Cell culture techniques

By immobilizing fructose-modified dendrimers on a polystyrene culture plate, the number of initially adhered hepatocytes on it was increased. Moreover, increasing the number of generations of fructose-modified dendrimer (fructose-dendrimer) increased the number. Urea synthesis per unit area also was increased, corresponding to the increase in the number of initially adhered hepatocytes. This result suggests that the fructose-dendrimers do not cause a decline in cell function. On the other hand, apoptosis of hepatocytes occurs during cultivation, and results in a decrease in the number of adhered cells and a decline in cell function. Fructose-dendrimers were found to suppress apoptosis of hepatocytes. This characteristic is considered to be responsible for the increase in the number of initially adhered hepatocytes without a decline in cell function. Fructose-dendrimers are shown to be very suitable scaffolds for use in a high-performance bioartificial liver support system.

This is the first report on the characterization and production optimization of poly-N-acetylglucosamine (PNAG) in Staphylococcus saprophyticus. A strain producing glucosamine exopolysaccharide was isolated and characterized by biochemical test and 16S rRNA gene sequence homology analysis and named as S. saprophyticus BMSZ711. The molecular mass of the purified exopolymer was about 12 kDa. Digestion of the PNAG with DispersinB proved that it has beta-1,6 linkage. BMSZ711 can only produce PNAG when grown in M1 minimal medium but not in nutrient rich medium with optimum temperature of 30 degrees C and pH of 7. Glycerol and ammonium sulfate were found to be the best carbon and nitrogen source, respectively. Maximum PNAG production was obtained when glycerol 100mM, ammonium sulfate 0.3%, yeast extract 1.5 g/L, sodium chloride 10 g/L and valine 2mM were used.

Vascular endothelial growth factor (VEGF) is a potent angiogenic peptide with biologic effects that include regulation of hematopoietic stem cell development, extracellular matrix remodeling, and inflammatory cytokine generation. To delineate the potential role of VEGF in patients with myelodysplastic syndrome (MDS), VEGF protein and receptor expression and its functional significance in MDS bone marrow (BM) were evaluated. In BM clot sections from normal donors, low-intensity cytoplasmic VEGF expression was detected infrequently in isolated myeloid elements. However, monocytoid precursors in chronic myelomonocytic leukemia (CMML) expressed VEGF in an intense cytoplasmic pattern with membranous co-expression of the Flt-1 or KDR receptors, or both. In situ hybridization confirmed the presence of VEGF mRNA in the neoplastic monocytes. In acute myelogenous leukemia (AML) and other MDS subtypes, intense co-expression of VEGF and one or both receptors was detected in myeloblasts and imma...

Triphenyltin acetate (TPTA), a triorganotin compound used in agriculture as a biocide, is immunotoxic in vivo and in vitro. The present study was undertaken to ascertain whether apoptosis might play a role in the TPTA toxicity in vitro. Mouse thymocyte primary cultures were exposed to 0, 4 and 8 micromol/L TPTA; methyl prednisolone (1 micromol/L) was used as a positive control. Cell aliquots were harvested after 0, 1, 2, 4, and 8 h and the presence of early or late apoptotic phenomena was checked by (a) morphological investigations; (b) spectrophotometric quantification of fragmented DNA and agarose gel electrophoresis; (c) cell flow cytofluorometry, using an annexin V-FITC kit; and (d) detection of in situ apoptosis by a colorimetric detection kit (Titer-Tacs). TPTA cytotoxicity was also evaluated using the trypan blue dye exclusion test. Morphological investigation indicated apoptosis and/or necrosis. After 8 h of incubation, cells exposed to 4 micromol/L TPTA showed an increase in DNA fragmentation (on electrophoresis), which was confirmed by spectrophotometry (p < 0.05). Flow cytofluorometry pointed out an early (p < 0.05) increase of annexin V-positive (apoptotic) cells in TPTA-exposed flasks, whereas at least partly contradictory, results were obtained with the Titer-Tacs kit. Overall, these results provide evidence that TPTA, at low concentrations (4 micromol/L) induces early and late apoptotic phenomena, whereas cells exposed to the highest concentrations (8 micromol/L) are likely to undergo necrosis rather than apoptosis.

We report here the isolation of a population of non-transformed pluripotent human cells from bone marrow after a unique expansion/selection procedure. This procedure was designed to provide conditions resembling the in vivo microenvironment that is home for the most-primitive stem cells. Marrow-adherent and -nonadherent cells were co-cultured on fibronectin, at low oxygen tension, for 14 days. Colonies of small adherent cells were isolated and further expanded on fibronectin at low density, low oxygen tension with 2% fetal bovine serum. They expressed high levels of CD29, CD63, CD81, CD122, CD164, hepatocyte growth factor receptor (cMet), bone morphogenetic protein receptor 1B (BMPR1B), and neurotrophic tyrosine kinase receptor 3 (NTRK3) and were negative for CD34, CD36, CD45, CD117 (cKit) and HLADR. The embryonic stem cell markers Oct-4 and Rex-1, and telomerase were expressed in all cultures examined. Cell-doubling time was 36 to 72 hours, and cells have been expanded in culture f...

Hyperprolactinemia caused by physiological or pathological conditions, such as those occurring during lactation and prolactinoma, respectively, results in progressive osteopenia. The underlying mechanisms, however, are controversial. Prolactin (PRL) may directly attenuate the functions of osteoblasts, since these bone cells express PRL receptors. The present study therefore aimed to investigate the effects of PRL on the expression of genes related to the osteoblast functions by using quantitative real-time PCR technique. Herein, we used primary osteoblasts that were derived from the tibiae of adult rats and displayed characteristics of differentiated osteoblasts, including in vitro mineralization. Osteoblasts exposed for 48 h to 1000 ng/mL PRL, but not to 10 or 100 ng/mL PRL, showed decreases in the mRNA expression of Runx2, osteoprotegerin (OPG), and receptor activator of nuclear factor κB ligand (RANKL) by 60.49%, 72.74%, and 87.51%, respectively. Nevertheless, PRL did not change ...