Time Of Cell Harvest Research Articles

Activity banding of human chromosomes as shown by histone acetylation.

The expression of genes in mammalian cells depends on many factors including position in the cell cycle, stage of differentiation, age, and environmental influences. As different groups of genes are expressed, their packaging within chromatin changes and may be detected at the chromosomal level. The organization of DNA within a chromosome is determined to a large extent by the positively charged, highly conserved histones. Histone subtypes and the reversible chemical modifications of histones have been associated with gene activity. Active or potentially active genes have been associated with hyperacetylated histones and inactive genes with nonacetylated histones. Sodium butyrate increases the acetylation levels of histones in cell cultures and acts as both an inducer of gene activity and as a cell-cycle block. We describe a method to label the interphase distribution of DNA associated with various histone acetylation stages on chromosomes. Nucleosomes from untreated and butyrate-treated HeLa cells were fractionated by their acetylation level and the associated DNA labeled, and hybridized to normal human chromosomes. In the sodium butyrate-treated cells the resulting banding patterns of the high- and low-acetylated fractions were strikingly different. DNA from low-acetylated chromatin labeled several pericentric regions, whereas hybridization with DNA from highly acetylated chromatin resulted in a pattern similar to inverse G-bands on many chromosomes. The results from noninduced cells at both high and low acetylation levels were noticeably different from their induced counterparts. The capture and hybridization of DNA from interphase chromatin at different acetylation states provides a "snapshot" of the distribution of gene activity on chromosomes at the time of cell harvest.

246 Comparison of two stem cell mobilization and harvesting regimens for peripheral blood stem cell (PBSC) transplantation in multiple myeloma (MM)

We have investigated two regimens for the mobilisation of stem cells prior to PBSC transplantation in MM. Regiman A: 125μg/m² GCSF b.d. for 7 days with harvest of stem cells days 5–8. Regimen B: 12–16μg/kg for 4 days with harvest days 4 and 5. For each regimen the median total cell count, CFU-GM and CD34 + counts and days to engraftment as measured by time to attainment of neutrophil count 500 were calculated and compared by the Mann-Whitney non-parametric test. <h3>Results</h3> A(n=25)B(n=13)Mann-Whitney p valueTotal cell count (×10⁸)6.6 (4.05–12.18)4.06(30–7.72)0.0002CFU-GM (×10⁴/kg)3.14 (0.44–24.5)3.35 (0.06–21.27)NSCD34+ (×10⁶)43.6(0.3–337.7)38.5(4.1–1187.7)NSDays to engraftmem12(6–21)12(8–23)NS <h3>Condusions</h3> Although Regimen A led to increased mononuclear cell count there was no difference in the CFU-GM or CD34+ counts or days to engraftment. Reduction of stem cell harvest time from 4 to 2 days has economic benefits and improves patient tolerability.

Expansion of human bone marrow progenitor cells in a high cell density continuous perfusion system.

We describe here a continuous perfusion bioreactor system that enables a population of unselected human mononuclear bone marrow cells obtained from adult donors to expand up to 20 to 25-fold over a two-week period. Colony-forming units of granulocyte-macrophage (CFU-GM) progenitor cells expand 10 to 30-fold. These expansions depend on the gas phase oxygen concentration, the seeding density and time of cell harvest. Under operating conditions that allow for good cell proliferation, 3 to 4 million mononuclear cells can be obtained per square centimeter, with 0.5 to 0.8% being progenitor cells. Autologous human sera supported cell expansion as efficiently as animal sera. Increasing the size of the perfusion system to produce a clinically meaningful number of CFU-GMs could have important applications in bone marrow transplantation therapies.

Chromosome aberration and sister chromatid exchange tests in chinese hamster ovary cells in vitro. V: Results With 46 Chemicals

Forty-six coded chemicals were tested for their ability to induce sister chromatid exchanges (SCEs) and chromosomal aberrations (ABs) in cultured Chinese hamster ovary (CHO) cells using a standard protocol with and without exogenous metabolic activation. Sixteen chemicals were negative and 15 were positive in both assays; 15 were positive for SCEs only (one chemical that was positive for SCEs was equivocal for ABs), and no chemicals induced ABs only. The effect of cell harvest time on the ability to detect the induction of ABs was examined for 18 chemicals. Seven chemicals produced a positive response using both standard and extended harvest times, five were positive only using an extended harvest time, and six were negative using both harvest times. The relationship between cell cycle delay and SCE induction was also examined, and the two appear to be unrelated.

Chromosome aberration and sister chromatid exchange tests in Chinese hamster ovary cells in vitro: II. Results with 20 chemicals.

Twenty chemicals were tested for their ability to induce sister chromatid exchanges (SCEs) and chromosomal aberrations (ABs) in cultured Chinese hamster ovary cells (CHO). These chemicals were tested with and without an added metabolic activation system (rat liver S9 fraction). Four chemicals were negative in both assays, 1 induced ABs only, and 15 were positive for SCEs; 6 of these 15 also induced ABs. The effect of cell harvest time on the ability to detect the induction of chromosomal aberrations was examined for six chemicals. Five of these had caused at least one of the following: cell cycle delay, aberrations observed in first division metaphase cells in the SCE assay, or a weak response in the standard AB assay (10-12-hr growth period). Three chemicals, chlorinated trisodium phosphate, 1,2-dibromo-3-chloropropane, and tetrakis(hydroxymethyl)phosphonium chloride, were positive using both the standard and extended harvest times. N-Nitrosodimethylamine and diphenhydramine HCl were only positive using an extended harvest time, and malonaldehyde was negative using both standard and extended harvest times.

Arachidonate metabolic pathways in cells harvested from rat pleural cavity at various times after carrageenan administration

Cells were harvested from rat pleural cavity before and during the inflammatory response stimulated by carrageenan injection. The conversion of [ 14C]arachidonate by intact cells into products of the cyclooxygenase and 5-lipoxygenase pathways was studied in the absence and presence of ionophore Incorporation of arachidonate into phosphatidic acid was also followed. In the absence of ionophore, the principal arachidonate metabolites of resident macrophages were the cyclooxygenase products, prostacyclin and thromboxane, while mobilized monocytes produced thromboxane as a major product, but very little prostacyclin. Arachidonate-metabolizing enzymes in mobilized monocytes were, in general, less active than those of resident macophages. Cells harvested at times when mobilized neutrophils were the predominant cell type were capable of converting arachidonate into thromboxane and prostaglandins, but not prostacyclin. These cells exhibited the most active turnover of arachidonate into phosphatidic acid, and the extent of this turnover appeared to be temporally related to the presence of edema in the pleural cavity at the time of cell harvest. Enzymatic formation of 5-lipoxygenase products was dependent on calcium and was markedly stimulated by ionophore A23187 in both resident and mobilized pleural cells. Among several non-steroidal drugs tested, cyclooxygenase inhibitors were the most effective in preventing the inflammatory response in the carrageenan model of inflammation.

The effect of cell kinetics and harvest time on SCE and NOR associations in Macaca fuscata lymphocytes.

The relationship between cell kinetics and cell harvest time on sister chromatid exchange (SCE) and NOR (marker chromosome) association was investigated in cultured Macaca fuscata lymphocytes. The average number of SCE's in individuals ranged from 3.77 to 9.07 per cell. The highest SCE rate was found in cultures harvested at 60 h. The data suggest a decrease in SCE frequency between 60 h and subsequent cell harvest at 72, 84, and 96 h. NOR association frequency was also affected by cell kinetics, as well as by cell harvest time. The highest association frequencies were at 48 h, when the number of first mitoses was greatest.