Reorganization of the system of intermediate filaments and detection of the organization centers after centrifugation of cells attached to a substrate

Cultured pig kidney epithelial cells were centrifuged at 20,000 gav so that the centrifugation force was oriented parallel to the substrate, fixed and processed for indirect immunofluorescent staining with tubulin and vimentin antibodies. After a 2 hour centrifugation vimentin filaments aggregated in the centripetal parts of the cells (probably, because of their association with floating lipid vesicles). Microtubule-organizing centers were found near the centripetal poles of the nuclei, which migrated in the direction of the centrifugal force. The distribution of the cytoplasmic microtubules did not change during centrifugation. The staining of the cultures one hour after centrifugation revealed vimentin-containing spots with radiating intermediate filaments in most of the cells. These spots were localized near the cell nuclei; double immunofluorescent staining with tubulin and vimentin antibodies showed that their position was identical to that of the microtubule-organizing centers. Similar foci of vimentin filaments were seen in the cells after a 3-4 hour centrifugation. Probably, these structures participate in organizing the intermediate filament cytoskeleton in cells.     

High density culture of hybridoma cells using a perfusion culture vessel with an external centrifuge

The influence of centrifugal force on the growth of cells was examined by exposing the cells of the mouse-human hybridoma X87 line to centrifugal force (100–500 G) for ten minutes twice a day and comparing the static culture with that of unexposed cells. In this experiment, both cell proliferation and specific antibody productivity were independent of the centrifugal effect, and gave the same results as in the case of no exposure to centrifugal force. High density cultivation of the mouse-human hybridoma X87 line was obtained by a perfusion system where the cells were separated from the culture medium by continuous centrifugation. In the serum-free culture, the maximum viable cell density exceeded 10⁷ cells/ml, and monoclonal antibody was stably produced for 37 days. The results in this culture were equivalent to those obtained by intermittent centrifugal cell separation from the culture medium, and separation by gravitational settlement.     

EFFECT OF CENTRIFUGATION ON THE DEVELOPMENT AND TIMING OF PREMITOTIC POSITIONING OF THE NUCLEUS IN ADIANTUM PROTONEMATA

When single-celled protonemata of Adiantum capillus-veneris L. were centrifuged immediately before transferring to darkness from continuous irradiation with red light, their nuclei were displaced basipetally. Both filamentous and branched protonemata were obtained. The stronger the centrifugal acceleration, the more frequently the branched protonemata were induced.
The effect of centrifugation at 1,300 x g for 15 min on nuclear displacement was different at different stages of the cell cycle. In early G₁ phase, the nucleus was easily displaced by centrifugation, but quickly returned to the original position after centrifugation. In late G₁ phase, the nucleus was displaced, but after centrifugation it never came back to the original position. In late G₂ and M phases, the nucleus was no longer displaced by the centrifugation. Premitotic positioning of the nucleus in cytokinesis took place about 5 hr before cell plate formation in all centrifugal treatments described above.     

Migration of the nucleus and cytoplasm along the pseudopodium of the differentiated neuroblastoma cell

Motility of neuroblastoma cells in the culture of cell line C-1300, clone N-18-A was investigated microcinematographically. In the course of morphological differentiation of the cells, after cytochalasin B treatment (1.8 mkg/ml for 24 hours), in some differentiated cells a special type of movement of the cytoplasmic mass together with the nucleus along elongated pseudopodia was detected. Such a type of movement has never been described. Sometimes, a shift in the nucleus position resulted in the complete change or reversion of cell polarity. The phenomenon of cell nucleus displacement relative to the cell configuration or reversion of the cell polarity can possibly play an important functional role for neural cells.     

Effect of cycloheximide on the ultrastructure and several metabolic processes of the cells of an SPEV culture. II

The action of cyclohexamide (in doses of 1 and 10 mkg/ml in the course of 24 hours) on porcine embryo kidney cells in culture is accompanied by a powerful suppression of protein and DNA synthesis, mitotic activity, a weaker suppression of RNA synthesis, a lowering of the activity of succinate-, lactate- and alpha-glycerophosphate-dehydrogenase, which leads to disorders in the ultrastructure of the cells. After incubation of cells in a fresh medium (in the course of 18, 24 hours) there occurs a total restoration of the ultrastructure of the nuclei, granular endoplasmatic reticulum, mitochondria, due to the repairing and strong intensification of synthetic processes, respiration and glycolysis, mitotic activity of the cells.     

Analysis of force vector field during centrifugation for optimizing cell sheet adhesion

A three-dimensional tissue was fabricated by layering cell sheets with centrifugation. In this system, an optimal centrifugal force promoted the adhesion between (a) a cell sheet and a culture dish, and (b) layered cell sheets, resulting in a significant decrease in the fabrication time of the tissue. However, negative effects like sliding/significant deformation of cell sheets were observed upon high rotational speed use. These negative effects inhibit the further shortening of the fabrication time. The sliding/deformation suggests that the centrifugal forces were applied on the cell sheets in unwanted directions. Studies on the force vector field applied to the object placed on the plate during centrifugation are not available, and thus, the reason for the occurrence of such negative effects is unclear. Here, we theoretically derived the spatial distribution of acceleration applied on a plate during centrifugation. Using this theory, we found that the negative effects were triggered by the centrifugal force in the direction parallel to the plate surface, which appeared due to an inclination of the plate surface against a horizontal plane. Therefore, by adding weights on the plate edge to maintain the plate surface in a horizontal position, we succeeded in eliminating the negative effects and in increasing the rotational speed, with the minimum risk of sliding/deformation of cell sheets. We succeeded in reducing the time to establish tight adhesion between a mouse myoblast sheet and a culture dish, and layered cell sheets by increasing the centrifugal force from 5 min to 1 min without significant cytotoxicity.     

Dynamics of changes in the surface of normal cultivated rat cells after a single exposure to the carcinogen 7,12-dimethylbenz(a)anthracene]

Changes in the cell surface after a single treatment with 7,12-dimethylbenz(a)anthracene (DMBA) of newborn rat carcass in cell culture have been studied by means of the agglutination reaction with concanavalin A. DMBA was shown to cause alterations in the cell surface. At 0.5 mkg/ml of DMBA, the difference in agglutinability of treated and untreated cells persists for 30 days. At 0.1 mkg/ml of DMBA, the agglutinability of drug-treated and control cells was similar on the 4th day after removal of carcinogen. A prolonged culturing of control cells results in an increased agglutinability of cells with concanavalin A, and in 2.5 months it becomes indistinguishable from the agglutinability level of tumor cells with concanavalin A. In 5 months, drastic karyotypic changes are registered in control cultures.     

In vitro induction of numerical changes in the karyotype of normal and transformed Djungarian and Chinese hamster cells]

The comparative study of the frequency of colcemid-induced aneuploidy and polyploidy in cultured normal and transformed cells of Djungarian hamster is described. The occurrence of variants with changed chromosome number is much higher in populations of SV40-transformed cell line (4/21) than in normal embryonic cultures. In transformed lines of Djungarian and Chinese hamsters (4/21 and V-79) the frequency of cells with changed chromosome number was found to be dependent on the culture density: the percentage of polyploids was 4-5-fold higher when the number of seeded cells was 2-fold lower. The highest number (18-29%) of hypermodal cells was produced at drug concentrations of 0.02-0.025 mkg/ml. The percengate of polyploids under these conditions reached 10-20. At further increase of colcemid concentrations the proportion of polyploid cells increased. In Djungarian hamster embryonic cell cultures there were single cells with changed chromosome numbers at a concentration of the drug of 0.015-0.1 mkg/ml.     

Centrifugal seeding of mammalian cells in nonwoven fibrous matrices

Three‐dimensional (3D) cell cultures have many advantages over two‐dimensional cultures. However, seeding cells in 3D scaffolds such as nonwoven fibrous polyethylene terephthalate (PET) matrices has been a challenge task in tissue engineering and cell culture bioprocessing. In this study, a centrifugal seeding method was investigated to improve the cell seeding efficiency in PET matrices with two different porosities (93% and 88%). Both the centrifugal force and centrifugation time were found to affect the seeding efficiency. With an appropriate centrifugation speed, a high 80?90% cell seeding efficiency was achieved and the time to reach this high seeding efficiency was less than 5 min. The seeding efficiency was similar for matrices with different porosities, although the optimal seeding time was significantly shorter for the low‐porosity scaffold. Post seeding cell viability was demonstrated by culturing colon cancer cells seeded in PET matrices for over 5 days. The centrifugal seeding method developed in this work can be used to efficiently and uniformly seed small fibrous scaffolds for applications in 3D cell‐based assays for high‐throughput screening. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Intact cell adhesion to glycan microarrays

A rapid and reproducible method was developed to detect and quantify carbohydrate-mediated cell adhesion to glycans arrayed on glass slides. Monosaccharides and oligosaccharides were covalently attached to glass slides in 1.7-mm-diameter spots (200 spots/slide) separated by a Teflon gasket. Primary chicken hepatocytes, which constitutively express a C-type lectin that binds to nonreducing terminal N-acetylglucosamine residues, were labeled with a fluorescent dye and incubated in 1.3-microL aliquots on the glycosylated spots. After incubating to allow cell adhesion, nonadherent cells were removed by immersing the slide in phosphate buffered saline, inverting, and centrifuging in a sealed custom acrylic chamber so that cells on the derivatized spots were subjected to a uniform and controlled centrifugal detachment force while avoiding an air-liquid interface. After centrifugation, adherent cells were fixed in place and detected by fluorescent imaging. Chicken hepatocytes bound to nonreducing terminal GlcNAc residues in different linkages and orientations but not to nonreducing terminal galactose or N-acetylgalactosamine residues. Addition of soluble GlcNAc (but not Gal) prior to incubation reduced cell adhesion to background levels. Extension of the method to CD4+ human T-cells on a 45-glycan diversity array revealed specific adhesion to the sialyl Lewis x structure. The described method is a robust approach to quantify selective cell adhesion using a wide variety of glycans and may contribute to the repertoire of tools for the study of glycomics.     

A new type of cytocentrifuge,the valve-centrifuge

Summary A new type of cytocentrifuge has been developed in which the sedimentation process of the cells onto the slides is separated from the draining of the sedimentation fluid. This is realised by electrically controlled valves which can be closed and opened while the centrifuge is running. Sedimentation is carried out with closed valves, draining of adhering medium with open valves.The preparations, freed of adhering medium by the centrifugal force can be taken out and the cells can be fixed. Alternatively the valves can be closed again and fixative can be introduced through a central well, the cells still being under the influence of the centrifugal force. With subsequent draining of the fixative and introduction of washing and staining solutions through the central well, the whole process from sedimentation to staining can be carried out in the running centrifuge. The process seems well suited for complete automation.Using dilution series from a suspension of human buffy coat cells counted in a Buerker chamber, the cell counts in the centrifuge preparations showed virtually total recovery of cells, with no apparent selection or specific distribution of cell types. Draining of the sedimentation and fixative fluids at a slow rate was found to be vital for optimal recovery of cells. The morphology of different cell types sedimented on the slide was excellent. The flattening of nuclei through gravity was studied by cytophotometry of Feulgen-stained leucocytes. The nuclear area of these cells was found to be approximately double that from cells in identically stained classical smears. With this type of valve-centrifuge a quantitative and unbiased recovery of uniformly spread and flattened cells on coverslips or slides may be obtained, thus making the procedure well suited to automated analysis based on cytophotometric principles and morphometric pattern recognition.     

Production of the mouse mammary tumor virus in cultures of BALB/cfC3H strain

The mouse mammary tumor virus (MTV) reproduces by a budding mechanism at the cell membrane of mouse mammary epithelial cells. In tissue culture, the tumor cells release their virions in the culture supernatant from which they can be removed by high speed centrifugation. Mammary tumor cells from the RIII, GR, and A strains of mice generally produce yields of virus which decrease after a few months. Cells derived from a spontaneous mammary tumor in a BALB/cfC3H mouse have shown the capability to shed relatively large amounts of virus continuously. A quantitative estimation by membrane immunofluorescence of the number of virus producing cells in one-year-old cultures revealed the presence of viral antigen on 80 to 90% of the cells; by comparison, cultures from other mouse strains had a ratio of only 10 to 15% virus producing cells. High speed centrifugation pellets obtained from 50 ml culture supernatant provided large amounts of mature virus particles which have been characterized by electron microscopy.     

Elimination of the nucleolus from the nucleus of a living cell by centrifugation (a literature review)

The following effects involving the nucleolus take place during centrifugation of living cells at centrifugal forces of several thousand g to several hundred thousand g: settling of the nucleolus in centrifugal direction on the nuclear envelope; pulling the latter as a long stalk with the nucleolus at its end (or alternatively an easy perforation of the nuclear envelope by the nucleolus); release of the nucleolus into the cytoplasm or its expulsion out of the cell; occasional stratification of the nucleolus in the nucleus; fusion of many nucleoli together under centrifugal pressure. The asymmetric topography of the nuclear envelope is considered to be one of the causes of its different resistance to the penetration of the nucleolus. Elimination of the nucleolus from cancer cell nuclei to test the nucleolar contribution to cell malignancy is suggested as one conceivable application of the centrifugal technique of cell enucleolation.     

High-yield culture and purification of Chlamydiaceae bacteria

Research on intracellular bacteria of the family Chlamydiaceae, and the diseases they cause, requires large amounts of infectious elementary bodies (EB). We describe an approach that maximizes the generation of Chlamydia pneumoniae, Chlamydia trachomatis, Chlamydia abortus, or Chlamydia pecorum EBs in several replication cycles over approximately 10 days or more in a saturated equilibrium monolayer cell culture system. Buffalo Green Monkey Kidney (BGMK) cells, Human Epidermoid Carcinoma-2 (HEp-2) cells, or mouse McCoy cells were tested. BGMK cells best supported C. pneumoniae replication when cultivated in Iscove's Modified Dulbecco's Medium. From day 1 to day 9 after inoculation, C. pneumoniae genomes per ml culture medium increased from 10(5.1) to 10(8.6) in BGMK, from 10(5.6) to 10(8.1) in HEp-2, and remained at 10(5.2) in McCoy cell cultures. Three-month pre-inoculation maintenance of BGMK cells in different culture media did not influence C. pneumoniae yields. Inoculation at multiplicities of infection (MOI) of 10 or higher and supplementation of the cell culture medium on day 7 after inoculation with 0.1% glucose enhanced C. pneumoniae EB yields in harvested cell culture medium. For purification, EBs in medium were concentrated by sedimentation, followed by low-speed centrifugation for removal of host cell nuclei, and by step-gradient centrifugation of the supernatant in a 30% RenoCal-76-50% sucrose step-gradient. Extensive sonication increased yield and infectivity of chlamydial EB. The combined method typically produced from 1000 ml infected BGMK culture medium 10 ml homogeneous, single-cell, highly infectious EB stock containing approximately 5x10(11) C. pneumoniae genomes equivalent to 4-5x10(11) inclusion forming units.     

Leukocytes Cultured from Small Inocula of Whole Blood and the Preparation of Metaphase Chromosomes by Treatment with Hypotonic KCL

Leukocytes were cultured from 0.2 ml of whole blood inoculated into 5 ml portions of a medium consisting of Eagle's basal amino acids and vitamins at double strength in Earle's balanced salt solution brought to pH 7.0 with 7.5% NaHCO₃, and containing additives: glutamine, 2 mM; penicillin, 100 units/ml; streptomycin, 100 μg/ml; phenol red, 7 μg/ml; fetal or newborn agammaglobulin bovine serum, 15%; phytohemagglutinin M, 2%; and U.S.P. heparin sodium, 20,000 units/liter. Cultures were incubated in closed 60 × 28 mm screw-cap vials, in a gas phase initially of room air, for 3 days at 37 C, with colchicine to make 0.2 μg/ml added for the final 3-5 hr. After incubation, the cells were separated from the medium by centrifugation, the medium replaced by 0.075 M KCI plus 16 U.S.P. units/ml of heparin sodium at 37 C, cells resuspended and allowed to incubate 10 min. Removal of the hypotonic KCI was followed by fixation in methanol-acetic acid, 3:1 (changed twice), spreading cells on slides by the air-drying method, and staining with 1% natural orcein (G. T. Gurr) in 60% acetic acid. Dehydration and covering completed the preparation. KCI, 0.075 M, has been used advantageously in the above way and for cells cultured by other means from skin and other organs of man and other mammals. Combined advantages of the method are: culture of leukocytes from small volumes of whole blood, with very few failures to obtain mitotic cells; a medium which can be stored frozen in culture vials, and which in a simpler form is usable for long term culture of other cell types; and the use of KCI for hypotonic treatment.     

Effect of lectins on induction of apoptosis in the populations of mammalian cells in vitro

The cytotoxic action of lectins different in origin and carboxyl specificity has been studied. It has been shown that all types of lectins at high concentrations (20 mkg/ml) were able to induce apoptosis in the in vitro populations of Chinese hamster cells two days after the treatment. In the case of Persa fluviatilis lectin this effect was detected immediately after the treatment and two days later as well. It was shown that Sambucus nigra lectin did not influence the frequency of apoptosis in the culture of human cells in contrast to the Lens culinaris and P. fluviatilis lectins. The tendency of stimulation of human cell proliferation under exposure to P. fluviatilis lectin at low concentration (0.2 microg/ml) has been registered.     

Evidence for the nucleus-centriole association in living cells obtained by ultracentrifugation

The association of centrioles with the interphase nuclei of L- and PE-cells has been studied using ultracentrifugation of a cell monolayer in a culture medium at 20 and 37 degrees C. Ultracentrifugation at 10 000 to 40 000 gav for 15 to 60 min did not cause any changes in the cell length or in the size of the nucleus, but entailed delocalization of nuclei. The distance between it and the centrioles hardly ever changes. Any nucleus-delocalizing centrifugation of non-treated cells also resulted in the centrioles being shifted towards the centripetal nucleus pole. After 30 to 60 min, at 40 000 gav, the cells remained viable and capable of mitosis. More intensive centrifugation (15 min at 70 000 gav) proved to be fatal to the cells. The distance between the centrioles and the nucleus became greater in the cells which were centrifuged after incubation with cytochalasin B. The results are interpreted as lending support to the previously demonstrated [28] association between the centrioles and the nucleus in the interphase cells.     

Spontaneous level of sister chromosome exchanges and their distribution in human cells]

Blood of practically healthy donors of both sexes (27 females and 23 males) was cultured under the standard conditions during 96 hours. Bromodeoxyuridine (BUdR) was added at the final concentration of 10 mkg/ml 28 hours before harvesting. The slides were stained with acridine orange and Giemsa for differential staining of chromatids. In each culture sister chromatid exchanges (SCE) were analysed in 50 cells, and the part of cells undergoing the first, second and third mitoses at the time of harvesting, was calculated. According to the mean number of SCE per cell, the distribution of individuals was consistent with the normal law, the mean being 6.525 and standard deviation--0.956. A significant heterogeneity in the speed of cell cycle of cultures was observed. The coefficient of variation for the part of cells undergoing the first mitosis was 50%, for the cells in the second mitosis--15%, and for the cells in the third mitosis--154%. Correlation analysis showed a positive dependence of the mean level of SCF upon the age of a donor and upon the part of cells in the second mitosis in this individual. No reliable correlation of the SCE level with the donor's sex was observed. The distribution of cells, obtained from the culture of one individual, was best approximated by beta-distribution, and the distribution of cells obtained from the cultures of different individuals--by gamma-distribution. In both there was obtained a satisfactory approximation by Pearson's distribution of the 1 type, and significant deviations were found from the normal, Poison's and the negative binomial distribution. The conditions were found of similarity of empirical distribution of SCE in cells to the normal one. For that, it is not the value of SCE for a separate cell that should be used as a unit of measurement, but the mean from the values of frequencies for 5-10 cells. Hence, it was shown that for the evaluation of the mean frequency of SCE with the precision of 1 exchange in separate individuals it is necessary to analyse 40 cells, and to observe the 15% increase of spontaneous SCE level under the action of deleterious factors--8 individuals are enough to analyse.     

Unequal cell division and chromatin differentiation in pollen grain cells

Pinus pollen grains, normally developing, were subjected to centrifugal force, low temperature and caffeine solution. In the former two treatments, daughter cells with some abnormal directions of division, abnormal volume and chromatin dispersion were induced in pollen grains treated. Regardless of the direction of division, of the two daughter cells produced by the unequal division, the larger one contained strongly dispersed chromatin and the smaller one weakly dispersed chromatin. In the two daughter cells produced by approximately equal division, the chromatin was dispersed strongly to a similar degree, and by halfway unequal division, chromatin in the larger cell was dispersed strongly and in the smaller one intermediately. Chromatin in bi-nucleate cells induced by caffeine treatment was dispersed strongly to an identical degree. It is suggested that for the occurrence of heteronomous chromatin configuration in natural pollen grains the unequal cell division was indispensable, although the axis of division didn't directly contribute. After both the treatments of centrifugation and low temperature during microspore and embryonal cell divisions, the affected daughter cells divided in terms of the certain fixed axis of division and chromatin dispersion, instead of exhibiting abnormal development.     

Cell differentiation in the hippocampus of the fetal rat in cell culture

Cell cultures from hippocampus of 16 and 17 days old embryonal rats were cultivated up to 4 weeks. After 24 hours in vitro on 18.4 percent of cells and after 5 days in vitro on 70 percent of cells processes could be recognized. These are neuroblasts. The cells reaggregated. Nerve fibers after 4 weeks in vitro are 200 to 300 mum long. Small and big neurons with 12 mum to 26 mum diameters of perikarya, bi- and multipolar neurons after 4 weeks in vitro were observed. In cultures and meningothel-monolayer developed. Maintenance and differentiation of cultures are possible only by sowing in at least 60,000 cells/ml medium. The advantage of cell culture opposite to organ culture exists in experiments with immediate selective influence.