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1.
Three different hybridoma cell lines, grown in serum-free media with different levels of Pluronic F-68, were subjected to a shear force of 0.6 N m-2. Some protective effect due to the polymer was found, indicating it to be a potentially useful adjuvant in serum-free media. Other observations of liquid and gas effects at the reactor level have been included here. A discussion of the difference between suspension and microcarrier cultures, in relation to hydrodynamic effects, is included. 相似文献
2.
High‐throughput miniaturized bioreactors for cell culture process development: Reproducibility,scalability, and control
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Shahid Rameez Sigma S. Mostafa Christopher Miller Abhinav A. Shukla 《Biotechnology progress》2014,30(3):718-727
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 相似文献
3.
Guozheng Wang Wenying Zhang Corey Jacklin David Freedman Lee Eppstein Avinoam Kadouri 《Cytotechnology》1992,9(1-3):41-49
This study describes two packed bed bioreactor configurations which were used to culture a mouse-mouse hybridoma cell line
(ATCC HB-57) which produces an IgG1 monoclonal antibody. The first configuration consists of a packed column which is continuously perfused by recirculating
oxygenated media through the column. In the second configuration, the packed bed is contained within a stationary basket which
is suspended in the vessel of a CelliGen™ bioreactor. In this configuration, recirculation of the oxygenated media is provided by the CelliGen Cell Lift impeller.
Both configurations are packed with disk carriers made from a non-woven polyester fabric. During the steady-state phase of
continuous operation, a cell density of 108 cells per cm3 of bed volume was obtained in both bioreactor configurations. The high levels of productivity (0.5 gram MAb per 1 of packed
bed per day) obtained in these systems demonstrates that the culture conditions achieved in these packed bed bioreactors are
excellent for the continuous propagation of hybridomas using media which contains low levels (1 %) of serum as well as serum-free
media.
These packed bed bioreactors allow good control of pH, dissolved oxygen and temperature. The media flows evenly over the cells
and produces very low shear forces. These systems are easy to set up and operate for prolonged periods of time.
The potential for scale-up using Fibra-cel carriers is enhanced due to the low pressure drop and low mass transfer resistance,
which creates high void fraction approaching 90% in the packed bed. 相似文献
4.
5.
Plant cell suspension cultures and hairy roots are potential sources of secondary metabolites and recombinant proteins. In
contrast to traditionally grown “whole wild plants” or “whole transgenic plants”, their production in bioreactors guarantees
defined controlled process conditions and therefore minimizes or even prevents variations in product yield and quality, which
simplifies process validation and product registration. Moreover, bioreactors and their configuration significantly affect
cultivation results by accomplishing and controlling the optimum environment for effective cell growth and production of bioactive
substances. This review highlights the main design criteria of the most widely used bioreactor types, both for plant cell
suspension cultures and for hairy roots, and outlines suitable low-cost disposable bioreactors which have found increasing
acceptance over the last 10 years.
Plants for human health in the post-genome era, PSE congress 26.8.2007–29.8.2007, Helsinki. 相似文献
6.
Moritz K. F. Wolf Andrea Müller Jonathan Souquet Hervé Broly Massimo Morbidelli 《Biotechnology and bioengineering》2019,116(8):1973-1985
The development of mammalian cell perfusion cultures is still laborious and complex to perform due to the limited availability of scale-down models and limited knowledge of time- and cost-effective procedures. The maximum achievable viable cell density (VCDmax), minimum cell-specific perfusion rate (CSPRmin), cellular growth characteristics, and resulting bleed rate at steady-state operation are key variables for the effective development of perfusion cultures. In this study, we developed a stepwise procedure to use shake tubes (ST) in combination with benchtop (BR) bioreactors for the design of a mammalian cell perfusion culture at high productivity (23 pg·cell−1·day−1) and low product loss in the bleed (around 10%) for a given expression system. In a first experiment, we investigated peak VCDs in STs by the daily discontinuous medium exchange of 1 reactor volume (RV) without additional bleeding. Based on this knowledge, we performed steady-state cultures in the ST system using a working volume of 10 ml. The evaluation of the steady-state cultures allowed performing a perfusion bioreactor run at 20 × 106 cells/ml at a perfusion rate of 1 RV/day. Constant cellular environment and metabolism resulted in stable product quality patterns. This study presents a promising strategy for the effective design and development of perfusion cultures for a given expression system and underlines the potential of the ST system as a valuable scale-down tool for perfusion cultures. 相似文献
7.
Ehrlich Kenneth C. Stewart Elaine Klein Elias 《In vitro cellular & developmental biology. Plant》1978,14(5):443-450
Summary Glucose, lactic-acid, and oxygen metabolism of BHK and L929 cells on artificial capillary perfusion units have been studied
using several different modes of perfusion. After 7 to 10 days, cells planted in the extracapillary compartment of culture
units containing 80 to 150 fibers reached populations that used 0.073±0.025 μmol per min glucose and 0.76±0.26 μl per min
oxygen and excreted 0.078±0.038 μmol per min lactic acid. From these data it is estimated that these units contain approximately
2×107 cells. The metabolic rate of cultures perfused through the capillaries or through the extracapillary compartment was not
affected significantly by change in flow rate except at perfusion flow rates ≤0.05 ml per min. The cell population, as measured
by metabolic activity, did not increase significantly when the serum content of the medium was ≤1%. No major differences were
found in glucose utilization rates of equal numbers of cells on artificial capillaries, on short-term suspension culture,
or as monolayers in plastic flasks. Artificial capillary perfusion may provide a simple system for studying metabolism of
mammalian cells in culture.
Research was supported by the U.S. Army Medical Research and Development Command, Washington, D.C. 20314, under Contract No.
DAMD 17-76-C-5075. 相似文献
8.
Understanding and modeling alternating tangential flow filtration for perfusion cell culture
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William Kelly Jennifer Scully Di Zhang Gang Feng Mathew Lavengood Jason Condon John Knighton Ravinder Bhatia 《Biotechnology progress》2014,30(6):1291-1300
Alternating tangential flow (ATF) filtration has been used with success in the Biopharmaceutical industry as a lower shear technology for cell retention with perfusion cultures. The ATF system is different than tangential flow filtration; however, in that reverse flow is used once per cycle as a means to minimize fouling. Few studies have been reported in the literature that evaluates ATF and how key system variables affect the rate at which ATF filters foul. In this study, an experimental setup was devised that allowed for determination of the time it took for fouling to occur for given mammalian (PER.C6) cell culture cell densities and viabilities as permeate flow rate and antifoam concentration was varied. The experimental results indicate, in accordance with D'Arcy's law, that the average resistance to permeate flow (across a cycle of operation) increases as biological material deposits on the membrane. Scanning electron microscope images of the post‐run filtration surface indicated that both cells and antifoam micelles deposit on the membrane. A unique mathematical model, based on the assumption that fouling was due to pore blockage from the cells and micelles in combination, was devised that allowed for estimation of sticking factors for the cells and the micelles on the membrane. This model was then used to accurately predict the increase in transmembane pressure during constant flux operation for an ATF cartridge used for perfusion cell culture. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1291–1300, 2014 相似文献
9.
Potential of cell retention techniques for large-scale high-density perfusion culture of suspended mammalian cells 总被引:4,自引:0,他引:4
Voisard D Meuwly F Ruffieux PA Baer G Kadouri A 《Biotechnology and bioengineering》2003,82(7):751-765
This review focuses on cultivation of mammalian cells in a suspended perfusion mode. The major technological limitation in the scaling-up of these systems is the need for robust retention devices to enable perfusion of medium as needed. For this, cell retention techniques available to date are presented, namely, cross-flow filters, hollow fibers, controlled-shear filters, vortex-flow filters, spin-filters, gravity settlers, centrifuges, acoustic settlers, and hydrocyclones. These retention techniques are compared and evaluated for their respective advantages and potential for large-scale utilization in the context of industrial manufacturing processes. This analysis shows certain techniques have a limited range of perfusion rate where they can be implemented (most microfiltration techniques). On the other hand, techniques were identified that have shown high perfusion capacity (centrifuges and spin-filters), or have a good potential for scale-up (acoustic settlers and inclined settlers). The literature clearly shows that reasonable solutions exist to develop large-scale perfusion processes. 相似文献
10.
Jean-Marc Bielser Jakub Domaradzki Jonathan Souquet Hervé Broly Massimo Morbidelli 《Biotechnology progress》2019,35(3):e2790
Perfusion cell culture, confined traditionally to the production of fragile molecules, is currently gaining broader attention in the biomanufacturing of therapeutic proteins. The development of these processes is made difficult by the limited availability of appropriate scale-down models. This is due to the continuous operation that requires complex control and cell retention capacity. For example, the determination of an optimal perfusion and bleed rate for continuous cell culture is often performed in scale-down bioreactors and requires a substantial amount of time and effort. To increase the experimental throughput and decrease the required workload, a semi-continuous procedure, referred to as the VCDmax (viable cell density) approach, has been developed on the basis of shake tubes (ST) and deepwell plates (96-DWP). Its effectiveness has been demonstrated for 12 different CHO-K1-SV cell lines expressing an IgG1. Further, its reliability has been investigated through proper comparisons with perfusion runs in lab-scale bioreactors. It was found that the volumetric productivity and the CSPRmin (cell specific perfusion rate) determined using the ST and 96-DWP models were successfully (mostly within the experimental error) confirmed in lab-scale bioreactors, which then covered a significant scale-up from the half milliliter to the liter scale. These scale-down models are very useful to design and scale-up optimal bioreactor operating conditions as well as screening for different media and cell lines. 相似文献
11.
E. V. Demidova O. V. Reshetnyak A. V. Oreshnikov A. M. Nosov 《Russian Journal of Plant Physiology》2006,53(1):134-140
The aim of the work was to study the growth characteristics of cultured cells of Panax japonicus var. repens, an endemic plant of the Primorski Krai of Russia, grown in laboratory bioreactors and to determine the content of basic ginsenosides under these conditions. An increase of the inoculum size of the culture produced higher biomass accumulation and economic coefficient but slightly reduced the specific growth rate. An increase in the auxin concentration in a medium by adding 2,4-D practically did not affect growth characteristics of the culture but significantly reduced the size of cell aggregates. In all treatments tested, all major ginsenosides (Rb1, Rc, Rb2, Rd, Rf, Rg1, and Re) were found in the culture. The total ginsenoside content was 2–3% per biomass dry weight. Meantime, ginsenosides of the Rg-series with protopanaxatriol as aglycone prevailed (70% of the total ginsenoside content). The culture conditions considerably affected the ratio of individual ginsenosides. In 2,4-D-containing medium, the preferential synthesis of Re ginsenoside was observed while both Rg1 and Re were synthesized in other treatments. 相似文献
12.
A problem nowadays tissue engineers encounter in developing sizable tissue implants is the nonuniform spread of cells and/or extracellular matrices. Research shows such a nutrients transport restriction may be improved by employing hydrodynamic culture systems. We propose a compact model for the simulation of cell growth in a porous construct under direct perfusion. Unlike the previous model proposed in the literature, which composes a cellular scaffold sandwiched between two culture media layers, the current model includes only the scaffold layer to simplify the mathematical and computational complex. Results show the present single-layer model can predict cell spreads and the nutrient and metabolic waste distribution as accurately as does the three-layer model. Only if the hydrodynamic aspects such as the pressure and viscous stress are prominent to know, should the more sophisticated analyses with the three-layer model be employed. The compact model provides comparable investigations for the tissue-engineering construct developments. 相似文献
13.
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. 相似文献
14.
Ioná W. Bettinardi Andreas Castan Ricardo A. Medronho Leda R. Castilho 《Biotechnology and bioengineering》2020,117(7):1915-1928
In this study, a hydrocyclone (HC) especially designed for mammalian cell separation was applied for the separation of Chinese hamster ovary cells. The effect of key features on the separation efficiency, such as type of pumphead in the peristaltic feed pump, use of an auxiliary pump to control the perfusate flow rate, and tubing size in the recirculation loop were evaluated in batch separation tests. Based on these preliminary batch tests, the HC was then integrated to 50-L disposable bioreactor bags. Three perfusion runs were performed, including one where perfusion was started from a low-viability late fed-batch culture, and viability was restored. The successive runs allowed optimization of the HC-bag configuration, and cultivations with 20–25 days duration at cell concentrations up to 50 × 106 cells/ml were performed. Separation efficiencies up to 96% were achieved at pressure drops up to 2.5 bar, with no issues of product retention. To our knowledge, this is the first report in literature of high cell densities obtained with a HC integrated to a disposable perfusion bioreactor. 相似文献
15.
Cultivation of animal cells for the production of recombinant proteins is an important method for manufacturing complex proteins requiring posttranslational processing. One of the often considered methods for cultivation is by immobilization of the cells in hollow fiber bioreactors (HFBRs). These systems allow the cells to grow to high densities in a shear protected environment; furthermore the product can be accumulated in high concentration in the case of ultrafiltration HFBRs. Operation and scale-up are constrained by nutrient and product transport with oxygen transfer to growing cells being the most critical parameter. Mathematical models describing HFBRs have proved to be useful in quantitating and understanding the constraints and guiding the scale-up of this approach to animal cell cultivation. 相似文献
16.
Evaluation and applications of optical cell density probes in mammalian cell bioreactors 总被引:2,自引:0,他引:2
Wu P Ozturk SS Blackie JD Thrift JC Figueroa C Naveh D 《Biotechnology and bioengineering》1995,45(6):495-502
On-line optical cell density probes were implemented to continuously monitor the cell densities in mammalian cell bioreactor and to achieve advanced bioreactor controls. We tested cell density probes from six manufacturers in high cell density bioreactors. When externally calibrated, Aquasant and Ingold backscattering probes produced the most linear probe responses (PR) versus cell density (CD), followed by the ASR and Cerex laser probes. Monitek and Wedgewood transmission probes had lower resolutions. All probes were tested in two murine hybridoma fermentations. Cell densities varied between 1 x 10(6) cells/mL to 20 x 10(6) cells/mL and the bioreactors were operated for 5 to 7 weeks. For our bioreactors, Aquasant, Ingold, ASR, Wedgewood, and Monitek probes gave satisfactory responses. Little fouling was observed with any probe at the end of 2 weeks. Fouling was a possibility after 3 weeks in one bioreactor but its effect can be easily corrected. Cell density control and specific perfusion control of bioreactors based on the Aquasant probe were achieved. Implementation of cell density probe based perfusion control, instead of "step perfusion adjustments" based on manual hemacytometer control, will result in smoother operation, healthier cultures, increased medium delivery efficiency, and reduced operational excursions. (c) 1995 John Wiley & Sons, Inc. 相似文献
17.
On-line biomass monitoring of CHO perfusion culture with scanning dielectric spectroscopy 总被引:7,自引:0,他引:7
In this work, dielectric spectroscopy was used to monitor two CHO perfusion culture experiments (B14 and B16). The capacitance of the cell suspension was recorded every 20 minutes over an excitation frequency range of 0.2 MHz to 10.0 MHz. A phase plot of the capacitance at a low excitation frequency vs. the value at a higher frequency proved to be an accurate indicator of the major transition points of the culture, i.e., maximum cell viability, end of lactate consumption, point of zero viability. For both experiments, the capacitance signal correlated very well (R(2) >0.98) with viable cell number up to concentrations of 1 x 10(7) cells/mL. Visual observation of the capacitance spectra indicated that changes in the capacitance relative to frequency were related to the cellular morphology. A multivariate model was developed using off-line data that could predict the median cell diameter within a single experiment (B14) with an error of 0.34 microm (2%). Upon extension to a subsequent experiment (B16), the predicted error was 1.18 microm (9%). 相似文献
18.
Vijay Singh 《Cytotechnology》1999,30(1-3):149-158
This work describes a novel bioreactor system for the cultivation of animal, insect, and plant cells using wave agitation
induced by a rocking motion. This agitation system provides good nutrient distribution, off-bottom suspension, and excellent
oxygen transfer without damaging fluid shear or gas bubbles. Unlike other cell culture systems, such as spinners, hollow-fiber
bioreactors, and roller bottles, scale-up is simple, and has been demonstrated up to 100 L of culture volume. The bioreactor
is disposable, and therefore requires no cleaning or sterilization. Additions and sampling are possible without the need for
a laminar flow cabinet. The unit can be placed in an incubator requiring minimal instrumentation. These features dramatically
lower the purchase cost, and operating expenses of this laboratory/pilot scale cell cultivation system. Results are presented
for various model systems: 1) recombinant NS0 cells in suspension; 2) adenovirus production using human 293 cells in suspension;
3) Sf9 insect cell/baculovirus system; and 4) human 293 cells on microcarrier. These examples show the general suitability
of the system for cells in suspension, anchorage-dependent culture, and virus production in research and GMP applications.
This revised version was published online in July 2006 with corrections to the Cover Date. 相似文献
19.
20.
This article presents a novel pumpless perfusion cell culture cap, the gravity‐driven flow rate of which is kept constant by the height difference of two parallel channel layers. Previous pumpless perfusion cell culture systems create a gravity‐driven flow by means of the hydraulic head difference (Δh) between the source reservoir and the drain reservoir. As more media passes from the source reservoir to the drain reservoir, the source media level decreases and the drain media level increases. Thus, previous works based on a gravity‐driven flow were unable to supply a constant flow rate for the perfusion cell culture. However, the proposed perfusion cell culture cap can supply a constant flow rate, because the media level remains unchanged as the media moves laterally through each channel having same media level. In experiments, using the different fluidic resistances, the perfusion cap generated constant flow rates of 871 ± 27 μL h?1 and 446 ± 11 μL h?1. The 871 and 446 μL h?1 flow rates replace the whole 20 mL medium in the petridish with a fresh medium for days 1 and 2, respectively. In the perfusion cell (A549 cell line) culture with the 871 μL h?1 flow rate, the proposed cap can maintain a lactate concentration of about 2200 nmol mL?1 and an ammonia concentration of about 3200 nmol mL?1. Moreover, although the static cell culture maintains cell viability for 5 days, the perfusion cell culture with the 871 μL h?1 flow rate can maintain cell viability for 9 days. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012 相似文献