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On the Evaluation of Gas—Liquid Interfacial Effects on Hybridoma Viability in Bubble Column Bioreactors
Abstract and Figures
Sparger aeration, with or without mechanical agitation is the simplest method of providing an oxygen supply. Although earlier workers have demonstrated the sensitivity of mammalian cells to air bubbles, recent successful applications of the airlift principle to hybridoma culture indicate that under some conditions, the cells can withstand these effects. Therefore, this work has as its objective the elucidation of the relationship between gas-liquid interfaces and the survival of mammalian cells. Simple, 0.5 litre bubble columns with sintered discs are used batch-wise to study the effects of sparging on hybridomas and other mammalian cells in suspension culture. The effects of bubble diameters, superficial gas velocities and the non-ionic surfactant, Pluriol PE 6800, are investigated in cultures grown in RPM1 1640 with 5% foetal calf serum and 6 ppm silicone antifoam. From these studies it has become apparent that cell viability and survival in the presence of bubbles depend on: Cell type--Some cell lines are shown to be particularly sensitive to the presence of bubbles, although no gross morphological differences are detected by electron microscopy. Bubble sizes--At a superficial gas velocity of 0.42 X 10(-4)m/s (5 cc/min gas flow rate), small bubbles are shown to be more detrimental to the cells than the larger ones. Bubble frequency/superficial gas velocities--Increasing superficial gas velocities (0.42 X 10(-4) to 8.5 X 10(-4) m/s) result in decreasing cell viability. Where the presence of bubbles is detrimental to cell growth, the addition of the non-ionic surfactant, Pluriol PE 6800, has a concentration dependent protective effect. Surface tension, viscosity and bubble diameter data for typical medium will be presented. A novel application of microscopy to which video film systems can be applied for direct visualisation of the cells in the bubble column has been developed. From these studies, it is indicative that both the geometry of the system and the cell type are important in mammalian cell culture scale-up strategy.
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... Thus the study by McQueen and Bailey (1989) which found that the turbulent shear stresses required to produce significant cell death for hybridomas, to be greater than 15 N m"^ is a useful target. It had been observed in certain medium compositions that cell damage occurs by cells being loosely absorbed to gas-liquid interfaces so that they are exposed to damage as a result of fluid shear (Handa et al, 1987(Handa et al, , 1987aChalmers and Bavarian, 1991 ;Bavarian et al, 1991;Orton and Wang, 1990). Although cells are damaged as bubbles form, burst and coalesce, bubble break-up is the main cause of cell damage (Hua et al, 1993;Cherry and Hulle, 1992, Tramper et al, 1991and Handa-Corrigan, 1989. ...
... An increase in the mass transfer is not required but a reduced coalescence leads to smaller sized bubble. These smaller sized bubbles cause more damage to cells at the gas disengagement zone and have a greater tendency to be carried over into the downcomer, where they can cause an increase in the amount of CO2 (Gray et al 1996;Aunins and Henzler, 1991;Handa et al 1987;Tramper et al, 1986;Chalmers and Bavarian, 1991;Bavarian et al, 1991 ;Orton and Wang, 1990;Newitt et al, 1954;Gamer et al, 1954;Gardner et al, 1990;Murhammer and Goochee, 1988). The base had a fiat rather than curved bottom. ...
This thesis concerns the concept of disposables for use in the manufacture of biopharmaceuticals. A disposable process is where all the equipment in contact with the process is disposable. Specifically disposable bioreactors for microbial fermentation and animal cell culture were investigated. For the disposable microbial bioreactor, the plunging jet reactor was proposed as the best option where the thesis details the design and manufacture for a disposable plunging jet bioreactor for use in a microbial fermentation. The 14 L disposable plunging jet bioreactor was compared with a 5 L stirred tank bioreactor. The comparison was made on both the physical performance of the bioreactors and the resultant performance of the fermentation. The maximum kLa in the plunging jet bioreactor was 0.13 to 0.15 s-1, which was comparable to kLa of 0.12 s-1 in the stirred tank bioreactor. This oxygen transfer was achieved with the stirred tank operating at a maximum power per unit volume of 11.2 to 12.7 kW m-3 whilst the plunging jet bioreactor operated at a lower maximum power per unit volume of 1.4 kW m-3. The plunging jet bioreactor incurred a higher maximum shear rate of 85000 s-1, compared to the stirred tank bioreactor with a maximum shear rate of 13000 to 135000 s-1. The mode of operation to maintain the DOT above 10 to 20 [percent] for the two bioreactors was different. For the plunging jet bioreactor the kLa increases with the OUR so that bioreactor was operated with a fixed power per unit volume. Operating the stirred tank bioreactor at a fixed power per unit volume results in a kLa that is fixed whilst the OUR increases. Thus for the stirred tank the power per unit volume was periodically increased resulting in an instantaneous increase in the kLa. For the plunging jet bioreactor the kLa was affected by the position of nozzle with respect to the outlet. For the plunging jet bioreactor operating at a power per unit volume of 1.4 kW m-3, with the Fab fermentation at an OUR of 5 mol L-1 hr-1; the kLa increased from 0.005 s-1 to 0.045 s-1 as a result of moving the nozzle angle from vertical (0°) to 5° away from the outlet (i.e. to an of angle of -5°). For both the Wild Type and Fab fermentation there were distinct differences between the resultant performance of the fermenations in the stirred tank bioreactor and the plunging jet bioreactor. For the Wild Type fermentation in the plunging jet bioreactor, lysis occurred within the first 140 minutes of growth, whilst in the stirred tank bioreactor the fermentation was grown as per the protocol. Unlike with the Wild Type fermentation, the Fab fermentation was grown in both the bioreactors without lysis, where the growth in the plunging jet bioreactor was attributed to the greater strength of Fab microbial cells compared to the Wild Type microbial cells. Despite the growth of the Fab fermentation in both bioreactors, there were distinct differences in the resultant performance of the fermentation. In the plunging jet bioreactor the Fab fermentation had both a lower maximum OUR and a lower final dry weight. In addition the RQ varied between 0.5 to 1.0, where addition of glycerol resulted in an increase in the RQ. In the stirred tank bioreactor the final concentration of the Fab product was 75 mg L-1 in the solid fraction and 14 to 22 mg L-1 in the supernatant. In the plunging jet bioreactor, the final concentration of the Fab product was 0.005 mg L-1 in the supernant and zero concentration in the solid fraction. This difference in the Fab concentration between the two bioreactors was attributed to shear in the plunging jet bioreactor stripping away the outer polysaccharide layer, so that the Fab product was released and subsequently degraded in the fermentation broth. The thesis suggests that these differences in the resultant performance of the Wild Type and Fab fermentation are attributed to the higher shear rate in the plunging jet bioreactor. Further work must be performed to determine whether reducing the shear rate of the nozzle whilst maintaining the mass transfer and mixing will result in a bioreactor whose resultant performance of the fermentation is comparable with the stirred tank bioreactor. The high turbulence nozzle, developed by Kenyres has been suggested as a potential method of reducing the shear rate in the bioreactor. The plunging jet reactor, bubble column reactor and the airlift reactor were all evaluated as potential disposable animal cell bioreactors. The comparison was made on the basis of measuring the kLa, CO2 stripping and mixing versus the power per unit volume for the three reactors. The targets for the kLa, CO2 stripping and mixing were measured in the airlift at a superficial velocity of 0.01 and 0.015 ms-1 which is the typical maximum superficial velocity range that a large scale industrial airlift operates during a biopharmaceutical process. The thesis concludes that the plunging jet reactor is not suitable as a disposable animal cell bioreactor. A 6 L plunging jet reactor was operating with a maximum power per unit volume of 0.01 kW m-3 which corresponded to a shear rate and stress of 15000 s-1 and 15N m-2 respectively. Whilst the upper kLa and mixing targets of 8.9 hr-1 and 13 seconds respectively were achieved below this maximum power per unit volume, operating at the maximum power per unit volume resulted in CO2 stripping of 0.030 hr-1, which was just within the range of the lower target. Whilst the 6 L plunging jet reactor performance was limited by its CO2 stripping, the principle reason for rejecting the reactor was that there were perceived to be problems in scaling up the reactor. Increasing the scale of the reactor would probably require an increase in the nozzle diameter and the use of multiple nozzles in order to maintain a low shear rate and prevent a 'foam' in the upper part of the bioreactor. Measuring the kLa, mixing and CO2 stripping for an airlift and bubble column both of 26 L scale, showed that at small scale the two reactors had comparable performance. In terms of construction the only difference between the bubble column reactor and the airlift reactor is the presence of a divider in the latter. The proposed design for the disposable airlift shown in section 7.3 has a square cross section so that the divider is diagonal. This insures that the divider is taut and is welded along the main seams of the disposable bag. Thus the airlift's construction is not sufficiently more complex than the bubble column and thus is the preferred option for a disposable animal cell bioreactor. This is because at large scale, published work has shown that the airlift has better mixing and aeration than the bubble column so that the airlift is more scaleable. Further work is required to determine whether the proposed design will result in a 7 day animal cell culture that is comparable with the same animal culture performed in a conventional airlift or stirred tank bioreactor.
... Admittedly, there seems a lack of consideration with regards to humidification control in previously developed systems. Furthermore, perfusion accelerates the evaporation ratio in a velocity-dependent manner (Handa et al., 1987;Sumino and Akiyama, 1987). The present study also disclosed notable medium loss during perfusion in the non-humidified condition, and this may potentially be detrimental to cell viability and growth. ...
Various perfusion bioreactor systems have been designed to improve cell culture with three-dimensional porous scaffolds, and there is some evidence that fluid force improves the osteogenic commitment of the progenitors. However, because of the unique design concept and operational configuration of each study, the experimental setups of perfusion bioreactor systems are not always compatible with other systems. To reconcile results from different systems, the thorough optimization and validation of experimental configuration are required in each system. In this study, optimal experimental conditions for a perfusion bioreactor were explored in three steps. First, an in silico modeling was performed using a scaffold geometry obtained by microCT and an expedient geometry parameterized with porosity and permeability to assess the accuracy of calculated fluid shear stress and computational time. Then, environmental factors for cell culture were optimized, including the volume of the medium, bubble suppression, and medium evaporation. Further, by combining the findings, it was possible to determine the optimal flow rate at which cell growth was supported while osteogenic differentiation was triggered. Here, we demonstrated that fluid shear stress up to 15 mPa was sufficient to induce osteogenesis, but cell growth was severely impacted by the volume of perfused medium, the presence of air bubbles, and medium evaporation, all of which are common concerns in perfusion bioreactor systems. This study emphasizes the necessity of optimization of experimental variables, which may often be underreported or overlooked, and indicates steps which can be taken to address issues common to perfusion bioreactors for bone tissue engineering.
... These create high strain rates and extensional stresses that injure cells [95,100]. Based on energy dissipation, smaller bubbles are more harmful to cells [22,101,102] with damage to insect cells being found to be inversely related to bubble size [99]. Tran conducted numerical simulations to investigate the effect of bubble diameter (0.5-6 mm) and surface tension (0.03-0.072 ...
Fluid forces and their effects on cells have been researched for quite some time, especially in the realm of biology and medicine. Shear forces have been the primary emphasis, often attributed as being the main source of cell deformation/damage in devices like prosthetic heart valves and artificial organs. Less well understood and studied are extensional stresses which are often found in such devices, in bioreactors, and in normal blood circulation. Several microfluidic channels utilizing hyperbolic, abrupt, or tapered constrictions and cross-flow geometries, have been used to isolate the effects of extensional flow. Under such flow cell deformations, erythrocytes, leukocytes, and a variety of other cell types have been examined. Results suggest that extensional stresses cause larger deformation than shear stresses of the same magnitude. This has further implications in assessing cell injury from mechanical forces in artificial organs and bioreactors. The cells’ greater sensitivity to extensional stress has found utility in mechanophenotyping devices, which have been successfully used to identify pathologies that affect cell deformability. Further application outside of biology includes disrupting cells for increased food product stability and harvesting macromolecules for biofuel. The effects of extensional stresses on cells remains an area meriting further study.
... affect cell growth. 13,50 Indeed, air bubbles are readily generated in perfusion bioreactors used for bone tissue engineering due to continuous flow, serum proteins acting as a surfactant and the microporous structure and hydrophobic nature of 3D scaffolds. [51][52][53][54] Therefore, Henry's law was applied to supress bubble formation completely. ...
The fatal determination of bone marrow mesenchymal stem/stromal cells (BMSC) is closely associated with mechano-environmental factors in addition to biochemical clues. The aim of this study was to induce osteogenesis in the absence of chemical stimuli using a custom-designed laminar flow bioreactor. BMSC were seeded onto synthetic microporous scaffolds and subjected to the subphysiological level of fluid flow for up to 21 days. During the perfusion, cell proliferation was significantly inhibited. There were also morphological changes, with F-actin polymerisation and upregulation of ROCK1. Notably, in BMSC subjected to flow, mRNA expression of osteogenic markers was significantly upregulated and RUNX2 was localised in the nuclei. Further, under perfusion, there was greater deposition of collagen type 1 and calcium onto the scaffolds. The results confirm that an appropriate level of fluid stimuli preconditions BMSC towards the osteoblastic lineage on 3D scaffolds in the absence of chemical stimulation, which highlights the utility of flow bioreactors in bone tissue engineering.
... Often serum and a shear protectant, like Pluronic F-68, are added to the cellular suspension to reduce the damages caused by force at the air/liquid interface [41]. They hinder the attachment of the cell on the rising bubble and strengthen the cell membrane [42,45,46]. Interestingly, the hydrophobic nature of shear protectants may also protect the cells by increasing the stability of the foam and lowering its draining [44]. ...
Foam formation in bioreactors (fermenters) and other types of reactors is a highly interesting topic that touches several disciplines. All of the phenomena involved in foam formation have been the subject of many studies, but their relationships are still not obvious to newcomers. This review aimed to give the reader a good background for understanding the various phenomena involved in foam formation, especially in bioreactors. Hopefully, this would give the reader the tools necessary to access any needed information about foaming, a task that can be difficult without such basic knowledge.
Mixing and aeration are normally used to provide a homogeneous, suitable environment for cells in a bioreactor. In spite of the large cumulative experience in the use of bioreactors in the biotechnology industry, there are still challenges when scaling‐up animal cell cultures. Remarkable advances have been achieved over the last two decades in designing and applying mixing and aeration strategies, as well as understanding the effects of hydrodynamic forces on cell cultures.
This section reviews the basic concepts and current practices for mixing and aeration in bioreactors, with focus on stirred tank bioreactors (STR), as most of the processes for recombinant proteins, antibodies, and vaccines involve the use of STR. Current methods of oxygen supply in STR include surface aeration and gas sparging, although the use of perfluorocarbons, oxygen carriers and membranes has been investigated. Sparger design and the role of CO 2 accumulation and removal are discussed. Also, a summary of the main aspects involved in mixing is presented, including tank characteristics, impeller geometry, and power drawn. Finally, this review explores several different approaches that have been taken in order to unveil the response of animal cells to the hydrodynamic forces generated inside bioprocessing devices.
Various types of single-cell analyses are now extensively used to answer many biological questions, and with this growth in popularity, potential drawbacks to these methods are also becoming apparent. Depending on the specific application, workflows can be laborious, low throughput, and run the risk of contamination. Microfluidic designs, with their advantages of being high throughput, low in reaction volume, and compatible with bio-inert materials, have been widely used to improve single-cell workflows in all major stages of single-cell applications, from cell sorting to lysis, to sample processing and readout. Yet, designing an integrated microfluidic chip that encompasses the entire single-cell workflow from start to finish remains challenging. In this article, we review the current microfluidic approaches that cover different stages of processing in single-cell analysis and discuss the prospects and challenges of achieving a full integrated workflow to achieve total single-cell analysis in one device.
This textbook provides an overview on current cell culture techniques, conditions, and
applications specifically focusing on human cell culture. This book is based on lectures,
seminars and practical courses in stem cells, tissue engineering, regenerative medicine and 3D cell culture held at the University of Natural Resources and Life Sciences Vienna BOKU and the Gottfried Wilhelm Leibniz University Hannover, complemented by contributions from international experts, and therefore delivers in a compact and clear way important theoretical, as well as practical knowledge to advanced graduate students on cell culture techniques and the current status of research. The book is written for Master students and PhD candidates in biotechnology, tissue engineering and biomedicine working with mammalian, and specifically human cells. It will be of interest to doctoral colleges, Master- and PhD programs teaching courses in this area of research.
The interactions that can occur between a cell and an adjacent biomaterial in culture are vast and depend on the biochemical, physical, and mechanical properties of the biomaterial. The present chapter will discuss the effects of key biomaterial properties on cell attachment, proliferation, migration, and differentiation in culture. These cell–surface interactions will be discussed in the context of the envisioned clinical application of the biomaterials to facilitate an improved understanding of the “big picture” considerations when studying biomaterial-based technologies in cell culture, with a particular focus on tissue engineering technologies.
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