A self-feeding roller bottle for continuous cell culture

The concept of a self-feeding roller bottle that delivers a continuous supply of fresh media to cells in culture, which is mechanically simplistic and works with existing roller apparatuses, is presented here. A conventional roller bottle is partitioned into two chambers; one chamber contains the fresh culture media reservoir, and the other contains the cell culture chamber. A spiroid of tubing inside the fresh media reservoir acts as a pump when the bottle rotates on its horizontal axis, continuously delivering fresh media through an opening in the partition to the cell culture chamber. The modified bottle proved capable of maintaining steady-state cell densities of a hybridoma cell line over the 10-day period tested, although at lower densities than reached during batch operation due to the continuous volume dilution. Steady-state density proved to be controllable by adjusting the perfusion rate, which changes with the rotation rate of the bottle. Specific antibody production rate is as much as 3.7 times the rate in conventional roller bottles operating with intermittent batch feeding.     

Cell growth optimization in microcarrier culture

Three monkey kidney cell lines and primary chicken embryo cells were grown in microcarrier culture. The carrier support was DEAE-Sephandex gel beads at low anion exchange capacity prepared according to a protocol developed at the Massachusetts Institute of Technology. The growth rate of the cells and the final cell density in microcarrier culture was dependent on the concentration of the beads in culture and on the size of the initial cell inoculum. A bead concentration of 1.0 to 2.0 mg of beads/ml of tissue culture medium and a cell inoculum of 20,000 cells/cm2 of bead surface appeared to be optimal. The efficiency of the microcarrier culture system was compared to that of stationary and roller bottle cultures. Stationary flasks gave cell densities about twofold higher than maximal densities in roller bottles and about threefold and twofold higher than cell densities in microcarrier culture at a bead concentration of 2.5 and 1.0 mg/ml, respectively. In terms of cell yield per milliliter of tissue culture medium, the microcarrier culture was superior to roller bottle and stationary cultures. An advantage of the microcarrier culture system is its suitability for scale up into large volume production units.     

Influence of a propeller on Saccharomyces cerevisiae fermentations in a pilot scale airlift bioreactor

The hydrodynamics (sectional gas holdup and liquid velocities) and oxygen transfer performance of a conventionally operated multiconfigurable pilot scale (0.25?m³) concentric airlift bioreactor containing baker's yeast were significantly improved by operating a marine propeller to draw liquid down the draft tube and aid recirculation at the base of the vessel. Propeller operation reduced the severe DOT heterogeneity of the reactor, which gave DOT values below 1% air saturation in the riser, by producing DOTs above 40% around the vessel at maximum energy dissipation rate. As a consequence the overall oxygen uptake rate (OUR) of the baker's yeast increased up to 3 fold with the total energy dissipation rate into the reactor until the lowest DOTs of the vessel were at or above 10%. The different degrees of heterogeneity generated by the two reactor configurations enabled the reactor to be used as a scale down tool to study the impact of heterogeneity on the physiology of fermentation broths. Comparison of the hydrodynamics and oxygen transfer between tall and short reactor heights revealed that the faster circulation times of the short reactor produced a greater improvement in the OUR with propeller operation even though similar DOT changes occurred around both sizes of reactor. This indicated that the yeast cells were responding to the rapid DOT changes around the vessel.     

Cell growth optimization in microcarrier culture

Summary Three monkey kidney cell lines and primary chicken embryo cells were grown in microcarrier culture. The carrier support was DEAE-Sephadex gel beads at low anion exchange capacity prepared according to a protocol developed at the Massachusetts Institute of Technology. The growth rate of the cells and the final cell density in microcarrier culture was dependent on the concentration of the beads in culture and on the size of the initial cell inoculum. A bead concentration of 1.0 to 2.0 mg of beads/ml of tissue culture medium and a cell inoculum of 20,000 cells/cm² of bead surface appeared to be optimal. The efficiency of the microcarrier culture system was compared to that of stationary and roller bottle cultures. Stationary flasks gave cell densities about twofold higher than maximal densities in roller bottles and about threefold and twofold higher than cell densities in microcarrier culture at a bead concentration of 2.5 and 1.0 mg/ml, respectively. In terms of cell yield per millitier of tissue culture medium, the microcarrier culture was superior to roller bottle and stationary cultures. An advantage of the microcarrier culture system is its suitability for a scale up into large volume production units.     

Hairy root culture in a liquid-dispersed bioreactor: characterization of spatial heterogeneity

A liquid-dispersed reactor equipped with a vertical mesh cylinder for inoculum support was developed for culture of Atropa belladonna hairy roots. The working volume of the culture vessel was 4.4 L with an aspect ratio of 1.7. Medium was dispersed as a spray onto the top of the root bed, and the roots grew radially outward from the central mesh cylinder to the vessel wall. Significant benefits in terms of liquid drainage and reduced interstitial liquid holdup were obtained using a vertical rather than horizontal support structure for the biomass and by operating the reactor with cocurrent air and liquid flow. With root growth, a pattern of spatial heterogeneity developed in the vessel. Higher local biomass densities, lower volumes of interstitial liquid, lower sugar concentrations, and higher root atropine contents were found in the upper sections of the root bed compared with the lower sections, suggesting a greater level of metabolic activity toward the top of the reactor. Although gas-liquid oxygen transfer to the spray droplets was very rapid, there was evidence of significant oxygen limitations in the reactor. Substantial volumes of non-free-draining interstitial liquid accumulated in the root bed. Roots near the bottom of the vessel trapped up to 3-4 times their own weight in liquid, thus eliminating the advantages of improved contact with the gas phase offered by liquid-dispersed culture systems. Local nutrient and product concentrations in the non-free-draining liquid were significantly different from those in the bulk medium, indicating poor liquid mixing within the root bed. Oxygen enrichment of the gas phase improved neither growth nor atropine production, highlighting the greater importance of liquid-solid compared with gas-liquid oxygen transfer resistance. The absence of mechanical or pneumatic agitation and the tendency of the root bed to accumulate liquid and impede drainage were identified as the major limitations to reactor performance. Improved reactor operating strategies and selection or development of root lines offering minimal resistance to liquid flow and low liquid retention characteristics are possible solutions to these problems.     

Human interferon production with diploid fibroblast cells grown on microcarriers.

A variety of diploid human fibroblast lines have been successfully grown to high densities (greater than 10(6) cell/ml) on recently developed microcarriers. Interferon induction using poly I.poly C and a superinduction procedure resulted in yields greater than 10,000 units/ml with one cell line. A direct comparison of microcarrier cultures to roller bottle cultures showed equivalent interferon yields on a per cell basis and some apparent differences relating to optimum inducer concentrations and kinetics of interferon accumulation.     

Pilot-scale culture ofCoffea arabica in a novel loop fluidised bed reactor

A novel bubble free loop fluidized bed reactor for plant cell cultures was developed and tested usingCoffea arabica as a model cell line. The effects of main operational parameters like morphology and size of inoculum, oxygen supply as well as recirculation of sparingly soluble gases on cell growth and alkaloid production rates in this reactor were studied and the results were compared with standard shake flask experiments. By on-line monitoring of biomass and oxygen uptake rates the main kinetic parameters for cell growth and alkaloid production were evaluated. It was demonstrated that the novel reactor is easy to run and is particularly adequate for measuring kinetic parameters necessary for scale up.     

Impact of nozzle operation on mass transfer in jet aerated loop reactors. Characterization and comparison to an aerated stirred tank reactor

The impact of mass transfer on productivity can become a crucial aspect in the fermentative production of bulk chemicals. For highly aerobic bioprocesses the oxygen transfer rate (OTR) and productivity are coupled. The achievable space time yields can often be correlated to the mass transfer performance of the respective bioreactor. The oxygen mass transfer capability of a jet aerated loop reactor is discussed in terms of the volumetric oxygen mass transfer coefficient k_La [h^?1] and the energetic oxygen transfer efficiency E [kgO₂ kW^?1 h^?1]. The jet aerated loop reactor (JLR) is compared to the frequently deployed aerated stirred tank reactor. In jet aerated reactors high local power densities in the mixing zone allow higher mass transfer rates, compared to aerated stirred tank reactors. When both reactors are operated at identical volumetric power input and aeration rates, local k_La values up to 1.5 times higher are possible with the JLR. High dispersion efficiencies in the JLR can be maintained even if the nozzle is supplied with pressurized gas. For increased oxygen demands (above 120 mmol L^?1 h^?1) improved energetic oxygen transfer efficiencies of up to 100 % were found for a JLR compared to an aerated stirred tank reactor operating with Rushton turbines.     

Stimulation of conversion rates and bacterial activity in a silage-fed two-phase biogas process by initiating liquid recirculation

The effects of liquid recirculation on a liquefaction-acidogenic reactor in an anaerobic two-phase digesting system operating with grass-clover silage was studied during 40 days after initiating recirculation of effluent from the methanogenic reactor to the liquefaction-acidogenic reactor. An increase in alkalinity and, thus, an increase in pH from 5.2 to 6.0 occurred in the liquefaction-acidogenic reactor. During the same period, a 10-fold increase (from 0.2 to 1.9 g·l^–1·h^–1) in the degradation rate of mannitol and an almost 9-fold increase in the activity of hydrogenotrophic methanogens was observed. The estimated number of these bacteria increased by one order of magnitude. The average degradation rate of lactate increased 3-fold, probably as a consequence of the more efficient hydrogen consumption by the hydrogenotrophic methanogens. An observed increase in net mineralization of organic nitrogen compounds was probably the main reason for an enhanced net production of organic acids (from 0.2 to 0.9 g·l^–1·d^–1). The liquefaction of cellulose and hemicellulose was low from the start of recirculation (3% and 20% reduction, respectively) and did not seem to be affected by the liquid recirculation. This was in accordance with the low number of cellulose degraders (4.0·10² counts·ml^–1) observed. The results from this investigation show that the initiation of liquid recirculation in silage-fed two-phase biogas processes will stimulate the activity of hydrogenotrophic methanogens in the liquefaction-acidogenic reactor. This will lead to more thermodynamically favourable conditions for acidification reactions which are dependent upon interspecies transfer of reducing equivalents.Abbreviations COD chemical oxygen demand - CSTR completely stirred tank reactor - HRT hydraulic retention time - LA-reactor liquefaction-acidogenic reactor - M-reactor methanogenic reactor - MPN most probable number - OLR organic loading rate - SMA specific methanogenic activity - SRT solids retention time - TKN total Kjeldahl nitrogen - ts total solids - tss total suspended solids - vs volatile solids - vss volatile suspended solids     

Stable hybridoma cultivation in a pilot-scale acoustic perfusion system: long-term process performance and effect of recirculation rate

Perfusion systems have the possibility to be operated continuously for several months. It is important that the performance of the cell retention device does not limit the operation time of a perfusion process used in the production of active pharmaceutical ingredients. Therefore, the aim of this study was to investigate the reliability and long-term stability of an acoustic perfusion process using the 200 L/d BioSep. As the BioSep is an external device, it is possible that dependent on the recirculation rate nutrient gradients occur in the external loop, which could affect the cell metabolism. Therefore, the effect of possible nutrient gradients on cell metabolism, viability and productivity was studied by varying the recirculation rate. In this study, it is shown that a perfusion process using a pilot-scale acoustic cell-retention device (200 L/d) is reliable and simple to operate, resulting in a stable 75-day cultivation of a hybridoma cell line producing a monoclonal antibody. The recirculation rate had a significant effect on the oxygen concentration in the external loop, with oxygen being depleted within the cell-retention device at recirculation rates below 6 m3/m(reactor)3.d (=600 L/d). The oxygen depletion at low circulation rates correlated with a slightly increased lactate production rate. For all other parameters no effect of the recirculation rate was observed, including cell death measured through the release of lactate dehydrogenase and specific productivity. A maximum specific productivity of 12 pg/cell.d was reached.     

Multisurface Glass Roller Bottle for Growth of Animal Cells in Culture

A multisurface glass roller bottle has been constructed for the growth of animal cells in culture. This bottle contains five concentrically placed glass cylinders that provide additional surfaces for the growth of animal cells. The bottle occupies the same space as a standard roller bottle, but it contains nine times the surface area of a standard bottle. L and HeLa cells can be grown in the bottle with cell yields 5- to 10-fold greater than in a standard bottle. L cells can be induced to produce interferon in the multisurface bottle.     

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.     

Root hairiness: effect on fluid flow and oxygen transfer in hairy root cultures

The effect of root hairiness on fluid flow and oxygen transfer in hairy root cultures was investigated using wild-type, transgenic and root-hair mutants of Arabidopsis thaliana. The root hair morphologies of the A. thaliana lines were hairless, short hairs, moderately hairy (wild-type) and excessively hairy, and these morphologies were maintained after transformation of seedlings with Agrobacterium rhizogenes. Filtration experiments were used to determine the permeability of packed beds of roots; permeability declined significantly with increasing root hairiness as well as with increasing biomass density. Hairy roots of wild-type A. thaliana grew fastest with a doubling time of 6.9 days, but the hairless roots exhibited the highest specific oxygen uptake rate. In experiments using a gradientless packed bed reactor with medium recirculation, the liquid velocity required to eliminate external mass transfer boundary layer effects increased with increasing root hairiness, reflecting the greater tendency towards liquid stagnation near the surface of roots covered with hairs. External critical oxygen tensions also increased with increasing root hairiness, ranging from 50% air saturation for hairless roots to ca. 150% air saturation for roots with excessive root hairs. These results are consistent with root hairs providing a significant additional resistance to oxygen transfer to the roots, indicating that very hairy roots are more likely than hairless roots to become oxygen-limited in culture. This investigation demonstrates that root hairiness is an important biological parameter affecting the performance of root cultures and suggests that control over root hair formation, either by use of genetically modified plant lines or manipulation of culture conditions, is desirable in large-scale hairy root systems.     

Improved fermentative production of Monascus pigments in roller bottle culture

The quantities and qualities of Monascus pigments produced by the filamentous fungus Monascus anka in batch submerged, agar surface, and roller bottle cultures were compared. In roller bottles, the fungus became attached to the wall of the culture vessels and produced a larger quantity of both intracellular (1508 absorbance units g⁻¹ cell mass) and extracellular (27 absorbance units g⁻¹ cell mass) Monascus red pigments, a yield that was about 10-fold greater than that of batch submerged and agar surface cultures. The optimum time required for maximum pigment production was reduced from 7 days in batch submerged or agar surface cultures to 4 days in roller bottle culture. In the roller bottle culture, the ratio of red to yellow pigments was also greatly increased. The advantage of the rotating vessel might be due to a combination of factors, including better gas exchange, higher medium pH, efficient pigment secretion, solid support for mycelium, and retarded conidiation.     

Influence of operating variables on liquid circulation in a 10.5-m(3) jet loop bioreactor

A 10.5-m(3) concentric tube jet loop reactor was used to study the influence of the working liquid volume, mean superficial air velocity, operating pressure, downcomer aeration, liquid jet velocity, and two ratios of draft tube/reactor diameter (D(t)/D) on liquid circulation time (T(c)). The experiments were carried out in a water-air system with the use of the acid pulse method. Results showed that circulation time was independent of the working liquid volume over a certain minimum liquid level, whereas downcomer aeration and D(t)/D ratio appeared as amenable parameters to achieve a high degree of control over liquid circulation and mixing efficiency, and to optimize the overall reactor performance. Increasing the operating pressure caused a reduction of the liquid circulation rate. However, ionger residence times of the air bubbles and the higher mass transfer driving force that result at higher pressures improve oxygen utilization. The relationship between T(c) and air load was independent of the operating pressure, provided the correlation is given as a function of the mean superficial air velocity. Neither liquid circulation nor gas holdup were significantly affected by liquid jet velocity. (c) 1995 John Wiley & Sons, Inc.     

Scale-up of enzymatic production of lactobionic acid using the rotary jet head system

Enzymatic oxidation of lactose to lactobionic acid (LBA) by a carbohydrate oxidase from Microdochium nivale was studied in a pilot-scale batch reactor of 600 L working volume using a rotary jet head (RJH) for mixing and mass transfer (Nordkvist et al., 2003, Chem Eng Sci 58:3877-3890). Both lactose and whey permeate were used as substrate, air was used as oxygen source, and catalase was added to eliminate the byproduct hydrogen peroxide. More than 98% conversion to LBA was achieved. Neither enzyme deactivation nor enzyme inhibition was observed under the experimental conditions. The dissolved oxygen tension (DOT) was constant throughout the tank for a given set of operating conditions, indicating that liquid mixing was sufficiently good to avoid oxygen gradients in the tank. However, at a given oxygen tension measured in the tank, the specific rate of reaction found in the RJH system was somewhat higher than previously obtained in a 1 L mechanically stirred tank reactor (Nordkvist et al., 2007, in this issue, pp. 694-707). This can be ascribed to a higher pressure in the recirculation loop which is part of the RJH system. Compared to mechanically stirred systems, high values of the volumetric mass transfer coefficient, k(L)a, were obtained when lactose was used as substrate, especially at low values of the specific power input and the superficial gas velocity. k(L)a was lower for experiments with whey permeate than with lactose due to addition of antifoam. The importance of mass transfer and of the saturation concentration of oxygen on the volumetric rate of reaction was demonstrated by simulations.     

Industrial choices for protein production by large-scale cell culture

Traditional barriers to large-scale mammalian culture have now been addressed, with the standard stirred-tank reactor emerging as industry's technology of choice. The issues of adapting cells to suspension culture, shear sensitivity and oxygen supply have been largely resolved. But for many low-volume and specialty applications, such as the production of viral vaccines and gene therapies, reactor technology remains diversified, with reactor types ranging from roller bottles to stacked plates and hollow fibers.     

Monitoring gradient formation in a jet aerated bioreactor

Jet aerated loop reactors (JLRs) provide high mass transfer coefficients (k_La) and can be used for the intensification of mass transfer limited reactions. The jet loop reactor achieves higher k_La values than a stirred tank reactor (STR). The improvement relies on significantly higher local power inputs (～10⁴) than those obtainable with the STR. Operation at high local turnover rates requires efficient macromixing, otherwise reactor inhomogeneities might occur. If sufficient homogenization is not achieved, the selectivity of the reaction and the respective yields are decreased. Therefore, the balance between mixing and mass transfer in jet loop reactors is a critical design aspect. Monitoring the dissolved oxygen levels during the turnover of a steady sodium sulfite feed implied the abundance of gradients in the JLR. Prolonged mixing times at identical power input and aeration rates (～100%) were identified for the JLR in comparison to the STR. The insertion of a draft tube to the JLR led to a more homogenous dissolved oxygen distribution, but unfortunately a reduction of mixing time was not achieved. In case of increased medium viscosities as they may arise in high cell density cultivations, no gradient formation was detected. However, differences in medium viscosity significantly altered the mass transfer and mixing performance of the JLR.     

Jet aeration as alternative to overcome mass transfer limitation of stirred bioreactors

In industrial biotechnology increasing reactor volumes have the potential to reduce production costs. Whenever the achievable space time yield is determined by the mass transfer performance of the reactor, energy efficiency plays an important role to meet the requirements regarding low investment and operating costs. Based on theoretical calculations, compared to bubble column, airlift reactor, and aerated stirred tank, the jet loop reactor shows the potential for an enhanced energetic efficiency at high mass transfer rates. Interestingly, its technical application in standard biotechnological production processes has not yet been realized. Compared to a stirred tank reactor powered by Rushton turbines, maximum oxygen transfer rates about 200% higher were achieved in a jet loop reactor at identical power input in a fed batch fermentation process. Moreover, a model‐based analysis of yield coefficients and growth kinetics showed that E. coli can be cultivated in jet loop reactors without significant differences in biomass growth. Based on an aerobic fermentation process, the assessment of energetic oxygen transfer efficiency [kgO₂ kW^?1 h^?1] for a jet loop reactor yielded an improvement of almost 100%. The jet loop reactor could be operated at mass transfer rates 67% higher compared to a stirred tank. Thus, an increase of 40% in maximum space time yield [kg m^?3 h^?1] could be observed.     

Optimized production of high-titer recombinant adeno-associated virus in roller bottles

Adeno-associated viral (AAV) vectors are used for in vivo gene transfer in a number of preclinical models of genetic diseases (including large-animal models) and are currently being tested in clinical trials for treatment of hemophilia B and cystic fibrosis. Protocols for production of AAV vectors in a helper virus-free system are available and are based on transient transfection of HEK-293 cells with multiple plasmids. Scale-up of vector production has been labor intensive and inefficient because of a lack of larger culture vessels suitable for growth of adherent cells, large-scale transfection, and vector production. Here we report efficient production of AAV vector in roller bottles, which represents a 10-fold scale-up from the conventional flask or plate method. Optimized production yielded greater than 10(13) vector genomes per bottle and was as cost effective as published protocols using plates. Successful vector production by this method was dependent on optimization of transfection by calcium phosphate precipitation, of monitoring of cell growth (by measurement of glucose consumption), of cell culture conditions, and CO2/air exchange with the culture vessel.