A new method to study shape recovery of red blood cells using multiple optical trapping.

In this new method for studying the shape recovery of deformed red blood cells, three optical traps ("optical tweezers") induce a parachute-shaped red cell deformation, which is comparable to the deformation in small capillaries. The shape recovery is recorded, and a relaxation time is obtained for each individual red blood cell. The sensitivity of this technique for the detection of differences in relaxation times is demonstrated on subpopulations of density-separated red blood cells: "young" cells have shorter (162 ms) and "old" cells have longer (353 ms) relaxation times compared with the total population (271 ms). The relaxation time is remarkably shorter (114 ms) when the plasma surrounding the cells is replaced by a phosphate-buffered saline solution. The main advantages of this technique are the relatively short measuring and preparation time and the physiological type of deformation and shape recovery in which all relevant cell properties play a role. Therefore, especially when automated further, the technique may be a powerful tool for the study of (sub)populations of pathological red blood 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.     

Glycolysis of red cells suspended in solutions of impermeable solutes. Intracellular pH and glycolysis.

The glycolytic rate human red cells suspended in a sucrose medium of low or physiological pH was higher than that of the cells suspended in Ringer's medium of the same. pH. The medium pHP-glycolytic rate curve of red cells suspended in soucrose media shifted to the acidic side by about one unit compared with that of cells suspended in Ringer's medium. Similarly, the pattern of glycolytic intermediates in red cells suspended in a sucrose medium resembled that in cells suspended in Ringer's solution of about one unit higher pH. These phenomena could be ascribed to the change of intracellular pH, which was measured by the 5,5'-dimethyl-oxazolidine-2,4-dione method. A similar stimulation of glycolysis was observed when sodium citrate was added to red cells suspended in Ringer's solution at constant pH. These observations indicate that membrane-impermeable non-electrolytes or anions stimulate glycolysis of red cells by elevation ofthe intracellular pH. Red cell glycolysis is influenced mainly by the intracellular pH rather than by the pH of the suspending medium.     

Digital image analysis of growth and starvation responses of a surface-colonizing Acinetobacter sp.

Surface growth of an Acinetobacter sp. cultivated under several nutrient regimens was examined by using continuous-flow slide culture, phase-contrast microscopy, scanning confocal laser microscopy, and computer image analysis. Irrigation of attached coccoid stationary-phase Acinetobacter sp. cells with high-nutrient medium resulted in a transition from coccoid to bacillar morphology. Digital image analysis revealed that this transition was biphasic. During phase I, both the length and the width of cells increased. In contrast, cell width remained constant during phase II, while both cell length and cell area increased at a rate greater than in phase I. Cells were capable of growth and division without morphological transition when irrigated with a low-nutrient medium. Rod-shaped cells reverted to cocci by reduction-division when irrigated with starvation medium. This resulted in conservation of cell area (biomass) with an increase in cell number. In addition, the changes in cell morphology were accompanied by changes in the stability of cell attachment. During phase I, coccoid cells remained firmly attached. Following transition in high-nutrient medium, bacillar cells displayed detachment, transient attachment, and drifting behaviors, resulting in a spreading colonization pattern. In contrast, cells irrigated with a low-nutrient medium remained firmly attached to the surface and eventually formed tightly packed microcolonies. It is hypothesized that the coccoid and bacillar Acinetobacter sp. morphotypes and associated behavior represent specialized physiological adaptations for attachment and colonization in low-nutrient systems (coccoid morphotype) or dispersion under high-nutrient conditions (bacillar morphotype).     

Tank-tread frequency of the red cell membrane: dependence on the viscosity of the suspending medium

Single human red cells were suspended in media with viscosities ranging from 12.9 to 109 mPa s and subjected to shear flow ranging from 1/s to 290/s in a rheoscope. This is a transparent cone-plate chamber adapted to a microscope. The motion of the membrane around red cells oriented in a steady-state fashion in the shear field (tank-tread motion) was videotaped. The projected length and width of the cells as well as the frequency of tank-tread motion were measured. One-thousand eight-hundred seventy-three cells of three blood donors were evaluated. The frequency increased with the mean shear rate in an almost linear fashion. The slope of this dependence increased weakly with the viscosity of the suspending medium. No correlation was found between the frequency and four morphological red cell parameters: the projected length and width of the cells as well as the ratio and the square root of the product of these quantities. The energy dissipation within the red cell membrane was estimated based on the measured parameters and compared to the energy dissipation in the undisturbed shear flow. At constant mean shear rate the rise of the energy dissipation with viscosity is slower whereas at constant viscosity the rise with the shear rate is steeper than in the undisturbed shear flow. A fit of the data collected in this work to a theoretical red cell model might allow one to determine intrinsic mechanical constants in the low deformation regime.     

Effect of pH on the regulatory characteristics of energy metabolism in human erythrocytes

The glucose consumption rate versus ATP content in human red cells (regulatory patterns of glycolysis) and ATP concentration versus glucose uptake rate in red cell suspension (regulatory patterns of total ATPases), when the rate of glucose uptake is constant and lower than the rate of glucose consumption at physiological conditions, were measured at different pH values. The shape of both types of kinetic curves was found to be dependent on the pH of the incubation medium but the same for the red cells taken from different donors. It is supposed that at alkaline pH, glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate kinase reactions become the rate-limiting steps of glycolysis instead of hexokinase and phosphofructokinase under physiological conditions.     

Factors affecting yield and survival of cells when suspensions are subjected to centrifugation

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.     

Studies on the mechanism of cell attachment to a substratum with serum in the medium: further evidence supporting a requirement for two biochemically distinct processes

Treatment of baby hamster kidney cells with cytochalasin B or omission of divalent cations from the culture medium are conditions resulting in an inhibition of cell attachment at rest; however, these conditions do not result in inhibition of cell attachment in a centrifugal field. In marked contrast, treatment of cells with trypsin or with tranquilizers such as fluphenazine results in an inhibition of cell attachment at rest or in a centrifugal field. The evidence is interpreted to indicate that cell adhesion involves at least two biochemical processes: formation of the adhesive bond per se (inhibited by tranquilizers or trypsin) and a mechanical process of cell-to-substratum contact and/or spreading (inhibited by cytochalasin B or omission of divalent cations from the medium).     

Effects of calcium and A23187 on deformability and volume of human red blood cells

Human red blood cells treated in vitro with Ca2+ plus A23187 in low K+ medium exhibited significantly decreased cell volume and deformability, the latter determined by ektacytometry. These effects of Ca2+ plus A23187 were prevented in the presence of high K+ medium. Increased K+ permeability mediated by increased intracellular Ca2+ (Gardos effect) was apparently responsible for decreased cell volume and deformability in low K+ medium. Although it is commonly accepted that Ca2+ accumulation and/or ATP depletion per se cause decreased red blood cell deformability, the present results demonstrate that acutely induced changes in red blood cell volume as promoted by Ca2+ are a more important determinant of red blood cell deformability.     

Rheological behaviors of bovine blood forming artificial rouleaux

A purpose of the present study is to make an artificial rouleau of bovine red blood cells which is not capable of rouleau formation under physiological condition. Rheological behaviors of bovine blood forming artificial rouleaux were examined. The modification of cell surface by enzyme trypsin induced rouleau formation, whereas the modification of cell surface by neuraminidase did not cause any aggregate formation. The drastic elevation of the fibrinogen content in bovine red blood cells suspension also brought about the formation of rouleau. The value of dynamic rigidity modulus G' of bovine red blood cells in saline solution containing high concentration of fibrinogen is somewhat smaller than that of trypsin treated bovine red blood cells in plasma. The value of G' of trypsin treated bovine red blood cells in plasma first increased to a maximum value and then decreased with the time. It is supposed that the removal of macro-molecules from the cell surface facilitates the mutual approach of cells and causes the formation of rouleau which seems to be the same as that of human and horse bloods.     

Changes in membrane properties of rat red blood cells induced by cadmium accumulating in the membrane fraction

When rat red blood cells were incubated in a cadmium (Cd)-free medium following 1-h pretreatment with 0.5 mM CdCl2, incorporated Cd was retained in the cell during 14-h incubation and progressively accumulated in the membrane fraction, especially in the cytoskeleton fraction. In parallel to this accumulation, red cell filterability decreased and echinocytic cells increased, although intracellular ATP was maintained at the control level. The echinocytic shape was maintained in ghosts and cytoskeletons prepared from the Cd-loaded cells. In addition, the association of bands 2.1, 3, 4.2, and 4.5 with cytoskeletons increased and dissociation of cytoskeletal networks at low ionic strength decreased as the incubation time increased. Pretreatment of red blood cells with Cd also induced a release of small vesicles. These vesicles contained hemoglobin but were depleted of spectrin and actin, showing a phospholipid composition similar to that of red cell ghosts. These results suggest that the organization of cell membranes, especially cytoskeletal networks, is altered by Cd accumulation in the cytoskeleton fraction, which results in acceleration of age-related changes of red blood cells such as shape change and decreased filterability.     

Red cell pH of lamprey (Lampetra fluviatilis) is actively regulated

Summary The red cell pH of lamprey (Lampetra fluviatilis) was measured using the DMO method (based on the passive distribution of the wead acid, DMO, across the red cell membrane). The measured red cell pH was higher than the pH of the incubation medium throughout the pH range (7.2–8.2) studied, and higher than the red cell pH calculated from the chloride distribution ratio. Treatment of cells with the metabolic inhibitors 2,4-dinitrophenol or KCN caused a drop in the red cell pH to values lower than the pH of incubation medium, and abolished the difference between the measured red cell pH and the pH calculated from the chloride distribution ratio. These data strongly suggest that the proton gradient across lamprey red cell membrane is actively maintained. Acid extrusion from lamprey red cells may require sodium, as indicated by the observation that when choline was substituted for sodium in the incubation medium, the intracellular pH decreased significantly.     

Cyclic loading on the red cell membrane in a shear flow: a possible cause of hemolysis

The fluid force acting on single human red cells in a high shear flow was analyzed. A two-dimensional elliptical microcapsule as a model of the deformed red cells was adopted to numerically calculate the distributions of the shear forces on both sides of the cell membrane. It is theoretically shown that the cell membrane undergoes an unsteady cyclic loading under the rotational motion around the interior. The mechanism leading to blood cell trauma is examined by repeatedly loading the continuously moving cell membrane.     

Na+ K+ pump and passive K+ transport in large and small red cell populations of anemic high and low K+ sheep

Reticulocytes, isolated by centrifugal elutriation from massively bled sheep and identified by cytometric techniques, were analyzed with respect to their cation transport properties. In sheep with genetically high K+ (HK) or low K+ (LK) red cells, two reticulocyte types were distinguished by conventional or fluorescence-staining techniques 5-6 days after hemorrhage: Large reticulocytes as part of a newly formed macrocytic (M) erythrocyte population, and small reticulocytes present among the adult red cell population (volume population III of normal sheep blood, Valet et al., 1978). Although cellular reticulin disappeared within a few days, the M-cell population persisted throughout weeks in the peripheral circulation permitting a transport study of in vivo maturation. At all times, M cells of LK sheep had lower K+ and higher Na+ contents than M cells of HK sheep. Regardless of the sheep genotypes, M cells apparently reduced their volume during their first days in circulation; however, throughout the observation period, they did not attain that characteristic for adult red cells. Both ouabain-sensitive K+ pump and ouabain-insensitive K+ leak fluxes were elevated in M cells of both HK and LK sheep. The increased K+ pump flux was mainly due to higher K+ pump turnover rather than to the modestly increased number of pumps as measured by [3H]ouabain binding. In contrast, small reticulocytes enriched from separated volume population III cells by a Percoll-density gradient exhibited transport parameters close to their prospective mature HK or LK red cells. The data support the concept that the M cells derived from emergency reticulocytes while the small reticulocytes represented precursors of normal red cell maturation. The Na+ and K+ composition found in M cells of HK and LK sheep, respectively, suggest development of the LK steady state at or prior to the reticulocyte state, a finding consistent with that of Lee and Kirk (1982) on low K+ dog red cells.     

Leukocyte kinetics in the microcirculation

The transport of leukocytes in the microcirculation is specific for the type, size, and the rheological and adhesive properties, the microanatomy of the host organ, and the hemodynamics. The adhesion to the endothelium is determined largely by the degree of activation via chemotactic factors. Leukocyte motion differs from that of red cells or platelets in several respects. When granulocytes enter into capillaries, they are deformed just like red cells. Under normal flow conditions, the time to deform at the entry to capillaries is typically 1,000 times larger than for the red cell, leading to temporary obstruction of the capillaries. After entry, granulocytes move with lower velocity than red cells which causes a cell train formation inside the capillary. At the venular side, the granulocyte is displaced from the center stream toward the endothelium by faster moving red cells. This process leads to systematic attachment of the granulocytes to the endothelium. At a reduced perfusion pressure or in the presence of locally elevated levels of chemotactic factors, the granulocytes may not be able to pass through the capillary network, which leads to microvascular obstruction. Organs with a narrow capillary network may thereby become filters for circulating granulocytes. This event is accompanied in many situations with damage to the host organ.     

An optical-manipulation technique for cells in physiological flows

We have developed a technique to manipulate human red blood cells (RBCs) in hydrodynamic flows. This method applies optical tweezers to trap and move microbead-attached RBCs in a liquid medium at various speeds, while it significantly minimizes laser heating and photon-induced stress for normal operation with laser-trapped cells. Computational fluid dynamics is applied to simulate flow-induced shear stress over the cell membrane and to correlate quantitatively the forces with the cell deformations. RBCs can be manipulated under physiological conditions by this approach, which may open an avenue to design principles for the next generation of cell sorting and delivery.     

Viscoelastic deformation response of red blood cells under conditions of oscillating centrifugal field

The red cell deformation under the conditions of oscillating centrifugal fields was studied. Experiments were carried out with a modified Cell-Elastometer operating in oscillating mode (0.02 to 0.30 Hz). Gravitational acceleration was sinusoidally modulated between 620 g and 2250 g. At low frequencies (below 0.08 Hz), native red cells followed the applied stress without delay. At 0.09 Hz and up, the cellular deformation was still periodical and included an additional perturbation due to intracellular movements. This perturbation was analysed and quantified. The influence of alterations on the erythrocyte membrane by diamide was analysed to verify the sensitivity of this method. On increasing the membrane stiffness with low concentrations of diamide, the response to oscillatory centrifugal stress was impaired characteristically in terms of amplitude deformation. Based on tangential and centrifugal accelerations, a physical model was developed that describes the basic observable changes on varying the oscillation frequency. From the data it can be concluded that viscoelastic properties of red cells can be analysed and quantified using oscillatory centrifugal accelerations. The described method can become a valid tool to differentiate between membrane alterations or intracellular viscous modifications.     

一种新的体外细胞牵张刺激装置

体外细胞机械刺激装置是研究细胞对机械牵张反应的一种研究设备。目前此类装置有多种,但是这些装置都存在一些不便之处。本文以商品化的Flexercell Strain Unit刺激装置为参照,设计一套离心力牵张装置。通过使贴壁细胞生长的培养板以一定的速度旋转,从而使心肌细胞受到固定大小的离心力的牵张作用。酶解分离3～5d SD大鼠心肌细胞,并分别给予12和24h机械刺激:传统的20%牵张刺激和180r/min的离心力刺激。以~3H-亮氨酸掺入量反映细胞的肥大指标,并测定牵张引起的血管紧张素Ⅱ的自分泌。同对照组相比,~3H-亮氨酸掺入在离心力牵张组显著升高[(1295.17±51.19)vs(1122.67± 51.63)in 12h;(1447.5±35.96)vs(1210.67±90.92)in 24h,P<0.05]。AngⅡ在离心力牵张组均比同期末牵张组均明显升高,12h为128%(P<0.05)和24h为139%(P<0.01)。经24h的牵张,培养液中乳酸脱氢酶活性在膜牵张组显著高于离心力牵张组[(14.5±8.7)U/Lvs (7.8±4.3)U/L,P<0.05]。新型改进的机械牵张装置能够有效刺激心肌细胞蛋白合成增加和AngⅡ的分泌增加,与FlexercellStrain Unit相比,离心力牵张装置对细胞的损害较轻微。     

EFFECT OF PROTEINS ON ELECTROPHORETIC MOBILITY AND SEDIMENTATION VELOCITY OF RED CELLS

The isoelectric point of normal red cells cannot be measured but is certainly lower than that of any plasma protein. Red cells are easily damaged so that they will adsorb proteins from low concentrations. Normal red cells do not adsorb protein even from concentrated solutions, as is evidenced by the finding that the ratio of the mobility of the cells to that of the proteins themselves is at least as high in concentrated casein, albumin, gelatin, or fibrinogen solutions as in dilute. The finding that the observed mobility of red cells is unchanged or only slightly decreased when bulk viscosity is increased by added protein is interpreted as indicating that the red cell surfaces are hydrated. The aggregating effect of certain proteins has been determined and is assumed to be due to their dehydrating effect on the cells. Some types of cells, as beef, are not aggregated, presumably because they are resistant to this dehydrating effect. The difference in the behavior of different types of red cells demonstrates the importance of the nature of the cell as well as of the medium in determining the rate of aggregation and therefore of sedimentation.     

Anin vitro study on the cytotoxicity of chlorhexidine digluconate to human gingival cells

Chlorhexidine digluconate is the active ingredient in mouthrinses used to prevent dental plaque and gingivitis. Thein vitro cytotoxicity of chlorhexidine was evaluated with the Smulow-Glickman (S-G) gingival epithelial cell line. The potency of chlorhexidine was dependent on the length of exposure and composition of the exposure medium. The midpoint cytotoxicity values for 1-, 24-, and 72-h exposures were 0.106, 0.011, and 0.0045 mmol/L, respectively. S-G cells exposed for 2 h to chlorhexidine and then maintained for 48 h in chlorhexidine-free medium were unable to recover from the initial insult. The adverse effects of chlorhexidine on the plasma membrane were suggested by the leakage of lactic acid dehydrogenase from chlorhexidine-treated S-G cells and by the increased permeability of chlorhexidine-treated liposomes to Ca²⁺. The toxicity of a 24-h exposure to chlorhexidine to the S-G cells was progressively lessened as the content of fetal bovine serum (FBS) in the exposure medium was increased from 2% to 8%. The potency of a 1-h exposure to chlorhexidine was reduced in medium amended with albumin, lecithin, and heat-killedEscherichia coli. These reductions in toxicity were presumably due to the binding of the cat onic chlorhexidine to the negatively charged chemical moieties of the components of FBS and of albumin and lecithin and of sites on the surfaces of bacteria. Combinations of chlorhexidine and carbamide peroxide were additive in their cytotoxicities.Abbreviations ANOVA analysis of variance - [Ca²⁺]i calcium concentration in internal medium of liposomes - DMEM Dulbecco's modified Eagle medium - EDTA ethylenediamine tetraacetic acid - FBS fetal bovine serum - Hepes N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid - HGF-1 human gingival fibroblast cell line - HSD honestly significant differences - KB cell line derived from a human epidermoid carcinoma in the mouth - LDH lactic acid dehydrogenase - NADH nicotinamide adenine dinucleotide, reduced form - NR neutral red - NR₅₀ concentration inhibiting neutral red uptake by 50% - PBS phosphate-buffered saline - SEM standard error of the mean - S-G Smulow-Glickman human gingival epithelial cell line