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Culture Initiation (culture + initiation)
Selected AbstractsMurine mesenchymal stem cells isolated by low density primary culture systemDEVELOPMENT GROWTH & DIFFERENTIATION, Issue 6 2006Mohamadreza Baghaban Eslaminejad Murine mesenchymal stem cells (mMSC) and the difficult task of isolation and purification of them have been the subject of rather extensive investigation. The present study sought to isolate these cells from two different mouse strains, one outbred and the other inbred, primarily through a relatively simple but novel approach, the most important feature of which was the low density primary culture of bone marrow cells. For this purpose, mononuclear cells from either NMRI or BALB/c bone marrow were plated at about 500 cells per well of 24-well plates and incubated for 7 days. At this point, the fibroblastic clones that had emerged were pooled together and expanded through several subcultures. To investigate the mesenchymal nature, we differentiated the cells into the osteoblastic, chondrocytic and adipocytic lineages in different subcultures up to passage 10. According to the results, 1 week after culture initiation, several clones each comprising several fibroblastic cells appeared in each plate. The cells from different passages were capable of differentiating into corresponding skeletal tissues. In the present investigation, the best culture condition for maximum proliferation and also the expression of certain surface marker on isolated cells were examined. In this term the two murine strains showed some differences. [source] Loss of FOXP3 expression in natural human CD4+CD25+ regulatory T cells upon repetitive in vitro stimulationEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 4 2009Petra Hoffmann Abstract The adoptive transfer of CD4+CD25+ natural regulatory T cells (Treg) is a promising strategy for the treatment of autoimmune diseases and the prevention of alloresponses after transplantation. Clinical trials exploring this strategy require efficient in vitro expansion of this rare cell population. Protocols developed thus far rely on high-grade purification of Treg prior to culture initiation, a process still hampered by the lack of Treg cell-specific surface markers. Depletion of CD127+ cells was shown to separate activated conventional T cells from natural Treg cell populations allowing the isolation of highly enriched FOXP3+ cells with all functional and molecular characteristics of natural Treg. Here, we demonstrate that upon in vitro expansion, CpG methylation in a conserved region within the FOXP3 gene locus increased in CD4+CD25+CD127low Treg, correlating with loss of FOXP3 expression and emergence of pro-inflammatory cytokines. Further analysis identified CD45RA,FOXP3+ memory-type Treg as the main source of converting cells, whereas CD45RA+FOXP3+ Treg from the same donors showed no conversion within 3,wk of in vitro expansion. Thus, Treg cell lineage differentiation does not seem to represent a final fate decision, as natural Treg can lose their cell-type-specific characteristics after repetitive TCR stimulation. [source] Effect of coexposure to 50 Hz magnetic fields and an aneugen on human lymphocytes, determined by the cytokinesis block micronucleus assayBIOELECTROMAGNETICS, Issue 3 2003G.R. Verheyen Abstract Interference of 50 Hz extremely low frequency magnetic fields (ELF-MF) with the known aneugen vinblastine (VBL) on micronucleus formation was tested with the in vitro cytokinesis block micronucleus assay in human lymphocyte cultures. Isolated lymphocyte cultures were prepared from 18 individuals. Three groups of quadruplicate cultures from six unrelated individuals were exposed to 50 Hz ELF-MF of background (bkg), 80 and 800 ,T, respectively, during the complete incubation period (72 h). Twenty-four hours after culture initiation, one replicate culture from each individual within each ELF-MF group was exposed to 0, 5, 10, or 15 ng/ml VBL. The isolated lymphocyte cultures were scored for the presence of micronuclei, the nuclear division index (NDI), and apoptosis. As expected, increased VBL concentration resulted in an increased micronucleus and apoptosis frequency and in a decreased NDI. In the presence of VBL, there was a systematic tendency for increased micronucleus and apoptosis frequency in the ELF-MF exposed groups compared to the bkg group. In the absence of VBL, we observed no statistically significant effect of ELF-MF on micronucleus induction or apoptosis frequency, but the NDI was significantly higher in the 800 ,T group compared to the other groups, suggesting an effect of ELF-MF on cell proliferation. An interaction between ELF-MF and VBL on NDI was observed. This interaction reflected the drastic decrease in NDI due to coexposure to VBL. Bioelectromagnetics 24:160,164, 2003. © 2003 Wiley-Liss, Inc. [source] An efficient method for the rapid establishment of Epstein-Barr virus immortalization of human B lymphocytesCELL PROLIFERATION, Issue 4 2003H.-M. Oh Several methods have been developed for the immortalization of B lymphocytes by Epstein-Barr virus (EBV). We developed an efficient method which reduces the time from culture initiation to immortalization and cryopreservation. Two infections of EBV to lymphocytes, and the use of phorbol ester-induced EBV stock significantly improved immortalization efficiency and reduced the time between initiation and immortalization and cryopreservation. The resulting cell bank was used to produce DNA for genetic studies focusing on the genes involved in immune and autistic disorders. [source] | ||||||||||