A Comparison of Low and High Dose-Rate Radiation For Recipient Mice In Spleen-Colony Studies

Over the last 15 years, endogenous spleen-colony formation in our mice, following lethal irradiation, has increased to an unacceptable level. It has been found necessary, therefore, to introduce a new method of preparing recipient mice for spleen-colony studies. Irradiation with low dose-rate ⁶⁰Cobalt γ rays has been compared with high dose-rate linear accelerator electrons, and their effects on endogenous spleen colony formation compared with earlier X and γ ray dose-response data. It was found that a large dose (13.5 Gy) of γ rays results in fewer endogenous colonies than 8.5 Gy of electrons, yet because of its low dose rate (14.1 × 10^?3 Gy/min) it has a marked sparing of the intestinal tissue as measured by the intestinal microcolony technique. This in turn permits better survival and, therefore, a ‘healthier’ animal for spleen-colony work. Exogenous colony formation is also lower in the low dose-rate, γ-irradiated recipients and this is shown to be due to a reduced spleen-seeding efficiency. It is concluded that very low dose-rate radiation is preferable for haemopoietic ablation, that a mouse colony requires constant monitoring for changes of endogenous spleen-colony formation and that the spleen-seeding efficiency of CFU-s depends on the irradiation technique used-there is no absolute value for a given strain of mouse.     

A comparison of low and high dose-rate radiation for recipient mice in spleen-colony studies

Over the last 15 years, endogenous spleen-colony formation in our mice, following lethal irradiation, has increased to an unacceptable level. It has been found necessary, therefore, to introduce a new method of preparing recipient mice for spleen-colony studies. Irradiation with low dose-rate 60Cobalt gamma rays has been compared with high dose-rate linear accelerator electrons, and their effects on endogenous spleen colony formation compared with earlier X and gamma ray dose-response data. It was found that a large dose (13.5 Gy) of gamma rays results in fewer endogenous colonies than 8.5 Gy of electrons, yet because of its low dose rate (14.1 X 10(-3) Gy/min) it has a marked sparing of the intestinal tissue as measured by the intestinal microcolony technique. This in turn permits better survival and, therefore, a 'healthier' animal for spleen-colony work. Exogenous colony formation is also lower in the low dose-rate, gamma-irradiated recipients and this is shown to be due to a reduced spleen-seeding efficiency. It is concluded that very low dose-rate radiation is preferable for haemopoietic ablation, that a mouse colony requires constant monitoring for changes of endogenous spleen-colony formation and that the spleen-seeding efficiency of CFU-s depends on the irradiation technique used--there is no absolute value for a given strain of mouse.     

REPOPULATION OF THE BONE-MARROW STEM-CELL COMPARTMENT AFTER IRRADIATION*

Bone-marrow repopulation has been followed as a function of time after a single whole-body dose of 150 rads X-rays to (C3H × C57B1)F₁ hybrid mice. The number of nucleated cells per bone shaft has been estimated by direct counting and the number of progenitor cells by the exogenous spleen-colony assay method. Vinblastine was also administered under various schedules of time and dose in an endeavour to distinguish between cycling and dormant cells in the progenitor pool. The depopulation of total marrow cells induced by Vinblastine proceeds at a comparable rate in irradiated and in normal mice. On the other hand, the depopulation of the colony-formers is faster in irradiated than in normal animals. The data are interpreted to show that the fraction of cycling progenitor cells is larger in a haemopoietic tissue undergoing numerical expansion.     

造血细胞活力冷冻损伤的可恢复性

人骨髓冻存后其造血祖细胞活力有一定程度下降,本研究对这种下降的可逆性作了初步观察。结果发现,用双层法和单层法作CFU-GM培养时,未冻存骨髓集落产率相近,冻存骨髓双层法的CFU-GM产率高于单层法。骨髓细胞用20％FM-CM、PHA-LYCM、PHA-PMCM预孵育2h后,分别测定其CFU-GM、BFU-E与CFU-Mix,发现这种孵育过程对未冻存骨髓的集落产率无明显影响,而冻存骨髓的集落产率在孵育后可升高(GEMmeg除外)。说明骨髓造血祖细胞对冻存的损伤反应不均一,部分受损细胞在一定条件下可以恢复其增殖活力。这对于用冻存骨髓作骨髓移植可能有一定意义。     

EFFECTS OF BACTERIAL ENDOTOXIN ON HEMOPOIETIC COLONY-FORMING CELLS IN THE SPLEENS OF NORMAL MICE AND MICE OF GENOTYPE S1/S1

Multiple doses of S. typhosa endotoxin caused an increase in the number of hemopoietic stem cells present in mouse marrow and spleen that could be detected using the spleen-colony assay. This increase was inhibited by Colcemid, and by the genetically-determined defect in hemopoiesis in mice of genotype S1/S1^d. However, the defect in S1/S1^d hosts did not prevent an endotoxin-induced increase in the number of cells capable of forming colonies in cell culture. The results support the view that bacterial endotoxin acts, via a genetically-controlled regulatory mechanism, to stimulate the proliferation of hemopoietic stem cells in the spleen.     

Hemopoietic progenitor cells with limited potential for differentiation: erythropoietic function of mouse marrow "lymphocytes"

Filtration of mouse marrow cell suspensions over columns of glass wool increased the frequency of small and medium-sized lymphocytes (SML) and of erythropoietic progenitor units (EPU) by about the same factor. Identical results were obtained when erythropoiesis was assayed by isotope uptake (⁵⁹FeCl₃ and ¹²⁵IUdR) or by the spleen-colony techniques. Transfusion of prospective donor mice with erythrocytes virtually eliminated morphologically recognizable erythroid cells from marrow without affecting the frequency of EPU. Injection of prospective donors with cortisol decreased the frequency of SML in marrow but not that of EPU or erythropoietin-sensitive cells. However, glass wool filtration of lymphocyte-poor marrow taken from mice pretreated with cortisol resulted in a similar increase in frequency of residual SML and of EPU. Therefore, it appears that a subpopulation of marrow SML are EPU. Whereas glass wool filtration increased the frequency of erythropoietic progenitor and colony-forming units, the filtration failed to change the frequency of leukopoietic progenitor or colony-forming units (assayed in mice hypertransfused with erythrocytes to suppress erythropoiesis). It follows that separate progenitor cells for erythropoiesis and leukopoiesis are present in bone marrow of adult mice, in addition to pluripotent stem cells.     

FREEZE ETCHING OF CELLS WITHOUT CRYOPROTECTANTS

The technique of spray-freeze etching was applied to unicellular organisms. The superior freezing rates obtainable with this method gave excellent cryofixation on Chlorella, Euglena, and spermatozoa without the use of antifreeze agents, and cell damage due to ice crystal formation was never observed. In many instances the resultant morphology differed significantly from that obtained from glycerol-treated, freeze-etched cells. Furthermore, viability studies of spray-frozen Chlorella compared favorably with cells frozen by other methods.     

Comparison of stem cell viability of bone marrow cryopreserved by two different methods

Two different cryogenic methods were used to study the preservation of murine bone marrow cells. Compared to the classical methods, in which separated mononuclear marrow cells in 10% dimethyl sulfoxide (DMSO) were cryopreserved in liquid nitrogen (-196 degrees C), a modified technique was carried out by cryopreservation of unfractionated marrow cells in a mixed protectant of 5% DMSO and 6% hydroxyethyl starch (HES) at -80 degrees C. Samples that were separately thawed after storage for 1, 4, 8, and 12 weeks were assayed for cell viability and recovery of CFU-GM and CFU-S. No macroscopic clumping of cells was noted either in fractionated or in unfractionated marrow cell cryopreservations. A mild damage, about 25% reduction of stem cells, was found at 1 week and did not deepen further. It seems that the greatest loss of stem cells occurred in the process of cryopreservation itself. Compared to prefreeze values, both a high number of cells that excluded trypan blue (87 +/- 3.4%) and a high recovery of CFU-GM (75 +/- 9.8%) and CFU-S (74 +/- 11.2) were observed in unfractionated marrow samples cryopreserved with the DMSO/HES mixture at -80 degrees C for 3 months and these results were very similar to those obtained from fractionated mononuclear marrow cells cryopreserved at -196 degrees C. The DMSO/HES protectant provides a simplified bone marrow cryopreservation technique that should be favorable to clinical application because of its high stem cell recovery and avoidance of cell-separation manipulation.     

Recovery of CFU-GM after freezing of normal human bone marrow: effect of three dilution techniques after thawing

In the cryopreservation procedures intended for autotransplantation of human bone marrow a controversial point is represented by the methods of reconstruction of the cellular suspension after thawing and before infusion into the patient. To evaluate how the dilution rate after thawing affects bone marrow viability, we cryopreserved the bone marrow from 16 hematologically normal patients in DMSO at a concentration of 10%. After thawing, the cells were diluted according to three different techniques and their viability was tested by the growth of CFU-GM in methylcellulose. The average recovery of CFU-GM, in comparison with that of fresh cells, was satisfactory and not affected by the type of dilution. In conclusion, if we accept that the resistance to osmotic stress due to the cryoprotectant is similar for stem cells and CFU-GM, we can maintain that a slow, gradual dilution is not a necessary condition to assume the staminality of bone marrow designed for autotransplantation.     

Isolation of spleen-colony forming cells (CFU-s) using wheat germ agglutinin and rhodamine 123 labeling

Mouse pluripotent hemopoietic stem cells could be enriched 100 to 200-fold by a procedure consisting of three steps: 1) equilibrium density centrifugation, 2) light-activated cell sorting on the basis of light scatter characteristics and fluorescence due to wheat germ agglutinin binding, 3) cell sorting after subsequent rhodamine 123 staining. The new isolation procedure does not make use of antibodies with mouse-strain restricted applicability, which were employed in earlier described methods. Therefore, it is more versatile. It is also faster due to diminished incubation time. Rhodamine 123 can also be used as a photosensitizer. The experimental conditions were, however, designed to prevent this action of the dye. Between 80% and 100% of the selected spleen-colony forming cells survived the labeling and sorting treatments. The procedure enriches for two types of stem cells. The rhodamine-dull fraction contains stem cells that form spleen colonies in lethally irradiated mice at 12-16 days and no spleen colonies at 8 days after transplantation. The rhodamine-bright fraction contains stem cells that give day-8 and day-12 spleen colonies. These latter cells, however, have a low radioprotective capacity and it can be argued that these are not self-renewing pluripotent stem cells. The heterogeneity of day-12 CFU-s (colony-forming unit spleen) that can be detected after labeling with rhodamine 123 has been observed earlier after treatment of bone marrow donor mice with 5-fluorouracil, and has led to the postulation of pre-CFU-s and a "generation-age" hypothesis for stem cells. Our presently sorted rhodamine dull cells resemble such pre-CFU-s.     

Method for concentrating marrow stem cells using the IBM 2991 washer. Necessary preparation before in vitro treatment of bone marrow by pharmacologic or immunologic means

The technique using the IBM 2991 blood cell processor is an effective technique for the concentration of mononuclear cells from large volumes of bone marrow. The marrow cells are layered on to Ficoll Metrizoate using the IBM processing set. The mononuclear cells and CFU-GM recoveries are in close relationship with the hematocrit of the cell suspension processed. Twenty two bone marrows have been collected and purified according to this protocol. The mononuclear cell recovery is an average of 78,3% (range: 44-92%) and the CFU-GM recovery is in average of 67,5% (range: 40-89%). At the end of the procedure the cell viability is satisfying (97,1% +/- 1,7 are trypan blue negatives). When it is necessary to remove from the bone marrow collected either malignant cells prior autologous bone marrow graft or T lymphocytes in an attempt to prevent GVHD in allogeneic BMT, the purity of marrow cell suspension become a fundamental parameter.     

Zymomonas mobilis cell viability: measurement method comparison

Comparison of three different cell viability methods: slide count, plate count and methylene blue staining techniques, applied onZymomonas mobilis cultures, was performed. The slide technique proved to be faster and more accurate than the plate count method, and both of them far more reliable than the standard methylene blue method which constantly overestimated theZymomonas cell viability. The slide technique is advantageous also because it gives information on the cell morphology changes, notably the abnormal cell elongation, in the ethanol fermentation.     

THE CELL SPECIFIC EFFECT OF THE GRANULOCYTE CHALONE DEMONSTRATED WITH THE DIFFUSION CHAMBER TECHNIQUE

The granulocytic chalone is secreted by mature granulocytes and inhibits ³H-thymidine incorporation of proliferating granulocytes in vitro . The effect and the cell line specificity of this chalone was assessed with the in vivo diffusion chamber culture technique. Tests were carried out on cultures from normal mouse bone marrow cells and mouse and rat blood leucocytes. The majority of the DNA synthesizing cells in marrow cultures were proliferating granulocytes. Macrophages and immunoblasts proliferated in rat leucocyte cultures, when the chambers had been carried for 5 days in host mice. Repeated chalone or control injections were given i.p. to the host mice during 6–7 hr prior to ³H-thymidine injection. Isotope uptake of proliferative granulocytes was reduced by the chalone treatment. No such effect was found on the rat immunoblasts and macrophages. The viability of cultured cells was apparently not affected by the chalone treatment.     

Advanced electrochemical sensors for cell cancer monitoring

The possibility of using minimally invasive analytical instruments to monitor cancerous cells and their interactions with analytes provide great advances in cancer research and toxicology. The real success in the development of a reliable sensor for cell monitoring depends on the ability to design powerful instrumentation that will facilitate efficient signal transduction from the biological process that occurs in the cellular environment. The resulting sensor should not affect cell viability and must function as well as adapt the system to the specific conditions imposed by the cell culture. Due to their performance, electrochemical biosensors could be used as an effective instrument in cell cancer research for studying biochemical processes, cancer development and progression as well as toxicity monitoring. Current research in this direction is conducted through high-throughput, compact, portable, and easy to use sensors that enable measurement of cells' activity in their optimum environment. This paper discusses the potential of a high-throughput electrochemical multisensor system, so-called the DOX system for monitoring cancerous cells and their interaction with chemical toxins. We describe the methodology, experiments, and the operation principle of this device, and we focus on the challenges encountered in optimizing and adapting the system to the specific cell-culture conditions. The DOX system is also compared with conventional cell-culture techniques.     

Application of multivariate analysis toward biotech processes: case study of a cell-culture unit operation

This paper examines the feasibility of using multivariate data analysis (MVDA) for supporting some of the key activities that are required for successful manufacturing of biopharmaceutical products. These activities include scale-up, process comparability, process characterization, and fault diagnosis. Multivariate data analysis and modeling were performed using representative data from small-scale (2 L) and large-scale (2000 L) batches of a cell-culture process. Several input parameters (pCO2, pO2, glucose, pH, lactate, ammonium ions) and output parameters (purity, viable cell density, viability, osmolality) were evaluated in this analysis. Score plots, loadings plots, and VIP plots were utilized for assessing scale-up and comparability of the cell-culture process. Batch control charts were found to be useful for fault diagnosis during routine manufacturing. Finally, observations made from reviewing VIP plots were found to be in agreement with conclusions from process characterization studies demonstrating the effectiveness of MVDA as a tool for extracting process knowledge.     

Life, death, and in-between: meanings and methods in microbiology

Determination of microbial viability by the plate count method is routine in microbiology laboratories worldwide. However, limitations of the technique, particularly with respect to environmental microorganisms, are widely recognized. Many alternatives based upon viability staining have been proposed, and these are often combined with techniques such as image analysis and flow cytometry. The plethora of choices, however, adds to confusion when selecting a method. Commercial staining kits aim to simplify the performance of microbial viability determination but often still need adaptation to the specific organism of interest and/or the instruments available to the researcher. This review explores the meaning of microbial viability and offers guidance in the selection and interpretation of viability testing methods.     

The r.b.e. of different energy neutrons as measured by the heamatopoietic spleen-colony technique.

The spleen-colony technique has been used for determining the relative biological effectiveness (r.b.e.) for several energies of neutron radiation. Donor mice were exposed for fission and accelerator-generated neutrons at a variety of doses and energies. Immediately after exposure, donor bone-marrow was removed from the hind legs, and standard amounts were injected intravenously into lethally X-irradiated recipients. After 7 days the recipients spleens were evaluated for surface colonies. Dose-response curves were obtained for each type of radiation and the Do was determined. The neutron r.b.e. values from the Do compared with 250kVp X-rays were: reactor 1.58, 252Cf 1:59, and accelerator varied from 2.85 at 1.0 Mev to 0.85 at 13.4 MeV.     

Effects of different immunolabeling techniques on the detection of small-cell lung cancer cells in bone marrow.

Recent reports have suggested that the immunodetection of tumor cells in bone marrow of small-cell lung cancer (SCLC) patients is by far more effective than traditional cytohistological methods and that this may be clinically relevant. This study aimed to evaluate whether the level of detection of tumor cells in bone marrow is affected by different immunostaining methods. Using two anti-NCAM monoclonal antibodies (MAbs), we compared four different "sandwich" methods on cytospin preparations of the N592 human SCLC cell line and of bone marrow aspirates from 37 SCLC patients. Our data indicate that the combination of the alkaline phosphatase-anti-alkaline phosphatase and streptavidin-biotin-alkaline phosphatase complex methods provides the best results in terms of sensitivity and specificity, and of intensity of immunoreaction and absence of staining background. Moreover, bone marrow micrometastases detected by this method were prognostically relevant and identified, among patients with apparently limited disease according to conventional staging procedures, a subgroup with shorter survival. We suggest that the choice of a sensitive immunostaining technique may significantly increase the detection rate of SCLC cells in bone marrow, mirroring the biological aggressiveness of the disease.     

Detecting the presence of infectious hepatitis A virus in molluscs positive to RT-nested-PCR

AIMS: The objective of this study was to determine the presence of infectious hepatitis A virus (HAV) in molluscs naturally contaminated with viral HAV-RNA. METHODS AND RESULTS: One hundred and forty-two mollusc samples were analysed for the presence of viral HAV-RNA using RT-nested-PCR; positive samples were then analysed with an integrated method, cell-culture RT-PCR, to detect infectious virus. Viral HAV-RNA was detected in 34.5% of the samples while 12.7% of the total samples were positive for the presence of infectious virus. CONCLUSIONS: The results demonstrate the validity of the screening method (RT-nested-PCR) and the necessity of applying a method that is capable of detecting the presence of infectious HAV. SIGNIFICANCE AND IMPACT OF THE STUDY: The study demonstrates that in any case, to determine the safety for human consumption, the results of RT-nested-PCR must be confirmed with an integrated cell-culture PCR method.     

Titration and Neutralization of Poliovirus in Micro Tissue Culture Under Increased Carbon Dioxide

Modification of the micro tissue culture technique, including incubation under increased CO(2), resulted in prolongation of the viability of the cells. As a consequence, satisfactory titrations of poliovirus and of poliovirus antiserum have been achieved by the micro method. The technique offers a number of advantages over conventional methods.