Deformation of human erythrocytes in a centrifugal field.

A new method for altering red cell morphology by high-speed centrifugation of cells through a physiological medium is described. Cell shape is preserved for microscopic analysis by allowing the sedimenting cells to pass from the physiological medium into a glutaraldehyde fixative solution. Examination of the deformed, fixed cells indicates that the vast majority resemble spheres with a flat, triangular tail. Measurements of the overall length of deformed cells show a nearly linear relationship between cell length and centrifugal force; average cell length increased from 8 to 11 micrometer as the centrifugal field was increased from 2,000 to 15,000 g. These data suggest that this centrifugal technique may be useful for evaluating cellular deformability and, potentially, the material properties of red cells.     

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.     

Centrifugal cell hybridization

Centrifugation of cell suspensions containing two or several cell types at forces up to 2 million g results in several basic events in succession: 1. Intracellular stratification. 2. Extrusion of the most dense cell parts (nuclei or cytoplasmic components) and their penetration into an adjacent cell in the compact sediment. Such introduction of protoplasmic elements from one cell into another is considered as centrifugal hybridization and fusion of cells. It differs from other methods of cell hybridization by its selectivity for cell components. 3. Further intermingling and mixing of cells into a fused protoplasmic mass. 4. With continuing increase of centrifugal force fractions of subcellular components are formed from the protoplasmic mass. These components are presumably viable since cells are not exposed to chemical treatment. Morphological demonstration of hybridization is based on centrifuging microscopy and on labeling donor cells or recipient cells by staining or fluorescence. Genetic evidence can be provided by cultivation of hybrid cellsin vitro and their cloningin vivo.     

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.     

Assessment of cell-substrate adhesion by a centrifugal method

Summary A centrifugal method has been evaluated for measuring the strength of Vero Green Monkey kidney cell adhesion to growth surfaces. The centrifugal force necessary to remove cells gave a quantitative measure of cell adhesion and hence the quality of the growth surface. After being subjected to high gravity forces, both the remaining attached cells and the detached cells were viable, indicating the detachment process did not simply rupture the cell. Electron microscope examination of growth surfaces after cell detachment suggested that remnants related to filopodia remained.     

Amyloplasts that sediment in protonemata of the moss Ceratodon purpureus are nonrandomly distributed in microgravity

Little is known about whether or how plant cells regulate the position of heavy organelles that sediment toward gravity. Dark-grown protonemata of the moss Ceratodon purpureus displays a complex plastid zonation in that only some amyloplasts sediment along the length of the tip cell. If gravity is the major force determining the position of amyloplasts that sediment, then these plastids should be randomly distributed in space. Instead, amyloplasts were clustered in the subapical region in microgravity. Cells rotated on a clinostat on earth had a roughly similar non-random plastid distribution. Subapical clusters were also found in ground controls that were inverted and kept stationary, but the distribution profile differed considerably due to amyloplast sedimentation. These findings indicate the existence of as yet unknown endogenous forces and mechanisms that influence amyloplast position and that are normally masked in stationary cells grown on earth. It is hypothesized that a microtubule-based mechanism normally compensates for g-induced drag while still allowing for regulated amyloplast sedimentation.     

Cumulative sedimentation analysis of Escherichia coli debris size

A new method to measure Escherichia coli cell debris size after homogenization is presented. It is based on cumulative sedimentation analysis under centrifugal force, coupled with Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) analysis of sedimented proteins. The effects that fermentation and homogenization conditions have on the resulting debris distributions were investigated using this method. Median debris size decreased significantly from approximately 0.5 mum to 0.3 mum as the number of homogenization passes increased from 2 to 10. Under identical homogenization conditions, uninduced host cells in stationary phase had a larger debris size than exponential cells after 5 homogenizer passes. This difference was not evident after 2 or 10 passes, possibly because of confounding intact cells and the existence of a minimum debris size for the conditions investigated. Recombinant cells containing protein inclusion bodies had the smallest debris size following homogenization. The method was also used to measure the size distribution of inclusion bodies. This result compared extremely well with an independent determination using centrifugal disc photosedimentation (CDS), thus validating the method. This is the first method that provides accurate size distributions of E. coli debris without the need for sample pretreatment, theoretical approximations (e.g. extinction coefficients), or the separation of debris and inclusion bodies prior to analysis. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioang 55: 556-564, 1997.     

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.     

Separation of hepatocytes from suspensions of mouse liver cells using programmed gradient sedimentation in gradients of ficoll in tissue sulture medium

Liver cells were obtained in suspension using a solution of lysozyme in Joklik's modification of minimum essential medium. Hepatocytes were separated in 74.2 ± 12.9% purity from other liver cells having different densities using isopycnic centrifugation, in 97.1 ± 1.9% purity from other liver cells having different diameters using velocity or rate-zonal centrifugation. A previously reported computer integration of the differential sedimentation equation was employed in determining the gradient design and the speed and duration of centrifugation which would permit purification of hepatocytes from other liver cells. More than 98% of the hepatocytes separated by velocity sedimentation excluded trypan blue. Velocity sedimentation is superior to isopycnic centrifugation for the separation of hepatocytes from liver cell suspensions because it gives more highly purified hepatocytes and because it requires lower centrifugal forces for shorter periods of time.     

A preparation technique for exfoliated and aspirated cells allowing different staining procedures

Quantitative cytology requires highly standardized preparation, fixation and staining techniques in order to obtain reproducible morphology (e.g., cell size, cell shape and chromatin distribution). We found centrifugal cytology best suited to this purpose. Therefore, we recently developed an improved bucket for centrifugation that permits sedimentation of cells in a fixative solution (2% polyethylene glycol in 50% ethanol) by using centrifugation at relatively high g forces. The cell quantity, the cell distribution and the flatness of the specimens thus prepared proved to be adequate for automated anlysis using the Leyden Television Analysis System (LEYTAS). Furthermore, different cytochemical and cytomorphologic staining procedures could be performed on different aliquots of the same cytologic sample without any change in the preparation or fixation technique.     

Characterization of a CV-1 cell cycle. V. An assessment of velocity sedimentation for cell separation and synchrony.

Velocity sedimentation at unit gravity has been used to enrich populations of logarithmically growing cells in different cell cycle phases. In order to evaluate the degree of synchrony obtained by this method of cell separation, synchronous populations of CV-1 cells, initially obtained by the selective detachment of mitotic cells from roller cultures, were separated by velocity sedimentation. It was found that although the mean cell volume increased linearly, the cells remained heterogeneous with respect to size during all phases of the cell cycle. Since the velocity sedimentation technique depends upon discrimination of cell size, the size heterogeneity of cells throughout the cycle limits the degree of synchrony which can be obtained by this method.     

Required minimum granule size in UASB reactor and characteristics variation with size

The objective of this study was to define minimum size of bioparticles that could be classified as granules, to offer all advantages of granular sludge. Based on the theory of sedimentation, the minimum diameter bioparticles, which should be considered as granules was found out for specific gravity of sludge ranging between 1.01 and 1.05. For example, for specific gravity of 1.035 the minimum diameter of granules required for better sludge retention was 0.34 mm. The diameter based on this theory was evaluated by carrying out settling column analysis of a granular sludge obtained from lab-scale UASB reactor and verified with microscopic observation. To find out the effect of granules size on the nature of biodegradability, specific methanogenic activity (SMA) was carried out. It was observed that SMA increased with size of bioparticles tested in the range of 0.27-3.03 mm. The change in VSS/SS ratio and specific gravity was observed with size of granules. Consideration of variation in specific gravity with size of granules increased the degree of validation of sedimentation theory for the calculation of granules diameter.     

Management of sedimentation in centrifugal counter-current distribution of sperm cells in an aqueous 2-phase system.

It is generally assumed that centrifugal counter-current distribution (CCCD) in aqueous two-phase systems cannot be employed for analyzing or fractioning cell populations, due to large particles of sediment in the system caused by enhanced gravity. The present work was undertaken to find out whether addition of Percoll to a two-phase system would be a useful method to avoid this cell sedimentation. The results obtained show that bull spermatozoa partition as a unique peak in a CCCD using a Dextran T500-poly(ethylene glycol) 6000 system, and that sedimentation takes place significantly in the upper phase during the process. Addition of increasing concentrations of Percoll made this unique peak wider and two different populations of bull spermatozoa were finally obtained when Percoll concentration rose to 13.6%. This management of cell sedimentation in CCCD could be of great interest for analyzing cell heterogeneity, since the shortening of the time required for counter-current distribution should prevent the loss of cell viability during the separation process. Finally, the results obtained suggest that an increase of viscosity rather than of density is the phase feature which has greater influence on managing cell sedimentation in CCCD.     

Sedimentation of a suspension in a centrifugal field.

To model centrifugal sedimentation of biological suspensions, the time history of sedimentation of particles in a centrifugal field was considered for two geometries: a tube and a cylindrical container. The Kynch theory for batch gravitational settling in Cartesian coordinates based on mass conservation was extended to include a centrifugal sedimentation force, cylindrical coordinates, and the Hawksley-Vand hindered settling model. The resulting quasi-linear partial differential equation was solved by the method of characteristics. The combination of radial dependence of the sedimentation force and cylindrical geometry in the centrifugal case results in several differences in the time-position history diagram of the sedimentation process compared to the gravitational case. First, instead of a region of uniform concentration equal to the initial concentration, a region of concentration that is continuously decreasing with time results. Second, in the region of particle accumulation, curved constant concentration contours result instead of straight lines. Finally, a secondary shock that is dependent upon the initial concentration and the radius ratio of the rotating vessel appears in the centrifugal case. The time history of the concentration for a particle suspension with an initial concentration typical of blood is presented.     

Reverse banding of Japanese quail microchromosomes.

Cells isolated from adult and fetal rat liver and ascites hepatoma were separated into distinct populations by velocity sedimentation at unit gravity. Normal adult liver ceils sediment with modal velocities ranging from 5 to 50 mm/h. Volume analysis using a Coulter-type counter demonstrated that the separation was based primarily on cell size. Appreciable differences were observed in the sedimentation velocity distribution of cells isolated from different normal lobes or regenerating liver. Most fetal rat liver cells sediment with velocities inferior to 12 mm/h. Ascites (Novikoff) hepatoma cells present a velocity distribution more similar to that of fetal than to normal adult liver cells. The results are discussed in terms of cell-size changes associated with liver maturation, regeneration or transformation.     

Fluid flow through a high cell density fluidized‐bed during centrifugal bioreactor culture

An increasing demand for products such as tissues, proteins, and antibodies from mammalian cell suspension cultures is driving interest in increasing production through high‐cell density bioreactors. The centrifugal bioreactor (CCBR) retains cells by balancing settling forces with surface drag forces due to medium throughput and is capable of maintaining cell densities above 10⁸ cells/mL. This article builds on a previous study where the fluid mechanics of an empty CCBR were investigated showing fluid flow is nonuniform and dominated by Coriolis forces, raising concerns about nutrient and cell distribution. In this article, we demonstrate that the previously reported Coriolis forces are still present in the CCBR, but masked by the presence of cells. Experimental dye injection observations during culture of 15 μm hybridoma cells show a continual uniform darkening of the cell bed, indicating the region of the reactor containing cells is well mixed. Simulation results also indicate the cell bed is well mixed during culture of mammalian cells ranging in size from 10 to 20 μm. However, simulations also allow for a slight concentration gradient to be identified and attributed to Coriolis forces. Experimental results show cell density increases from 0.16 to 0.26 when centrifugal force is doubled by increasing RPM from 650 to 920 at a constant inlet velocity of 6.5 cm/s; an effect also observed in the simulation. Results presented in this article indicate cells maintained in the CCBR behave as a high‐density fluidized bed of cells providing a homogeneous environment to ensure optimal growth conditions. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Centrifugal elutriation: separation of spermatogenic cells on the basis of sedimentation velocity.

Various types of cells from the testes of mice and hamsters were separated according to differences in sedimentation velocity by centrifugal elutriation, a counterflow centrifugation technique. Approximately 3 times 10(8) cells, prepared from six mouse testes or from one hanster testis, were separated into 11 fractions in less than two hours as compared to the 4--5 hours required for sedimentation at unit gravity ("Staput"). Fractions enriched in elongated spermatids and spermatozoa (100%), stages 1--8 spermatids (69%) and pachytene spermatocytes (58%) were obtained from mouse testis dispersions. Similarly enriched fractions were obtained from hamster cells. A single fraction enriched in stages 1--8 spermatids (mouse) was prepared in less than 30 minutes. As many as 2 times 10(9) cells were separated in a single procedure. Spermatogenic cells exhibited no evidence of structural damage with trypan blud and phase microscopy, and recovery was essentially 100%. Centrifugal elutriation had no effect on sperm motility or on the plating efficiency of CHO cells.     

ERYTHROPOIETIN SENSITIVITY OF RAT BONE MARROW CELLS SEPARATED BY VELOCITY SEDIMENTATION

Rat bone marrow cells have been separated on the basis of their sedimentation at unit gravity. The cell population most responsive to erythropoietin in vitro was found to have a sedimentation velocity of about 6.6 mm/hr. In the process of becoming hemoglobin-synthesizing cells, it undergoes cell division and its sedimentation velocity decreases to 3.9 mm/hr and then to 2.1 mm/hr, the sedimentation velocity of mature red blood cells.     

The effect of centrifugal accelerations on the polarity of statocytes and on the graviperception of cress roots

The structural polarity of statocytes of Lepidium sativum L. is converted to a physical stratification by a root-tip-directed centrifugal acceleration. Sedimentation of amyloplasts and nucleus to the centrifugal (distal) cell pole and the lateral displacement of the distal endoplasmic reticulum (ER) complex occur after centrifugation for 20 min at an acceleration of 50 g. With higher doses (20 min, 100-2,000 g), smaller organelles become increasingly displaced. From the centrifugal to the centripetal cell pole, the following stratification is observed: 1) amyloplasts with mitochondria; 2) nucleus with mitochondria and a few dictyosomes, as well as laterally located ER; 3) dictyosomes with a few mitochondria; 4) vacuoles; and 5) lipid droplets. Within the first 7.5 min, after the roots have been returned to 1 g, the original arrangement of the amyloplasts sedimented on the underlying ER complex is reestablished in 66% of the statocytes. When roots previously centrifuged in an apical direction are exposed in a horizontal position to 1 g, the latent period of the graviresponse is increased by 7.5 min relative to the non-centrifuged controls. The kinetics of the response are identical to the controls. Roots centrifuged first in an apical direction and then for 2 h in a lateral direction (1,000 g) have statocytes with a physical stratification perpendicular to the root axis. A gravitropic curvature does not take place during the lateral centrifugation. These results support the hypothesis that the distal ER complex is necessary and sufficient for graviperception.     

How to activate a plant gravireceptor. Early mechanisms of gravity sensing studied in characean rhizoids during parabolic flights

Early processes underlying plant gravity sensing were investigated in rhizoids of Chara globularis under microgravity conditions provided by parabolic flights of the A300-Zero-G aircraft and of sounding rockets. By applying centrifugal forces during the microgravity phases of sounding rocket flights, lateral accelerations of 0.14 g, but not of 0.05 g, resulted in a displacement of statoliths. Settling of statoliths onto the subapical plasma membrane initiated the gravitropic response. Since actin controls the positioning of statoliths and restricts sedimentation of statoliths in these cells, it can be calculated that lateral actomyosin forces in a range of 2 x 10(-14) n act on statoliths to keep them in place. These forces represent the threshold value that has to be exceeded by any lateral acceleration stimulus for statolith sedimentation and gravisensing to occur. When rhizoids were gravistimulated during parabolic plane flights, the curvature angles of the flight samples, whose sedimented statoliths became weightless for 22 s during the 31 microgravity phases, were not different from those of in-flight 1g controls. However, in ground control experiments, curvature responses were drastically reduced when the contact of statoliths with the plasma membrane was intermittently interrupted by inverting gravistimulated cells for less than 10 s. Increasing the weight of sedimented statoliths by lateral centrifugation did not enhance the gravitropic response. These results provide evidence that graviperception in characean rhizoids requires contact of statoliths with membrane-bound receptor molecules rather than pressure or tension exerted by the weight of statoliths.