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Factors affecting yield and survival of cells when suspensions are subjected to centrifugation. Influence of centrifugal acceleration, time of centrifugation, and length of the suspension column in quasi-homogeneous centrifugal fields.

The goals of the centrifugation of cell suspensions are to obtain the maximum yield of cells with minimum adverse effects of centrifugation. In the case of mechanically sensitive cells such as mouse sperm, the two goals are somewhat contradictory in that g-forces sufficient to achieve high yields are damaging, and g-forces that yield high viability produce low yields. This paper mathematically analyzes the factors contributing to each goal. The total yield of pelleted cells is determined by the sedimentation rate governed by Stokes' Law, and depends on the relative centrifugal force, centrifugation time, size and shape of the cells, density of the cells and medium, viscosity of the medium, and the length of the column of suspension. Because in the situation analyzed the column is short relative to the rotor radius, the analysis considers the centrifugal field to be quasi-homogeneous. The assumption is that cells are not damaged during sedimentation, but that they become injured at an exponential rate once they are pelleted, a rate that will depend on the specific cell type. The behavior is modeled by the solution of coupled differential equations. The predictions of the analysis are in good agreement with experimental data on the centrifugation of mouse sperm.

Animals↗

Centrifugal cytology. III. The utilization of centrifugal cytology for the preparation of fixed stained dispersions of cells separated by bovine serum albumin bouyant density centrifugation.

This paper describes the modification of Centrifugal Cytology for the preparation of permanent, fixed, stained dispensions for both light and scanning electron microscopy of cells which have been isolated on bovine serum albumin (BSA) boyant density gradients. The principal problem with BSA gradient fractions is that the albumin which is present even after dilution is precipitated by the glutaraldehyde fixative. This problem has been solved by the layering of an intermediate D2O solution under the BSA and subsequent removal of the BSA solution and the underlaying with D2O containing glutaraldehyde. A special layering machine facilitates and expedites these operations. This technique has also been applied to BSA-seperated guinea pig and chicken bone marrow cells, as well as Ehrlich ascites tumor cells, hen and human blood cells. The number of celll present in each area of the slide is maintained at a constant value by utlizing a table of dilution factors. This table was generated by a computer program which calculates the concentration of cells present in the rractions and divides it by the number of celll desired.

Animals↗

Observations on centrifugation: application to centrifuge development.

This report outlines the background to the development of an automated, serial, discrete centrifuge, reporting on the criteria considered essential in such an instrument. We established the criteria by examining the detailed logistics of centrifuge operation in a hospital laboratory. The mean sample load per run, using six centrifuges, was 13.6 samples, and the user-selectable cycle time ranged from 00:01:10 to 00:12:33 (hours:minutes:seconds) with a fixed g value of 1050. During the laboratory working window, (0900-1700), only 50% of the centrifuge capacity was utilized and more than one-third of the sample workload was delayed for >5 min because the centrifuges were not emptied promptly. In addition, 35% of the sample workload was centrifuged for less than the time prescribed in the operational specifications. Based on these findings, we designed a new continuous, serial centrifuge to overcome some of the deficiencies noted in the logistics study. The centrifuge operates continuously, nominally treating 150 samples/h, with a cycle time of 5 min at 1,000 g. The cycle time and g value are variable between limits, and their selection governs the throughput rate. Each sample is centrifuged separately in individual rotors mounted in a sturdy carousel with a periphery that traverses a load/unload station. There is no sample delay because of operator absence, and the capacity is fully utilized. The centrifuge can operate in a stand-alone capacity or has the capability of being integrated into a sample preparation system or as a direct front end for high-throughput analyzers.

Centrifugation↗

Centrifugation of human spermatozoa induces sublethal damage; separation of human spermatozoa from seminal plasma by a dextran swim-up procedure without centrifugation extends their motile lifetime.

While washing of human sperm cells by centrifugation and resuspension is a procedure in widespread use, there have been indications that this procedure per se may be harmful to the cells. The objective of this study was to investigate this question. To this end, a method for the clean separation of motile human spermatozoa from seminal plasma in the absence of centrifugation was developed, using a modified swim-up procedure, in which liquefied semen was mixed with an equal volume of 30 mg/ml dextran in medium, and the mixture overlaid with medium containing 5 mg/ml bovine serum albumin, forming two discreet layers with stable interface. The percentage of motile cells in a given sample was consistently > 80% immediately after recovery. Damage to the cells was assessed by loss of motile cells during incubation up to 96 h post-recovery. Comparison of aliquots of spermatozoa obtained by the dextran swim-up procedure showed that the aliquot subjected to centrifugation had 4 +/- 3% motile cells after 48 h, while the untreated aliquot had 52 +/- 12%. The aliquots showed no difference 1 h post-recovery. Similar results were obtained with spermatozoa that had been centrifuged in seminal plasma and resuspended in fresh plasma, then recovered by dextran swim-up. The delayed onset of motility loss in the centrifuged samples implies that this treatment induces sublethal damage in the cells. Comparison of the standard swim-up and Percoll gradient methods for sperm recovery, both of which involve centrifugation steps, showed decline in motility of the samples similar to that seen with dextran swim-up of centrifuged cells. We conclude that centrifugation per se induces sublethal damage in human spermatozoa, independently of treatment method, and suggest that recovery methods for human spermatozoa which avoid centrifugation might partially alleviate the damage incurred by these cells during cryopreservation.

Adult↗

The optimum relative centrifugal force and centrifugation time for improved sensitivity of smear and culture for detection of Mycobacterium tuberculosis from sputum.

Direct microscopy is the only available method for diagnosis of tuberculosis in most centres in developing countries. Methods to improve the sensitivity of direct smear are an urgent requirement. Sputum specimens artificially seeded with known concentrations of Mycobacterium tuberculosis were liquefied and decontaminated with sodium hydroxide-sodium citrate-N-actyl-L-cysteine solutions. They were subjected to different centrifugation forces and centrifugation times after which the centrifuged deposits were examined by smear and culture. Statistical analysis of results was carried out using EpiInfo version 6.0. The optimum relative centrifugal force (RCF) and centrifugation time combination was 4000 g for 15 min. The sensitivity of detection at an RCF of 4000 g for 15 min was 5000 organisms/mL and 500 organisms/mL for smear and culture, respectively. When results of 163 clinical samples were analyzed after centrifugation at 4000 g for 15 min sensitivity of the direct smear improved from 63% to 92% (P < 0.05) and negative predictive value from 30.5% to 45% (P < 0.05) when culture was considered the 'gold standard'. With the concentrated smear there was a reduction in specificity from 82% to 60% (P > 0.05). As most laboratories are equipped with a simple centrifuge, smear sensitivity can be improved with this simple modification. The other advantage is that the same centrifuged deposit can be cultured, in contrast to when sodium hypochlorite is used for liquefaction.

Bacteriological Techniques↗

Colloidal silica--aluminum modified--PVP density gradient centrifugation: centrifuge tube wall cell adherence, aggregation, separation properties and comparison to BSA and Ficoll.

A method is described for the inexpensive and easy preparation of colloidal silica--aluminum modified--polyvinylpyrrolidone (CS-AM-PVP) density gradient centrifugation medium. Using density gradient centrifugation, several cell separation and biochemical characteristics were studied: centrifuge tube wall cell adherence, mouse spleen cell density distribution, rebanding properties, mitogen response and cell aggregation. Cell adherence to the centrifuge tube wall using density gradients of CS-AM-PVP was compared with density gradients of bovine serum albumin and Ficoll. Few cells adhered to the centrifuge tube wall when CS-AM-PVP was used as a gradient medium; whereas, significant cell adherence to the centrifuge tube cell wall occurred when bovine serum albumin or Ficoll was used as a gradient medium. The CS-AM-PVP gradient medium did not inhibit the response to mitogens of mouse spleen cells which had been separated into density subpopulations in a discontinuous CS-AM-PVP density gradient, caused a minimum amount of cell aggregation, and was found to be non-toxic.

Aluminum↗

Centrifugal recovery and dissolution of recombinant Gly-IGF-II inclusion-bodies: the impact of feedrate and re-centrifugation on protein yield.

The impact of centrifuge feedrate and multiple centrifuge passes on protein yield following recombinant Gly-Insulin-like Growth Factor II (Gly-IGF-II) inclusion-body dissolution has been investigated. Altering centrifuge feedrate did not significantly improve the overall protein yield following dissolution. Improved centrifuge recovery at a low feedrate was offset by poorer inclusion body paste purity. This reduced purity resulted in a significant loss of protein during inclusion-body dissolution due to proteolysis. Multiple centrifuge passes improved the inclusion-body paste purity. This resulted in a net improvement in the overall protein yield following dissolution. This work demonstrates that a strong interaction exists between centrifuge performance and inclusion-body dissolution for protease-sensitive products such as Gly-IGF-II.

Bioreactors↗

Concept designs of nonrotating-type centrifugal blood pump and basic study on output characteristics of the oscillating disk-type centrifugal pump.

When designing a turbo-type blood pump as an artificial heart, the gap between a rotating shaft and a pump housing should be perfectly sealed to prevent any leakage or contamination through a seal. In addition, blood coagulation in a blood chamber must be avoided. To overcome these problems, we proposed five different nonrotating-type turbo pumps: a caudal-fin-type axial-flow pump, a caudal-fin-type centrifugal pump, a nutating-column-type centrifugal pump, a nutating-collapsible-tube-type centrifugal pump, and an oscillating-disk-type centrifugal pump. We selected and developed the oscillating-disk-type centrifugal pump that consists of a disk, a driving rod, a seal, an oscillation mechanism, and a pump housing. The disk is mounted on the end of the rod, which is connected to a high-speed DC motor through an oscillation mechanism. The rod and the disk do not rotate, but they oscillate in the pump housing. This movement of the disk generates forward fluid flow around the axis (i.e., the rotational fluid flow). Centrifugal force due to fluid rotation supports the pressure difference between the outlet and the inlet. The diameter of the disk is 39 mm, the maximum inner diameter of the pump housing is 40 mm, and the volume of the blood chamber for 25 degrees' oscillation is 16.9 ml. The performance of the pump was tested in a mock circulatory system.(ABSTRACT TRUNCATED AT 250 WORDS)

Equipment Design↗

Effect of relative centrifugal force and centrifugation time on sedimentation of mycobacteria in clinical specimens.

Optimum relative centrifugal force (RCF) and centrifugation time to concentrate mycobacteria in clinical specimens were determined by processing split samples of sputa and urines containing mycobacteria with combinations of different RCFs and centrifugation times. Although individual test results showed considerable variation in the recovery rates of mycobacteria in the sediment, the data indicated that higher recovery rates occurred as centrifugation speed and time were increased. With a 15- to 20-min centrifugation time, on the average, 67 to 71% of mycobacteria were recovered at an RCF of 2,074 X g, and 76 to 80% were recovered at 3,005 or 3,895 X g at maximum radius. The remainder of mycobacteria was mostly recovered from the supernatant, but culturing of supernatant was not profitable. Increasing RCF had a negligible effect on acid-fast bacillus smear sensitivity. The smear sensitivity for about 25,000 clinical specimens processed with an RCF of 3,800 X g for 20 min was 71% compared with 69% as determined for over 30,000 specimens processed in a similar manner but an RCF of 2,000 X g. An RCF of 3,000 X g applied for 15 min, or an RCF of about 2,000 to 2,500 X g applied for 20 min, is considered adequate to concentrate mycobacteria in clinical specimens.

Bacteriuria↗

Incidence of electrocardiographic changes during cytapheresis using an intermittent flow centrifuge, haemonetics V50 and a continuous flow centrifuge, AS104.

Electrocardiographic (ECG) monitoring was performed during cytapheresis and the incidence of ECG changes was compared between methods of intermittent flow centrifugation using the Haemonetics V50 with apheresis donors and continuous flow centrifugation with the AS104 for peripheral blood stem cell (PBSC) collection from patients. ST depression, inverted T wave and other ECG changes were found in 49 (14.3%) of the 342 cytaphereses carried out by the intermittent flow centrifugation method and in 8 (4.7%) of the 169 PBSC collections by the continuous flow centrifugation method. This difference was significant (P < 0.05). The hemodynamic changes in the procedure with the AS104 seem to be small. This is considered to be important for prevention of serious ST and T wave changes.

Blood Component Removal↗

Purification of Synechococcus lividus by equilibrium centrifugation and its synchronization by differential centrifugation.

A culture of the thermophilic, unicellular, blue-green alga Synechococcus lividus was freed from two thermophilic bacteria by use of equilibrium (isopycnic) centrifugation in Ficoll density gradients. After removal of the bacteria, the alga would grow only on agar plates in a high carbon dioxide atmosphere. Intermittent illumination, equilibrium centrifugation, and differential centrifugation were tested as techniques for obtaining synchronized cultures of the alga. Daughter cells selected by differential centrifugation in Ficoll density gradients yielded the best synchrony, and a cross-wall index of 85% was observed during the period of cell division.

Agar↗

The consistency of ameba cytoplasm and its bearing on the mechanism of ameboid movement. II. The effects of centrifugal acceleration observed in the centrifuge microscope.

Three species of common, free-living amebae, Amoeba proteus, Amoeba dubia, and Chaos chaos were directly observed and photographed while exposed to a range of centrifugal accelerations in two types of centrifuge microscopes. Cytoplasmic inclusions in all three species are displaced discontinuously (at a variable velocity) in apparently all parts of the cell, suggesting non-Newtonian behavior and/or heterogeneous consistency. The ectoplasm of all species shows the highest yield point of any region in the cell; the posterior ectoplasm is less rigid than that in the anterior part of the cell. The axial part of the endoplasm shows evidence of structure (a sharp viscosity transition if not a true yield point) by its: (a) resistance to the displacement of particles carried in that region of the cell, (b) hindrance to the passage through the cell of inclusions displaced from other regions, and its (c) support without visible back-slip of inclusion being resuspended in the axial endoplasm in a centripetal direction at accelerations as high as 170 g. At this acceleration, each crystal "weighs" the equivalent reduced weight of seven times its volume in gold at 1 g. The only regions of the normal, moving cell which show clear evidence of low apparent viscosity are the "shear zone" (see Fig. 8) and the "recruitment zone." Possible reasons for low apparent viscosity in these regions are discussed. A new scheme of ameba "structure" is presented on the basis of the combined results of velocity profile analysis and the present centrifugation study.

Acceleration↗

Counterflow centrifugation apheresis for the collection of autologous peripheral blood stem cells from patients with malignancies: a comparison with a standard centrifugation apheresis procedure.

Two apheresis methods used to collect hematopoietic stem cells from peripheral blood were compared in eight patients with a variety of malignancies. The standard lymphocyte collection method was alternated with the counterflow centrifugation or lymphocyte surge protocol. The number of clonogenic cells (CFU-GM and BFU-E), the red cell volume, and the number of mononuclear cells in each collection were assessed as well as the changes in circulating leukocytes, platelets, and blood hemoglobin produced by each apheresis procedure. There was no statistically significant difference found in the number of clonogenic cells collected with either method, but the number of mononuclear cells collected with the standard procedure was significantly higher (P = 0.001). The red cell volume collected with the standard procedure was significantly higher, (P = 0.0001), but corrected for the number of mononuclear cells the difference was not significant. The counterflow centrifugation apheresis produced significantly less thrombocytopenia (P = 0.005). The counterflow centrifugation apheresis procedure used collected fewer mononuclear cells than the standard procedure, however, with less red cell contamination but a comparable number of CFU-GM and BFU-E in four hour apheresis procedures. Each collection method resulted in a comparable amount of anaemia and leukopenia but the lymphocyte surge method produced less thrombocytopenia following the collection.

Adult↗

Centrifugal elutriation (counterstreaming centrifugation) of cells.

Lindahl first described the separation of cells by velocity sedimentation utilizing a special technique (counterstreaming centrifugation) that was later modified slightly and renamed centrifugal elutriation. Centrifugal elutriation has been applied, with variable degrees of success, to the separation of hemopoietic cells, mouse tumor cells, testicular cells, and a variety of other specialized cells as well as cells in particular phases of the cell cycle. The capacity of the elutriator to separate large numbers of cells is its chief advantage. The purities of the separated cells have not been compared with the purities of cells separated by other methods in most cases; such comparisons would permit more sophisticated comparison of elutriation with other techniques for velocity sedimentation.

Animals↗

Large scale isolation of human blood monocytes by continuous flow centrifugation leukapheresis and counterflow centrifugation elutriation for adoptive cellular immunotherapy in cancer patients.

The increasing interest in mononuclear phagocytes for adoptive cellular immunotherapy (ACI) trials in cancer patients led us to define a procedural approach to harvest reproducibly highly purified single-cell suspensions of large numbers of functional human circulating blood monocytes (Mo). A semiclosed counterflow centrifugal elutriation (CCE) system has been developed, using a new large capacity Beckman JE 5.0 rotor with one interchangeable 40 ml or 5 ml separation chamber, to purify Mo from mononuclear cell (MNC) concentrates of healthy donors and cancer patients obtained by continuous flow centrifugation leukapheresis (CFCL). This method does not require a Ficoll density gradient centrifugation step. A total of 115 leukapheresis procedures were carried out in 35 patients and in 30 healthy donors by either Cobe 2997 or Cobe Spectra, with a similar efficiency in MNC apheresis. The average yield per leukapheresis procedure was 5.6 x 10(9) MNC of purity 90-100% (25-45% Mo, 40-65% lymphocytes). The average yields per elutriation procedure (R/O fraction) were 1.1 x 10(9) cells (purity 93% Mo) using the 5 ml separation chamber, and 1.5 x 10(9) cells (purity 91%) using the 40 ml separation chamber, with a respective recovery of 82 +/- 7% and 78 +/- 8% Mo. In vitro analysis of the viability and function of the purified Mo shows that neither morphological integrity nor physiological activity was compromised by this two-step isolation procedure, which additionally provides highly purified human Mo suspensions, in a quantity suitable for ACl of cancer patients.

Cell Separation↗

A straight path centrifugal blood pump concept in the Capiox centrifugal pump.

This article describes comparative studies of a newly developed "straight path" centrifugal pump (Capiox centrifugal pump) targeted for open-heart surgery and circulatory support. A unique straight path design of the rotor was very effective in reducing the pump's rotational speed and prime volume. This pump was evaluated for hydraulics, hemolysis, depriming characteristics, cavitation, and heat generation. Two commercially available centrifugal pumps, the Biomedicus cone-type pump and the Sarns 3M impeller-type pump, were used as controls. The new pump required the lowest pump speed to produce the same flow rates under the same pressure loads and demonstrated the lowest hemolysis and the lowest temperature rise with the outlet clamped. The air volume required to deprime the new pump was one-third to one-half that for the other pumps, and no sign of cavitation was observed even if a small amount of air was introduced to the pump inlet under a negative pressure of 200 mm Hg.

Animals↗