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The 5'-nucleotidase considered as a marker enzyme of the plasma membranes shows a highly specific acitvity in both nuclear and mitochondrial-lysosomal fractions of the rat yolk-sac. In isopycnic sucrose gradient fractionation of the mitchondrial-lysosomal fraction a relative accumulation of this enzyme could be shown in the density level of the phagolysosomes (d = 1,16--1,20).
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Certain embryonic tissue masses and cell aggregates behave like deformable solids during brief experimental manipulations but like viscous liquids in long-term organ cultures. To investigate these seemingly paradoxical physical properties, we have mechanically deformed cell aggregates derived from several embryonic chick organs by centrifuging them against solid substrata. Aggregate shapes during brief centrifugation were observed directly in a microscope-centrifuge. In addition, techniques were devised for fixing cell aggregates during prolonged centrifugation. Evidence presented here shows that these fixative-injection procedures accurately preserve the prefixation shapes of living centrifuged aggregates. According to a simple viscous-liquid model for cell aggregates, cohering cells slide past one another when external forces and/or tissue surface tensions cause gradual rearrangements in aggregate conformations. In earlier experiments, 2 types of behaviour predicted from this model were confirmed for several embryonic chick tissues subjected to prolonged centrifugation. First, initially flat aggregates rounded up against the centrifugal force to adopt the same shapes that initially round aggregates reached by flattening. Second, the relative roundness of centrifuged aggregates of different tissues at shape equilibrium correlated with the relative positions that these tissues assumed when they were combined in aggregate-spreading and cell-sorting experiments. By contrast, the brief centrifugation experiments described here provide some support for a simple elastic-solid model in which aggregate shape changes are accompanied by cell deformations rather than cell redistributions. In particular, since cell migration tends to occur quite slowly, the very rapid aggregate flattening observed during the first few minutes of centrifugation presumably requires cell stretching. Moreover, since they do also round up very rapidly following brief centrifugation, these aggregates exhibit considerable elasticity that presumably reflects the swift relaxation of cell stretching as the centrifugal force is removed. Athough both elastic-solid and viscous-liquid properties can be recognized in cell aggregates, we note that, in the prolonged centrifugation experiments described here, rapid initial aggregate flattening is followed by much more gradual, continued flattening. Similarly, after prolonged centrifugation, rapid partial aggregate rounding-up is also followed by much more gradual, continued rounding-up during subsequent culture at Ig. Such rapid-then-slow shape changes contradict both simple elastic-solid and simple viscous-liquid models for cell aggregates. These bimodal shape changes are instead consistent with both compound-viscoelastic-solid and elasticoviscous-liquid models for cell aggregates, although only the latter can also account for long-term liquid-like aggregate behaviour...
A high-cell-density perfusion culture process, using a novel centrifuge, was developed. The centrifuge has spiral multiple settling zones to separate cells from culture medium. Because of the multiple zones, the separation area can be efficiently increased without enlarging the diameter of the centrifuge. The centrifuge used in this study had a separation capacity of 2600 ml culture medium min -1 at 100 g of the centrifugal force. A new cell separation and withdrawal method was also developed. The cells separated in the centrifuge can be withdrawn easily from the centrifuge with no cell clogging by feeding a liquid carrier such as a perfluorocarbon into the centrifuge and pushing the cells out with the liquid carrier. By this culture process, monoclonal antibodies were produced with mouse-human hybridoma X87X at a cell density of about 8 x 10(6) cells ml -1 for 25 days. This centrifuge culture shows promise as a large-scale perfusion culture process. (Himmelfarb et al. 1969; Feder and Tolbert 1983) usually have the problem of filter clogging, and perfusion culture processes with gravitational cell settling (Tokashiki and Arai 1989) are limited in scale-up because of low separation efficiency. Recently centrifugal separation of cells from culture medium has been used for perfusion culture processes (Hamamoto et al. 1989; Tokashiki et al. 1990; Apelman 1992; Jäger 1992). This process essentially has a high separation efficiency and can avoid further operational problems, such as filter clogging, by the type of filtration. To investigate the scale-up feasibility of the centrifuge culture system, we have developed a new type of centrifuge with four layers of spiral cell-settling zones to increase the separation area, and a novel cell separation and withdrawal method using a perfluorocarbon.
During the 1998 Neurolab mission (STS-90), four astronauts were exposed to interaural centripetal accelerations (Gy centrifugation) of 0.5 g and 1 g during rotation on a centrifuge, both on Earth and during orbital space flight. Subjects were oriented either left-ear out or right-ear out, facing or back to motion. Binocular eye movements were measured in three dimensions using a video technique. On Earth, tangential centrifugation that produces 1 g of interaural linear acceleration combines with gravity to tilt the gravitoinertial acceleration (GIA) vector 45 degrees in the roll plane relative to the head vertical, generating a summed vector of 1.4 g. Before flight, this elicited mean ocular counterrolling (OCR) of 5.7 degrees. Due to the relative absence of gravity during flight, there was no linear acceleration along the dorsoventral axis of the head. As a result, during in-flight centrifugation, gravitoinertial acceleration was strictly aligned with the centripetal acceleration along the interaural axis. There was a small but significant decrease (mean 10%) in the magnitude of OCR in space (5.1 degrees). The magnitude of OCR during postflight 1 g centrifugation was not significantly different from preflight OCR (5.9 degrees). Findings were similar for 0.5 g centrifugation, but the OCR magnitude was approximately 60% of that induced by centrifugation at 1 g. OCR during pre- and postflight static tilt was not significantly different and was always less than OCR elicited by centrifugation of Earth for an equivalent interaural linear acceleration. In contrast, there was no difference between the OCR generated by in-flight centrifugation and by static tilt on Earth at equivalent interaural linear accelerations. These data support the following conclusions: (1) OCR is generated predominantly in response to interaural linear acceleration; (2) the increased OCR during centrifugation on Earth is a response to the head dorsoventral 1 g linear acceleration component, which was absent in microgravity. The dorsoventral linear acceleration could have activated either the otoliths or body-tilt receptors that responded to the larger GIA magnitude (1.4 g), to generate the increased OCR during centrifugation on Earth. A striking finding was that magnitude of OCR was maintained throughout and after flight. This is in contrast to most previous postflight OCR studies, which have generally registered decreases in OCR. We postulate that intermittent exposure to artificial gravity, in the form of the centripetal acceleration experienced during centrifugation, acted as a countermeasure to deconditioning of this otolith-ocular orienting reflex during the 16-day mission.
In Xenopus laevis, the dorsal structures normally develop from regions of the egg opposite the side of sperm entry. Gravity is known to affect this topographic relationship in eggs inclined obliquely from their normal vertical orientation in the period before first cleavage. This effect has been explored in detail, making use of low-speed centrifugation (10-50 g) for short durations (4 min). Eggs were immobilized in gelatin and oriented with their animal-vegetal axes 90 degrees to the force vector, with the sperm entry point (SEP) side of the egg either toward or away from the center of the rotor. It has been found that the egg shows three distinct periods of response to centrifugal force in the interval from fertilization to first cleavage: Prior to 0.4 (40% of the first cleavage interval), the egg is very sensitive to centrifugal force and develops dorsal structures from its centrifugal side, regardless of the position of the SEP in the centrifugal field. Thus, the dorsal structures of the embryo are reversed from normal in eggs centrifuged with the SEP away from the center of the rotor. In the period 0.4 to 0.7, the egg is still very sensitive to centrifugal force and develops dorsal structures from its centripetal side, regardless of the position of the SEP in the centrifugal field. Thus, the dorsal structures of the embryo are reversed from normal in eggs centrifuged with the SEP toward the center of the rotor. In the period 0.7-1.0, the egg becomes increasingly resistant to centrifugal force and forms dorsal structures at the normal position opposite the SEP side. This resistance can be overcome in some egg clutches by 50 g centrifugation followed by prolonged 90 degrees off-axis inclination at 1g. Midway in the second cell cycle, there is a brief period of sensitivity to centrifugal force. These These results are discussed in terms of the types of cytoplasmic rearrangements occurring in the egg at different times of the cell cycle, and in terms of the process of cytoplasmic localization of determinants of dorsal axial development.
Centrifugation was introduced during virus adsorption to Vero E6 cells to improve the infectivity of hantavirus. Centrifugal adsorption of a stock solution of Hantaan virus strain 76-118 to a monolayer of Vero E6 cells enhanced virus infectivity depending on the centrifugation time and the centrifugal force. The maximum level of infectivity (3.1 x 10(6) FFU/ml) was enhanced after a 2 h centrifugation at 671 x g, which was almost 9-times higher than that of conventional adsorption of the virus at 37 degrees C for 1 h. Vero E6 cells were inoculated with a new hantavirus strain, KI-91-40, isolated with a low infectious titer (400 FFU/ml) from an urban rat and adsorbed by centrifugation. A higher virus titer was detected sooner compared to when using conventional adsorption. To analyze the mechanism of the enhancement, the centrifugation was carried out before and after virus adsorption. The infectivity was reduced when Vero E6 monolayers were centrifuged before virus inoculation. When the centrifugation proceeded after inoculation, the infectivity was almost equal to that without centrifugation. The infectivity was only enhanced when centrifugation was carried out during inoculation. These results indicate that centrifugation promotes a very early event of infection, probably attachment of the virus to cells.
As part of an effort to develop a suspension-culture perfusion-based process with high flow rate without the fouling and antibody retention inherent to filter-based cell-separation devices, we have evaluated and contributed to the development of the Centritech Lab centrifuge for the perfusion culture of hybridoma cells in protein-free medium. Culture start-ups showed that cell growth and monoclonal-antibody (MAb) production rates were similar in both a spinner flask and continuous centrifugation coupled to a bioreactor. The centrifuge efficiently separated viable cells from dead ones. Viable-cell recoveries were never below 98%, whereas dead-cell recoveries were usually around 80%. The cell content of the centrifuge supernatant and concentrate was strongly determined by the total amount of cells, viable and dead, in the culture broth, but an influence of the centrifugation parameters (feed rate, times of separation and discharge, and rotor speed) was observed. This understanding of the separation process inside the centrifuge is important and may apply to other similar devices. Monoclonal antibodies were not retained in the bioreactor during centrifugation perfusion. However, whereas similar growth rates were obtained in perfusion cultures using either continuous centrifugation or filtration, MAb concentrations were 35% lower in the former case. Utilization of the centrifuge in an intermittent fashion decreased the daily cell residence time outside the bioreactor, the daily pelleted-cell residence time in the centrifuge, and the frequency of cell passage to the centrifuge. This led to higher viable-cell numbers in the bioreactor and an accompanying increase in MAb concentrations, 225-250 mg of IgM L-1, equal to the performance of filter-based perfusion systems with the same cell line. It was hypothesized that having cells periodically packed at the bottom of the centrifuge insert (up to 800 x 10(6) cells mL-1) is deleterious to the culture by exposing the pelleted cells to prolonged nutrient limitations.
Functional enucleation is removal or denaturation of an oocytes DNA without piercing the zona pellucida. Two experiments were conducted in this study to determine the effects of centrifugation, and ultraviolet (UV) light on metaphase II bovine oocytes. Experiment 1 evaluated the effects of centrifugation (12,000 x g for 4 min) on the cleavage rate of in vitro matured oocytes. Centrifugation decreased (P < 0.05) the cleavage rate of oocytes (79.5 vs 70.4%). In addition, it was noted that there were two types of ooplasm after centrifugation, stratified and granular. Developmental potential, as represented by cleavage percent, of the two types of ooplasm was not significantly different. Experiment 2 was conducted to determine the interactive effects of centrifugation (as above) and UV light (254 nm) on cleavage rate of oocytes exposed as metaphase II oocytes. The UV light decreased (P < 0.07) oocyte cleavage rates (35.4 vs 25.2%). Centrifuging metaphase II oocytes also decreased (P < 0.07) cleavage rates (34.1 vs 26.5%). In addition, we determined the fate of chromosomes of oocytes centrifuged and(or) exposed to UV light. Both centrifugation and UV light alone affected (P < 0.05) chromosome placement at 42 +/- 3 h after fertilization. Furthermore, centrifugation and UV light interactively increased (P < 0.05) the percentage of non-cleaved oocytes with their DNA located in the perivitelline space (17.4, 15.5, 13.1, and 49.2, respectively, for control, UV exposed, centrifuged, and UV *centrifuged). Collectively, these data indicate that bovine oocytes at the metaphase II stage can be functionally enucleated with centrifugation and exposure to UV light; however, developmental potential may be diminished by those techniques.
25 female Sprague-Dawley rats were placed on a 3.66 m radius centrifuge and subsequently exposed almost continuously for 810 days to 2.76 G. Compared to normal gravity controls, the most noticeable effect of hypergravity was the inhibition of growth of the centrifuged animals. The rats exposed to hypergravity showed on average a smaller femur length (-6.5%), a smaller cross-sectional area (-7.7%, when expressed linearly, i. e. (area/pi)1/2), and smaller outer and inner cross-sectional radii (linearly -9.3% and -12.3%) at the mid-shaft of the femoral bones. The growth inhibition of 3 hind-leg muscles was on average significantly less ranging from (-3.5% to -4.1%), compared to the growth inhibition of the linear dimension of the femur. Statistically there was no difference in the slope and elevation of the regression of the square-root of the cross-sectional area divided by pi on the length of the femur between centrifuged animals and their 16 age matched controls. In the weight control group of 24 animals, comprised of 34,74, and 102 day old rats, the corresponding regression line was parallel and lower in elevation by -12.8%, compared to the line for the centrifuged and age control groups. But, compared to the regression derived from all control animals ranging from 34 to 840 days of age, the cross-sectional area at the mid-shaft of the femur was 8.4% greater in the rats exposed to 2.76 G for 810 days. The slopes of the regression of the outer radius at mid-shaft on the length of the femur were the same in the centrifuged group and in the weight and age control groups of animals. But, the regression lines differed in elevation by -4.4% on average between the centrifuged and age control animals. The line for the regression of the inner radius at the mid-shaft on the length of the femur was parallel and lower in elevation by -7.6% in the centrifuged animals compared to the line for the age controls. But, compared to all control animals living at normal gravity, the outer radius was increased by 3.0% and the inner radius was decreased by 5.7% in the animals exposed to 2.76 G for 810 days. Since the centrifuged animals were all 840 days old, while the controls were from 34 to 840 days old, only further experiments comparing centrifuged and control animals of the same age at various growth stages will be able to furnish evidence for an unambiguous bone hypertrophy. The regressions of the cube-root of body weight on length of the femur deviate significantly in the 3 groups of animals. The heavier rats of the age control group have relatively shorter femurs than the lighter animals. The opposite applies to the centrifuged and the weight control groups of rats. Although the rats on the centrifuge are markedly smaller in overall body size than the controls, they exhibit on average the same absolute muscle weights as the animals at earth gravity, if rats of the same overall body size are compared...
OBJECTIVE: To determine the centrifugal force required to pellet sperm. DESIGN: Prospective, in vitro study. SETTING: Tertiary referral center. PATIENT(S): Men undergoing semen processing or postvasectomy semen analyses. INTERVENTION(S): In Phase I, postvasectomy semen samples were centrifuged at 600 x g, and the pellets were examined. Supernatants from samples with no visible sperm underwent repeat centrifugation at 1,000 x g; the supernatants were removed and centrifuged at 3,000 x g. Pellets from both centrifugations were examined. Phase II examined nonazoospermic semen that was divided into 3 aliquots and centrifuged at 500, 1,000, and 3,000 x g. The supernatants were examined for the presence of sperm. MAIN OUTCOME MEASURE(S): Sperm presence in centrifuged pellets or seminal supernatant. RESULT(S): Phase I: After centrifugation at 1,000 x g and 3,000 x g, sperm were noted in 12% and 0% of samples, respectively. Phase II: Sperm were noted in the supernatant in 100% of samples subjected to 500 x g and 1,000 x g, and in 92% of samples subjected to 3,000 x g centrifugation. CONCLUSION(S): If sperm are in the seminal plasma, they will also be present in the pellet following centrifugation at a force of 1,000 x g or greater. Semen samples that appear azoospermic upon initial wet mount microscopy should be centrifuged at a minimum of 1,000 x g for 15 minutes.
Current techniques of sperm preparation for in vitro fertilization or intrauterine insemination require centrifugation of human semen to separate spermatozoa from the seminal plasma. Centrifugation increases reactive oxygen species (ROS) formation in semen. Moreover, high levels of ROS are associated with sperm membrane injury through spontaneous lipid peroxidation, which may alter sperm function. We investigated the relationship between centrifugation variables (time and g-force) and ROS production to establish an optimal centrifugation protocol for sperm preparation techniques. Semen from 38 men (24 patients and 14 normal volunteers) was evaluated for the formation of ROS before centrifugation and after centrifugation at 200 g for 2 or 10 min and after 500 g for 2 or 10 min. The absence of white blood cells in semen which can also produce ROS was determined with the myeloperoxidase technique (Endtz test). All specimens were negative (< 1 x 10(6)/ml) by the Endtz test. The formation of ROS was measured by chemiluminescence. ROS formation was regarded as high (positive) when the chemiluminescence response was at least 10 x 10(4) counted photons/min (cpm). The sperm concentration in each sample was adjusted to 15-20 x 10(6) cells/ml before analysis. Eight specimens (7 patients and 1 donor) exhibited high levels of ROS before centrifugation. All 8 showed further, significant increases in ROS formation regardless of g-force or time. The increase in ROS was significantly less when semen was centrifuged for 2 as compared to 10 min (p < 0.001). Six specimens previously ROS-negative became ROS-positive after centrifugation for 10 min at 200 and 500g. We conclude that the time of centrifugation is more important than g-force for inducing ROS formation in semen. Based on these results, we recommend a shorter centrifugation period in the preparation of sperm for assisted reproductive techniques.
Current protocols for boar sperm cryopreservation require the centrifugation of semen in order to separate sperm cells from the seminal plasma. This study evaluated the influence of different centrifugation regimes on both sperm recovery and yield (percentage of viable sperm with an intact acrosome relative to the initial sperm population) after centrifugation (experiment 1) as well as the influence of different centrifugation regimes on boar sperm cryosurvival (experiment 2). In both experiments, sperm-rich fractions from 3 boars were diluted, pooled, and cooled to 17 degrees C before centrifugation. In experiment 1, the g-forces tested were 400, 800, 1600, and 2400 x g for 3 or 5 minutes, using the standard regime (800 x g for 10 minutes) as a reference. Sperm recovery (Bürker Chamber) and yield (triple fluorescent stain of PI/R123/FITC-PNA [DNA-specific fluorochrome propidium iodide/mitochondria-specific fluorochrome rhodamine-123/acrosome-specific fluorochrome fluorescein isothiocyanate-labeled peanut (Arachis hypogaea) agglutinin]) were calculated. The highest recovery and yield (P <.05) values were achieved using 2400 x g for 5 or 3 minutes and 1600 x g for 5 minutes, which showed no differences (P >.05) from the reference in terms of sperm yield. In experiment 2, cooled semen was centrifuged using 3 different regimes: C1 (2400 x g for 3 minutes), C2 (1600 x g for 5 minutes), and C3 (800 x g for 10 minutes). Pellets were diluted in lactose-egg yolk (LEY)-glycerol-Equex STM (1 x 10(9) cells/mL) and frozen in 0.5-mL straws. After thawing, sperm quality was assessed after 30 and 150 minutes of incubation (37 degrees C). Centrifugation regimes C1 and C2 showed significantly (P <.05) higher postthaw sperm motility (assessed with a computer-assisted semen analysis system), viability (evaluated as for experiment 1), and percentage of uncapacitated sperm (assessed with a chlortetracycline assay) than did C3. In addition, C1 had the highest (P <.05) oocyte penetrating ability (assessed with the homologous in vitro penetration test performed with immature oocytes). Malondialdehyde production, assessed with the thiobarbituric acid reactive species test, was unaffected (P >.05) by the centrifugation regime used. We conclude that high g-force (2400 x g) and short centrifugation time (3 minutes) do not affect sperm recovery and yield and that, moreover, they have a positive effect on the cryosurvival of boar sperm. Therefore, we recommend the use of short-term centrifugation with a relatively high g-force (2400 x g for 3 minutes) in boar sperm cryopreservation protocol.