Experimental evaluation of laminar shear stress on the behaviour of hybridoma mass cell cultures,producing monoclonal antibodies against mitochondrial creatine kinase

In biotechnology, the interest in mass cultures of animal cells rises constantly; thus knowledge of the conditions influencing the cultures becomes important. Suspension cell cultures in bioreactors are subject to considerable shear forces, when stirring devices are employed. In order to get reliable data on the influence of shear forces on proliferation and antibody production of hybridoma cells, we tested cells, producing monoclonal antibodies against mitochondrial creatine kinase (Mi-CK), under laminar flow conditions in a rotating viscosimeter. Flow conditions in the annular gap were characterized by measuring the speed of revolution of the inner cylinder and the torque. From these data the shear stress could be determined. To confirm the laminar flow condition the velocity profile was determined by laser Doppler anemometry (LDA). After exposure to shear stress, cells were tested for viability, growth rate, antibody production as well as for the glucose uptake and lactate production rates. The data showed that cell death increases as a function of shear stress. The cells, remaining viable after exposure to shear stress showed growth and production rates similar to untreated cells.     

Crossflow microfiltration of animal cells

Laminar shear is the primary mechanism of cell damage, limiting flow rate (and hence flux) in crossflow microfiltration of animal cells. Sensitivity to hydrodynamic and interfacial stress is reduced by the addition of 0.1% Pluronic polyol. A critical average wall shear rate of 3000 s(-1) (above which damage occurs) is found for several cell types, including mammalian and insect cells. Hydrodynamic stress also limits the maximum tip speed in a rotary lobe pump to less than 350 cm/s. Turbulent flow in the recirculation loop piping at Reynolds numbers of up to 71,000 does not cause cell damage. Maximum sustainable flux decreases with cell concentration and increases with cell size (in qualitative agreement with the hydrodynamic lift model). A flux of 30 to 75 L/m(2) h (depending on cell size) can be sustained during 20-fold concentration from 2.5 x 10(6) cells/ml, while maintaining high cell viability.     

Kinetic analysis of hybridoma cells viability under mechanical shear stress with and without serum protection

The effect of a well-defined mild shear stress on hybridoma cell viability (HB-8852) in a serum-free culture medium has been analysed, and the role as shear protector of different concentrations of fetal bovine serum have been studied. Samples harvested from cultures in their late exponential growth phase, were subjected in a rheometer to a constant shear stress of 0.41 ± 0.02 Pa, and the evolution of viable and total cell concentrations was determined and compared with static controls. A simple segregated kinetic model for the viable and dead cells was used to know the effect of serum concentration on the specific cell growth and death rate of the cells.     

Reactive oxygen species induced by shear stress mediate cell death in Bacillus subtilis

Exposure of Bacillus subtilis to a shear rate of 1,482/s leads to a rapid loss of cell viability after 10 h of growth. Biochemical and molecular evidences provided below strongly suggest that cell death under high shear results from an apoptosis-like process similar to that described in eukaryotes, with activation of a caspase-3-like protease (C(3)LP) followed by DNA fragmentation. Shear stress leads to an increase in specific intracellular reactive oxygen species (siROS), possibly through activation of NADH oxidase (NOX). The formation of siROS precedes the activation of C(3)LP and DNA fragmentation, thus establishing siROS as the molecular link between shear stress and apoptosis-like cell death. A model is proposed in which NOX is viewed as being strategically placed on the plasma membrane of B. subtilis that senses and converts a mechanical force arising from shear stress into a chemical signal leading to activation of C(3)LP, DNA fragmentation, and thus, apoptosis-like cell death.     

Modeling shear‐induced CHO cell damage in a rotary positive displacement pump

Rotary lobe pumps are commonly used in the biotechnology industry for a variety of purposes. Shear damage to animal cells within the rotary lobe pump can adversely affect the product yield or purity during, for example, cell concentration via cross‐flow filtration. In this research, CHO cells grown in 20‐L bioreactors were fed to a rotary lobe pump in both single pass and recycle experiments were conducted at different RPMs and “slip” conditions. The results indicate that the slip flow rate more severely impacts the viability of the CHO cells than the pump RPM. A novel mathematical modeling approach is presented that predicts shear rates in all of the positive displacement pump's slip regions, and then predicts cell death vs. operating conditions. This model accounts for the complex flow situation that results from changes to RPM, backpressure and pump geometry (i.e., clearances). © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Modeling of flow‐induced shear stress applied on 3D cellular scaffolds: Implications for vascular tissue engineering

Novel tissue‐culture bioreactors employ flow‐induced shear stress as a means of mechanical stimulation of cells. We developed a computational fluid dynamics model of the complex three‐dimensional (3D) microstructure of a porous scaffold incubated in a direct perfusion bioreactor. Our model was designed to predict high shear‐stress values within the physiological range of those naturally sensed by vascular cells (1–10 dyne/cm²), and will thereby provide suitable conditions for vascular tissue‐engineering experiments. The model also accounts for cellular growth, which was designed as an added cell layer grown on all scaffold walls. Five model variants were designed, with geometric differences corresponding to cell‐layer thicknesses of 0, 50, 75, 100, and 125 µm. Four inlet velocities (0.5, 1, 1.5, and 2 cm/s) were applied to each model. Wall shear‐stress distribution and overall pressure drop calculations were then used to characterize the relation between flow rate, shear stress, cell‐layer thickness, and pressure drop. The simulations showed that cellular growth within 3D scaffolds exposes cells to elevated shear stress, with considerably increasing average values in correlation to cell growth and inflow velocity. Our results provide in‐depth analysis of the microdynamic environment of cells cultured within 3D environments, and thus provide advanced control over tissue development in vitro. Biotechnol. Bioeng. 2010; 105: 645–654. © 2009 Wiley Periodicals, Inc.     

Fluid shear effects on suspension cultures of Morinda citrifolia

The shear susceptibility of cell suspension cultures of the plant cell Morinda citrifolia was investigated by subjecting the cells to the well-defined shear field generated in turbulent flow through a capillary. Suspensions were circulated using a peristaltic pump and average shear stresses between 25 and 350 N m(-2) were generated in the capillary test section. Control experiments were performed to assess the possible contribution of the peristaltic pump to the observed cell damage. There was clear evidence of pump-induced damage at the more severe test conditions and all viability measurements were corrected accordingly. Both shake flask suspension cultures (aged between 9 and 15 days) and repeated batch fermentation cultures, grown in a stirred tank reactor (STR) under a variety of controlled agitation conditions, were tested in the capillary shear loop. The cell damage incurred was evaluated in terms of suspension viability, as determined by a dye exclusion technique. Viability loss was found to conform closely to a first-order model in which the rate constant was observed to increase with the imposed shear stress. Furthermore, a linear relationship was identified between the specific death constant and the cumulative energy dissipated. Post-shear morphological measurements showed that the chain length distribution is shifted toward markedly lower values. In comparison with shake flask cultures, repeated batch fermentation cultures exhibited a marked increase in sensitivity to capillary shear. Based upon the determined morphological characteristics, this result is primarily attributable to the increased chain lengths characteristic of the repeated batch cultures. (c) 1995 John Wiley & Sons, Inc.     

Controlled shear filtration: A novel technique for animal cell separation.

A novel rotary microfiltration technique specifically suited for the separation of animal cells has been developed. The concept allows the independent adjustment of wall shear stress, transmembrane pressure, and residence time, allowing straightforward optimization of the microfiltration process. By using a smooth, conically shaped rotor, it is possible to establish a controlled shear field in which animal cells experience a significant hydrodynamic lift away from the membrane surface. It is shown in preliminary experiments that shear-induced cell-rupture speeds up membrane clogging and that cell debris poses the most significant problem in harvesting of BHK cell cultures by dynamic microfiltration. However, a threshold value of shear stability exists which depends on the frequency of passing the shear field, the residence time in the shear field, as well as on cell status. By operating close to this threshold value, cell viability can be maintained while concentration polarization is efficiently minimized. By applying this concept, it is possible to attain flux rates several times higher compared to conventional crossflow filtration. Controlled shear filtration (CSF) can be used for batch harvesting as well as for cell retention in high cell density systems. In batch harvesting of hIL-2 from rBHK cell culture, a constant flux rate of 290 L h-1 m-2 has been adjusted without indication of membrane clogging or fouling.     

Shear-induced intracellular loading of cells with molecules by controlled microfluidics

This study tested the hypothesis that controlled flow through microchannels can cause shear-induced intracellular loading of cells with molecules. The overall goal was to design a simple device to expose cells to fluid shear stress and thereby increase plasma membrane permeability. DU145 prostate cancer cells were exposed to fluid shear stress in the presence of fluorescent cell-impermeant molecules by using a cone-and-plate shearing device or high-velocity flow through microchannels. Using a syringe pump, cell suspensions were flowed through microchannels of 50-300 microm diameter drilled through Mylar sheets using an excimer laser. As quantified by flow cytometry, intracellular uptake and loss of viability correlated with the average shear stress. Optimal results were observed when exposing the cells to high shear stress for short durations in conical channels, which yielded uptake to over one-third of cells while maintaining viability at approximately 80%. This method was capable of loading cells with molecules including calcein (0.62 kDa), large molecule weight dextrans (150-2,000 kDa), and bovine serum albumin (66 kDa). These results supported the hypothesis that shear-induced intracellular uptake could be generated by flow of cell suspensions through microchannels and further led to the design of a simple, inexpensive, and effective device to deliver molecules into cells. Such a device could benefit biological research and the biotechnology industry.     

A versatile assay for the accurate,time-resolved determination of cellular viability

A convenient and versatile method for the accurate, time-resolved determination of cellular viability has been developed. The conventional viability indicator fluorescein diacetate (FDA), which is converted to the fluorescent compound fluorescein in living cells, was employed as a viability probe. Fluorescence emission from cells was measured using a spectrofluorimeter equipped with a magnetic stirrer. Using this assay cell suspensions exhibiting densities in the range 0.5 x 10(5) to 2.0 x 10(5) cells displayed a linear response when FDA concentrations less than 12 micro M were employed. To calibrate the method, viability standards were elaborated using different proportions of living and dead cells, and a correlation coefficient for the viability of tobacco BY-2 suspensions was calculated as 0.998. This viability assay was also found to be applicable to Chlamydomonas reinhardtii and Arabidopsis thaliana cultured cells. Using this cell viability assay, kinetic analyses of cell death could be performed. Using the proteinaceous elicitor from Phytophthora cryptogea, cryptogein, to induce cell death in tobacco cell suspensions, values for the maximum velocity of death induction rate (V(max)) and the LD50 (half-maximal velocity or k(1/2)) were calculated as 17.2 (% death/h) and 65 nM, respectively.     

Effect of shear stress on expression of a recombinant protein by Chinese hamster ovary cells

A flow chamber was used to impart a steady laminar shear stress on a recombinant Chinese hamster ovary (CHO) cell line expressing human growth hormone (hGH). The cells were subjected to shear stress ranging from 0.005 to 0.80 N m(-2). The effect of shear stress on the cell specific glucose uptake, cell specific hGH, and lactate productivity rates were calculated. No morphological changes to the cells were observed over the range of shear stresses examined. When the cells were subjected to 0.10 N m(-2) shear in protein-free media without Pluronic F-68, recombinant protein production ceased with no change in cell morphology, whereas control cultures were expressing hGH at 0.35 microg/10(6 )cells/h. Upon addition of the shear protectants, Pluronic F-68 (0.2% [w/v]) or fetal bovine serum (1.0% [v/v] FBS), the productivity of the cells was restored. The effect of increasing shear stress on the cells in protein-free medium containing Pluronic F-68 was also investigated. Cell specific metabolic rates were calculated for cells under shear stress and for no-shear control cultures performed in parallel, with shear stress rates expressed as a percentage of those obtained for control cultures. Upon increasing shear from 0.005 to 0.80 N m(-2), the cell specific hGH productivity decreased from 100% at 0.005 N m(-2) to 49% at 0.80 N m(-2) relative to the no-shear control. A concurrent increase in the glucose uptake rate from 115% at 0.01 N m(-2) to 142% at 0.80 N m(-2), and decreased lactate productivity from 92% to 50%, revealed a change in the yield of products from glucose compared with the static control. It was shown that shear stress, at sublytic levels in medium containing Pluronic F-68, could decrease hGH specific productivity.     

Laminar shear stress enhances endothelial cell survival through a NADPH oxidase 2-dependent mechanism

The beneficial effects of laminar shear stress (LSS) due to blood flow include inhibition of endothelial cell death, but the associated mechanism is not well understood. This issue was addressed in the present study. In a normal growth medium, the endothelial cell death rate was below 5%, but this value increased beyond 30% when the serum was depleted. However, when cells were exposed to LSS during the serum depletion period, cell viability recovered to the levels of the serum-provided cells. The pro-survival effect of LSS was not affected by l-arginine methyl ester, but it was abrogated by apocynin, indicating that NADPH oxidases (NOX) play key roles in the mechanism. The pro-survival effect of LSS was reduced by NOX2 siRNA, but not by NOX4 siRNA. LSS increased the expressions of p47^phox and p67^phox, the subunits of NOX2 complex. These observations suggest that LSS prevents apoptotic death of endothelial cells through a NOX2-dependent mechanism.     

Evaluation of blood antioxidant defense and apoptosis in peripheral lymphocytes on exogenous administration of pineal proteins and melatonin in rats

In view of the significant health impact of oxidative stress and apoptosis dysfunction, and further, because of suggestions that administration of antioxidants might reduce apoptosis rate through up-regulation of body antioxidant defense systems, therefore the purpose of this study was to compare the effect of buffalo (Bubalus bubalis) pineal proteins (PP at 100 μg/kg BW, i.p.) with melatonin (MEL at 10 mg/kg BW, i.p.) on blood (erythrocytes) antioxidant defense system and apoptosis in isolated peripheral blood lymphocytes of female Wistar albino rats. The cell viability index (%) and apoptosis index (%), which are directly related to the apoptosis rate of the cells, were used as dependent measures for inferring PP and MEL activity. The total cell viability index did not differ between rats treated with MEL and PP from control animals. The percentage of apoptotic cell death through fluorescence microscopy also did not change in MEL and PP groups as compared with control. DNA fragmentation as an index of apoptosis was detected with propidium iodide staining and assessed by flow cytometry. Pineal proteins and MEL administration caused significant (p < 0.05) reduction in lipid peroxidation and increased level of catalase, superoxide dismutase, glutathione peroxidase, and glutathione in erythrocytes as compared with control. Interestingly, we did not observe increase in the non-viable cells and percentage of apoptotic cell death in PP-treated group, controls or in animals in which MEL had been administered. Therefore, the present study confirmed the up-regulation of erythrocytes (blood) antioxidant defense systems and absence of adverse effect on rate of apoptosis in PP and MEL-administered rats under absence of stress or toxicant exposure. Hence, these test agents can be tested for further therapeutic values against adverse apoptosis rate under stress or toxicants exposures.     

Cell size and water permeability as determining factors for cell viability after freezing at different cooling rates

This work studied the viabilities of five types of cells (two yeast cells, Saccharomyces cerevisiae CBS 1171 and Candida utilis; two bacterial strains, Escherichia coli and Lactobacillus plantarum; and one human leukemia K562 cell) as a function of cooling rate during freezing. The range of investigated cooling rates extended from 5 to 30,000 degrees C/min. Cell viability was classified into three ranges: (i) high viability for low cooling rates (5 to 180 degrees C/min), which allow cell water outflow to occur completely and do not allow any intracellular crystallization; (ii) low viability for rapid cooling rates (180 to 5,000 degrees C/min), which allow the heat flow to prevail over water outflow (in this case, cell water crystallization would occur as water was flowing out of the cell); (iii) high viability for very high cooling rates (>5,000 degrees C/min), which allow the heat flow to be very rapid and induce intracellular crystallization and/or vitrification before any water outflow from the cell. Finally, an assumption relating cell death to the cell water crystallization as water is flowing out of the cell is made. In addition, this general cell behavior is different for each type of cell and seems to be moderated by the cell size, the water permeability properties, and the presence of a cell wall.     

Microbubble-induced sonoporation involved in ultrasound-mediated DNA transfection in vitro at low acoustic pressures

In the present work, human breast cancer cells MCF-7 mixed with polyethylenimine: deoxyribonucleic acid complex and microbubbles were exposed to 1-MHz ultrasound at low acoustic driving pressures ranging from 0.05 to 0.3 MPa. The sonoporation pores generated on the cell membrane were examined with scanning electron microscopy. The transfection efficiency and cell viability were evaluated with flow cytometry. The results showed that ultrasound sonication under the current exposure condition could generate cell pores with mean size ranging from about 100 nm to 1.25 μm, and that larger sonoporation pores would be generated with the increasing acoustic pressure or longer treatment time, leading to the enhancement of transfection efficiency and the reduction of cell viability. The simulations based on the Marmottant model were performed to test the hypothesis that the microstreaming-induced shear stress might be involved in the mechanisms of the low-intensity ultrasound induced sonoporation. The calculated shear stress resulting from the micro-streaming ranged from 15 to 680 Pa corresponding to the applied acoustic pressures 0.05-0.3 MPa, which is sufficient to induce reversible sonoporation. This study indicates that the shear stress related bio-effects may provide a base for strategies aimed at targeted drug delivery.     

Optimising Cell Aggregate Expansion in a Perfused Hollow Fibre Bioreactor via Mathematical Modelling

The need for efficient and controlled expansion of cell populations is paramount in tissue engineering. Hollow fibre bioreactors (HFBs) have the potential to meet this need, but only with improved understanding of how operating conditions and cell seeding strategy affect cell proliferation in the bioreactor. This study is designed to assess the effects of two key operating parameters (the flow rate of culture medium into the fibre lumen and the fluid pressure imposed at the lumen outlet), together with the cell seeding distribution, on cell population growth in a single-fibre HFB. This is achieved using mathematical modelling and numerical methods to simulate the growth of cell aggregates along the outer surface of the fibre in response to the local oxygen concentration and fluid shear stress. The oxygen delivery to the cell aggregates and the fluid shear stress increase as the flow rate and pressure imposed at the lumen outlet are increased. Although the increased oxygen delivery promotes growth, the higher fluid shear stress can lead to cell death. For a given cell type and initial aggregate distribution, the operating parameters that give the most rapid overall growth can be identified from simulations. For example, when aggregates of rat cardiomyocytes that can tolerate shear stresses of up to are evenly distributed along the fibre, the inlet flow rate and outlet pressure that maximise the overall growth rate are predicted to be in the ranges to (equivalent to to ) and to (or 15.6 psi to 15.7 psi) respectively. The combined effects of the seeding distribution and flow on the growth are also investigated and the optimal conditions for growth found to depend on the shear tolerance and oxygen demands of the cells.     

Shear stress effects on human embryonic kidney cells in Vitro

Human embryonic kidney cells grown as an attached, confluent monolayer on a flat substrate were subjected to steady, uniform laminar flow of medium in a specially designed chamber in which flow patterns and shear stress are accurately defined and controlled. Experiments were performed for shear stress levels ranging from 0.2 to 6.0 N/m(2) with times of exposure to the shear stress ranging from 2 to 24 h. The influence of the shear field was slight at low shear stress (0.26 N/m(2)). Higher stress levels (0.65 N/m(2) and higher) had significant effects on cell morphology, and on the post-shear release of urokinase enzyme. Still higher stress levels (2.6 N/m(2) and higher) caused marked reduction in cell viability. These results may be of interest in addressing practical problems in developing commercial biosynthesis reactors.     

Effects of pH and trace minerals on long-term starvation of Leuconostoc mesenteroides

Laboratory experiments have definitively shown that exopolymer-producing bacteria have the potential to modify the flow of fluids in oil reservoirs to enhance oil production. Once injected into the reservoir, they will be subjected to a wide range of pH values and to starvation resulting from nutrient depletion. For successful field implementation it is necessary to have a fundamental understanding of these effects on the viability of bacteria. This paper addresses the effects of pH and trace minerals on cell viability of Leuconostoc mesenteroides during carbon source depletion. Two different carbon sources were used to grow cells before transferring the cells to starvation conditions: sucrose and a combination of glucose and fructose. These substrates were chosen because L. mesenteroides produces a significant amount of water-insoluble exopolymers (dextran) under sucrose-fed conditions, which may enhance cell survival under harsh conditions. The effects of dextran on the cell viability were tested at different pH values with and without trace minerals. The rate of cell death followed an exponential-decay law for different values of the solution pH. The optimal solution pH for survival was pH 5, whereas cells died rapidly at pH 3 and below and at pH 13 and above. The sucrose-fed cells showed a greater viability than cells fed glucose and fructose for all pH ranges tested. The results indicated that water-insoluble exopolymers help cells survive for longer periods of time under starvation conditions. The effects of trace minerals on cell culturability were tested at two pH values, 4.5 and 7. For both cases, cells showed a greater culturability (smaller decay rate constant) in the presence of trace minerals than without trace minerals. It was also found that the effects of trace minerals on cell culturability were greater for glucose-fructose-fed cells than for sucrose-fed cells. The Michaelis pH function theory was used for comparing the relationships between the cell decay rate and pH.     

Insulin-like growth factor I rescues SH-SY5Y human neuroblastoma cells from hyperosmotic induced programmed cell death

Insulin-like growth factor I (IGF-I) and the type I IGF receptor are widely distributed in developing and adult mammalian nervous systems. In vitro, IGF-I is a mitogen for primary neurons and also for cells from the SH-SY5Y human neuroblastoma cell line, a well-characterized model system of neuronal growth. In the current study, we examined the effects of osmotic stress on SH-SY5Y cell viability and the mechanism by which IGF-I serves as a neuronal osmoprotectant. Within 24 hr, exposure of SH-SY5Y cells to hyperosmotic serum-free media decreased (1) the number of viable cells, (2) the rate of ³H-thymidine incorporation, and (3) cell cycle progression. The inclusion of 10 nM IGF-I with hyperosmotic media prevented the loss of cell viability. The osmoprotective effects of IGF-I were inhibited by α-IR_J, a blocking antibody of the type I IGF receptor. The observed loss of SH-SY5Y cell viability following hyperosmotic shock was due to an induction of programmed cell death as determined by flow cytometry and gel electrophoresis. Our results suggest that IGF-I can protect SH-SY5Y cells from hyperosmotic induced programmed cell death. © 1996 Wiley-Liss, Inc.     

Induction of cell-cycle arrest and apoptosis in human cholangiocarcinoma cells by pristimerin

Pristimerin, a triterpenoid isolated from Celastraceae and Hippocrateaceae, is known to induce cytotoxicity in several cancer cell lines. However, whether pristimerin can induce apoptosis in cholangiocarcinoma cells and the underlying mechanism remain unexplored. We assessed the function of human cholangiocarcinoma QBC and RBE cell lines using various experimental methods such as the cell viability assay to elucidate the viability of cells, flow cytometry to detect the death rate of cells, and Western blot analysis to evaluate the expression of cell cycle-related proteins and autophagy-related proteins. Human cholangiocarcinoma QBC cells were transplanted to nude mice to establish an animal model, and the effect of pristimerin on tumor growth in this model was observed. QBC and RBE cell lines treated with pristimerin (0, 5, 10, and 20 μmol/L) demonstrated the induction of apoptosis in a dose-dependent manner. The cell viability assay revealed a reduction in the cell viability with an increase in the pristimerin concentration. Similarly, flow cytometry revealed a gradual increase in the cell death rate with an increase in the pristimerin concentration. In addition, pristimerin significantly lowered the expression of apoptosis-related proteins (Bcl-2, Bcl-xL, and procaspase-3), but increased the Bax expression. Furthermore, pristimerin resulted in the G0/G1 cell-cycle arrest, reducing the expression of cell cycle-related proteins (cyclin E, CDK2, and CDK4), and increased the expression of autophagy-related proteins (LC3) in QBC cell line. Treatment with pristimerin could inhibit tumor growth in the nude mouse model. Overall, this study suggests the potential effect of pristimerin on the cell-cycle arrest and apoptosis in human cholangiocarcinoma cells.