Perfusion Mode Research Articles

Mechanical assistance of the left ventricle: Acute effect on cardiac performance and coronary flow of different perfusion patterns

Mechanical circulatory assistance by ventricular assist devices provides an opportunity to influence the aortic pressure pattern, which may affect ventricular loading and coronary perfusion. The effect of synchronous, pulsatile coronary perfusion of an assist device-supported left ventricle has not been studied. To analyze the effect of different perfusion patterns on left ventricular performance and on coronary flow, independent of pressure and volume loading, we used three different modes of aortic perfusion in an isometric, contracting, isolated canine heart model. The effect of nonpulsatile, counter-pulsatile, and copulsatile coronary perfusion was analyzed in four subgroups to simulate different, clinically relevant situations (using two different ventricular end-diastolic volumes [normal and high] and two mean perfusion pressures [normal and critically low]). Our experiments demonstrated that total coronary flow is optimized by making the perfusion pressure pulsatile and by synchronously timing the pump systole with ventricular diastole (counterpulsation). Under identical conditions of preload and mean perfusion pressure, coronary flow and left ventricular contractility were decreased during non-pulsatile and copulsatile aortic perfusion when compared with counterpulsatile flow. There were no significant differences between the nonpulsatile and copulsatile modes. We conclude from these data that a nonejecting, but contracting, left ventricle will have improved systolic function and coronary blood flow if the coronary perfusion pressure is synchronized in a counterpulsatile manner. This is a significant implication for mechanical left ventricular assist devices when used to promote myocardial recovery.

The metabolism of fructose in the bivascularly perfused rat liver

The metabolism of fructose was investigated in the bivascularly and hemoglobin-free perfused rat liver. Anterograde and retrograde perfusions were performed. In anterograde perfusion, fructose was infused at identical rates (19 μmol min −1 g −1) via the portal vein (all liver cells) or the hepatic artery (predominantly perivenous cells); in retrograde perfusion fructose was infused via the hepatic vein (all liver cells) or the hepatic artery (only periportal cells). The cellular water spaces accessible via the hepatic artery were measured by means of the multiple-indicator dilution technique. The following results were obtained, (i) Fructose was metabolized to glucose, lactate and pyruvate even when this substrate was infused via the hepatic artery in retrograde perfusion: oxygen consumption was also increased. (ii) When referred to the water spaces accessible to fructose via the hepatic artery in each perfusion mode, the rate of glycolysis was 0.99 ± 0.14 μmol min −1 ml −1 in the retrograde mode; and, 2.05 ± 0.19 μmol min −1 ml −1 in the retrograde mode ( P = 0.002). (iii) The extra oxygen uptake due to fructose infusion via the hepatic artery was 1.09 ± 0.16 μmol min −1 ml −1 in the retrograde mode; and, 0.51 ± 0.18 μmol min −1 ml −1 in the anterograde mode ( P = 0.005). (iv) Glucose production from fructose via the hepatic artery was 2.18 ± 0.18 μmol min −1 ml −1 in the retrograde mode; and, 1.83 ± 0.16 μmol min −1 ml −1 in the anterograde mode ( P = 0.18). (v) Glucose production and extra oxygen uptake due to fructose infusion did not correlate by a single factor in all perfusion modes. It was concluded that: (a) rates of glycolysis are lower in the periportal area, confirming previous views; (b) extra oxygen uptake due to fructose infusion is higher in the periportal area; (c) a predominance of glucose production in the periportal area could not be demonstrated; and (d) extra oxygen uptake due to fructose infusion is not a precise indicator for glucose synthesis.

A cellular automation model for the growth of anchorage-dependent mammalian cells used in vaccine production

In the production of human vaccines, the kinetics of anchorage-dependent cell growth on microcarriers is complicated by their spatial environment. As the cells begin to cover the surface area available for growth, the growth rate gradually decreases until the entire surface is covered, and growth ceases. This contact inhibition of cell growth makes it difficult to accurately determine key kinetic parameters such as the specific growth rate of a culture. In this paper, a simple, but useful cellular automation model is proposed which is capable of decoupling the effect of contact inhibited cell growth from the overall kinetics of the culture. The automaton model is a good qualitative representation of cellular growth on microcarrier cultures. Moreover, quantitative comparison with experimental data is very good. The model is capable of fitting growth curves initiated at different inoculation levels by taking into account the initial cell attachment distribution for microcarrier cultures. Information generated by the automation on the dynamic nature of the specific growth rate of a culture has been coupled to a kinetic model describing the dynamics of key nutrients and metabolites in both the batch and perfusion modes of bioreactor operation, and these results are compared to experimental data.

Effects of amrinone and isoproterenol on mechanoenergetics of blood-perfused rabbit heart.

To compare the effects of amrinone (AMR) and isoproterenol (Iso) on left ventricular contractility and energetics, we assessed Emax (ventricular contractility index) and the relation between oxygen consumption per beat (VO2) and systolic pressure-volume area (PVA, a measure of left ventricular total mechanical energy) in isolated cross-circulated (blood-perfused) rabbit hearts during infusion of AMR or Iso in either a constant-flow (CF) or constant-pressure (CP) perfusion mode. Both Emax and the VO2 intercept of the linear VO2-PVA relation increased significantly during AMRCP (increase in Emax 15% and increase in VO2 intercept 11%), ISOCF (49 and 43%), and ISOCP (55 and 54%) but not during AMRCF. However, neither drug changed the slope of the VO2-PVA relation (reciprocal of contractile efficiency) in either perfusion mode. Furthermore, with both drugs the relation between increases in Emax and the VO2 intercept fell on a single regression line (r = 0.92). We conclude that 1) although the mechanism of action and inotropic potency of the two drugs differ, their effects on cardiac energetic cost are essentially the same, i.e., both drugs increase the nonmechanical oxygen cost in proportion to the increase in contractility without changing contractile efficiency, and 2) a significant portion of the inotropic effect of AMR in the whole ventricle is likely due to increased coronary blood flow, i.e., Gregg's phenomenon.

High-performance gel permeation chromatographic analysis of immunoglobulin M produced by hybridoma cell culture

A high-performance gel permeation chromatographic (HPGPC) method, using TSK-Gel SW XL 3000 and TSK-Gel SW XL 4000 columns installed in series, was developed for the analysis of immunoglobulin M (IgM) produced by hybridoma cell culture. The detection of this protein was achieved using ultraviolet absorption at 225 nm, yielding a detection limit of ca. 0.3 μg ml −1. IgM-containing samples obtained from different culture systems (T-flask, roller bottle, spinner flask, bioreactor operated in batch, fed-batch or perfusion modes) and media (Dulbecco's Modified Eagle Medium or Protein-Free Hybridoma Medium) were evaluated by this chromatographic technique and also by conventional enzyme-linked immunosorbent assay (ELISA). Concentrations of IgM in culture samples determined by both techniques were always in the same range (±10%). The main advantages of HPGPC over ELISA included improved reproducibility (relative average deviation of 1−3% compared with 10−20% for ELISA), high linearity range between the signal and the concentration [at least three decades (0.3−500 μg ml −1) compared with one decade for ELISA (25−200 ng ml −1)] and ease of operation.

The medium cycle bioreactor (MCB): monoclonal antibody production in a new economic production system.

The perfusion mode of a continuous cell culture bioreactor was modified to establish a closed loop system. Eighty percent of the spent medium was re-used twice. The medium cycle bioreactor unit was operated sterile and uncomplicated without a technical retention system for the high molecular weight substances. Therefore, only 20% of the actual medium was necessary to run the recycling process. During seven days culture time in a two liter scale 5 grams of IgG1 type monoclonal antibody was produced. During that period the cell specific productivity was constant. Renewal of proteins was omitted because the protein content in the system persisted at a high level. Therefore, self-conditioning substances of the cells were retained in the system as well as the expensive medium components (proteins with catalytic or stimulating function). Seventy to 80% of medium costs and medium quantity were saved for each medium recycling step. Only cheap metabolites that are consumed by the cells had to be supplemented. Uptake rates of glucose and amino acids were calculated to establish a suitable supplementation mixture for the recirculated medium.

Optimization of serum-free fermentation processes for antibody production.

Serum free fermentation procedures of cell cultures have got a wide application in production of biochemicals. But, cells cultured in serum free media in general are more sensitive to changes in culture condition, especially to nutrient limitation. There are no substances from serum which can support the cells when conditions are changing. In this study special attention is directed to amino acid utilization of mouse hybridoma in batch, chemostat and perfusion fermentations. Detailed data are presented which show the considerable difference of amino acid consumption rates in different fermentation modes. Already, in batch mode there are differences of the two investigated mouse hybridoma cell lines, although they are derived from the same myeloma line. In chemostat running at a dilution rate representing maximal growth rate most of the consumption rates are significant higher than in batch. On the other hand, in perfusion mode the rates are lower than in batch. This indicates clearly the different conditions of the fermentation modes. Therefore, it is necessary to develop serum free processes under the desired production conditions. An accurate analysis of the process is strongly recommended.

Pulmonary lobar vascular resistances during constant and pulsatile flows

We measured arterial and venous occlusion pressure profiles in left lower lobes of dog lungs perfused in constant flow and pulsatile flow. Initially, pulmonary arterial pressure, flow rate, and venous pressure were recorded during a series of inflow and outflow occlusions effected during constant flow perfusion. The perfusion was then made pulsatile and while keeping the same flow rate, a second series of vascular occlusions was performed. The arterial occlusion pressures measured with the two modes of perfusion were similar. During pulsatile perfusion, the variation in arterial pressure following an arterial occlusion varied with the timing of occlusion in the pressure cycle. The arterial pressure drop could be recovered by selecting the time-averaged pulmonary arterial pressure as the upstream pressure. During both types of perfusion, the venous occlusion curves were similar. The arterial and venous pressure drops were approximately 4.8 mm Hg and 4.2 mm Hg, respectively. We conclude that the longitudinal distribution of pulmonary vascular pressures can be inferred from occlusion measurements obtained in pulsatile flow.

Myogenic autoregulation of flow may be inversely related to endothelium-derived relaxing factor activity

The influence of basal endothelium-derived relaxing factor (EDRF) activity on autoregulation was studied under conditions of controlled-flow and controlled-pressure perfusion in the isolated rabbit ear, a weakly autoregulating vascular bed. Hemoglobin and NG-monomethyl-L-arginine were used to inhibit EDRF activity, and in some experiments resting tone was increased by serotonin. The diameters of five generations of resistance arteries (ranging from 70 to 1,000 microns in size) were measured at different flow rates by X-ray microangiography. Diameter-flow (D-Q) relationships were correlated with pressure-flow (P-Q) and conductance-flow (G-Q) relationships. In the presence of EDRF activity no autoregulation was observed, P-Q relationships being linear and G-Q and D-Q relationships common both to controlled-flow and to controlled-pressure modes of perfusion. After inhibition of EDRF activity in constricted preparations, P-Q relationships became sigmoidal in shape in controlled-pressure perfusion mode, reflecting a range of perfusion pressures/flow rates over which they were able to "autoregulate" flow. Over this autoregulatory range the corresponding G-Q and D-Q relationships exhibited regions of negative slope. Autoregulation was not observed in controlled-flow perfusion mode even in the absence of EDRF activity. The findings imply that flow- or pressure-dependent constriction can mediate autoregulation in controlled-pressure mode when not overridden by basal EDRF activity, as normally appears to be the case in these preparations. Differences in autoregulation in different organs may be inversely related to EDRF activity, which is known to differ between vascular beds.

Biologische Erfahrung mit einem blasenfreien Begasungssystem (CHEMCELL)

AbstractThe increased interest in large scale production of biologically active molecules, as for example monoclonal antibodys, hormones, proteins and enzymes, has stimulated a rapid development of different methods to cultivate eukaryotic cells. Further progress in this field of modern biotechnology is expected not only from the selection of more productive cell lines and more efficient cultivation techniques, but also from the improvement in new bioreactor design and operation, which guarantees increased productivity per unit volume and reduces the downstream processing. Most important factors for a new reactor design in future will be the energy‐ and mass transfer, shear stress and scaleability. The provision of an adequate oxygen supply to large scale reactors is the most critical barrier to scale up. If oxygen is limited in a small degree, the result is inhibition of cell density and cellular efficiency in the production of the desired biomolecules. In addition, when methodologies are used which allow high cell densities and high metabolic active cells, the oxygen transfer becomes more important and, at the same time, more difficult. Since direct sparging of air into the cell‐containing medium causes problems such as shear forces and foaming, new, efficient methods in bubble free aeration must be utilized. A new aeration system is presented, which is suitable to bubble‐free aeration as well to separation of microcarrier‐anchered cells from the harvested medium when running in a continuous perfusion mode. The efficiency of the CHEMCELL System regarding aeration and cell retention is demonstrated by the growth kinetics of BHK 21‐cells in batch and perfusion mode.

Microprocessor control of perfusion during extracorporeal purification of physiological data

The authors describe microprocessor perfusion monitoring system, which allows one to regulate the input of the perfusate, to measure and control the parameters to visualize information on the linear display, to estimate the parameters quantitatively in the digital form, manual and automatic measuring of the given and controlled values of the parameters, alarms, dialogue forms to set the perfusion modes.

Pulse oximetry during low cardiac output and hypothermia states immediately after open heart surgery

Two commercially available pulse oximeters were found to read reliably early during periods of low cardiac index (CI) and low peripheral temperature (Tp). The lowest mean CI and mean Tp values at which a reading could be obtained were 2.4 L/min.m2 and 26.5 degrees C, respectively. The highest mean systemic vascular resistance index was 2930 dyne.sec/cm5.m2. The reliability of one monitor was equally good both in a normal and low perfusion mode (i.e., when the monitor displayed a low-quality signal message). The use of locally applied vasodilating creme widened the physiologic limits within which reliable measurements could be obtained. All oximeters and sensors overestimated slightly the saturation as compared to the hemoximeter readings, possibly because of a high mean carboxyhemoglobin concentration that was found to be present.

Mechanism of improvement in pulmonary gas exchange during growth in awake piglets.

Previous studies have shown a lower arterial PO2 (PaO2) in infants and young animals than in adults. To investigate the mechanism of this impairment of gas exchange we studied 13 piglets from 12 to 65 days of age. Two days after instrumentation we measured the distribution of ventilation-perfusion ratios (VA/Q) by use of the multiple inert gas technique on awake animals. We showed that PaO2 is lower in young animals, increasing from 72 +/- 11.5 Torr before 2 wk to 102 Torr at 2 mo. This hypoxemia is due to an enlarged alveolar-arterial O2 pressure difference that significantly decreases with age. This impairment in gas exchange is not due to shunting (0.6 +/- 1.3%). Mean dead space (36 +/- 11%) was not related to age. Mean modes of perfusion and ventilation did not differ significantly between age groups. However, the dispersion of perfusion as expressed by its logSD decreased significantly with age, whereas dispersion of ventilation remained constant. Furthermore, in the young animals only, a significant difference was evidenced between measured alveolar-arterial PO2 gradient and the value predicted by the inert gas model. We therefore conclude that the impairment of gas exchange in piglets is due to two mechanisms: VA/Q mismatch and diffusion limitation for O2.

An extension set for 81Rb- 81mKr solution generators for lung ventilation studies

For 81Rb- 81mKr solution generators, an extension set has been developed, which strips the 81mKr from the liquid eluate with a stream of air for use in lung ventilation studies. Via a three-way valve the 81Rb- 81mKr solution generator can be operated alternately in the perfusion mode or in the ventilation mode. Measurements and calculations have been performed to get more insight into the parameters of interest for an optimum design of the extension set and its operation conditions.

Activation of glycogenolysis by the reduction in the extracellular calcium concentration in verapamil-prefused rat liver

In an attempt to elucidate the role of Ca 2+ flux in the initial events of hepatic glycogenolysis, extracellular Ca 2+ concentration was manipulated in rat liver perfused Ca 2+ antagonistic drugs. After the liver had been perfused with a buffer containing verapamil and 1 mM CaCl 2, either the addition of ethylneglycol-bis-( rmβ-aminoethyl ether)- N, N′-tetraacetic acid to the perfusate or the replacement of the perfusate with Ca 2+-free buffer caused a rapid increase in glucose output as well as 45Casu2+ efflux. Substitution of diltiazem, but not 5–20 mM LaCl 2, for verapamil also stimulated glucose output and 45Ca 2+ efflux. However, when Ca +-free buffer was used througout the experiment, any modes of verapamil or diltiazem perfusion were without significant effects on glucose output or Ca 2+ efflux. The increases in glucose output and 45Ca 2+ efflux were not affected by either 20 μM phentolamine or 300 μM ouabain, but they were inhibited significantly by 10–100 μM trifluoperazine. These results indicate that rapid decline in the extracellular Ca 2+ concentration in verapamil- or diltiazem-perfused liver initiates the change in Ca 2+ equilibirum on or across plasma membrane and activates glycogenolysis through a Ca 2+-dependent mechanism.

Ventilation-perfusion relationships in children.

Ventilation-perfusion relationships (Va/Q) were studied in nine lung-healthy children, 7-15 years old, in whom a diagnostic heart catheterization was performed. VA/Q was assessed by a multiple inert-gas elimination technique. In subjects above 10 years of age, single, narrow modes of ventilation and perfusion were seen centred upon a VA/Q ratio of one. In three of the four youngest children, 10 years old or less, additional; 'high' VA/Q regions were observed. Neither shunt, nor 'low' VA/Q regions were seen in any child. Arterial PO2 was above 12 kPa in all except one and no age dependence was observed.

Reflex control of canine hindlimb resistance under different modes of perfusion.

The carotid sinus control of hindlimb hemodynamics was determined in halothane-anesthetized vagotomized dogs. The carotid sinuses were bilaterally isolated and perfused under controlled conditions. The hindlimb was perfused under conditions of 1) physiological inflow, 2) constant pressure, and 3) constant flow. The variation of hindlimb resistance with mean carotid sinus perfusion pressure was largest under conditions of constant-pressure perfusion and smallest with physiological inflow. Values for constant-flow perfusion were intermediate. The effects of different values of constant-flow perfusion on the carotid sinus control of hindlimb hemodynamics was also determined. Values of hindlimb resistance increased with decreasing flow rate as did the variation with carotid sinus perfusion pressure. No evidence of a contribution of local control mechanisms related to changes in perfusion pressure or bed inflow could be demonstrated. The differences in the variation of hindlimb resistance with carotid sinus pressure under the various perfusion conditions can be explained on the basis of the mechanics of vascular smooth muscle. The amount of lumen constriction associated with changes in degree of activation depends on the conditions under which shortening occurs as well as upon the initial muscle length. It appears that local factors do not contribute to the control of hindlimb hemodynamics under the conditions in which these studies were performed.

Artificial capillary perfusion cell culture: Metabolic studies

Glucose, lactic-acid, and oxygen metabolism of BHK and L929 cells on artificial capillary perfusion units have been studied using several different modes of perfusion. After 7 to 10 days, cells planted in the extracapillary compartment of culture units containing 80 to 150 fibers reached populations that used 0.073 +/- 0.025 mumol per min glucose and 0.76 +/- 0.26 microliter per min oxygen and excreted 0.078 +/- 0.038 mumol per min lactic acid. From these data it is estimated that these units contain approximately 2 x 10(7) cells. The metabolic rate of cultures perfused through the capillaries or through the extracapillary compartment was not affected significantly by change in flow rate except at perfusion flow rates less than or equal to 0.05 ml per min. The cell population, as measured by metabolic activity, did not increase significantly when the serum content of the medium was less than or equal to 1%. No major differences were found in glucose utilization rates of equal numbers of cells on artificial capillaries, on short-term suspension culture, or as monolayers in plastic flasks. Artificial capillary perfusion may provide a simple system for studying metabolism of mammalian cells in culture.

Influence of the blood pressure level and the mode of contraction on the strength of drug-induced vasoconstriction.

1. Under constant pressure or constant flow conditions noradrenaline (NA) was infused into the superior mesenteric artery of the cat in such a manner that there always resulted a local concentration of 2·10−7 g NA/ml. In the case of constant pressure perfusion an electrical coupling was necessary between the perfusing roller pump and the continuously adjustable infusion pump. 2. For the condition of constant pressure perfusion the constrictor response caused by NA is expressed by the resulting flow reduction ΔI. With rising pressure levels this value increases at first in a steep manner, then it reaches a flat maximum. If the reduction of flow is related to the initial flow by the expression ΔI/I, the appearance of a maximum is still more obvious. 3. Under constant flow perfusion the NA-induced pressure increase ΔP first rises with rising initial pressure and also shows a clear maximum. Only after this maximum the often described negative regression lines related to the resting intraluminal pressure are approximately valid. The existence of an optimum in the physiological pressure range strongly limits the “law of initial value” of Wilder, but is in good agreement with results obtained on the isolated vascular smooth muscle. 4. A comparison of the constrictor responses during constant pressure or constant flow perfusion is made by the increase of the respective resistances. Thus larger values result under constant pressure perfusion at initial pressures above 80 mm Hg. Below this pressure level the constant flow perfusion leads to larger values with a maximum at 60 mm Hg. The cause of the different optima for both perfusion modes is discussed. 5. The evidence for the appearance of an optimum for vasoconstrictions as dependent on the intraluminal pressure can be offered only if the experiments are extended over a very wide range of pressures. Thus the discrepancy with former investigations is interpreted. Now an optimum of constrictions could be found under both constant pressure and constant flow perfusion, the former equalling more isotonic conditions, the latter more isometric conditions.

A comparison of pulsatile and nonpulsatile pumping for ex vivo renal perfusion

Dog kidneys were perfused in an ex vivo circuit using a 10-minute interval of autoperfusion (with a slightly damped natural pulse) as a control mode and similar periods of pulsatile and nonpulsatile pumping, randomly alternated, as experimental perfusion modes. In one group of 10 experiments, flow was regulated to the preceding control flow with an occlusive finger pump. In a second group of 10 experiments, pressure was regulated to the preceding control pressure by means of a pressure-controlled servo pump. Pulsation resulted in equal flow at significantly lower pressure in the “adjusted pressure” group. In addition, a degree of autoregulation persisted. Constant pressure appeared superior to constant flow as a means of regulating perfusion, since this provided automatic adjustment to the progressive rise in renal resistance that occurred. Edema did not develop during the five-hour periods of perfusion, indicating the basic soundness of both regulating methods. The most logical explanation for the superiority of pulsatile flow is to be found in the viscoelastic properties of the renal vessels which impart a capacitive flow effect that is augmented by pulsatility. The higher frequency, “natural,” pulse was superior to both experimental pump pulses indicating the desirability of further defining the characteristics of an optimal pulse under various conditions of storage including hypothermia.