Channing R. Robertson

The immobilization of whole cells: Engineering principles

Abstract The immobilization of whole cells involves the retention of catalytically active cells within a restricted region of a bioreactor. Techniques which have been used to immobilize whole cells include adsorption, aggregation, confinement and entrapment. These techniques can be applied to essentially all of the viable or non-viable whole cell systems of potential interest: microorganisms, animal and plant cells. The fact that immobilized cells may be living leads to unique effects in this form of heterogeneous catalysis. These include the impact of immobilization on cell physiology and cell mobility, physical interactions of immobilized cells with the support, and the creation of a microenvironment. The theory of mass-transfer and reaction in these types of systems is well understood, but schemes capable of predicting substrate and product diffusivities and the intrinsic kinetics in the aggregate must still be developed. New experimental approaches are being used to elucidate the basic mechanisms that determine these physical and metabolic properties of immobilized cells. Several reactor configurations have been successfully used with immobilized cells, and many more have been proposed. The choice of a particular design for a given process depends on the requirements for mass-transfer, the growth behaviour of the cells, and the structural properties of the aggregate.

Permselectivity of the Glomerular Capillary Wall: FACILITATED FILTRATION OF CIRCULATING POLYCATIONS

To examine the electrostatic effects of fixed negative charges on the glomerular capillary wall, polydisperse [(3)H]DEAE dextran, a polycationic form of dextran, was infused into 10 Munich-Wistar rats. Fractional clearances of DEAE ranging in radius from 18 to 44A were determined in these rats, together with direct measurements of the forces and flows governing the glomerular filtration rate of water. These results were compared with data previously obtained in Munich-Wistar rats receiving tritiated neutral dextran (D) and polyanionic dextran sulfate (DS). Measured values for the determinants of the glomerular filtration rate of water in rats given DEAE were found to be essentially identical to those in rats given either D or DS. In addition, DEAE was shown to be neither secreted nor reabsorbed. Fractional clearances of polycationic DEAE were increased relative to both D and DS, the increase relative to D being significant for effective molecular radii ranging from 24 to 44A. Fractional DEAE clearances were also measured in a separate group of six Munich-Wistar rats in the early autologous phase of nephrotoxic serum nephritis (NSN). Fractional DEAE clearances in NSN rats were reduced significantly, relative to values measured in normal rats, for effective DEAE radii ranging from 18 to 42A. Moreover, in NSN rats, fixed negative charges on the glomerular capillary wall were greatly reduced, relative to non-NSN rats, as evidenced by a reduction in intensity of colloidal iron staining. Thus, in NSN rats, DEAE clearances were essentially indistinguishable from values obtained with both neutral D and polyanionic DS.

Permselectivity of the glomerular capillary wall to macromolecules. II. Experimental studies in rats using neutral dextran

To determine the permselectivity characteristics of the glomerular capillary wall, known molecular size fractions of [3H]dextran, prepared by gel chromatography, were infused into normally hydrated Wistar rats, thus permitting simultaneous measurement of Bowmans space/plasma water (BS/P) and urine/plasma water (U/P) concentration ratios, along with glomerular pressures and flows. Since (BS/P)inulin = 1.01 +/- 0.01 SE(n = 34, radius = approximately 14 A) and since (BS/P)dextran/(BS/P)inulin equaled (U/P)dextran/(U/P)inulin for dextrans ranging in molecular radius from 21 to 35 A, these findings validate that dextrans are neither secreted nor reabsorbed. For dextran radii of 20, 24, 28, 32, 36, 40, and 44 A, (U/P)dextran/(U/P)inulin averaged 0.99, 0.92, 0.69, 0.42, 0.19, 0.06, and 0.01, respectively. In accord with theoretical predictions that these fractional dextran clearances should vary appreciably with changes in glomerular transcapillary pressures and flows, an increase in glomerular plasma flow rate, induced in these same rats by plasma volume expansion, resulted in a highly significant lowering of fractional clearance of all but the smallest and largest dextrans studied. These findings emphasize that fractional solute clearances alone are inadequate to describe the permselective properties of the glomerular capillary wall unless glomerular pressures and flows are also known. This sensitivity of fractional dextran clearance to changes in plasma flow indicates that dextrans are transported across the capillary not only by bulk flow but also to an important extent by diffusion.

Protein adsorption on crosslinked polydimethylsiloxane using total internal reflection fluorescence

Abstract Total internal reflection fluorescence (TIRF) is used to examine the adsorption of bovine serum albumin (BSA) and bovine fibrinogen on crosslinked polydimethylsiloxane (silicone rubber) surfaces from flowing solutions. By comparing experimentally observed adsorption rates with the predictions of a convection/diffusion model, it is shown that the adsorption of both BSA and fibrinogen on silicone rubber is diffusion-controlled over the range of solution concentrations (0.01

Dynamics of Glomerular Ultrafiltration in the Rat. IV. DETERMINATION OF THE ULTRAFILTRATION COEFFICIENT

Pressures and flow rates were measured in accessible surface glomeruli of mutant Wistar rats under conditions deliberately designed to prevent achievement of filtration pressure equilibrium, that is, the equalization of transcapillary hydrostatic and oncotic pressures by the efferent end of the glomerulus as typically observed in the normal hydropenic rat. Disequilibrium was obtained at elevated levels of glomerular plasma flow (GPF) brought about by acute expansion of plasma volume with a volume of rat plasma equal to 5% of body weight. Glomerular hydrostatic and oncotic pressures measured at high GPF were used to calculate the ultrafiltration coefficient, K(f), the product of effective hydraulic permeability and surface area. GPF was then either lowered (by aortic constriction) or raised (by carotid occlusion) in order to examine the dependence of K(f) on GPF. The value of K(f) per glomerulus, 0.08 nl/(s.mm Hg), was found not to vary over an approximately twofold range of GPF. This finding, taken together with data from previous studies from this laboratory, leads us to conclude that plasma-flow dependence of glomerular filtration rate (GFR) results primarily from flow-induced changes in mean ultrafiltration pressure, rather than large changes in K(f).

Total internal reflection fluorescence: a technique for examining interactions of macromolecules with solid surfaces

Abstract Total internal reflection fluorescence (TIRF) is a technique that may be used to examine the interaction of macromolecules with solid surfaces. In this report, a TIRF apparatus and its application to the adsorption of proteins from flowing solutions on polymer films are described. Bovine serum albumin (BSA)/crosslinked polydimethylsiloxane (silicone rubber) is the protein/surface pair considered herein. To relate TIRF fluorescence signals to actual surface concentrations, a calibration technique using BSA labeled with both tritium and fluorescein isothiocyanate (FITC) was developed. Unambiguous calibration results require a linear relationship between observed fluorescence intensity and protein surface concentration, reproducible evanescent field intensities, and constant fluorescence quantum efficiencies. The contribution of bulk solution fluorescence to the total TIRF signal must be ascertained to ensure accurate quantitative interpretation of TIRF data. There are two sources of solution fluorescence: one arising from excitation of fluorescent solution protein by scattered light, the second from solution protein within the evanescent field. Solution protein within the evanescent field is distinguished from reversibly adsorbed proteins by comparing its exchange dynamics with that of nonadsorbing FITC-dextran together with the predictions of a hydrodynamic model. By removing the solution protein contributions to the total TIRF signal, surface-adsorbed protein fluorescence, and thus the amount of protein adsorbed, can be obtained. Bulk solution contribution to the total TIRF signal increases with solution concentration. Experiments were conducted at solution concentrations of 2 and 200 mg%, both corresponding to an equilibrium surface concentration of 0.14 μg/cm 2 , the saturated surface concentrations for BSA adsorbing on silicone rubber. Proteins in the 2 mg% solution contributed negligibly to the total fluorescence, while fluorescence from the 200 mg% solution constituted a significant fraction of the total TIRF signal.

Permselectivity of the glomerular capillary wall to macromolecules. I. Theoretical considerations.

The transport of macromolecules across the renal glomerular capillary wall has been described theoretically using flux equations based on (a) restricted transport through small pores, and (b) the Kedem-Katchalsky formulation. The various assumptions and limitations inherent in these two approaches are discussed. To examine the coupling between macromolecular solute transport and the determinants of glomerular filtration rate, these flux equations were combined with mass balance relations which allow for variations in the transmembrane driving forces along a glomerular capillary. It was predicted, using both pore theory and the Kedem-Katchalsky equations, that fractional solute clearance should be strongly dependent on the determinants of glomerular filtration rate when convection and diffusion both contribute to solute transport. When convection becomes the sole mechanism for transcapillary solute transport, however, fractional solute clearance is essentially independent of changes in the determinants of glomerular filtration rate. Consequently, unless diffusion is absent, fractional solute clearances alone are insufficient to characterize the permselective properties of the glomerular capillary wall, since these values may be altered by changes in glomerular pressures and flows as well as changes in the properties of the capillary wall per se.

Mechanisms of the Puromycin-Induced Defects in the Transglomerular Passage of Water and Macromolecules

To investigate the mechanism(s) of increased filtration of serum proteins after glomerular injury, polydisperse samples of uncharged [(3)H]dextran (D) or anionic [(3)H]dextran sulfate (DS) were infused into 14 control and 16 puromycin aminonucleoside- (PAN) treated Munich-Wistar rats. Fractional clearances of D or DS ranging in radius from 18 to 42A were determined in these rats, together with direct measurements of the forces governing the glomerular filtration rate of water. Whole kidney and single nephron glomerular filtration rates were approximately 40% lower in PAN-treated rats, relative to controls, due mainly to a marked reduction in the glomerular capillary ultrafiltration coefficient and, to a lesser extent, to a small reduction in glomerular plasma flow rate as well. In PAN-treated rats, as in normal controls, inulin was found to permeate the glomerular capillary wall without measurable restriction, and both D and DS were shown to be neither secreted nor reabsorbed. Fractional clearances of uncharged D were reduced after PAN administration, falling significantly for effective D radii from 22 to 38A. Utilizing a theory based on macromolecular transport through pores, these results indicate that in PAN-treated rats, effective pore radius is the same as in controls, approximately 44A. In PAN nephrosis, however, the ratio of total pore surface area/pore length, a measure of pore density, is reduced to approximately one-third that of control, due very likely to a reduction in filtration surface area. In contrast to the results with uncharged D, fractional clearances of DS were found to increase after PAN administration for all DS radii studied. These results with D and DS suggest that proteinuria in PAN nephrosis is due, not to an increase in effective pore radius or number of pores, but rather to a diminution of the electrostatic barrier function of the glomerular capillary wall, thereby allowing increased passage of polyanions such as DS and albumin.

Mechanisms of polarization and fouling of ultrafiltration membranes by proteins

Unstirred, constant-pressure, batch-cell ultrafiltration of dilute (0-6 g/l) aqueous bovine serum albumin (BSA) solutions through high permeability, asymmetric ultrafiltration membranes yields the classical linear dependence of permeate volume with (time)1/2, as predicted by osmotic (Vilker and Colton) and “gel-polarization” (Trettin and Doshi) models of the solute polarization process. However, step-changes in pressure during ultrafiltration reveal a linear dependence of flux on pressure, which is inconsistent with either of these models. A simple particle filtration model, based on the build-up of a BSA filter cake of constant hydraulic permeability, adequately correlates the observed results Of particular importance is the surprising observation that, following interruption of flow for extended time-periods (up to 16 hr), there is relatively little rebound of flux on repressurization, indicating only slight diffusional relaxation of the polarization layer. This implicates the existence of strong intermolecular attractive interactions within the BSA gel. Stirring of the solution overlying the polarized membrane (under zero-permeation conditions) is accompanied by a rapid removal of the BSA gel-layer (as evidenced by a rapid increase in pure-solvent permeability of the membrane with agitation time). For hydrophobic membranes which display strong adsorptivity of BSA from solution (e.g., polysulfone), the rate of BSA-layer removal by agitation is much slower than that observed with non BSA-sorbing, hydrophilic membranes (regenerated cellulose). This finding indicates that molecular interactions between BSA and the membrane surface are of central importance to membrane-fouling by this (and undoubtedly other) macrosolutes, and suggests that surface treatments to minimize solute adsorption should be highly beneficial to the reduction of fouling during ultrafiltration.

Lateral diffusion of bovine serum albumin adsorbed at the solid-liquid interface

Abstract The lateral mobility of eosin isothiocyanate-labeled bovine serum albumin irreversibly adsorbed to poly(methylmethacrylate) (PMMA) and poly(dimethylsiloxane) (PDMS) surfaces from aqueous solution was measured by a combination of total internal reflection fluorescence and fluorescence recovery after pattern photobleaching techniques. Lateral mobility over distances of several micrometers was probed by photobleaching and monitoring fluorescence with the fringe pattern formed by two intersecting coherent laser beams in total internal reflection. The period of the fringes determined the characteristic length for transport on the surface and was varied by changing the angle of intersection. The dependence of the fluorescence recovery time on the period of the fringes indicates that lateral migration of the adsorbed protein on PMMA is by surface diffusion with D = (1.2 ± 0.3) × 10−9cm2/s, while the extent of fluorescence recovery reveals the coexistence of a mobile population and a population that is apparently immobile over 20 min. For comparison, D = (2.6 ± 0.1) × 10−9cm2/s on PDMS.