Animal cell culture in stirred bioreactors: observations on scale-up

The scale-up of stirred tank bioreactors from 0·02 m³ to a 0·3 m³ commercial plant is discussed for hybridoma suspension cultures. Schemes for dissolved oxygen control with sparged air in serum containing media are described, as well as mechanical breakage of foam in small and large bioreactors. Porous metal spargers (180–200 × 10⁻⁶ m) were found to produce foams which were hard to control. Aeration with larger (≥ 0·001 m) multihole spargers is recommended.

Combined cell damage due to foam formation and control, and possible damage at mechanical seals or submerged bearings, were found to have no measurable effect on cell growth relative to roller bottle production. Hybridomas are shown to withstand significant impeller tip speed ( > 1 m s⁻¹) and fluid turbulence as evidenced by impeller Reynolds numbers in excess of 10⁵. The size of the energy-dissipating terminal eddies was calculated to be greater than ten-fold that of the hybridoma cells. The specific fluid turnover rate was employed as the scale-up criterion.     

Suspension culture of hematopoietic stem cells in stirred bioreactors

Hematopoietic stem cells have applications in bone marrow transplantations for the treatment of hematopoietic disorders. When murine hematopoietic stem cells were cultured in 50 ml stirred bioreactors for 14 d, stem-cell-antigen-1 positive cells (hematopoietic primitive progenitor cells) and long-term culture-initiating cells (hematopoietic stem cells) grew by 5-fold and 4-fold, respectively. These results show the possibility of growing hematopoietic stem cells using a stirred bioreactor.     

Mammalian cell retention devices for stirred perfusion bioreactors

Within the spectrum of current applications for cell culture technologies, efficient large-scale mammalian cell production processes are typically carried out in stirred fed-batch or perfusion bioreactors. The specific aspects of each individual process that can be considered when determining the method of choice are presented. A major challenge for perfusion reactor design and operation is the reliability of the cell retention device. Current retention systems include cross-flow membrane filters, spin-filters, inclined settlers, continuous centrifuges and ultrasonic separators. The relative merits and limitations of these technologies for cell retention and their suitability for large-scale perfusion are discussed. This revised version was published online in August 2006 with corrections to the Cover Date.     

Mammalian cell culture: engineering principles and scale-up

If biological products such as monoclonal antibodies, interferons, vaccines, plasminogen activators and many others are to be obtained at an economic cost from mammalian cells, a number of engineering problems must be solved (Tables 1 and 2). The two most imposing barriers to the scale-up of this technology from those listed are the inability to provide sufficient oxygen to high density cultures of mammalian cells grown in large volumes, and the high cost of serum usage. This review focuses on: (i) techniques used to cultivate mammalian cells; (ii) technical barriers to scale-up; and (iii) comparing methods of producing cells with regards to their ability to overcome these barriers.     

Scale-up of mouse embryonic stem cell expansion in stirred bioreactors

The aim of this study was to develop a robust, quality controlled and reproducible large-scale culture system using serum-free (SF) medium to obtain vast numbers of embryonic stem (ES) cells as a starting source for potential applications in tissue regeneration, as well as for drug screening studies. Mouse ES (mES) cells were firstly cultured on microcarriers in spinner flasks to investigate the effect of different parameters such as the agitation rate and the feeding regimen. Cells were successfully expanded at agitation rates up to 60 rpm using the SF medium and no significant differences in terms of growth kinetics or metabolic profiles were found between the two feeding regimens evaluated: 50% medium renewal every 24 h or 25% every 12 h. Overall, cells reached maximum concentrations of (4.2 ± 0.4) and (5.6 ± 0.8) ×10(6) cells/mL at Day 8 for cells fed once or twice per day; which corresponds to an increase in total cell number of 85 ± 7 and 108 ± 16, respectively. To have a more precise control over culture conditions and to yield a higher number of cells, the scale-up of the spinner flask culture system was successfully accomplished by using a fully controlled stirred tank bioreactor. In this case, the concentration of mES cells cultured on microcarriers increased 85 ± 15-fold over 11 days. Importantly, mES cells expanded under stirred conditions, in both spinner flask and fully controlled stirred tank bioreactor, using SF medium, retained the expression of pluripotency markers such as Oct-4, Nanog, and SSEA-1 and their differentiation potential into cells of the three embryonic germ layers.     

The scale-up of plant cell culture: Engineering considerations

The enormous versatility of plants has continued to provide the impetus for the development of plant tissue culture as a commercial production strategy for secondary metabolites. Unfortunately problems with slow growth rates and low products yields, which are generally non-growth associated and intracellular, have made plant cell culture-based processes, with a few exceptions, economically unrealistic. Recent developments in reactor design and control, elicitor technology, molecular biology, and consumer demand for natural products, are fuelling a renaissance in plant cell culture as a production strategy. In this review we address the engineering consequences of the unique characteristics of plant cells on the scale-up of plant cell culture.Abbreviations a gas-liquid interfacial area per volume - C dissolved oxygen concentration - C^* liquid phase oxygen concentration in equilibrium with the partial pressure of oxygen in the bulk gas phase - K_L overall mass transfer coefficient - k_L liquid film mass transfer coefficient - m_O2 cell maintenance coefficient for oxygen - OTR oxygen transfer rate - OUR oxygen uptake rate - pO₂ partial pressure of oxygen - STR stirred-tank reactor - v.v.m. volume of gas fed per unit operating volume of reactor per minute - X biomass concentration - Y_x/O2 biomass yield coefficient for oxygen - specific growth rate     

Monitoring online biomass with a capacitance sensor during scale-up of industrially relevant CHO cell culture fed-batch processes in single-use bioreactors

Bioprocess and Biosystems Engineering - In 2004, the FDA published a guideline to implement process analytical technologies (PAT) in biopharmaceutical processes for process monitoring to gain...     

Derivation of iPSCs in stirred suspension bioreactors

Induced pluripotent stem cells (iPSCs) are typically derived in adherent culture. Here we report fast and efficient derivation of mouse iPSCs in stirred suspension bioreactors, with and without the use of c-Myc. Suspension-reprogrammed cells expressed pluripotency markers, showed multilineage differentiation in vitro and in vivo, and contributed to the germline in chimeric mice. Suspension reprogramming has the potential to accelerate and standardize iPSC research.     

Physical mechanisms of cell damage in microcarrier cell culture bioreactors

The negative effects of excessive agitation on tissue cells in microcarrier culture have often been ascribed to "shear." Analysis of the fluid mechanics occurring suggests that there are actually three potential damage mechanisms: collisions of a cell-covered microcarrier with other beads, collisions with parts of the reactor (primarily the impeller), and interaction with turbulent eddies the size of the microcarrier beads. Review of the available quantitative information on agitation effects in cell cultures does not establish which mechanism is predominant; the range of experimental variables reported emphasizes power input over the other reactor and impeller parameters. The bead-bead collision model is tentatively supported by the available data, but the other mechanisms may still be significant in some systems. The formation of bead aggregates by cellular bridging provides a parallel means of damaging cells. Breaking of these bridges by any of the three means identified earlier can cause cell destruction and/or the net transfer of cells to formerly bare beads. High concentrations of bridges are favored by lower agitation rates, presumably because the bridges are not as quickly destroyed after formation.     

Aerobic bioreactors: condensers,evaporation rates,scale-up and scale-down

Biotechnology Letters - Hydrodynamics, mixing and shear are terms often used when explaining or modelling scale differences, but other scale differences, such as evaporation, can arise from...     

Scale-up of cell culture bioreactors using biomechatronic design

Scale-up of cell culture bioreactors is a challenging engineering work that requires wide competence in cell biology, mechanical engineering and bioprocess design. In this article, a new approach for cell culture bioreactor scale-up is suggested that is based on biomechatronic design methodology. The approach differs from traditional biochemical engineering methodology by applying a sequential design procedure where the needs of the users and alternative design solutions are systematically analysed. The procedure is based on the biological and technical functions of the scaled-up bioreactor that are derived in functional maps, concept generation charts and scoring and interaction matrices. Basic reactor engineering properties, such as mass and heat transfer and kinetics are integrated in the procedure. The methodology results in the generation of alternative design solutions that are thoroughly ranked with help of the user needs. Examples from monoclonal antibodies and recombinant protein production illuminate the steps of the procedure. The methodology provides engineering teams with additional tools that can significantly facilitate the design of new production methods for cell culture processes.     

Oxygen mass transfer and scale-up studies in baffled roller bioreactors

Oxygen mass transfer was studied in conventional, bead mill and baffled roller bioreactors. Using central composite rotational design, impacts of size, rotation speed and working volume on the oxygen mass transfer were evaluated. Baffled roller bioreactor outperformed its conventional and bead mill counterparts, with the highest k _L a obtained in these configurations being 0.58, 0.19, 0.41 min^?1, respectively. Performances of the bead mill and baffled roller bioreactor were only comparable when a high bead loading (40 %) was applied. Regardless of configuration increase in rotation speed and decrease in working volume improved the oxygen mass transfer rate. Increase in size led to enhanced mass transfer and higher k _L a in baffled roller bioreactor (0.49 min^?1 for 2.2 L and 1.31 min^?1 for 55 L bioreactors). Finally, the experimentally determined k _L a in the baffled roller bioreactors of different sizes fit reasonably well to an empirical correlation describing the k _L a in terms of dimensionless numbers.     

Packed-bed bioreactors for mammalian cell culture: bioprocess and biomedical applications

This article describes the development history of packed-bed bioreactors (PBRs) used for the culture of mammalian cells. It further reviews the current applications of PBRs and discusses the steps forward in the development of these systems for bioprocess and biomedical applications. The latest generation of PBRs used in bioprocess applications achieve very high cell densities (>10(8) cells ml(-1)) leading to outstandingly high volumetric productivity. However, a major bottleneck of such PBRs is their relatively small volume. The current maximal volume appears to be in the range of 10 to 30 l. A scale-up of more than 10-fold would be necessary for these PBRs to be used in production processes. In biomedical applications, PBRs have proved themselves as compact bioartificial organs, but their metabolic activity declines frequently within 1 to 2 weeks of operation. A main challenge in this field is to develop cell lines that grow consistently to high cell density in vitro and maintain a stable phenotype for a minimum of 1 to 2 months. Achieving this will greatly enhance the usefulness of PBR technology in clinical practice.     

Low protein serum-free medium for antibody-production in stirred bioreactors

Summary A chemically defined serum-free medium was modified with respect to protein content by substituting PEG (polyethylen glycol) for BSA-FA (bovine serum albumin fatty acid complex). The outstanding advantage of this low-protein medium is the excellent support of hybridoma-growth and MAb (monoclonal antibody)-production in stirred bioreactors even when inoculated at low cell concentrations. Using this medium, the secreted MAbs were a major part of the total protein. Addition of lipoproteins was in most cases not found to be necessary, and when they were added at higher concentrations even a toxic effect was observed.     

Turbulent breakage of protein precipitates in mechanically stirred bioreactors

Experimental data relating to the breakage of isoelectric Soya protein precipitates in a mechanically agitated bioreactor are provided and examined in the light of a proposed mechanistic model which relates the size of the maximum attainable aggregate diameter to the energy dissipation rate in the vessel. The analysis suggests that protein precipitation results in the formation of scale-invariant fractal aggregates with a dimensionality of 2.2. Comparing the fractal dimensionality of the protein precipitates with reported values based on computer simulation studies suggests that the aggregates undergo considerable restructuring during agitation.List of Symbols A Hamaker constant (J) - D impeller diameter (m) - d _p primary particle diameter (m) - d _f maximum aggregate diameter (m) - G shear rate (s^–1) - H ₀ separation distance between two primary particles (m) - k constant in Eq. (5) - K constant in Eq. (6) - N impeller speed (rpm or rps) - r radial position in an aggregate, measured from the centre (m) - t time of exposure to shear (mins) - T _e eddy period (s^–1) - v _f aggregate volume (m³) Greek Symbols aggregate dimensionality constant - energy dissipation rate (W/kg) - dynamic viscosity of particle-free liquid (kg/ms) - kinematic viscosity of particle-free liquid (m²/s) - collision probability (–) - _p aggregate density (kg/m³) - _p continuous phase density (kg/m³) - aggregate mechanical strength (N/m²) - shear stress (N/m²) - particle concentration in an aggregate (m³/m³) - (r) porosity at radial position, r     

A novel scale-up method for mammalian cell culture in packed-bed bioreactor

A novel method for the scale-up culture of Chinese hamster ovary (CHO) cells in a packed-bed bioreactor is developed wherein microcarriers, attached with CHO cells in a microcarrier culture system, are inoculated directly into the packed-bed bioreactor. Cells continue to grow after inoculation and the maximum cell density reaches about 2×10⁷ cells ml^–1. The method provides a new technique for the scale-up of a packed-bed culture while decreasing the labour cost and ensuring the safety of operation.     

An engineering analysis of rotating sieves for hybridoma cell retention ins stirred tank bioreactors

The use of internal rotating sieves for perfused hybridoma culture offers unique advantages but has been up to now largely empirical. Calculations have been performed on a 15 l spinfilter stirred tank in order to have an idea of hydrodynamic conditions inside and outside the rotating sieve. The large peripheral velocity value, resulting from sieve rotation (compared to axial and radial velocities) is expected to affect strongly sieve surface colonization by cells; this is confirmed by lab scale experiments, showing that cell colonization is prevented providing sieve rotation exceeds a defined value (around 0.6 m.s.1 tip speed); the fluid removal force calculated under these conditions appears to be in the range of 10 pN, similar to the adhesion force already reported for mammalian cells attached to inorganic substrata.