells with the capacity to be differentiated into several cell lineages are promising sources for cell therapy and tissue engineering nowadays. Today most of culturing media are supplemented with fetal bovine serum (FBS). But FBS containing culturing media may raise the possibility of zoonotic infections and immunological reactions in cell therapy conditions. Numerous investigations have been performed to assess the use of FBS-free culturing systems for bone marrow derived mesenchymal stromal cell isolation. The present investigation aimed to assess the effect of serum-free media on growth and differentiating capacity of adipose tissue- derived MSCs. Approximately, 1cm3 surgically waste sterile adipose tissue was digested with collagenase-I leading to a single cell suspension. The isolated cells were cultured in Ultra Culture media supplemented with 2% Ultroser G. MSC's isolation was confirmed with respect to morphology, flowcytometry, adipogenic and osteogenic differentiation potentials. The isolated cells showed adherent spindle shaped morphology, expanded rapidly and revealed expected MSC flowcytometric characteristics; they were positive for CD73, CD90, CD105, CD44, CD166, CD44 and negative for hematopoietic antigen such as CD45, CD34 and CD14. They could also be differentiated successfully into osteoblast and adipocyte, being confirmed by using Alizarin Red and Oil red O staining, respectively. According to the results of the present study, it can be concluded that adipose derived MSCs can be cultured in serum-free media with no change in their differentiating capacity. This finding gives us a hope for future cell therapy studies and trials with little concern about zoonotic infections or immunological reaction.Iranian Red Crescent medical journal. 04/2013; 15(4):324-329.
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ABSTRACT: We aim to identify current in vitro research exploring platelet-rich plasma (PRP) effects in human Mesenchymal Stem Cells (MSCs) that may encourage or limit the clinical application of MSCs along with PRP. After a systematic search, we identified 57 in vitro studies, focused on optimization of MSC manufacturing, and expanding knowledge about how PRP modifies MSCs behavior for translational purposes. Influences of PRP on proliferation, migration, stemness, preservation of MSC immune-modulatory properties and appearance of senescence phenotype have been explored. Overall PRP stimulates MSC proliferation, preserves MSCs multipotency and does not interfere with any lineage differentiation. PRP (as platelet lysate or releasate) preserves the immune-privileged potential of MSCs and may delay the appearance of the senescent phenotype. Currently there are few data linking precise molecules and biological mechanisms. Various gaps of knowledge need to be addressed in order to obtain enough useful information for translational purposes.Muscles, ligaments and tendons journal. 4(1):52-62.