Acoustic cell filter: a proven cell retention technology for perfusion of animal cell cultures

This article is a review highlighting the application of the acoustic filter as a reliable cell retention device during the long-term perfusion of animal cell cultures. Critical operating parameters such as duty cycle, perfusion and re-circulation flow rates, acoustic power and backflush frequency are discussed with regard to influence on the separation efficiency and optimal operating ranges have been identified. Perfusion data gathered from the literature have been complemented with original data from a series of perfusion experiments carried out in the context of industrial projects for industrially relevant cell lines including NS0, HEK-293, SP2-derived hybridoma and insect cells in different serum-supplemented and serum-free media at different perfusion rates and acoustic chamber volumes. Finally, scale-up potential of the acoustic filter for large-scale industrial applications is discussed.     

Development of a shaking bioreactor system for animal cell cultures

The feasibility of using shake flasks to culture animal cells was evaluated using various sizes of cylindrical shaped vessels as bioreactors. It was found that conditions can be optimized so that hybridoma, Chinese Hamster Ovary cells, and insect cells can be efficiently cultured in the shaking reactors to cell densities comparable to that obtained with stirred-jar bioreactors, and the system is scalable to larger volumes for the production of recombinant proteins or cell mass production in the laboratory.     

An on-line method for the reduction of fouling of spin-filters for animal cell perfusion cultures

The main limitation in the use of spin-filters during perfusion cultures of animal cells was revealed to be filter fouling. This phenomenon involves cell-sieve interactions as well as cell attachment to, and growth on, the filter surface. The cell attachment effect has been analysed in the present study during long-term perfusion simulations with CHO animal cells. It was demonstrated that at low filter acceleration, below 6.2 m/s2, a high perfusion rate of 25 cm/h induced rapid filter pore clogging within 3 days, whereas increasing the filter acceleration to 25 m/s2 increased filter longevity from 3 to 25 days, for filters with a pore size of 8.5 microm. Increasing the filter pore size to 14.5 microm improved filter longevity by 84% with less viable and dead cell deposits on the filter surface. However, it was demonstrated that filter longevity was not necessarily dependent on the amount of cell deposit on the filter surface. In the second part of this study, ultrasonic technology was used to reduce filter fouling. Filter vibration, induced by a piezo actuator, improved filter longevity by 113% during CHO cells perfusion cultures.     

Baculovirus expression system scaleup by perfusion of high-density Sf-9 cell cultures

A perfusion system based on a 4-L stirred tank bioreactor and a custom-designed tangential (cross-flow) filter was assembled to realize a scaleup of the Baculovirus Expression Vector System (BEVS). When perfused with 1 to 1.5 vol/day, Spodoptera frugiperda (Sf-9) insect cell cultures grew from 4 x 10(6) to 15 x 10(6) cells/mL over 3 to 4 days. The possibility of maintaining high specific production of recombinant VP6 protein (from bovine rotavirus) after baculovirus infection of the high-density cultures was then assessed. The process consisted of a growth phase in TNMFH + 10% FBS, followed by infection with Bac-BRV6L recombinant baculovirus and a shift to a low-serum (0 to 1%) medium for perfusion during the production phase. Multiple runs were executed, each including a battery of shaker flask controls at various cell densities and serum concentrations. On average, specific rVP6 production in the bioreactor amounted to 76% of that found in 20-mL shaker cultures simulatingthe bioreactor's high cell density, low serum concentration, and medium renewal rate. Mechanical stress generated by cell/medium separation in theperfusion process reduced cell growth rate but had minimal effect on rVP6production. Our results also indicated that serum concentration during the infection phase affected the rVP6 specific production in a cell density-dependent fashion. Although the feasibility of the cell density scale up was demonstrated, optimization is still needed to achieve a truly cost-effective process.     

Hybridoma perfusion system using a sedimentation device

Summary A novel sedimentation method with a spiral decanter was utilized with a bioreactor for propagation of hybridoma cells at high densities. The live cell concentration was increased and cell lysis was greatly reduced in this system compared to a tangential flow hollow fiber perfusion system. The specific monoclonal antibody productivity was higher than that obtained using a hollow fiber perfusion system or in a batch culture. Cell specific productivity usually declined over time in long term experiments. The use of the sedimentation device eliminated progressive deterioration of reactor performance usually associated with a perfusion device.     

A novel scale-down mimic of perfusion cell culture using sedimentation in an automated microbioreactor (SAM)

Continuous upstream processing in mammalian cell culture for recombinant protein production holds promise to increase product yield and quality. To facilitate the design and optimization of large-scale perfusion cultures, suitable scale-down mimics are needed which allow high-throughput experiments to be performed with minimal raw material requirements. Automated microbioreactors are available that mimic batch and fed-batch processes effectively but these have not yet been adapted for perfusion cell culture. This article describes how an automated microbioreactor system (ambr15) can be used to scale-down perfusion cell cultures using cell sedimentation as the method for cell retention. The approach accurately predicts the viable cell concentration, in the range of about 1 × 10⁷ cells/mL for a human cell line, and cell viability of larger scale cultures using a hollow fiber based cell retention system. While it was found to underpredict cell line productivity, the method accurately predicts product quality attributes, including glycosylation profiles, from cultures performed in bioreactors with working volumes between 1 L and 1,000 L. The spent media exchange method using the ambr15 was found to predict the influence of different media formulations on large-scale perfusion cultures in contrast to batch and chemostat experiments performed in the microbioreactor system. The described experimental setup in the microbioreactor allowed an 80-fold reduction in cell culture media requirements, half the daily operator time, which can translate into a cost reduction of approximately 2.5-fold compared to a similar experimental setup at bench scale.     

High cell density perfusion cultures of anchorage-dependent Vero cells in a depth filter perfusion system

A depth filter perfusion system (DFPS) with polypropylene fibers had been demonstrated to support high density cultures of anchorage-independent hybridoma cells. The DFPS provides advantages of high surface-to-volume ratio of 450–600 cm²/cm³, low cost set-up, easy operation and scale-up. To test the feasibility of using DFPS for high density cultures of anchorage-dependent cells, Vero cells were cultivated in the DFPS. Gelatin coating on polypropylene fibers in the DFPS was necessary to promote cell attachment and growth. Dissolved oxygen (DO) concentrations could be controlled by sparging air into the reservoir vessel through a filter sparger. When DO concentration was controlled above 40% of air saturation in the DFPS with 40 m pore size, the maximum cell concentration as estimated on specific lactate production rate, was 3.81×10⁷ cells/ml of the total reactor volume. This viable cell concentration is approximately 18 times higher than that obtained in a T-flask batch culture. Taken together, the results obtained here showed the potential of DFPS for high-density cultures of anchorage-dependent cells.     

Mass spectrometry: A tool for on-line monitoring of animal cell cultures

The magnetic sector mass spectrometer is able to detect oxygen uptake and carbon dioxide production rates from animal cell cultivations performed in 101 biorectors. Such data have not been available with the use of classic exhaust gas analysis applying paramagnetic analyzers and infra-red sensors due to the insensitivity of the apparatus available. In the course of the present work we were able to demonstrate, that the oxygen uptake rate correlates to the number of viable cells. Additionally oxygen uptake rates supplied on-line information about the actual physiology of the cells: When the rates changed during the cultivation process, this immediately indicated the occurrence of limitations of components in the medium. The information could be useful in timing key events, such as performing splits or harvesting the bioreactor.Abbreviations OUR oxygen uptake rate - CDPR carbon dioxide production rate - RQ respiratory quotient This publication is dedicated to the 65 ^th birthday of Prof. Dr. F. Wagner, University of Braunschweig.     

Mini-scale bioprocessing systems for highly parallel animal cell cultures

Animal cells have been used extensively in therapeutic protein production. The growth of animal cells and the expression of therapeutic proteins are highly dependent on the culturing environments. A large number of experimental permutations need to be explored to identify the optimal culturing conditions. Miniaturized bioreactors are well suited for such tasks as they offer high-throughput parallel operation and reduce cost of reagents. They can also be automated and be coupled to downstream analytical units for online measurements of culture products. This review summarizes the current status of miniaturized bioreactors for animal cell cultivation based on the design categories: microtiter plates, flasks, stirred tank reactors, novel designs with active mixing, and microfluidic cell culture devices. We compare cell density and product titer, for batch or fed-batch modes for each system. Monitoring/controlling devices for engineering parameters such as pH, dissolved oxygen, and dissolved carbon dioxide, which could be applied to such systems, are summarized. Finally, mini-scale tools for process performance evaluation for animal cell cultures are discussed: total cell density, cell viability, product titer and quality, substrates, and metabolites profiles.     

Recombinant antibody production by perfusion cultures of rCHO cells in a depth filter perfusion system

Recombinant Chinese hamster ovary cells, producing recombinant antibody against the human platelet, were cultivated in a depth filter perfusion system (DFPS). When perfusion cultures with working volume of 1 L were operated at perfusion rates of 5/d and 6/d, volumetric antibody productivities reached values 28 and 34 times higher than that of batch suspension culture in Erlenmeyer flasks and 43 and 53 times higher than that of batch culture in a controlled stirred tank reactor, respectively. Perfusion cultures in the DFPS showed stable antibody production over the whole culture period of up to 20 days. In the DFPS, inoculated cells in suspension were entrapped in a few hours within the depth filter matrix by medium circulation and retained there until the void space of the filter matrix was saturated by the cultured cells. After cells in the depth filter matrix reached saturation, overgrown viable cells at a perfusion rate of 5/d or 6/d were continuously collected into waste medium at a density of 2-4 x 10(5) cells/mL, which resulted in stable operation at high perfusion rates, maintaining values of process parameters such as glucose/lactate concentration, pH, and dissolved oxygen concentration. Because the DFPS overcomes most drawbacks observed with conventional perfusion systems, it is preferable to be used as a key culture system to produce monoclonal antibody stably for a long culture period.     

Control of IgG glycosylation in CHO cell perfusion cultures by GReBA mathematical model supported by a novel targeted feed,TAFE

The N-linked glycosylation pattern is an important quality attribute of therapeutic glycoproteins. It has been reported by our group and by others that different carbon sources, such as glucose, mannose and galactose, can differently impact the glycosylation profile of glycoproteins in mammalian cell culture. Acting on the sugar feeding is thus an attractive strategy to tune the glycan pattern. However, in case of feeding of more than one carbon source simultaneously, the cells give priority to the one with the highest uptake rate, which limits the usage of this tuning, e.g. the cells favor consuming glucose in comparison to galactose.We present here a new feeding strategy (named ‘TAFE’ for targeted feeding) for perfusion culture to adjust the concentrations of fed sugars influencing the glycosylation. The strategy consists in setting the sugar feeding such that the cells are forced to consume these substrates at a target cell specific consumption rate decided by the operator and taking into account the cell specific perfusion rate (CSPR). This strategy is applied in perfusion cultures of Chinese hamster ovary (CHO) cells, illustrated by ten different regimes of sugar feeding, including glucose, galactose and mannose. Applying the TAFE strategy, different glycan profiles were obtained using the different feeding regimes. Furthermore, we successfully forced the cells to consume higher proportions of non-glucose sugars, which have lower transport rates than glucose in presence of this latter, in a controlled way.In previous work, a mathematical model named Glycan Residues Balance Analysis (GReBA) was developed to model the glycosylation profile based on the fed carbon sources. The present data were applied to the GReBA to design a feeding regime targeting a given glycosylation profile. The ability of the model to achieve this objective was confirmed by a multi-round of leave-one-out cross-validation (LOOCV), leading to the conclusion that the GReBA model can be used to design the feeding regime of a perfusion cell culture to obtain a desired glycosylation profile.     

A fiber-bed bioreactor for anchorage-dependent animal cell cultures: part II. Scaleup potential

A simple hydrodynamic model is introduced to describe the airlift fiber-bed bioreactor, which can enhance the volumetric productivity of anchorage-dependent animal cell cultures. By applying the model, liquid flow rates and volumetric mass transfer coefficients are predicted and are in agreement with experimental measurements. Consequently, the optimal reactor configuration giving the maximal oxygen supply is derived. Also, theoretical scaleup potential of this concentric internal loop reactor is considered for volumes ranging from 10 to 67,000 L with which cell densities of 5.1 x 10(7) and 1.2 x 10(7) cells/cm(3), respectively, can be maintained.     

A novel system for dynamic stretching of cell cultures reveals the mechanobiology for delivering better negative pressure wound therapy

Biomechanics and Modeling in Mechanobiology - Serious wounds, both chronic and acute (e.g., surgical), are among the most common, expensive and difficult-to-treat health problems. Negative pressure...     

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.     

Importance of circadian rhythms in animal cell cultures

Circadian rhythms are a characteristic feature of many cell and organ cultures. Such rhythms may be important in the interpretation of data from cells in culture. More examples of circadian rhythms in tissue culture are badly needed to understand this phenomena. However, they will only be expressed under optimum and well-understood conditions.     

A novel high resolution micro-radiotherapy system for small animal irradiation for cancer research

Micro-radiotherapy (micro-RT) system is specially designed for small animal (cancer cell) irradiation for basic and translational cancer research. We use carbon nanotube (CNT) field emission technology to develop a novel micro-RT system for image-guided high precision irradiation that is similar to the state of the art radiotherapy which our cancer patients receive today at mouse scale. Through the field emission control of its individually addressable x-ray pixel beams the micro-RT system electronically shapes the radiation field and forms intensity modulation pattern. In this paper, we present the development of a carbon nanotube field emission cathode array chip--a key component for our novel micro-RT system. The prototype micro-RT CNT field emission cathode array chip has 5 x 5 individually addressable cathode pixels that are 1 mm in diameter and 2 mm in pitch. An individual CNT cathode pixel is predicted to generate a dose rate in the order of 100 cGy/min at the center of the irradiated mouse based on our Monte Carlo simulation. The temporal and spatial resolutions of the micro-RT system are expected to be approximately ms level and < 2 mm respectively.     

Physical modeling of animal cell damage by hydrodynamic forces in suspension cultures

Physical damage of animal cells in suspension culture, due to stirring and sparging, is coupled with complex metabolic responses. Nylon microcapsules, therefore, were used as a physical model to study the mechanisms of damage in a stirred bioreactor and in a bubble column. Microcapsule breaskage folowed first-order kinetices in all experiments Entrainment of bubbles into the liquid phase in the stirred bioreactor gave more microcapsule breakage. In the bubble column, the bubble bursting zone at gas-liquid interface was primarilu responsible for microcapsule breakage. The forces on the microcapsules were equivalent to an external pressure of approximately 4 x 10(4) N . m(-2), based on the critical microcapsule diameter for survival of 190 mum. A stable foam layer, however, was found to be effective in protecting microcapsules from damage. The microcapsule transport to the gas-liquid interface and entrainment into the foam phase was consistent with flotation by air bubbles. This result implies that additives and operation of bioreactors should be selected to minimize flotation of cells. (c) 1992 John Wiley & Sons, Inc.