Cell Washout Research Articles

Antibacterial peritoneal defence in automated peritoneal dialysis: Advantages of tidal over continuous cyclic peritoneal dialysis?

In tidal peritoneal dialysis (TPD) only a part of the infused dialysate is drained with each exchange, leaving a residual volume on top of which fresh fluid is cycled. As the persistent presence of a buffered intraperitoneal reserve volume might favour peritoneal macrophage (PMO) function, PMO obtained from eight patients during a 3-h continuous cyclic peritoneal dialysis (CCPD) or TPD session were studied in a randomized cross-over trial. PMO were studied for uptake of E. coli (complement-dependent) and S. epidermidis (antibody-dependent), as well as for their killing capacity and peak chemiluminescence response. In addition, dialysate was sampled during both treatment sessions and studied for pH, osmolality, and effect on the viability of donor phagocytes and mesothelial cells. TPD-derived PMO were significantly better able to phagocytose E. coli than CCPD-PMO (48 +/- 8 versus 33 +/- 6% uptake, P < 0.05), whereas the other tested functional capacities revealed no significant difference between TPD- and CCPD-PMO. During TPD dialysate pH ranged from 6 to 7 as compared to a pH range from 5 to 7 in CCPD. The presence of a residual dialysate volume resulted in less wash-out of cells and opsonins early in the treatment, and to some extent blunted the noxious effects of fresh dialysis solutions. Overall, however, tidal PD appeared to have no advantage over CCPD regarding preservation of peritoneal defences.

Hydrodynamic influences on interannual chlorophyll variability in an estuary: Upper San Francisco Bay-Delta (California, U.S.A.)

Diversion of estuarine water for human use, in conjunction with river inflow, is the main underlying cause of interannual chlorophyll variability in the landward portion of the San Francisco Bay-Delta estuary, i.e. Suisun Bay and the Sacramento-San Joaquin Delta. The principal feature of year-to-year change is high chlorophyll variability within the specific conductance range of 2–10 mS cm−1 (salinity 1–6), the approximate boundaries of an estuarine turbidity maximum (ETM). This variability stems from two separate hydrodynamic forces: (1) the strength of flow into the ETM, which affects primary productivity, losses to benthic macroinvertebrates, washout of phytoplankton cells, and chlorophyll loading; and (2) export of water from the estuary for human use, which independently affects the loading of chlorophyll from upstream sources into the ETM. In addition to the evidence for (1) presented in previous, largely descriptive studies, the simultaneous existence of both pathways is supported here by statistical models and material budgets. The source of phytoplankton organic matter from upstream was investigated further. Estimates were made for primary productivity levels in the Delta. These estimates are consistent with a mass balance for chlorophyll that suggests the Delta usually augments the flow of phytoplankton biomass from the rivers. If water were diverted from upstream of the Delta, rather than from within the Delta, the mass balance implies that both chlorophyll outflow from the Delta into the Bay, as well as primary productivity within the Delta, would increase. The primary productivity estimates also indicate that, on average, most of the phytoplankton emerging from the Delta is actually produced within the Delta, not upstream in the rivers; furthermore, most of the Delta primary productivity is respired or consumed within the Delta. A second, smaller mode of variability is as a result of phytoplankton blooms in the San Joaquin River, which were quite large and variable prior to 1978 but have been relatively small since that time. The underlying processes have not been identified in detail, but are suspected to be hydrodynamic in nature.

Mammalian cell retention in a spinfilter perfusion bioreactor

A spinning cylindrical filter is often used to retain mammalian cells in a continuous perfusion bioreactor. This device, known as a spinfilter, has typically been with pore size smaller than the cell particles (single cells or aggregates) in order to achieve cell separation. For single cells in suspension, such an operation cannot be sustained over a long period of time because of clogging of the filter surface. Recently, screens with openings larger than the average cell size have been used to reduce the incidence of clogging. In this article, we have investigated how the screen size affects cell retention. We also showed why it is necessary to optimize the rotational speed of the spinfilter in order to achieve cell retention and reduce screen clogging. Effects of bulk mixing and perfusion rate on screen fouling cell retention, and cell washout were also investigated.

Operational range for a continuous stirred-tank bioreactor degrading an inhibitory substrate

An analysis of various models of a continuous stirred-tank bioreactor (CSTB) showed that those based on the specific uptake rate of substrate described the dynamics of the CSTB satisfactorily for controlling the parameters after a disturbance so that all substrate introduced into the bioreactor was utilized. The maximum values of dilution rate D max and/or substrate concentration S 0 max in the feed after a step increase in D and/or S 0 respectively which could be achieved without causing cell washout were measured and compared with those calculated using a “short-cut” method. It was found that the calculated values of D max and S 0 max were lower by approximately 20% than the experimental results. Thus the values of D max and S 0 max predicted by the short-cut method were not substantial underestimates and can be considered safe for the operation of a CSTB utilizing inhibitory substrates.

Evaluation of production of recombinant human interleukin‐2 in fluidized bed bioreactor

The production of recombinant human interleukin-2 in a fluidized bed bioreactor containing porous glass carriers is described. Cultivations were carried out with different medium formulations over 80 days. Maximal cell densities and product yield could be maintained even when protein free medium was perfused, with less than 10% cell washout. Due to this effective immobilization of the cells in the reactor, continuous operation was easy to perform. Final cell densities on the order of 3.8 x 10(8) mL(-1) intrasphere volume were reached while the interleukin-2 production rate was 0.75 mg L(-1) d(-1). The production rate showed a maximum of a 1.9 fold decrease compared with a homogeneous stirred bubble-free aerated system. This result was in contrast to that achieved with hybridoma cell lines, where better performance was obtained with the fluidized bed bioreactor. The situation may reflect the problems caused by the dense cell culture with adherent cells, as previously shown in a hollow-fiber bioreactor with the same cell line.

Palytoxin induces an increase in the cation conductance of red cells.

Palytoxin (PTX), isolated from the marine soft coral Palythoa tuberculosa, increases the cation conductance of human red cell membranes. In the presence of 10(-10) M PTX and 10(-5) M DIDS, the membrane potential approximates the equilibrium potential for Na+ or K+ rather than Cl-. Even in the absence of DIDS, the Na+ and K+ conductances were greater than the Cl- conductance. The selectivity of the PTX-induced cation conductance is K+ greater than Rb+ greater than Cs+ greater than Na+ greater than Li+ much greater than choline+ greater than TEA+ much greater than Mg2+. Measurements of K+ efflux revealed two apparent sites for activation by PTX, one with a Kal of 0.05 nM and a maximum flux, nu max1, of 1.4 mol/liter of cells per h and another with a Ka2 of 98 nM and a nu max2 of 24 mol/liter of cells per h. These effects of PTX are completely blocked by external ouabain (300 microM) and prevented by internal vanadate (100 microM). When the PTX channels are open, the Na,K pumps do not catalyze ATP hydrolysis. Upon thorough washout of cells exposed to about five molecules of PTX/pump, the Na,K pump of these cells operates normally. Blockage of the positively charged NH2 terminus of PTX with a p-bromobenzoyl group reduces the potency of the compound to induce Na and K fluxes by at least a factor of 100, and to compete with the binding of [3H]ouabain by at least a factor of 10. These data are consistent with the conclusion that PTX binds reversibly to the Na,K pumps in the red cell membrane and opens a (10-pS) channel equally permeable to Na and K at or near each pump site.

Performance characterization of a model bioreactor for the biodegradation of trichloroethylene by Pseudomonas cepacia G4

Pseudomonas cepacia G4 grown in chemostats with phenol demonstrated constant specific degradation rates for both phenol and trichloroethylene (TCE) over a range of dilution rates. Washout of cells from chemostats was evident at a dilution rate of 0.2 h-1 at 28 degrees C. Increased phenol concentrations in the nutrient feed led to increased biomass production with constant specific degradation rates for both phenol and TCE. The addition of lactate to the phenol feed led to increased biomass production but lowered specific phenol and TCE degradation rates. The maximum potential for TCE degradation was about 1.1 g per day per g of cell protein. Cell growth and degradation kinetic parameters were used in the design of a recirculating bioreactor for TCE degradation. In this reactor, the total amount of TCE degraded increased as either reaction time or biomass was increased. TCE degradation was observed up to 300 microM TCE with no significant decreases in rates. On the average, this reactor was able to degrade 0.7 g of TCE per day per g of cell protein. These results demonstrate the feasibility of TCE bioremediation through the use of bioreactors.

Enhancement of red cell washout from blood clots by alteration of gel pore size and red cell flexibility

Plasma clots formed in the presence of erythrocytes have pores large enough to allow significant perfusion induced washout of red cells. Erythrocyte washout is a function of gel pore size and erythrocyte flexibility. This study evaluated the possibility of increasing cell washout by enhancing gel pore size and/or erythrocyte flexibility. Addition of increasing concentrations of Dextran 40 resulted in gels composed of large fibers with large intervening pores. Gel fiber mass-to-length ratios increased from 2.6 to 8.1 x 10(13) Da/cm as Dextran 40 concentration increased from 0 to 12 mg/ml. Over the same dextran concentration range, perfusion measurements revealed an increase in gel pore diameter from 1.4 to 3.5 x 10(-4) cm and an increase in erythrocyte washout from 8.2 to 30.4% during a 1.5 gel volume exchange. Pentoxifylline was added to blood to enhance erythrocyte flexibility. Whereas turbidity measurements indicated minimal direct impact of pentoxifylline (50 micrograms/ml) on fibrin assembly or structure, perfusion measurements indicated an increase in erythrocyte washout from 8.1 to 13.4% after a 1.5 gel volume exchange. Efforts to evaluate pentoxifylline effects at higher concentrations were hampered by decreased adhesion of the gel to the sides of the perfusion chamber. These results indicate that it may be possible to enhance erythrocyte washout and thereby increase diffusion and perfusion of materials through in situ clots.

In situ specific loss and growth rates of purple sulfur bacteria in Lake Cisó

Growth rates and population dynamics of phototrophic bacteria in Lak Cisó were analysed by measuring bacterial abundances and determining specific rates of growth and loss. Net growth rates were calculated from actual changes in biomass assuming exponential growth. Values ranged between −0.072 and 0.037 per day (d −1) for Chromatium, and between −0.043 and 0.022 d −1 for Amoebobacter. Exponential loss rates through sedimentation, decomposition and washout were determined independently. Values ranged between 0 and 0.025 d −1 in the case of Chromatium and between 0 and 0.015 d −1 in the case of Amoebobacter. Finally, gross growth rates were calculated by adding net growth to losses. Maximal values were 0.063 d −1 for Chromatium and 0.037 d −1 for Amoebobacter. In the case of Chromatium, population growth rates were found to be correlated with the amount of light available per unit of growing biomass. It was concluded that growth of phototrophic bacteria in Lake Cisó was limited by light availability. Altogether, purple sulfur bacteria seemed to maintain a very large biomass with very slow growth, thanks to very slow losses during stratification. During holomixis the situation was more dynamic. Washout of cells and disappearance of algal cells allowed more light to reach the bacteria. Therefore, high growth rates were found towards the end of the winter. A similar pattern repeated itself from year to year. These are the first estimates of in situ growth rates for populations of phototrophic bacteria.

In situ specific loss and growth rates of purple sulfur bacteria in Lake Cisó

Growth rates and population dynamics of phototrophic bacteria in Lak Ciso were analysed by measuring bacterial abundances and determining specific rates of growth and loss. Net growth rates were calculated from actual changes in biomass assuming exponential growth. Values ranged between −0.072 and 0.037 per day (d−1) for Chromatium, and between −0.043 and 0.022 d−1 for Amoebobacter. Exponential loss rates through sedimentation, decomposition and washout were determined independently. Values ranged between 0 and 0.025 d−1 in the case of Chromatium and between 0 and 0.015 d−1 in the case of Amoebobacter. Finally, gross growth rates were calculated by adding net growth to losses. Maximal values were 0.063 d−1 for Chromatium and 0.037 d−1 for Amoebobacter. In the case of Chromatium, population growth rates were found to be correlated with the amount of light available per unit of growing biomass. It was concluded that growth of phototrophic bacteria in Lake Ciso was limited by light availability. Altogether, purple sulfur bacteria seemed to maintain a very large biomass with very slow growth, thanks to very slow losses during stratification. During holomixis the situation was more dynamic. Washout of cells and disappearance of algal cells allowed more light to reach the bacteria. Therefore, high growth rates were found towards the end of the winter. A similar pattern repeated itself from year to year. These are the first estimates of in situ growth rates for populations of phototrophic bacteria.

Analysis of continuous cultures of recombinant methylotrophs

Static and dynamic characteristics of continuous cultures of recombinant methylotrophs, which are designed to improve the selectivity of plasmid-bearing cells and the plasmid stability, are investigated in detail. Operational regions in which coexistence (survival of plasmid-bearing and plasmid-free cells) operation is feasible have been identified in the entire space of kinetic parameters and operating variables. The stability characteristics of each steady state are examined. The existence of oscillatory states around the coexistence steady state is investigated using the dynamic (Hopf) bifurcation analysis. For proper startup of the continuous culture operation, it is critical to identify the sets of initial conditions, if any, which lead to transients that ultimately result in washout of plasmid-bearing cells and avoid such conditions. For the numerical illustrations presented, the coexistence steady state happens to be locally stable over much of its region of existence, particular for the operating conditions corresponding to maximum productivity.

Continuous Production of Lactic Acid from Whey Perméate by Free and Calcium Alginate Entrapped Lactobacillus helveticus

Lactobacillus helveticus strain milano was used for the continuous fermentation of lactic acid in cheese whey-yeast extract permeate medium. The best productivity of lactic acid was with the free cell system, which was 9.7g/L per h at a dilution rate of .352h−1. Under such conditions, lactose conversion was 87.5%, based on the lactose concentration of 37.4g/L in feed. Under high dilution rates, the cells were elongated to several times their normal size, resulting in wall growth. The cell growth on the fermentor wall caused system instability; however, it prevented cell wash-out under high dilution rates. The packed bed column system using Ca alginate entrapped cells is not suitable for practical application. Nonuniform pH control, plugging of the column by leaked cells, and decalcification of Ca alginate beads were major drawbacks of the packed bed system.

CONTROL OF ALGAL BIOMASS BY INFLOW NITROGEN

ABSTRACT Dilution water has entered Moses Lake since 1977, usually in amounts exceeding 100 106m3, which has amounted to exchange rates of around 10 percent day−1in 8 percent of the lake volume nearest the input. The principal cause for the marked reduction in algal biomass and increased transparency, prior to the Mount St. Helen's ashfall, was the dilution of inflow concentrations of NO3. Average summer chlorophyll a was closely correlated with flow-weighted mean inflow NO3. Increased NO3–N:SRP in undiluted inflow water following the ashfall caused P limit growth in the lake. Increased dilution water input in 1982, compared to the 2 post-ash years, reduced inflow NO3to biomass-controlling levels in spite of continued high ratios of soluble N:P in the lake. Dilution water was pumped to previously undiluted Pelican Horn in 1982 at a rate of 1.4 m3s−1during July through August. Algal biomass was reduced largely because of cell washout, because undiluted water was pumped after July, but chl a actually increa...

Characterization of Nitrosomonas europaea immobilized in calcium alginate

Nitrosomonas europaea cells have been immobilized in calcium alginate and the resulting preparation was used as a biocatalyst for the oxidation of NH + 4 to NO − 2. Characterization of this immobilized biocatalyst was done according to the guidelines recommended by the Working Party on Immobilized Biocatalysts of the European Federation of Biotechnology. The most important indications obtained from the results are: (a) at low concentrations of substrate, either ammonium ions or oxygen, diffusion limitation will play a role; (b) inhibition by nitrite ions accumulating in the support is not rapidly controlling the efficiency of the immobilized cells; (c) accumulation of hydrogen ions is a rate-limiting factor, especially in unbuffered solutions; (d) the activity of immobilized N. europaea can increase as a result of growth in the support under conditions which would cause washout of free cells. This last result shows the potential of immobilized N. europaea for nitrification of wastewater. The development of a system applying a cheaper and more stable support is, however, a prerequisite for this application.

Small vessel and total coronary blood volume during intracoronary adenosine.

The effect of a maximally dilating dose of intracoronary adenosine on total (CBV) and small-vessel blood volume (MSVBV, an index of open-capillary density), hematocrit (MSVHct), and related parameters of O2 supply-demand ratio was examined in left ventricular myocardium of anesthetized open-chest dogs. CBV was measured from washout of 51Cr-labeled red blood cells (RBC) and MSVBV and MSVHct from contents of 51Cr-RBC and plasma label, either 131I-serum albumin or 59Fe-siderophilin, in samples of myocardium and blood. Coronary blood flow (CBF) was measured by electromagnetic flowmeter. Myocardial oxygen consumption (MVO2) was computed with the Fick equation. Myocardial oxygen tension (MPO2) was measured with bare-tipped platinum electrodes. Adenosine raised CBV 75%, CBF, 574%, and MPO2 122%, but did not affect significantly MSVBV, MSVHct, or MVO2. These results indicate that infusion of adenosine into a coronary artery perfused at constant pressure causes relaxation of smooth muscle of arteriolar resistance vessels and of other vessels larger than 100 micrometers diam, but not that of the precapillary sphincters. This may be explained by the opposing action of increased MPO2 on the sphincters when flow increases. MSVHct was consistently much less than large-vessel Hct. This warrants combined use of red blood cell and plasma labels for accurate measurements of MSVBV.

THE DILUTION/FLUSHING TECHNIQUE IN LAKE RESTORATION1

ABSTRACT: Dilution/flushing has been documented as an effective restoration technique to restore eutrophic Moses and Green Lakes in Washington State. The dilution water added to both lakes was low in nitrogen and phosphorus content relative to the lake or normal input water. Consequently, lake nutrient content dropped predictably. Dilution or flushing rates were about ten times normal during the spring‐summer periods in Moses Lake and three times normal on an annual basis in Green Lake. Improvement in quality (nutrients, algae, and transparency) was on the order of 50 percent in Moses Lake and even greater in Green Lake. The facilities for supplying dilution water were largely in place for the cited lakes; thus, costs for water transport were minimal. Available facilities, and therefore, costs, for water transport would usually vary greatly, however. Achieving maximum benefit from the technique may be more limited by availability of low nutrient water rather than facilities costs. Quality improvement may occur from physical effects of algal cell washout and water column instability if only high nutrient water is available.

Experimental assessment of prostacyclin in the harvesting of kidneys for transplantation.

Prostacyclin is a potent vasodilator and the most potent inhibitor of platelet aggregation yet discovered. A possible role for prostacyclin in the harvesting of kidneys in experimental canine transplantation has been investigated. Prostacyclin gives a 52.6% increase in renal blood flow prenephrectomy despite inducing a 23% drop in mean arterial blood pressure, without changing the rate of urine output. There is an associated drop in renal vascular resistance of 50.2% and an increase in renal blood volume of 25%. The value of heparin treatment before donor nephrectomy is confirmed as it improves the flow of flushing solution through the kidney after nephrectomy and improves red blood cell washout from the kidney. Prostacyclin and heparin together improve these parameters further. After a warm ischemic period of 45 min, autotransplanted kidneys in dogs pretreated with prostacyclin had normal renal function within 48 hr as judged by the serum creatinine whereas untreated dogs had permanent impairment of function.

Development of a nonthrombogenic collagenous blood-prosthetic interface.

Investigations to develop an implantable assist pump for prolonged circulatory support have been impeded by accumulation of friable thrombus on the prosthetic interface, with subsequent embolization. To circumvent this problem, the textured, fibril surface of a polyurethane pump chamber (mat thickness 430 microns) was inoculated with cultured bovine fetal fibroblasts (labelled with thymidine-14C) prior to animal implantation. The pneumatically actuated device (stroke volume 75 ml), maintained a pulsatile blood flow throughout each study. In 20 calf experiments, extending up to 335 days, 30 X 10(6) fibroblasts (in 50 ml media) derived from a single Holstein fetus were distributed on the urethane surface (360 +/- 50 cells/mm2) by rotation of a sealed device for three hours (12 revolutions/hour). Following connection to the circulation, cell washout was minimal. Resultant biologic linings, examined after animal sacrifice, were densely adherent to the underlying polymer matrix, and varied in thickness from 250 micron-1.5 mm. Microscopically, fibroblasts were identified from the surface to base, accompanied by numerous collagen bundles and abundant ground substance. Amino acid analysis in 10/20 pumps implanted for 31--335 days, revealed 50 +/- 5 Hydroxyproline residues/1000 residues (50% collagen) and scant elastin. Donor fibroblasts were identified by radioautography and karyotyping. Lack of immunologic response in 12 Hereford pump recipients as confirmed by serial fibroblast cytotoxicity assays. In conclusion, an induced collagenous-blood interface permitted prolonged mechanical circulatory support in animals without thromboembolic complications.

Compartmental analysis of circulation of erythrocytes through the rat spleen.

Control, polycythemic, and anemic spleens of Sprague-Dawley rats surgically removed under pentobarbital sodium anesthesia (0.06 mg/g ip) were perfused in vitro by methods similar to those reported by Song and Groom (Am. J. Physiol. 220: 779-784, 1971). Red Blood cell (RBC) washout curves obtained for each group could be described adequately by a series of three exponentials--fast, intermediate, and slow--representing functional compartments in the spleens. Best-fit estimates of the parameters were used to characterize the flow distributions, cellular capacities, and t1/2 for washout of these components. It is thought that the fast compartment represents flow through intrasplenic vessels (including shunts) that bypass the red pulp, the intermediate compartment represents RBCs that flow through less impeded passageways in the red pulp, and the slow compartment consists of more tortuous, convoluted pathways through the red pulp. During polycythemia and anemia, inflow to the intermediate component was decreased, which suggested decreased filtration. In the slow compartment, the shutdown during polycythemia suggested RBC storage whereas during anemia control levels of inflow but increased t1/2 for washout suggested sequestration or erythropoiesis.

Steady-State Growth and Chemical Composition of the Marine Chlorophyte Dunaliella tertiolecta in Nitrogen-Limited Continuous Cultures

The marine chlorophyte Dunaliella tertiolecta was grown in continuous cultures under NH(4)-N, NO(2)-N, NO(3)-N, and urea-N limitations. The effect of the nitrogen cell quota (Q(n)) on the steady-state growth rate (mu) was the same regardless of the N source. The relationship between mu and Q(n) was well described by the Droop equation, but only up to the true maximum growth rate ;mu (= cell washout rate). The ratio between the minimum cell quota (k(Q)) and the maximum cell quota (Q(m)) was 0.19. Hence, there is no substitute for determining ;mu experimentally. That there was no difference in growth response to different N sources suggests that no internal pooling of inorganic nitrogen occurred. Both the carbon (Q(c)) and phosphorus (Q(p)) cell quotas under N limitation increased with increasing mu in a threshold fashion: virtually no change in either cell quota up to approximately 0.8 ;mu, followed by a rapid and large increase up to ;mu. In addition, in the region of low mu, there was an increase in Q(p) with a decreasing medium N/P ratio of between 15 and 5 (by atoms). The results generally indicate the physiological limits in cellular constituency under N limitation. The usefulness of this information, however, in describing the response of natural populations of marine phytoplankton to transient nutrient exposures on the temporal and spatial microscales that most likely exist is of limited value.