The effect of ethanol on lateral and rotational mobility of plasma membrane vesicles isolated from cultured mar 18.5 hybridoma cells

Intramolecular excimer formation of 1,3-di(1-pyrenyl)propane (Py-3-Py) and fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH) were used to evaluate the effect of ethanol on the rate and range of the lateral mobility and the range of the rotational mobility of bulk bilayer structures of the plasma membrane vesicles (ATCC-PMV) isolated from cultured hybridoma cells (ATCC TIB 216). In a concentration-dependent manner, ethanol increased the excimer to monomer fluorescence intensity ratio (I/I) of Py-3-Py in the ATCC-PMV and decreased the anisotropy (r), limiting anisotropy (r) and order parameter (S) of DPH in the ATCC-PMV. This indicates that ethanol increased both the lateral and rotational mobility of the probes in the ATCC-PMV. Selective quenching of DPH by trinitrophenyl groups was utilized to examine the range of transbilayer asymmetric rotational diffusion of the ATCC-PMV. The anisotropy (r), limiting anisotropy (r ) and order parameter (S) of DPH in the inner monolayer were 0.024, 0.032, and 0.069, respectively, greater than calculated for the outer monolayer of the ATCC-PMV. Selective quenching of DPH by trinitrophenyl groups was also used to examine the transbilayer asymmetric effects of ethanol on the range of the rotational mobility of the ATCC-PMV. Ethanol had a greater increasing effect on the range of the rotational mobility of the outer monolayer as compared to the inner monolayer of the ATCC-PMV. It has been proven that ethanol exhibits a selective rather than nonselective fluidizing effect within the transbilayer domains of the ATCC-PMV.This paper was supported in part by a research grant from the Korea Science and Engineering Foundation (KOSEF 88-1013-01) and from the Korea Research Foundation (1991–1993).     

Cell aggregation and neurite growth in gels of extracellular matrix molecules

Components of the extracellular matrix are believed to guide both nerve cells and neurites to their targets during embryogenesis and, therefore, might be useful for controlling regeneration of nervous tissue in adults. To study the influence of extracellular conditions on neurite outgrowth and cell motility, PC12 cells were suspended in three-dimensional gels containing (i) collagen (0.4 to 2 mg/mL), (ii) collagen (1 mg/mL) with added fibronectin or laminin (1 to 100 mug/mL), and (iii) agarose (7 mg/mL) with added collagen (0.001 to 1 mg/mL). Neurite outgrwoth was stimulated with nerve growth factor (NGF) and both the extent of neurite outgrowth ad cell aggregation were quantitated over 10 to 12 days in culture. The extent of neurite outgrowth was greatest at the lowest collagen concentration tested (0.4 mg/mL) and decreased with increasing concentration. The addition of laminin or fibronectin altered the extent of neurite outgrowth in collagen gels, but the differences were small. Although no neurite growth was observed in pure agarose gels, considerable neurite outgrowth occurred with the addition of small amounts (>/=0.01 mg/mL) of collagen. Mean aggregate size increased more quickly in gels with lower concentrations of collagen. For cells in 1.0 mg/mL collagen, a four- to fivefold increase in aggregate volume was seen between days 2 and 10 o the culture period, whereas the increase in DNA content during this same period was less than twofold, suggesting that the cells were aggregating, not multiplying. These results suggest that the composition of the matrix supporting nerve cells has a significant effect on both neurite outgrowth and cell motility. (c) 1994 John Wiley & Sons, Inc.     

Continuous, real-time monitoring of the oxygen uptake rate (OUR) in animal cell bioreactors

A new method for real-time monitoring of the oxygen uptake rate (OUR) in bioreactors, based on dissolved oxygen (DO) measurement at two points, has been developed and tested extensively. The method has several distinct advantages over known techniques.It enables the continuous and undisturbed monitoring of OUR, which is conventionally impossible without gas analyzers. The technique does not require knowledge of k(L)a. It provides smooth, robust, and reliable signal. The monitoring scheme is applicable to both microbial and mammalian cell bioprocesses of laboratory or industrial scale. The method was successfully used in the cultivation of NSO-derived murine myeloma cell line producing monoclonal antibody. It was found that while the OUR increased with the cell density, the specific OUR decreased to approximately one-half at cell concentrations of 16 x 10(6) cells/mL, indicating gradual reduction of cell respiration activity. Apart from the laboratory scale cultivation, the method was applied to industrial scale perfusion culture, as well as to processes using other cell lines. (c) 1994 John Wiley & Sons, Inc.     

Effects of substratum morphology on cell physiology

Among the host of substratum properties that affect animal cell behavior, surface morphology has received relatively little attention. The earliest effect of surface morphology on animal cells was discovered almost a century ago when it was found that cells became oriented in response to the underlying topography. This phenomenon is now commonly known as contact guidance. From then until very recentrly, little progress has been made in understanding the role of surface morphology on cell behavior, primarily due to a lack of defined surfaces with uniform morphologies. This problem has been solved recently with the development of photolithographic techniques to prepare substrata with well defined and uniform surface morphologies. Availability of such surfaces has facilitated systematic in vitro experiments to study influence of surface morphology on diverse cell physiological aspects such as adhesion, growth, and function. For example, these studies have shown that surfaces with uniform multipls parallel grooves can enhance cell adhesion by confining cells in grooves and by mechanically interlocking them. Several independent studies have demosterated that cell shape is a major determinant of cell growth and function. Because surface morphology has been shown to modulate the extent of cell spreading and cell shape, its effects on cell growth and function appear to be mediated via this biological coupling between cell shape and function. New evidence in the cell biology literature is emerging to suggest that surface morphology could affect other cell behavioral properties such as post-translational modifications. Further elucidation of such effects will enable better designs for implant and cell culture substrata.     

Foaming and cell flotation in suspended plant cell cultures and the effect of chemical antifoams

Foam development and stability in Atropa belladonna suspensions were investigated as a function of culture conditions. Foaming was due mainly to properties of the cell-free broth and was correlated with protein content; effects due to presence of cells increased towards the end of batch culture. Highest foam levels were measured 11 days after inoculation. Air flow rate was of major importance in determining foam volume; foam volume and stability were also strongly dependent on pH. Foam flotation of plant cells was very effective. After 30 min foaming, ca. 55% of cells were found in the foam; this increased to ca. 75% after 90 min. Polypropylene glycol 1025 and 2025, Pluronic PE 6100, and Antifoam-C emulsion were tested as chemical antifoams. Polypropylene glycol 1025 and Antifoam C at concentrations up to 600 ppm had no adverse effect on growth in shake flasks; Pluronic PE 6100 has an inhibitory effect at all levels tested. Concentrations of polypropylene glycol 2025 and Pluronic PE 6100 as low as 20 ppm reduced foam volumes by a factor of ca. 10. Addition of antifoam reduced k(L)a values in bubble-column and stirred-tank bioreactors. After operation of a stirred reactor for 2 days using Antifoam C for foam control, cell production was limited by oxygen due to the effect of antifoam on mass transfer. Theoretical analysis showed that maximum cell concentrations and biomass levels decline with increasing reactors working volume due to greater consumption of antifoam to prevent foam overflow. The results indicate that when chemical foam control is used in plant cell cultures, head-space volume and tolerable foam levels must be considered to optimize biomass production. (c) 1994 John Wiley & Sons, Inc.     

Preliminary validation of an activation assay for ex vivo activated T cells utilized in cancer immunotherapy

An in vitro assay that measures the activation level of ex vivo activated (EVA) T cells currently being used in the adoptive immunotherapy of metastatic renal cell carcinoma has been developed. This assay is based on the ability of activated, but not resting. T cells to proliferate in response to the protein kinase C activator, phorbol myristate (PMA). To utilize this assay for in-process monitoring and control, we have begun an initial validation of the overall reproducibility of this assay. The proliferation of activated T cells in response to PMA, as measured by the mean cpm values of (3)H-thymidine incorporated, was demonstrated to have intra-assay coefficients of variation (cv's) for individual analysts that were typically less than 10% and rarely exceeded 20%. Activated T cells could be frozen and stored for at least 6 weeks with little or no deterioration in their ability to proliferate in response to PMA. Using these cells, inter-assay cv's that were typically less than 15% were obtained by individual analysts, and overall cv's of 10% to 25% were obtained for different samples assayed by different analysts at different times. This level of variability is very reasonable for a cellular assay. Furhter validation of this assay will address the issues of sensitivity, linearity and selectivity. To date, this assay has been used to analyze over 90 patient EVA cell samples and has revealed a broad range of proliferative responses to PMA. Taken together, these results suggest that this assay may be useful in defining the potency of the activated T cell used therapeutically.     

Characterization of a recombinant antibody produced in the course of a high yield fed-batch process

Many mammalian cell fed-batch processes rely on maintaining the cells in a viable and productive state for extended periods of time in order to reach high final concentrations of secreted protein. In the work described herein, a nonamplified NSO cell line was transfected with a vector expressing a recombinant human anti-HIV gp 120 monoclonal antibody (Mab) and a selectable marker, glutamine synthetase. A fed-batch process was developed which improved product yields tenfold over the yields reached in batch culture. In this case, the clone was cultured for a period of 22 days and produced 0.85 g Mab/L. To gauge the effect of extended culture lifetime on product quality, biochemical characteristics of MAb isolated from different time points in the fed-batch culture were determined. The apparent molecular weight of the MAb was constant throughout the course of the culture. Isoelectric focusing revealed four major charged species, with a fifth more acidic species appearing later in the culture. The antigen binding kinetics were constant for MAb isolated throughout the culture period. Glycosylation analysis, on the other hand, revealed that MAb produced later in the culture contained greater percentages of truncated N-acetylglucosamine and highmannose N-glycans. Possible contributions to this underglycosylated material from either cell lysis or synthesis from noviable cells were found to be negligible. Instead, the viable cells appeared to be secreting more truncated and high mannose MAb glycoforms as the culture progressed. (c) 1994 John Wiley & Sons, Inc.     

Predictions for oxygen supply control to enhance population stability of engineered production strains

The simultaneous growth and product formation in a microbial culture is an important feature of several laboratory, industrial, and environmental bioprocesses. Metabolic burden associated with product formation in these bioprocesses may lead to growth advantage of a nonproducing mutant leading to a loss of the producing population over time. A simple population dynamics model demonstrates the extreme sensitivity of population stability to the engineered productivity of a strain. Here we use flux balance analysis to estimate the effects of the metabolic burden associated with product secretion on optimal growth rates. Comparing the optimal growth rates of the producing and nonproducing strains under a given processing condition allows us to predict the population stability. In order to increase stability of an engineered strain, we determine processing conditions that simultaneously maximize the growth rate of the producing population while minimizing the growth rate of a nonproducing population. Using valine, tryptophan, and lysine production as specific examples, we demonstrate that although an appropriate choice of oxygenation may increase culture longevity more than twofold, total production as governed by economic criterion can be increased by several orders of magnitude. Choice of optimal nutrient and oxygen supply rates to enhance stability is important both for strain screening as well as for culture of engineered strains. Appropriate design of the culture environment can thus be used to enhance the productivity of bioprocesses that use engineered production strains. (c) 1994 John Wiley & Sons, Inc.     

Scaleup of spinfilter perfusion bioreactor for mammalian cell retention

A spinning cylindrical filter, known as a spinfilter, permits the mammalian cell bioreactor operation at high perfusion rates leading to very high cell densities (10(7) mL(-1)). Filter screens with openings (25 mum) slightly larger than the average cell size have been used to retain single cells in suspension over a long period of operation without clogging. We have previously shown why it is necessary to optimize the rotational speed of the spinfilter in order to achieve efficient cell retention and avoid potential screen clogging. Effects of bulk mixing and perfusion rate on screen fouling and cell retention were also investigated. Based on this analysis, in this article, we suggest strategies for scaleup of spinfilters. Experimental data from 12- and 175-L (working volume) bioreactors is shown in support of the scaleup analysis. (c) 1994 John Wiley & Sons, Inc.