31-Dec-2018 12:00 AM
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Purpose: Development and application of techniques for safe derivation of cells from osteoblast and osteoclast lineages from progenitors of different sources and origin. Methods: Osteoblasts and osteoclasts with human origin were obtained from adult human bone marrow stem/stromal cells. Normal human mesenchymal stromal/stem cells (MSCs) were derived from human bone marrow material. Isolated nucleated cells were washed twice and resuspended in basic α-MEM (Minimum Essential Medium, alpha modification), supplemented with 15% heat-inactivated Fetal Bovine Serum (FBS), 50µg/ml freshly prepared Ascorbic acid (Vitamin C) and antibiotics (100µg/ml Penicillin G). For further cell differentiation in osteoblasts, separated cell sub-populations were pre-cultured in the presence of 10-8M Dexamethazone and 10mM β-Glycerophosphate, previously added to the basic α-MEM, described above. For osteoclast differentiation, other sub-populations of the same progenitors were pre-cultivated in the presence of 25ng/ml M-CSF and 50ng/ml RANKL (receptor activator of NF-κB ligand). Analogically, separate sub-populations from 3T3 fibroblast cell line, derived from Balb/c mouse embryos, were de novo-pre-incubated in the presence of supplemented cultural fluids, in which mouse malignant myeloma cells were previously cultivated, after centrifugation and filtration of the picked up and preserved media. After their centrifugation and filtration, the described cultural media, conditioned by incubation of both cell types, separately or subsequently both one and the other type, were used for treatment of new monolayer of de novo-incubated 3T3 mouse embryonic fibroblast sub-populations, after formation of semi-confluent cell monolayers. Sub-populations from the so pre-cultured cells were then freezed at -800C in the presence of cryo-protector Dymethylsulfoxide (DMSO) for 2-4 weeks, after which the laboratory in vitro-incubation was continued by the procedures, described above. For investigation the influence of the derived cells from both types to each other, co-cultivation osteoblast-like and osteoclast-like cells was made. In this case, cell suspensions, containing cultural fluid plus the derived cells of the respective lineages were used. Results: In both cases of human bone marrow material used, derivation of mature osteoclasts and osteoblasts was proved, when respective appropriate conditions/growth factors were present. Probably, separated stem/progenitor cells in the general 3T3 line differentiated in each one of the respective lineages, when appropriate cultivation conditions are available. In incubation in conditioned cultural fluid, derived after incubation of malignant myeloma cells, signs of early myeloid differentiation were noted. After addition of DMSO and consequent and thawing, appearance of separated osteoclast-like cells was noticed. Similarly, in treated on the same way 3T3 cell sub-populations, pre-incubated subsequently in the presence of media, supplemented from normal 3T3 cells and from malignant myeloma cells, signs of osteoblast-like cell morphology were observed. In co-cultivation of the so derived from mouse embryonic progenitors osteoblast-like and osteoclast-like cells (suspensions of cultural fluids plus cells from both types), zones of destroyed osteoblast-like cells monolayer were noted. Conclusions: Besides the factors of cultivation and the components of extra-cellular matrix, the role of cryo-protector DMSO as a stimulator of both cell differentiation in the two cell types, but also of intra-cellular fusion in the formation of osteoclast-like cells, was suggested.
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