Effect of shear stress on anthraquinones production by Rubia tinctorum suspension cultures

Suspension cultures of Rubia tinctorum, an anthraquinones (AQs) producer, were grown both in Erlenmeyer flasks at 100 rpm and in a 1.5 L mechanically stirred tank bioreactor operating at 450 rpm. The effect of hydrodynamic stress on cell viability, biomass, and AQs production was evaluated. Cell viability showed a transient decrease in the bioreactor during the first days, returning to the initial values toward the end of the culture time. The biomass obtained in the bioreactor was 29% lower than that attained in the Erlenmeyer flasks. The H2O2 production in the bioreactor (with peaks at 7 and 10 days) was about 15 times higher than that obtained in the flasks. A clear relationship exists between the maximum concentration of H2O2 generated and AQs produced. The AQs content in the bioreactor was 233% higher than that in the Erlenmeyer flasks. The AQs specific productivity in the stirred tank and in the Erlenmeyer flasks was 70.7 and 28.5 micromol/g FW/day, respectively. This production capability was maintained in the regrowth assays. On the other hand, the negative effects of hydrodynamic stress on viability and biomass concentration observed in the bioreactor culture were reverted in the regrowth cultures. It can be concluded that R. tinctorum suspension cultures are able to grow in stirred tanks at 450 rpm responding to the hydrodynamic stress with higher concentrations of AQs, which suggest the possibility of a technological approach taking advantage of this phenomenon.     

A shaken disposable bioreactor system for controlled insect cell cultivations at milliliter‐scale

While wave‐mixed and stirred bag bioreactors are common devices for rapid, safe insect cell culture‐based production at liter‐scale, orbitally shaken disposable flasks are mainly used for screening studies at milliliter‐scale. In contrast to the two aforementioned bag bioreactor types, which can be operated with standard or disposable sensors, shaker flasks have not been instrumented until recently. The combination of 250 mL disposable shake flasks with PreSens's Shake Flask Reader enables both pH and dissolved oxygen to be measured, as well as allowing characterization of oxygen mass transfer. Volumetric oxygen transfer coefficients (k_La‐values) for PreSens 250 mL disposable shake flasks, which were determined for the first time in insect cell culture medium at varying culture volumes and shaker frequencies, ranged between 4.4 and 37.9/h. Moreover, it was demonstrated that online monitoring of dissolved oxygen in shake flasks is relevant for limitation‐free growth of insect cells up to high cell densities in batch mode (1.6×10⁷ cells/mL) and for the efficient expression of an intracellular model protein.     

Cultivation of transgenicNicotiana tabacum suspension cells in bioreactors for the production of mGM-CSF

TransgenicNicotiana tabacum cells were cultivated for the production of murine granulocyte macrophage-colony stimulating factor (mGM-CSF) in both a stirred, tank biore|actor and an airlift bioreactor with draft tube. Cell growth and mGM-CSF production in the airlift bioreactor were found to be better than those achieved in the stirred tank bioreactor. In the airlift bioreactor. 9.0 g/L of cells and 2.2 ng/mL of mGM-CSF were obtained (11.0 g/L and 2.4 ng/mL, respectively in shake flasks). Although the lag period was prolonged and mGM-CSF production was lowered by 33% in the stirred tank bioreactor as compared to the control culture, the maximum cell density was increased up to 12.0 g/L due to better mixing by agitation at the higher cell density.     

Advances in clone selection using high‐throughput bioreactors

Effective clone selection is a crucial step toward developing a robust mammalian cell culture production platform. Currently, clone selection is done by culturing cells in well plates and picking the highest producers. Ideally, clone selection should be done in a stirred tank bioreactor as this would best replicate the eventual production environment. The actual number of clones selected for future evaluation in bioreactors at bench‐scale is limited by the scale‐up and operational costs involved. This study describes the application of miniaturized stirred high‐throughput bioreactors (35 mL working volume; HTBRs) with noninvasive optical sensors for clone screening and selection. We investigated a method for testing several subclones simultaneously in a stirred environment using our high throughput bioreactors (up to 12 clones per HTBR run) and compared it with a traditional well plate selection approach. Importantly, it was found that selecting clones solely based on results from stationary well plate cultures could result in the chance of missing higher producing clones. Our approach suggests that choosing a clone after analyzing its performance in a stirred bioreactor environment is an improved method for clone selection. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Effect of bioreactor configuration on the growth and maturation of Picea sitchensis somatic embryo cultures

Somatic embryo suspension cultures of Picea sitchensis (Sitka spruce) derived from two cell lines, SS03 and SS10, were grown in shake flasks, air-lift, bubble, stirred tank and hanging stirrer bar bioreactors. Cell line SS03 yielded freely suspended and individual stage 1 embryos, while the embryos of SS10 were present in large aggregates. Compared to shake flasks, proliferation in bioreactors resulted in increased biomass; however, cell line morphology influenced the effect of different bioreactor configurations on growth and maturation of embryo cultures. Somatic embryos grown in shake flasks and bioreactors were matured on gelled solid medium and in submerged culture where gelled solid medium was covered with a layer of liquid medium. The number of stage 3 (mature) embryos produced from SS03 in the bubble bioreactor was significantly higher than those from stirred tank and hanging stirrer bar bioreactors with both solid medium and submerged culture. Submerged culture was unsuitable for SS10 embryo maturation. This revised version was published online in June 2006 with corrections to the Cover Date.     

High‐throughput miniaturized bioreactors for cell culture process development: Reproducibility,scalability, and control

Decreasing the timeframe for cell culture process development has been a key goal toward accelerating biopharmaceutical development. Advanced Microscale Bioreactors (ambr?) is an automated micro‐bioreactor system with miniature single‐use bioreactors with a 10–15 mL working volume controlled by an automated workstation. This system was compared to conventional bioreactor systems in terms of its performance for the production of a monoclonal antibody in a recombinant Chinese Hamster Ovary cell line. The miniaturized bioreactor system was found to produce cell culture profiles that matched across scales to 3 L, 15 L, and 200 L stirred tank bioreactors. The processes used in this article involve complex feed formulations, perturbations, and strict process control within the design space, which are in‐line with processes used for commercial scale manufacturing of biopharmaceuticals. Changes to important process parameters in ambr? resulted in predictable cell growth, viability and titer changes, which were in good agreement to data from the conventional larger scale bioreactors. ambr? was found to successfully reproduce variations in temperature, dissolved oxygen (DO), and pH conditions similar to the larger bioreactor systems. Additionally, the miniature bioreactors were found to react well to perturbations in pH and DO through adjustments to the Proportional and Integral control loop. The data presented here demonstrates the utility of the ambr? system as a high throughput system for cell culture process development. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:718–727, 2014     

Demonstrating the Manufacture of Human CAR‐T Cells in an Automated Stirred‐Tank Bioreactor

Chimeric antigen receptor T‐cell (CAR‐T) therapies have proven clinical efficacy for the treatment of hematological malignancies. However, CAR‐T cell therapies are prohibitively expensive to manufacture. The authors demonstrate the manufacture of human CAR‐T cells from multiple donors in an automated stirred‐tank bioreactor. The authors successfully produced functional human CAR‐T cells from multiple donors under dynamic conditions in a stirred‐tank bioreactor, resulting in overall cell yields which were significantly better than in static T‐flask culture. At agitation speeds of 200 rpm and greater (up to 500 rpm), the CAR‐T cells are able to proliferate effectively, reaching viable cell densities of >5 × 10⁶ cells ml‐1 over 7 days. This is comparable with current expansion systems and significantly better than static expansion platforms (T‐flasks and gas‐permeable culture bags). Importantly, engineered T‐cells post‐expansion retained expression of the CAR gene and retained their cytolytic function even when grown at the highest agitation intensity. This proves that power inputs used in this study do not affect cell efficacy to target and kill the leukemia cells. This is the first demonstration of human CAR‐T cell manufacture in stirred‐tank bioreactors and the findings present significant implications and opportunities for larger‐scale allogeneic CAR‐T production.     

Monoterpenoid oxindole alkaloid production by Uncaria tomentosa (Willd) D.C. cell suspension cultures in a stirred tank bioreactor

Cell growth, monoterpenoid oxindole alkaloid (MOA) production, and morphological properties of Uncaria tomentosa cell suspension cultures in a 2-L stirred tank bioreactor were investigated. U. tomentosa (cell line green Uth-3) was able to grow in a stirred tank at an impeller tip speed of 95 cm/s (agitation speed of 400 rpm), showing a maximum biomass yield of 11.9 +/- 0.6 g DW/L and a specific growth rate of 0.102 d(-1). U. tomentosa cells growing in a stirred tank achieved maximum volumetric and specific MOA concentration (467.7 +/- 40.0 microg/L, 44.6 +/- 5.2 microg/g DW) at 16 days of culture. MOA chemical profile of cell suspension cultures growing in a stirred tank resembled that of the plant. Depending on culture time, from the total MOA produced, 37-100% was found in the medium in the bioreactor culture. MOA concentration achieved in a stirred tank was up to 10-fold higher than that obtained in Erlenmeyer flasks (agitated at 110 rpm). In a stirred tank, average area of the single cells of U. tomentosa increased up to 4-fold, and elliptical form factor increased from 1.40 to 2.55, indicating enlargement of U. tomentosa single cells. This work presents the first report of U. tomentosa green cell suspension cultures that grow and produce MOA in a stirred tank bioreactor.     

Verification of energy dissipation rate scalability in pilot and production scale bioreactors using computational fluid dynamics

Suspension mammalian cell cultures in aerated stirred tank bioreactors are widely used in the production of monoclonal antibodies. Given that production scale cell culture operations are typically performed in very large bioreactors (≥ 10,000 L), bioreactor scale‐down and scale‐up become crucial in the development of robust cell‐culture processes. For successful scale‐up and scale‐down of cell culture operations, it is important to understand the scale‐dependence of the distribution of the energy dissipation rates in a bioreactor. Computational fluid dynamics (CFD) simulations can provide an additional layer of depth to bioreactor scalability analysis. In this communication, we use CFD analyses of five bioreactor configurations to evaluate energy dissipation rates and Kolmogorov length scale distributions at various scales. The results show that hydrodynamic scalability is achievable as long as major design features (# of baffles, impellers) remain consistent across the scales. Finally, in all configurations, the mean Kolmogorov length scale is substantially higher than the average cell size, indicating that catastrophic cell damage due to mechanical agitation is highly unlikely at all scales. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:760–764, 2014     

New milliliter‐scale stirred tank bioreactors for the cultivation of mycelium forming microorganisms

A novel milliliter‐scale stirred tank bioreactor was developed for the cultivation of mycelium forming microorganisms on a 10 milliliter‐scale. A newly designed one‐sided paddle impeller is driven magnetically and rotates freely on an axis in an unbaffled reaction vessel made of polystyrene. A rotating lamella is formed which spreads out along the reactor wall. Thus an enhanced surface‐to‐volume ratio of the liquid phase is generated where oxygen is introduced via surface aeration. Volumetric oxygen transfer coefficients (k_La) > 0.15 s^?1 were measured. The fast moving liquid lamella efficiently prevents wall growth and foaming. Mean power consumption and maximum local energy dissipation were measured as function of operating conditions in the milliliter‐scale stirred tank bioreactor (V = 10 mL) and compared to a standard laboratory‐scale stirred tank bioreactor with six‐bladed Rushton turbines (V = 2,000 mL). Mean power consumption increases with increasing impeller speed and shows the same characteristics and values on both scales. The maximum local energy dissipation of the milliliter‐scale stirred tank bioreactor was reduced compared to the laboratory‐scale at the same mean volumetric power input. Hence the milliliter impeller distributes power more uniformly in the reaction medium. Based on these data a reliable and robust scale‐up of fermentation processes is possible. This was demonstrated with the cultivation of the actinomycete Streptomyces tendae on both scales. It was shown that the process performances were equivalent with regard to biomass concentration, mannitol consumption and production of the pharmaceutical relevant fungicide nikkomycin Z up to a process time of 120 h. A high parallel reproducibility was observed on the milliliter‐scale (standard deviation < 8%) with up to 48 stirred tank bioreactors operated in a magnetic inductive drive. Rheological behavior of the culture broth was measured and showed a highly viscous shear‐thinning non‐Newtonian behavior. The newly developed one‐sided paddle impellers operated in unbaffled reactors on a 10 milliliter‐scale with a magnetic inductive drive for up to 48 parallel bioreactors allows for the first time the parallel bioprocess development with mycelium forming microorganisms. This is especially important since these kinds of cultivations normally exhibit process times of 100 h and more. Thus the operation of parallel stirred tank reactors will have the potential to reduce process development times drastically. Biotechnol. Bioeng. 2010; 106: 443–451. © 2010 Wiley Periodicals, Inc.     

Production of anti‐inflammatory compounds in Sphaeralcea angustifolia cell suspension cultivated in stirred tank bioreactor

Sphaeralcea angustifolia is a plant used for the treatment of inflammatory processes. Scopoletin, tomentin, and sphaeralcic acid were identified as the compounds with anti‐inflammatory and immunomodulatory effects. Successful establishment of the cell culture in Erlenmeyer flasks has been reported previously. The aim of this study was to evaluate the ability of cells in suspension from S. angustifolia grown in a stirred tank bioreactor and demonstrate their capacity to produce bioactive compounds. Cells in suspension grown at 200 rpm reached a maximal cell biomass in dry weight at 19.11 g/L and produced 3.47 mg/g of sphaeralcic acid. The mixture of scopoletin and tomentin was only detected at the beginning of the culture (12.13 μg/g). Considering that the profile of dissolved oxygen during the cultures was lesser than 15%, it is possible that the low growth at 100 rpm could be due to oxygen limitations or to cell sedimentation. At 400 rpm, a negative effect on cell viability could be caused by the increase in the hydrodynamic stress, including the impeller tip, average shear rate, and Reynolds number. The sphaeralcic acid content in the cell suspension of S. angustifolia obtained in the bioreactor was two orders of magnitude greater than that reported for the culture grown in Erlenmeyer flasks.     

Fast dynamic response of the fermentative metabolism of Escherichia coli to aerobic and anaerobic glucose pulses

The response of Escherichia coli cells to transient exposure (step increase) in substrate concentration and anaerobiosis leading to mixed‐acid fermentation metabolism was studied in a two‐compartment bioreactor system consisting of a stirred tank reactor (STR) connected to a mini‐plug‐flow reactor (PFR: BioScope, 3.5 mL volume). Such a system can mimic the situation often encountered in large‐scale, fed‐batch bioreactors. The STR represented the zones of a large‐scale bioreactor that are far from the point of substrate addition and that can be considered as glucose limited, whereas the PFR simulated the region close to the point of substrate addition, where glucose concentration is much higher than in the rest of the bioreactor. In addition, oxygen‐poor and glucose‐rich regions can occur in large‐scale bioreactors. The response of E. coli to these large‐scale conditions was simulated by continuously pumping E. coli cells from a well stirred, glucose limited, aerated chemostat (D = 0.1 h^?1) into the mini‐PFR. A glucose pulse was added at the entrance of the PFR. In the PFR, a total of 11 samples were taken in a time frame of 92 s. In one case aerobicity in the PFR was maintained in order to evaluate the effects of glucose overflow independently of oxygen limitation. Accumulation of acetate and formate was detected after E. coli cells had been exposed for only 2 s to the glucose‐rich (aerobic) region in the PFR. In the other case, the glucose pulse was also combined with anaerobiosis in the PFR. Glucose overflow combined with anaerobiosis caused the accumulation of formate, acetate, lactate, ethanol, and succinate, which were also detected as soon as 2 s after of exposure of E. coli cells to the glucose and O₂ gradients. This approach (STR‐mini‐PFR) is useful for a better understanding of the fast dynamic phenomena occurring in large‐scale bioreactors and for the design of modified strains with an improved behavior under large‐scale conditions. Biotechnol. Bioeng. 2009; 104: 1153–1161. © 2009 Wiley Periodicals, Inc.     

Evaluation of a novel pneumatic bioreactor system for culture of recombinant Chinese hamster ovary cells

Single use culture systems are a tool in research and biotechnology manufacturing processes and are employed in mammalian cell-based manufacturing processes. Recently, we characterized a novel bioreactor system developed by PBS Biotech. The Pneumatic Bioreactor System? (PBS) employs the Air-wheel?, which is a mixing device similar in structure to a water wheel but is driven by the buoyant force of gas bubbles. In this study, we investigated the physical properties of the PBS system, with which we performed biological tests. In 2 L PBS, the mixing times ranged from 6 (30 rpm, 0.175 vvm) to 15 sec (10 rpm, 0.025 vvm). The k_La value reached upto 7.66/h at 0.5 vvm, even without a microsparger, though this condition is not applicable for cell cultures. Also, when a 10 L PBS equipped with a microsparger was evaluated, a k_La value of upto approximately 20/h was obtained particularly in mild cell culture conditions. We performed cultivation of Chinese hamster ovary (CHO) cells in 2 and 10 L PBS prototypes. Results from the PBS were compared with those from an Erlenmeyer flask and conventional stirred tank type bioreactor (STR). The maximum cell density of 10.6 × 106 cells/mL obtained fromthe 2 L PBSwas about 2 times higher than that from the Erlenmeyer flask (5.6 × 10⁶ cells/mL) andwas similar to the STR (9.7 × 10⁶ cells/mL) when the CHO-S cells were cultured. These results support the general suitability of the PBS system using pneumatic mixing for suspension cell cultivation as a novel single-use bioreactor system.     

Scale-up and optimization of culture conditions of a human heterohybridoma producing serotype-specific antibodies to Pseudomonas aeruginosa

Summary Three different stirred bioreactors of 0.5 to 12 l volume were used to scale up the production of a human monoclonal antibody. Inoculation density and stirrer speed were evaluated in batch cultures, whereas dilution rate and pH were optimized in chemostat cultures with respect to high specific antibody production rate and high antibody yield per time and reactor volume. The cell line used for the experiments was a heterohybridoma, producing immunoglobulin M (IgM) against lipopolysaccharide of Pseudomonas aeruginosa. Cells were cultured in spinner flasks of 500 ml liquid volume for adaptation to stirred culture conditions. Subsequently cells were transferred to the 1.5-1 KLF 2000 bioreactor and to the 12-1 NLF 22 bioreactor for pilot-scale cultures. Chemostat experiments were done in the 1.5-1 KLF bioreactor. Cell density, viability, glucose and lactate and antibody concentration were measured during culture experiments. In batch cultures in all three stirred bioreactors, comparable maximal cell densities and specific growth rates were achieved. Chemostat experiments showed that at a pH of 6.9 and a dilution rate of 0.57 per day the specific antibody production rate was threefold higher than similar experiments done at pH 7.2 with a dilution rate of 0.36 per day. By optimizing pH and dilution rate in chemostat cultures the daily yield of human IgM increased nearly threefold from 6 to 16 mg/day and per litre of reactor volume. The yield per litre of medium increased twofold. Correspondence to: U. Schürch     

Scale‐up analysis for a CHO cell culture process in large‐scale bioreactors

Bioprocess scale‐up is a fundamental component of process development in the biotechnology industry. When scaling up a mammalian cell culture process, it is important to consider factors such as mixing time, oxygen transfer, and carbon dioxide removal. In this study, cell‐free mixing studies were performed in production scale 5,000‐L bioreactors to evaluate scale‐up issues. Using the current bioreactor configuration, the 5,000‐L bioreactor had a lower oxygen transfer coefficient, longer mixing time, and lower carbon dioxide removal rate than that was observed in bench scale 5‐ and 20‐L bioreactors. The oxygen transfer threshold analysis indicates that the current 5,000‐L configuration can only support a maximum viable cell density of 7 × 10⁶ cells mL^?1. Moreover, experiments using a dual probe technique demonstrated that pH and dissolved oxygen gradients may exist in 5,000‐L bioreactors using the current configuration. Empirical equations were developed to predict mixing time, oxygen transfer coefficient, and carbon dioxide removal rate under different mixing‐related engineering parameters in the 5,000‐L bioreactors. These equations indicate that increasing bottom air sparging rate is more efficient than increasing power input in improving oxygen transfer and carbon dioxide removal. Furthermore, as the liquid volume increases in a production bioreactor operated in fed‐batch mode, bulk mixing becomes a challenge. The mixing studies suggest that the engineering parameters related to bulk mixing and carbon dioxide removal in the 5,000‐L bioreactors may need optimizing to mitigate the risk of different performance upon process scale‐up. Biotechnol. Bioeng. 2009;103: 733–746. © 2009 Wiley Periodicals, Inc.     

CO2 accumulation in bioreactor suspension cultures of Cyclamen persicum Mill. and its effect on cell growth and regeneration of somatic embryos

CO₂ accumulation in different culture systems containing embryogenic cell suspension cultures of cyclamen (Cyclamen persicum Mill.) was analyzed. In bioreactors equipped with a bubble-free or a bubble aeration system, CO₂ mole fractions in the gas phase of more than 10% were determined whereas in Erlenmeyer flasks, CO₂ mole fractions were below 2%. CO₂ accumulation in bioreactors was severely growth inhibiting in comparison to the flasks. By removing CO₂ in the aeration gas of a bubble-free aerated bioreactor, cell growth comparable to that in flasks was achieved. The regeneration ability of cell suspensions after being cultured in bioreactors with CO₂ accumulation was better than those after culture in bioreactors without CO₂ accumulation or in flasks. Received: 16 June 1998 / Revision received: 13 August 1998 / Accepted: 1 December 1998     

IPTG can replace lactose in auto‐induction media to enhance protein expression in batch‐cultured Escherichia coli

Auto‐induction media containing glucose, lactose, and glycerol are a simple and efficient approach for high‐throughput protein expression in Escherichia coli with lac‐derived expression systems. Its principle is based on inducer exclusion between glucose and lactose, preventing the induction by lactose before the depletion of glucose. Isopropyl‐β‐d ‐1‐thiogalactopyranoside (IPTG)—at least in typically used millimolar concentrations—is thought to be unsuitable for this purpose since it can enter the cell by diffusion independently of inducer exclusion. In this study, using parallel batch cultivations in stirred‐tank bioreactors on a milliliter scale, we show that the induction by micromolar concentrations of IPTG is prevented in the presence of glucose. With up to 40 μM IPTG, full induction and heterologous protein expression start only after the depletion of glucose. Thus, auto‐induction is possible with either lactose or IPTG, and the expression greatly depends on the type and concentration of the inducer. The best expression of enhanced green fluorescent protein was achieved with 40 μM IPTG in stirred‐tank bioreactors on a milliliter scale. The IPTG‐based auto‐induction was also reproduced in shaking flasks. Therefore, IPTG can be used in auto‐induction media for protein expression in batch‐cultured E. coli. Furthermore, we show that acetate or arabinose can have significant effects on the auto‐induction mechanism.     

Shear rate and microturbulence effects on the synthesis of proteases by Jacaratia mexicana cells cultured in a bubble column, airlift, and stirred tank bioreactors

Cysteine proteases from Jacaratia mexicana, an endemic Mexican plant, could compete in industrial applications with papain. Currently the only way to obtain these proteases is by extracting them from the wild plant. An alternative source of these enzymes is by J. mexicana suspension culture. In this work, this culture was carried out in airlift, bubble column and stirred tank bioreactors, and the effects of shear rate and microturbulence on cell growth, protein accumulation and proteolytic activity were determined. The shear rates in the stirred tank, bubble column and airlift bioreactors were 274 1/s, 13 1/s and 36 1/s respectively, and microturbulences (symbolized by λ, in units of μm) were 46, 79, and 77 μm, respectively. Protein levels and proteolytic activity were linearly correlated with both shear rate and microturbulence. A higher shear rate and a more intensive microturbulence occurred in the stirred tank, producing higher protein accumulation and higher proteolytic activity compared with those of the other two bioreactor systems. Higher shear rate and microturbulence had an elicitor effect on protease synthesis, because microturbulence in stirred tank bioreactors was lower than the average length of J. mexicana cells. Furthermore, cells in the stirred tank were smaller and thinner than those grown in shake flask, bubble column and airlift bioreactors. In summary, proteases were produced by J. mexicana cell cultures in a stirred tank under conditions of high shear rate and intensive microturbulence, which are similar to those which occur in industrial stirred tanks. These results encourage continuation of the process development for large scale production of these proteases by this technology.     

Novel bioreactors for the culture and expansion of aggregative neural stem cells

Neural stem cells have been cultured as three-dimensional aggregates in a number of different types of bioreactors. The design and configuration of the bioreactor are shown to be crucial factors for the successful propagation of the cells. A novel bioreactor with liquid re-circulation and a working volume of 200 ml has been designed, tested and shown to be able to produce a higher cell vitality compared to those produced in multi-well plates, shake flasks and stirred flasks. The novel reactor was able to produce a total density of cells of 3.5 x 10(6) cells/ml consisting of a larger number of smaller and proliferative aggregates, compared to only 1.8 x 10(6) cells/ml produced in a multi-well plate. Shake flasks and stirred flasks commonly used for facilitating mass transfer in the culture of micro-organisms are shown to be unsuitable for the propagation of neural stem cells.     

Production of oncolytic adenovirus and human mesenchymal stem cells in a single‐use,Vertical‐Wheel bioreactor system: Impact of bioreactor design on performance of microcarrier‐based cell culture processes

Anchorage‐dependent cell cultures are used for the production of viruses, viral vectors, and vaccines, as well as for various cell therapies and tissue engineering applications. Most of these applications currently rely on planar technologies for the generation of biological products. However, as new cell therapy product candidates move from clinical trials towards potential commercialization, planar platforms have proven to be inadequate to meet large‐scale manufacturing demand. Therefore, a new scalable platform for culturing anchorage‐dependent cells at high cell volumetric concentrations is urgently needed. One promising solution is to grow cells on microcarriers suspended in single‐use bioreactors. Toward this goal, a novel bioreactor system utilizing an innovative Vertical‐Wheel? technology was evaluated for its potential to support scalable cell culture process development. Two anchorage‐dependent human cell types were used: human lung carcinoma cells (A549 cell line) and human bone marrow‐derived mesenchymal stem cells (hMSC). Key hydrodynamic parameters such as power input, mixing time, Kolmogorov length scale, and shear stress were estimated. The performance of Vertical‐Wheel bioreactors (PBS‐VW) was then evaluated for A549 cell growth and oncolytic adenovirus type 5 production as well as for hMSC expansion. Regarding the first cell model, higher cell growth and number of infectious viruses per cell were achieved when compared with stirred tank (ST) bioreactors. For the hMSC model, although higher percentages of proliferative cells could be reached in the PBS‐VW compared with ST bioreactors, no significant differences in the cell volumetric concentration and expansion factor were observed. Noteworthy, the hMSC population generated in the PBS‐VW showed a significantly lower percentage of apoptotic cells as well as reduced levels of HLA‐DR positive cells. Overall, these results showed that process transfer from ST bioreactor to PBS‐VW, and scale‐up was successfully carried out for two different microcarrier‐based cell cultures. Ultimately, the data herein generated demonstrate the potential of Vertical‐Wheel bioreactors as a new scalable biomanufacturing platform for microcarrier‐based cell cultures of complex biopharmaceuticals. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1600–1612, 2015