Limited use of Centritech Lab II Centrifuge in perfusion culture of rCHO cells for the production of recombinant antibody

Perfusion cultures of recombinant Chinese hamster ovary cells, producing recombinant antibody against the S surface antigen of Hepatitis B virus, were carried out in continuous and intermittent mode using a Centritech Lab II Centrifuge. In the continuous perfusion process, despite the absence of shear stress from the pump head, long-term operation was not possible because of continuously repeated exposure to oxygen limitation and low temperature, as well as shear stress from centrifugal force. In the intermittent perfusion processes, the frequency of cell-passage through the centrifuge was substantially reduced, compared with the continuous perfusion mode; however, the degree of reduction could not guarantee stable long-term operation. Although various operating parameters were applied in the intermittent perfusion cultures, high cell densities could not be maintained stably. In a single bioreactor culture system, a specific cell that is returned from the centrifuge to the bioreactor could be transferred from the bioreactor to the centrifuge again in the next cycle. These repetitive damages, caused by shear stress from the pump head and centrifugal force, as well as exposure to suboptimal conditions such as oxygen limitation and low temperature below 37 degrees C, were more serious at higher perfusion rates. Subsequently, damaged cells and dead cells were continuously accumulated in the bioreactor. Culture temperature shift from 37 to 33 degrees C increased antibody concentrations but showed inhibitory effects on cell growth. The negative effects of lowering culture temperature on cell growth overwhelmed the positive effects on antibody production. To protect cells from shear stress, Pluronic F-68 was 2-fold concentrated in the culture medium; nevertheless, a significantly higher concentration of Pluronic F-68 (2 g/L) may have inhibitory effects on cell growth.     

MECHANISMS OF FLUID SHEAR‐INDUCED INHIBITION OF POPULATION GROWTH IN A RED‐TIDE DINOFLAGELLATE1

Net population growth of some dinoflagellates is inhibited by fluid shear at shear stresses comparable with those generated during oceanic turbulence. Decreased net growth may occur through lowered cell division, increased mortality, or both. The dominant mechanism under various flow conditions was determined for the red‐tide dinoflagellate Lingulodinium polyedrum (Stein) Dodge. Cell division and mortality were determined by direct observation of isolated cells in 0.5‐mL cultures that were shaken to generate unquantified fluid shear. Larger volume cultures were exposed to quantified laminar shear in Couette‐flow chambers (0.004–0.019 N·m ^{? 2} shear stress) and to unquantified flow in shaken flasks. In these larger cultures, cell division frequency was calculated from flow cytometric measurements of DNA·cell^?1. The mechanism by which shear inhibits net growth of L. polyedrum depends on shear stress level and growth conditions. Observations on the isolated cells showed that shaking inhibited growth by lowering cell division without increased mortality. Similar results were found for early exponential‐phase cultures exposed to the lowest experimental shear stress in Couette‐flow chambers. However, mortality occurred when a late exponential‐phase culture was exposed to the same low shear stress and was inferred to occur in cultures exposed to higher shear stresses. Elevated mortality in those treatments was confirmed using behavioral, morphological, and physiological assays. The results predict that cell division in L. polyedrum populations will be inhibited by levels of oceanic turbulence common for near‐surface waters. Shear‐induced mortality is not expected unless shear‐stress levels are unusually high or when cellular condition resembles late exponential/stationary phase cultures.     

Effects from shear stress on morphology and growth of early stages of Norway spruce somatic embryos

The shear stress effect on directional expansion of pro embryogenic masses (PEMs) and suspensor cell development of somatic embryos of Norway spruce (Picea abies) at the proliferation stage was studied by a direct and quantitative image analysis system. The experimental system allowed for detailed observations of the effect of hydrodynamic shear stress in rotating and deforming liquid cultures of proliferating Norway spruce somatic embryos. Briefly, somatic embryos at an early development stage comprised only of clusters of meristematic cells without suspensor cells were fixed on an alginate film. The alginate film was affixed on the bottom of a flow cell and the somatic embryos were subjected to laminar flow through the chamber of the flow cell. Magnified images of the cell clusters were collected every 24 h. The image data was processed based on a normalized cross‐correlation method, capable of measuring morphological and size features of individual cell clusters in both temporal and spatial domains. No suspensor cells developed in the cell clusters under shear stress of 140 s^?1 for the duration of the experiments. Cell clusters in the control cultured in stationary liquid conditions developed suspensor cells after 5–9 days in culture. Furthermore, the radial growth of meristematic cell clusters was inhibited by shear rates of 86 and 140 s^?1, corresponding to shear stress of 0.086 and 0.14 N/m², compared to growth under stationary conditions. The shear rate showed a significant negative correlation to growth rate. Control group showed no preference for direction during growth under static conditions. Biotechnol. Bioeng. 2010; 105: 588–599. © 2009 Wiley Periodicals, Inc.     

Effects of shear stress on the growth kinetics of human aortic smooth muscle cells in vitro

After cardiovascular intervention, smooth muscle cells (SMC) are directly exposed to blood flow and thus their behavior might be affected by fluid hemodynamic forces. The aim of this study was to determine the effect of fluid shear stress on the growth rate of SMC. Human aortic smooth muscle cells (hASMC) were seeded on fibronectin-coated glass slides and were exposed to different levels of shear stress using parallel plate flow chambers. After 24 h, cell numbers in the stationary and sheared cultures were measured by a Coulter counter. Results demonstrated that increasing shear stress significantly reduces the proliferation rate of hASMC (P < 0.05). Comparable lactate dehydrogenase levels in the media of stationary and flow cultures provided evidence that the reduction of cell number was not due to cell injury. Proliferating cell nuclear antigen (PCNA) immunofluorescence studies indicated that the cell cultures were not growth arrested 24 h after exposure to shear stress, and that the differences in PCNA staining between stationary control and flow cultures were comparable to the cell counts. (c) 1996 John Wiley & Sons, Inc.     

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.     

Mechanical agitation of hybridoma suspension cultures: Metabolic effects of serum, pluronic F68, and albumin supplements

Metabolic effects of the medium supplements, fetal bovine serum (FBS), Pluronic F68, and bovine serum albumin (BSA) were compared for agitated bioreactor cultures of hybridoma cells. Agitation speeds up to 600 rpm, without entrainment of gas bubbles by sparging or vortex formation, allowed examination of cell interactions with turbulent fluid forces. For cultures in FBS-supplemented RPMI media, there was no significant effect of intense turbulent fluid shear on cell growth, metabolism, or antibody, production. Serum-free cultures (Pluronic F68 or BSA supplements) at 600 rpm demonstrated greatly increased glycolysis rates during exponential growth relative to controls. Nutrient limitations caused increased rates of decline of the viable cell concentrations and a reduction in final antibody titers by around 70%. The Pluronic F68 and BSA supplements did not lead to cell protection by modifying metabolism under conditions of intense turbulent fluid shear. Supplementing the protein-free medium with FBS reduced glycolysis rates in exponential growth phase, but this did not prevent a high rate of viable cell decline and low antibody titers. We concluded that FBS does not have a metabolic effect on cells subjected to intense turbulent fluid shear. Although the agitation conditions employed in this study were more intense than generally required for agitated bioreactor culture of hybridomas, we have demonstrated the importance of considering metabolic effects of turbulent fluid forces on cultures using nutrient-rich basal media, in addition to the considerations of gas bubble effects described by other workers. (c) 1992 John Wiley & Sons, Inc.     

Flow mechanotransduction regulates traction forces, intercellular forces, and adherens junctions

Endothelial cells respond to fluid shear stress through mechanotransduction responses that affect their cytoskeleton and cell-cell contacts. Here, endothelial cells were grown as monolayers on arrays of microposts and exposed to laminar or disturbed flow to examine the relationship among traction forces, intercellular forces, and cell-cell junctions. Cells under laminar flow had traction forces that were higher than those under static conditions, whereas cells under disturbed flow had lower traction forces. The response in adhesion junction assembly matched closely with changes in traction forces since adherens junctions were larger in size for laminar flow and smaller for disturbed flow. Treating the cells with calyculin-A to increase myosin phosphorylation and traction forces caused an increase in adherens junction size, whereas Y-27362 cause a decrease in their size. Since tugging forces across cell-cell junctions can promote junctional assembly, we developed a novel approach to measure intercellular forces and found that these forces were higher for laminar flow than for static or disturbed flow. The size of adherens junctions and tight junctions matched closely with intercellular forces for these flow conditions. These results indicate that laminar flow can increase cytoskeletal tension while disturbed flow decreases cytoskeletal tension. Consequently, we found that changes in cytoskeletal tension in response to shear flow conditions can affect intercellular tension, which in turn regulates the assembly of cell-cell junctions.     

Effect of different concentrations of ortho-phthalaldehyde on biofilms formed by Pseudomonas fluorescens under different flow conditions

The effectiveness of different concentrations of ortho-phthalaldehyde (OPA) in controlling biofilms of Pseudomonas fluorescens formed on stainless steel slides, using flow cell reactors under laminar and turbulent flow, was investigated by determining the variation in mass and respiratory activity. The physical stability of the biofilm with and without exposure to OPA was studied in a rotating device as variation in the mass of the biofilm on the surface after exposure to different rotation velocities. The activity of OPA against bacterial suspended cultures was evaluated in the presence and absence of bovine serum albumin (BSA) in order to evaluate the interference of proteins on the activity of the biocide. The results showed that biofilms formed under different flow conditions had different properties and reacted differently after biocide application. Biofilms formed under laminar flow were more easily inactivated than those formed under turbulent conditions. However, OPA did not promote the detachment of biofilms from the surface. The exposure of biofilms to different shear stress conditions after OPA treatment enhanced removal from the surface, indicating that OPA may weaken the biofilm matrix. The biocide was more effective on suspended cells than on cells grown in biofilms. This fact may be explained by the reaction of the biocide with proteins of the polymeric matrix of the biofilm as suggested by the significant reduction of biocide action on suspended cells in the presence of BSA.     

Effect of Different Concentrations of Ortho-phthalaldehyde on Biofilms Formed by Pseudomonas fluorescens Under Different Flow Conditions

The effectiveness of different concentrations of ortho-phthalaldehyde (OPA) in controlling biofilms of Pseudomonas fluorescens formed on stainless steel slides, using flow cell reactors under laminar and turbulent flow, was investigated by determining the variation in mass and respiratory activity. The physical stability of the biofilm with and without exposure to OPA was studied in a rotating device as variation in the mass of the biofilm on the surface after exposure to different rotation velocities. The activity of OPA against bacterial suspended cultures was evaluated in the presence and absence of bovine serum albumin (BSA) in order to evaluate the interference of proteins on the activity of the biocide. The results showed that biofilms formed under different flow conditions had different properties and reacted differently after biocide application. Biofilms formed under laminar flow were more easily inactivated than those formed under turbulent conditions. However, OPA did not promote the detachment of biofilms from the surface. The exposure of biofilms to different shear stress conditions after OPA treatment enhanced removal from the surface, indicating that OPA may weaken the biofilm matrix. The biocide was more effective on suspended cells than on cells grown in biofilms. This fact may be explained by the reaction of the biocide with proteins of the polymeric matrix of the biofilm as suggested by the significant reduction of biocide action on suspended cells in the presence of BSA.     

Effects of stirring and sparging on cultured hybridoma cells

The response of hybridoma cells to fluid shear caused by stirring and sparging has been investigated in a 2-L turbine-agitated bioreactor. Viable cell count, lactate dehydrogenase (LDH) release, and antibody secretion were measured over the course of batch culture experiments under varied conditions of stirring and gas sparging. The effectiveness of Pluronic F68 as a protective agent in sparged cultures was also studied. Growth was found to be unaffected by stirring of the culture under surface aerated conditions, but gas sparging had a significant detrimental effect on growth and antibody production. The effect of sparging was reduced when cultures were supplemented with Pluronic at a level of 0.4% (w/v). Experimental data were analyzed through formulation of models for LDH release and antibody production. Rates of cell lysis could be estimated by correlating extracellular LDH levels through the model for LDH release. The lysis rate estimated for sparged conditions was sufficiently large to approximately account for the observed decrease in the specific growth rate of the culture. The presence of Pluronic apparently interfered with the LDH release mechanism, so precise estimation of lysis rates under these conditions was not possible. Sparging was found not to have a detrimental effect on antibody production in cultures without Pluronic added. Specific antibody production rates in cultures supplemented with Pluronic were about 25% higher than in sparged cultures without Pluronic added.     

Recruitment of endothelial caveolae into mechanotransduction pathways by flow conditioning in vitro

The luminal surface of rat lung microvascular endothelial cells in situ is sensitive to changing hemodynamic parameters. Acute mechanosignaling events initiated in response to flow changes in perfused lung microvessels are localized within specialized invaginated microdomains called caveolae. Here we report that chronic exposure to shear stress alters caveolin expression and distribution, increases caveolae density, and leads to enhanced mechanosensitivity to subsequent changes in hemodynamic forces within cultured endothelial cells. Flow-preconditioned cells expressed a fivefold increase in caveolin (and other caveolar-residing proteins) at the luminal surface compared with no-flow controls. The density of morphologically identifiable caveolae was enhanced sixfold at the luminal cell surface of flow-conditioned cells. Laminar shear stress applied to static endothelial cultures (flow step of 5 dyn/cm2), enhanced the tyrosine phosphorylation of luminal surface proteins by 1.7-fold, including caveolin-1 by 1.3-fold, increased Ser1179 phosphorylation of endothelial nitric oxide synthase (eNOS) by 2.6-fold, and induced a 1.4-fold activation of mitogen-activated protein kinases (ERK1/2) over no-flow controls. The same shear step applied to endothelial cells preconditioned under 10 dyn/cm2 of laminar shear stress for 6 h and induced a sevenfold increase of total phosphotyrosine signal at the luminal endothelial cell surface enhanced caveolin-1 tyrosine phosphorylation 5.8-fold and eNOS phosphorylation by 3.3-fold over static control values. In addition, phosphorylated caveolin-1 and eNOS proteins were preferentially localized to caveolar microdomains. In contrast, ERK1/2 activation was not detected in conditioned cells after acute shear challenge. These data suggest that cultured endothelial cells respond to a sustained flow environment by directing caveolae to the cell surface where they serve to mediate, at least in part, mechanotransduction responses.     

Generation of reactive oxygen and antioxidant species by hydrodynamically stressed suspensions of Morinda citrifolia

The generation of reactive oxygen species (ROS) by plant cell suspension cultures, in response to the imposition of both biotic and abiotic stress, is well-documented. This study investigated the generation of hydrogen peroxide by hydrodynamically stressed cultures of Morinda citrifolia, over a 5-h period post-stress imposition. Suspensions were exposed to repeated passages through a syringe, under laminar flow conditions, corresponding to cumulative energy dissipation levels of approximately 3-6 J kg-1. Extracellular hydrogen peroxide was detected using a luminol-based chemiluminescence assay. The addition of exogenous hydrogen peroxide facilitated the detection of low levels of hydrogen peroxide in the presence of antioxidants. Immediately after shear exposure, there was evidence of significant antioxidative capacity in the sheared cell cultures, which potentially masked any oxidative burst (OB), but which decreased over the following 40 min. This antioxidative capacity was determined to derive from the shearing process. Trials in which ground cellular debris was added to control suspensions suggested that some of the antioxidative capacity observed in stressed suspensions was directly associated with debris generated by the shearing process. Using UV-vis spectrophotometry and HPLC, stress-related increases in the levels of phenolic compounds were detected in suspension filtrates. Under the stress conditions investigated, maximum hydrogen peroxide levels of 11.5 muM were observed, 5 h after shear exposure. This study emphasizes the importance of considering both oxidative and antioxidative capacities as part of a holistic approach to the determination of the OB in hydrodynamically stressed plant cell suspension cultures.     

Determination of a "critical shear stress level" applied to adherent mammalian cells.

An apparatus for the detailed investigation of the influence of shear stress on adherent BHK cells was developed. Shear forces between 0.0 and 2.5 N m-2 were studied. The influence on cell viability, cell morphology, cell lysis, and cell size was determined. Increasing shear forces as well as increasing exposure duration caused increasing changes in cell morphology and cell death. A "critical shear stress level" was determined.     

Impact of aeration strategy on CHO cell performance during antibody production

Stirred tank bioreactors using suspension adapted mammalian cells are typically used for the production of complex therapeutic proteins. The hydrodynamic conditions experienced by cells within this environment have been shown to directly impact growth, productivity, and product quality and therefore an improved understanding of the cellular response is critical. Here we investigate the sub‐lethal effects of different aeration strategies on Chinese hamster ovary cells during monoclonal antibody production. Two gas delivery systems were employed to study the presence and absence of the air–liquid interface: bubbled direct gas sparging and a non‐bubbled diffusive silicone membrane system. Additionally, the effect of higher gas flow rate in the sparged bioreactor was examined. Both aeration systems were run using chemically defined media with and without the shear protectant Pluronic F‐68 (PF‐68). Cells were unable to grow with direct gas sparging without PF‐68; however, when a silicone membrane aeration system was implemented growth was comparable to the sparged bioreactor with PF‐68, indicating the necessity of shear protectants in the presence of bubbles. The cultures exposed to increased hydrodynamic stress were shown by flow cytometry to have decreased F‐actin intensity within the cytoskeleton and enter apoptosis earlier. This indicates that these conditions elicit a sub‐lethal physiological change in cells that would not be detected by the at‐line assays which are normally implemented during cell culture. These physiological changes only result in a difference in continuous centrifugation performance under high flow rate conditions. Product quality was more strongly affected by culture age than the hydrodynamic conditions tested. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2013.     

Influence of serum level,cell line,flow type and viscosity on flow-induced lysis of suspended mammalian cells

An investigation was conducted of the parametric dependence of cell lysis observed when mammalian cells growing in suspension are subjected intermittently to intense hydrodynamic forces. Two flow devices were tested: one consisting of a sudden contraction into a short length of capillary tubing, in which turbulent flow is obtained, and another consisting of a smoothly converging and diverging tube, in which laminar flow is obtained. Changes in the cell line and the serum level in which the cells were grown and subjected to flow trauma both affected the specific lysis rate (fraction of cells lysed per pass through the flow device) in the turbulent flow device. The threshold value of the average wall shear stress level was approximately the same in the turbulent and laminar flow devices (1500–1800 dyn/cm²). Increasing the viscosity of the medium with 70,000 MW dextran had no effect on the specific lysis rate in either flow device.     

Dynamic adhesion of eryptotic erythrocytes to endothelial cells via CXCL16/SR-PSOX

Suicidal death of erythrocytes, or eryptosis, is characterized by cell shrinkage and cell membrane scrambling leading to phosphatidylserine exposure at the cell surface. Eryptosis is triggered by increase of cytosolic Ca2+ activity, which may result from treatment with the Ca2+ ionophore ionomycin or from energy depletion by removal of glucose. The present study tested the hypothesis that phosphatidylserine exposure at the erythrocyte surface fosters adherence to endothelial cells of the vascular wall under flow conditions at arterial shear rates and that binding of eryptotic cells to endothelial cells is mediated by the transmembrane CXC chemokine ligand 16 (CXCL16). To this end, human erythrocytes were exposed to energy depletion (for 48 h) or treated with the Ca2+ ionophore ionomycin (1 μM for 30 min). Phosphatidylserine exposure was quantified utilizing annexin-V binding, cell volume was estimated from forward scatter in FACS analysis, and erythrocyte adhesion to human vascular endothelial cells (HUVEC) was determined in a flow chamber model. As a result, both, ionomycin and glucose depletion, triggered eryptosis and enhanced the percentage of erythrocytes adhering to HUVEC under flow conditions at arterial shear rates. The adhesion was significantly blunted in the presence of erythrocyte phosphatidylserine-coating annexin-V (5 μl/ml), of a neutralizing antibody against endothelial CXCL16 (4 μg/ml), and following silencing of endothelial CXCL16 with small interfering RNA. The present observations demonstrate that eryptotic erythrocytes adhere to endothelial cells of the vascular wall in part by interaction of phosphatidylserine exposed at the erythrocyte surface with endothelial CXCL16.     

Fabrication and use of a transient contractional flow device to quantify the sensitivity of mammalian and insect cells to hydrodynamic forces

A microfluidic device was fabricated via photolithographic techniques which can create transient elongational and shear forces ranging over three orders of magnitude while still maintaining laminar flow conditions. The contractional fluid flow inside the microfluidic device was simulated with FLUENT (a computational fluid dynamics computer program) and the local deformation forces were characterized with the scalar quantity, local energy dissipation rate. The sensitivities of four cell lines (CHO, HB-24, Sf-9, and MCF7) were tested in the device. The results indicate that all four cell lines are able to withstand relatively intense energy dissipation rates (up to 10(4)-10(5) kW/m(3)), which is orders of magnitude higher than the maximum local energy dissipation rates generated by impellers in bioreactors, but comparable to that associated with small bursting bubbles. While the concept that suspended animal cells are relatively robust with respect to purely hydrodynamic forces in bioprocess equipment is well known, these results quantitatively demonstrate these observations.     

Characterization of flow conditions in 2 L and 20 L wave bioreactors® using computational fluid dynamics

Characterization of flow conditions is of great importance to control cell growth and cell damage in animal cell culture because cell viability is influenced by the flow properties in bioreactors. Alternative reactor types like Wave Bioreactors® have been proposed in recent years, leading to markedly different results in cell growth and product formation. An advantage of Wave Bioreactors® is the disposability of the Polyethylenterephthalet‐bags after one single use (fast setup of new production facilities). Another expected advantage is a lower shear stress compared to classical stirred‐tank reactors, due to the gentle liquid motion in the rocking cellbag. This property would considerably reduce possible cell damage. The purpose of the present study is to investigate in a quantitative manner the key flow properties in Wave Bioreactors®, both numerically and experimentally. To describe accurately flow conditions and shear stress in Wave Bioreactors® using numerical simulations, it is necessary to compute the unsteady flow applying Computational Fluid Dynamics (CFD). Corresponding computations for two reactor scales (2 L and 20 L cellbags) are presented using the CFD code ANSYS‐FLUENT®. To describe correctly the free liquid surface, the present simulations employ the Volume of Fluid (VOF) method. Additionally, experimental measurements have been carried out to determine liquid level, flow velocity and liquid shear stress, which are used as a validation of the present CFD simulations. It is shown that the obtained flows stay in the laminar regime. Furthermore, the obtained shear stress levels are well below known threshold values leading to damage of animal cells. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010