PubMed · 9276268
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D W Williams, D Wynford-Thomas. 1997. Human thyroid epithelial cells.. https://doi.org/10.1385/0-89603-441-0%3A163
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OBJECTIVE: The preservation of Streptococcus pneumoniae by standard freezing methods for subsequent tests--such as serotyping and antibiotic susceptibility--is not possible or is difficult in many developing countries because of the high cost of equipment, inadequate equipment maintenance, and irregular power supply. We evaluated alternative low-cost methods, by comparing different culture media and storage temperatures. METHODS: Clinical isolates of five capsular types (1, 5, 7, 19, and 23) of S. pneumoniae were preserved in rabbit blood, sheep blood, skimmed milk, or glycerol-chocolate broth, and stored at -20 degrees C or -70 degrees C. The cultures were also preserved by lyophilization or sand desiccation, followed by storage at room temperature and 4 degrees C. The viability of the preserved cultures was determined by making serial colony counts on day 0 and after 1 week, 4 weeks, 4 months and 16 months. The viability of cultures preserved by sand desiccation and storage at 4 degrees C was also determined every 6 months for up to 68 months. FINDINGS: Irrespective of the media used, cultures maintained at -20 degrees C became nonviable by the fourth month, while those maintained at -70 degrees C were still viable at 16 months. Cultures preserved by lyophilization or sand desiccation lost their viability by the fourth month when maintained at local room temperature (30-42 degrees C), but remained viable when stored at 4 degrees C for up to 68 months. CONCLUSIONS: Our results confirm that freezing at -70 degrees C, or lyophilization and storage at 4 degrees C are the ideal methods for the preservation of S. pneumoniae. In laboratories where lyophilization is not feasible, sand desiccation and storage at 4 degrees C offers an alternative low-cost method for the long-term preservation of S. pneumoniae.
To modulate alloreactivity after hematopoietic stem cell transplantation, suicide gene-expressing donor T cells can be administered with an allogeneic T-cell-depleted bone marrow graft. Immune competence of such cells is a critical issue. The impact of the ex vivo gene transfer protocol (12-day culture period including CD3/interleukin-2 [IL-2] activation, retroviral-mediated gene transfer, and G418-based selection) on the anti-Epstein-Barr virus (EBV) potential of gene-modified cells has been examined. Cytotoxic (pCTL) and helper (pTh) cell precursor limiting dilution assays, interferon-gamma enzyme-linked immunospot, or fluorescence-activated cell sorter analysis after tetrameric HLA-A2/EBV peptide complexes revealed that the frequency of anti-EBV T cells was lower in gene-modified cells (GMCs) than in similarly cultured but untransduced T cells and was even lower than in fresh peripheral blood mononuclear cells, demonstrating both an effect of the culture and of the transduction or selection. The culture-dependent loss of EBV-reactive cells resulted from the preferential induction of activation-induced cell death in tetramer(+) cells. Replacing the initial CD3/IL-2 activation by CD3/CD28/IL-2 partially restored the anti-EBV response of GMCs by reducing the initial activation-induced cell death and enhancing the proliferation of EBV-tetramer(+) cells. Moreover, the G418 selection, and not the transduction, was directly toxic to transduced tetramer(+) cells. Replacing the G418 selection by an immunomagnetic selection significantly prevented the selection-dependent loss of EBV-specific cells. Overall, ex vivo gene modification of primary T cells can result in a significant reduction in EBV-reactive T cells through both culture-dependent and selection-dependent mechanisms. Improving immune functions of GMCs through modifications of the cell culture conditions and transduction/selection processes is critical for further clinical studies.
Membranes of the obligate methylotroph Methylobacillus flagellatus KT contained hemes B, O, and C and cytochromes b, o, and c both in batch and in continuous cultures. Neither heme A nor heme D was detected in the membranes. The cytochromes o and bb were the main components reversibly binding carbon monoxide (CO) in the terminal part of the respiratory chain. The alpha-region and especially the alpha-peaks at 568 and 573 nm and the alpha-troughs at 586 and 592 on the CO-difference spectra were diagnostic for the cytochromes o and bb, respectively. The cytochrome o content increased up to 1.8 times upon increasing the dilution rate of the culture from 0.15 to 0.55 h(-1) under methanol limitation. By contrast, the level of the CO-binding cytochrome bb was not affected by methanol concentration but its content increased up to 1.9 times when the level of oxygen decreased from 95 to 21 microM under the constant dilution rate (mu = 0.55 h(-1)). The maximum ratio between the cytochromes o and bb reached 2 during continuous cultivation under methanol-limited conditions (mu = 0.55 h(-1)), whereas the minimum ratio between them was about 0.7 during batch cultivation at stationary phase of growth. The synthesis of the CO-binding cytochrome bb but not of the cytochrome o in M. flagellatus KT was assumed to depend on the ambient redox potential of the medium. The cytochrome o synthesis was supposed to depend on the transmembrane gradient of protons (Delta(mu)H+).