Gas Bubbles and Their Influence on Microorganisms

Animal-cell damage in sparged bioreactors

Animal cells are affected by hydrodynamic forces that occur in culture vessel, transfer piping, and recovery operations such as microfiltration. Depending on the type, intensity, and duration of the force, and the specifics of the cell, the force may induce various kinds of responses in the subject cells. Both biochemical and physiological responses are observed, including apoptosis and purely mechanical destruction of the cell. This review examines the kinds of hydrodynamic forces encountered in bioprocessing equipment and the impact of those forces on cells. Methods are given for quantifying the magnitude of the specific forces, and the response thresholds are noted for the common types of cells cultured in free suspension, supported on microcarriers, and anchored to stationary surfaces.

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Hydrodynamic damage to animal cells.

Plant cells are perceived to be sensitive to the hydrodynamic environment in conventional bioreactors. Heightened sensitivity, relative to most bacterial cultures, is frequently attributed to larger plant cell sizes, extensive vacuolization and aggregation patterns. Early studies of shear sensitivity focused on cell lysis and/or loss of viability. More recently, an extensive ar- ray of sub-lethal responses has been identified. A fuller understanding of these sub-lytic ef- fects may assist in the optimization of large-scale cultivation conditions. This paper reviews recent work on the hydrodynamic shear sensitivity of plant cell systems, under cultivation conditions and in purpose-built shearing devices. The relevance of different approaches to the characterization of the intensity of a given hydrodynamic environment is discussed. Indicators of cell response to hydrodynamic stress are evaluated. The potential significance of cellular defense mechanisms, observed in response to mechanical stimulants, is identified.

Effects of Hydrodynamic and Interfacial Forces on Plant Cell Suspension Systems

To evaluate the antimicrobial activity of a newly developed foam mouthwash containing a modified lactoperoxidase system in vitro. Biofilms of five bacterial species were developed on hydrophobic and hydrophilic surfaces whilst salivary-based biofilm was grown on tooth enamel. Each surface was exposed to the foam mouthwash or saline in vitro. Optical density and scanning electron microscopy (SEM) was used to determine retention of the biofilm following 5 or 30 s exposure time. The foam mouthwash was active against biofilms formed by S. aureus, K. rhizophila, M. thailandicus, E. coli, and C. violaceum and eliminated significant amount of biofilm from each surface; immature 4 h biofilm was less resistant than 24 h biofilm. A 30 s rinse showed best performance, with removal of up to 66% of biofilm from the hydrophilic surface. SEM imaging confirmed oral biofilm removal from the enamel surface after a 5 s rinse with the foam mouthwash. Foam mouthwash demonstrated a significant impact on growing biofilm when compared against saline solution. Growing biofilms were more susceptible to the action of the foam mouthwash, which justifies after-meal use of the mouthwash when traditional dentifrices may not be accessible. Foam mouthwash can be a convenient on-the-go format of oral care products that can be used after meals or when needed to reduce the risk of biofilm-associated oral conditions.

/pdf/the-antibacterial-efficacy-of-a-foam-mouthwash-and-its-38warv85hj.pdf

The antibacterial efficacy of a foam mouthwash and its ability to remove biofilms.

........................................................................................................ IV NOMENCLATURE ............................................................................................ VI ABBREVIATIONS AND ACRONYMS ........................................................ VI UNITS AND MEASURES ........................................................................... VIII LIST OF FIGURES .............................................................................................. X LIST OF TABLES ........................................................................................ XXIII CHAPTER 1: GENERAL INTRODUCTION ........................................................ 1 1.1 Therapeutic Monoclonal Antibodies .................................................................. 2 1.1.1 Monoclonal Antibodies and Disease Targets ......................................... 2 1.1.2 Mechanisms of Action of Monoclonal Antibodies ................................ 3 1.1.3 Monoclonal Antibody Market ................................................................ 6 1.1.4 Development of Monoclonal Antibodies ............................................... 7 1.1.4.1 Overview of Industrial Cell Culture Process Development ............... 7 1.1.4.2 Cell Line Development ...................................................................... 7 1.1.4.3 Cell Lines used for Monoclonal Antibody Production ...................... 8 1.1.4.4 NS0 Cells ............................................................................................ 9 1.2 Large-Scale Monoclonal Antibody Production ............................................... 13 1.2.1 The Monoclonal Antibody Production Process ................................... 13 1.2.2 Cell Culture in Large-Scale Bioreactors .............................................. 15 1.2.3 Sources of Cellular Damage in Bioreactors ......................................... 17 1.2.4 The Cellular Responses to Damage ..................................................... 20 1.3 Lab-Scale Cell Culture Models ........................................................................ 21 1.3.1 Shake Flasks (Non-Baffled and Baffled) ............................................. 21 1.3.2 Small-Scale Bioreactors ....................................................................... 22 1.4 Methods of Exposing Cells to Damage ............................................................ 24 1.4.1 Laminar Shear Devices ........................................................................ 25 1.4.2 Hydrodynamic Stress Devices ............................................................. 26 1.4.3 Quantification of Damage .................................................................... 27 1.5 Protective Additives for Industrial Cell Culture .............................................. 29 1.5.1 Pluronic F-68 ........................................................................................ 30 1.5.1.1 Pluronic F-68 Protective Mechanisms of Action ............................. 32 1.5.1.2 Issues Associated with Pluronic F-68............................................... 35 1.5.1.3 Alternatives to Pluronic F-68 ........................................................... 37 1.5.2 Serum and BSA .................................................................................... 37 1.5.2.1 Serum Offers Physical and Biological Protection ............................ 38 1.5.2.2 Potential Protective Mechanisms of Action of Serum ..................... 39 1.5.2.3 Serum Albumin ................................................................................ 40 1.5.3 Problems Associated with Serum and Serum Derived Proteins .......... 42 1.6 Study Aims and Objectives .............................................................................. 43 CHAPTER 2: MATERIALS AND METHODS ................................................... 46 2.1 Materials ........................................................................................................... 47 2.1.1 Cell Lines ............................................................................................. 47 2.1.2 Cell Culture Reagents .......................................................................... 47 2.1.3 Media Formulations ............................................................................. 48 2.2 Methods ............................................................................................................ 48 2.2.1 Cell Culture Conditions ....................................................................... 48 2.2.2 AMBRTM15 Mini-Bioreactor System .................................................. 49 2.2.3 NS0 Cell Culture in Baffled Shake Flasks ........................................... 50 2.2.4 Lactate Dehydrogenase Activity Measurement ................................... 54 2.2.5 Nuclear Magnetic Resonance Analysis ................................................ 55 2.2.6 NS0 Cell Lysate Formation .................................................................. 55 2.2.7 SDS-PAGE and Western Blotting ....................................................... 56 2.2.8 BSA Immunoprecipitation (IP) ............................................................ 56 2.2.9 Amplex Red Cholesterol Quantification Assay ................................... 57 2.2.10 Laminar Shear Device .......................................................................... 57 2.2.11 Media Surface Tension Measurement .................................................. 59 2.2.12 Media Viscosity Measurement ............................................................. 59 2.2.13 Determination of Bubble Stabilisation ................................................. 59 2.2.14 Membrane Hydrophobicity Measurement ........................................... 60 2.2.15 Heat Denaturation of BSA ................................................................... 61 2.2.16 Circular Dichorism ............................................................................... 61 CHAPTER 3: DEVELOPMENT OF LAB-SCALE MODELS TO TEST THE PROTECTIVE EFFECT(S) OF BSA ON NS0 CELLS ....................................... 63 3.1 Abstract ............................................................................................................ 64 3.2 Introduction ...................................................................................................... 66 3.3 Investigations into the effects of BSA on NS0 cells under various cell culture conditions in industrial cell culture media ............................................................. 69 3.3.1 BSA does not impact the growth of NS0 cells in non-baffled shake flasks under standard cell culture conditions ....................................... 69 3.3.2 BSA does not impact NS0 cell growth in a sparged and agitated minibioreactor system ................................................................................. 71 3.3.3 NS0 cell growth is reduced in baffled shake flasks at high orbital shaker speeds ........................................................................................ 73 3.4 Testing the effects of reduced Pluronic F-68 concentrations on NS0 cell growth under various cell culture conditions ......................................................... 75 3.4.1 Reduced concentrations of Pluronic F-68 do not impact NS0 cell growth in standard non-baffled shake flask conditions........................ 75 3.4.2 BSA promotes NS0 cell growth in an AMBR15 mini-bioreactor system .............................................................................................................. 77 3.4.3 Increased orbital shaker speeds reduce NS0 cell growth in baffled shake flasks .......................................................................................... 80 3.4.4 The presence of baffles in the shake flasks causes the reduction in NS0 cell growth ............................................................................................ 83 3.4.5 Model 1: Moderate hydromechanical stress ........................................ 85 3.4.6 NS0 cells experience rapid damage at high orbital shaker speeds in baffled shake flasks .............................................................................. 87 3.4.7 Exposure time and initial seeding density affect the damage experienced by NS0 cells under extreme hydromechanical stress in baffled shake flasks .............................................................................. 90 3.4.8 The damage experienced by NS0 cells at high orbital speeds is due to the presence of baffles in the shake flasks ........................................... 92 3.4.9 Model 2: Extreme hydromechanical stress .......................................... 94 3.5 Evaluation of the protective effect(s) of BSA on NS0 cells experiencing hydromechanical stress .......................................................................................... 96 3.5.1 BSA promotes NS0 cell growth under moderate hydromechanical stress in baffled shake flasks ................................................................ 96 3.5.2 BSA affords dose dependent protection for NS0 cells exposed to the extreme hydromechanical stress model ............................................... 99 3.5.3 The protective effect of BSA is redundant in media with high concentrations of Pluronic F-68 ......................................................... 102 3.6 Confirming the protective effect of BSA on mAb-producing NS0 cells experiencing hydromechanical stress ................................................................... 105 3.6.1 BSA offers protection to mAb-producing NS0 cells in industrial media with reduced concentrations of Pluronic F-68 ................................... 105 3.7 Discussion ...................................................................................................... 110 CHAPTER 4: INVESTIGATIONS INTO THE PROTECTIVE MECHANISM(S) O

Gas Bubbles and Their Influence on Microorganisms

Citations

Animal-cell damage in sparged bioreactors

Hydrodynamic damage to animal cells.

Effects of Hydrodynamic and Interfacial Forces on Plant Cell Suspension Systems

The antibacterial efficacy of a foam mouthwash and its ability to remove biofilms.

Characterisation of the fundamental function(s) of bovine serum albumin in the culture of NS0 cells

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