Mammalian Cell Bioreactors Research Articles

Phenotype of Hepatocyte Spheroids Behavior within Thermo-Sensitive Poly(NiPAAm-co-PEG-g-GRGDS) Hydrogel as a Cell Delivery Vehicle

Aggregates (spheroids) of specific cells are often regarded as a better form in artificial organs and mammalian cell bioreactors for improved cell-specific functions. Freshly harvested primary rat hepatocytes, cultivated as spheroids and entrapped in an adhesion molecules of Arg-Gly-Asp (RGD)-conjugated extracellular matrix, have been examined for differentiated morphology and enhanced liver-specific functions. A copolymer of RGD conjugated p(NiPAAm-co-PEG) hydrogel was used to entrap hepatocytes in the forms of spheroids. Over 28 days, entrapped the spheroids had a higher viability and produced albumin and urea at constant rates, while there was slight increase in cell numbers and reduction of albumin secretion in single cell culture in the hydrogel. Hepatocytes cultured in this way are a potentially useful three-dimensional cell system for application in a bioartificial liver device and bioreactor.

Phenotype of hepatocyte spheroids in Arg-GLY-Asp (RGD) containing a thermo-reversible extracellular matrix.

The spheroid of specific cells is often regarded as the better form in artificial organs and mammalian cell bioreactors for improved cell-specific functions. In this study, freshly harvested primary rat hepatocytes, which had been cultivated as spheroids and entrapped in a synthetic thermo-reversible extracellular matrix, were examined for differentiated morphology and enhanced liver-specific functions as compared to a control set (hepatocytes in single-cell form). A copolymer of N-isopropylacrylamide (98 mole % in the feed) and acrylic acid (poly(NiPAAm-co-AAc)), and the adhesion molecule, an Arg-Gly-Asp (RGD)-incorporated thermo-reversible matrix, were used to entrap hepatocytes in the form of either spheroids or single cells. In a 28-day culture period, the spheroids in the RGD-incorporated gel maintained higher viability and produced albumin and urea at constant rates, while there was lower cell viability and less albumin secretion by the spheroids in p(NiPAAm-co-AAc). Hepatocytes cultured as spheroids in the RGD-incorporated gel would constitute a potentially useful three-dimensional cell system for application in a bio-artificial liver device.

Phenotype of hepatocyte spheroids in synthetic thermo-reversible extracellular matrix

Aggregates of specific cells are often regarded as a better form in artificial organs and mammalian cell bioreactors in terms of cell-specific functionality. In this study, the morphology and liver-specific functions of freshly harvested primary rat hepatocytes, which were cultivated as spheroids and entrapped in a synthetic thermo-reversible extracellular matrix, were examined and compared to a control (hepatocytes in single cell form). A copolymer of N-isopropylacrylamide (98 mole % in feed) and acrylic acid (poly(NiPAAm-co-AAc)), a thermo-reversible copolymer gel matrix, was used to entrap hepatocytes either in spheroids or single cells. During a 7-day culture period, the spheroids maintained higher viability and produced albumin and urea at a relatively constant rate, while the single cell culture showed a slight increase in cell numbers and a reduction in albumin secretion. Hepatocytes cultured as spheroids present a potentially useful three-dimensional cell culture system for application in a bioartificial liver device.

On-line electrocatalyzed luminol chemiluminescence determination of D-glucose and dissolved oxygen in simulated mammalian cell bioreactor perfusion fluid using solid phase reagent modules

On-line instrumentation and methods for the chemiluminescence based real-time monitoring of d-glucose and O2 levels in mammalian cell bioreactor perfusion fluid are described. The unit processes required for the analysis include: pH adjustment using solid phase flow-through modules, immobilized enzyme catalyzed oxidation of glucose by molecular oxygen to produce hydrogen peroxide, controlled release of luminol using a solid phase flow-through module, electrocatalyzed luminescence using gold electrodes, and photodetection of chemiluminescent emissions. Calibration curves for d-glucose and dissolved O2 in simulated bioreactor perfusion fluid have been generated using fully integrated reagentless test systems from 0–800 mg l−1 and 0–10 mg l−1, respectively.

Fibroblast aggregation by suspension with conjugates of poly(ethylene glycol) and RGD

Cell aggregates may be useful components of artificial organs and mammalian cell bioreactors, but many cells do not naturally aggregate. In a previous report,(4) we described a method for promoting neural cell aggregation by addition of water-soluble conjugates of cell adhesion peptides, containing the three amino acid sequence Arg-Gly-Asp (RGD), and poly(ethylene glycol) (PEG). Here, we examined the mechanism of conjugate-induced aggregation using fibroblasts and a variety PEG-peptide conjugates. Aggregation was monitored during rotation culture of fibroblasts in the presence of unconjugated GRGDY and PEG; monofunctional (PEG-GRGDY) and bifunctional (GRGDY-PEG-GRGDY) conjugates; and bifunctional conjugates produced with a similar, but non-cell-binding, peptide (GRGEY-PEG-GRGEY). GRGDY-PEG-GRGDY conjugates induced rapid and pronounced fibroblast aggregation that was dose-dependent; at the highest concentration tested (5 mg/mL GRGDY-PEG-GRGDY), cell aggregates were produced more quickly ( approximately 1 h) and were significantly larger at 24 h (mean radius approximately 66 mum) than at slightly lower concentrations (1.7 and 3.3 mg/mL). Aggregation with GRGDY-PEG-GRGDY was completely inhibited by dissolved GRGDY (1.7 mg/mL). Neither unmodified GRGDY, unmodified PEG, PEG-GRGDY, nor GRGEY-PEG-GRGEY conjugates led to significant aggregation. The extent of aggregation depended on PEG molecular weight: conjugates with 3400 M(w) PEG produced aggregates with significantly larger mean radius than conjugates with 20,000 M(w) PEG. When 1N-8A fibroblasts, genetically engineered to produce recombinant nerve growth factor (NGF), were aggregated with GRGDY-PEG-GRGDY, aggregated cells produced more NGF per cell than nonaggregated cells. Aggregation of cells may lead to improved cell function, such as the increase in NGF production observed here, which could be useful in large-scale cell culture and construction of artificial organs or tissue transplants for tissue engineering. (c) 1996 John Wiley & Sons, Inc.

The use of particle-mediated gene transfer for the study of promoter activity in somatic tissues.

Research in molecular genetic studies and in transgenic biotechnology has resulted in excellent advancements in exploiting gene transfer techniques. Basic research in transcriptional and/or translational control, protein engineering, cellular immunological mechanisms, and developmental genetic processes using transient or stable transgenic approaches have generated the main body of information in our current understanding of various molecular biology and cell biology disciplines. Parallel with this development, the gene transfer and transgene expression techniques have also been extremely useful in their application to plant and animal (including mammalian) biotechnologies. Specifically, analyses of promoter activity and usage have played important roles in generation of genetically engineered plants, transgenic animals, and recombinant or transgenic protein products in various microbial fermenters or plants or in mammalian cell bioreactors. More recently, various gene transfer and promoter usage technologies have also been evaluated extensively for application to gene therapy research.

Rapid calibration of near-infrared spectroscopic measurements of mammalian cell cultivations.

Near-infrared (NIR) spectroscopy is a flexible method that can be employed to noninvasively monitor the concentrations of multiple nutrients and wastes in mammalian cell bioreactors. Development of suitable calibrations can be a labor- and time-intensive process that must be repeated when process conditions are altered significantly. To address this difficulty, we have produced a new approach for generating NIR spectroscopic calibrations that requires significantly less time compared with standard calibration schemes. This method reduces development time from the present level of several weeks to several hours. A small number of experimentally collected spectra serve as inputs to a computational procedure that yields a large number of simulated spectra, each containing both analyte-specific and analyte-independent information. Such simulated spectra may be employed as a calibration set for quantifying analytes in experimentally collected spectra. Spectroscopic measurements of the concentrations of five components (ammonia, glucose, glutamate, glutamine, and lactate) can be accomplished with levels of error similar to those obtained with full experimental calibrations. A key to this process is the utilization of random numbers, which randomizes the influence of natural variations, present in each experimentally collected spectrum, on the resultant composite spectrum. This approach may increase the feasibility of employing NIR spectroscopy to monitor bioreactors and other biological processes subjected to varying operating conditions.

Investigating the Performance of Intensive Mammalian Cell Bioreactor Systems Using Magnetic Resonance Imaging and Spectroscopy

Investigating the Performance of Intensive Mammalian Cell Bioreactor Systems Using Magnetic Resonance Imaging and Spectroscopy

Transgenic bioreactors

Since the generation of the first transgenic mice in 1980, transgene technology has also been successfully applied to large farm animals. Although this technology can be employed to improve certain production traits of livestock, this approach has not been very successful so far owing to unwanted effects encountered in the production animals. However, by using tissue-specific targeting of the transgene expression, it is possible to produce heterologous proteins in the extracellular space of large transgenic farm animals. Even though some recombinant proteins, such as human hemoglobin, have been produced in the blood of transgenic pigs, in the majority of the cases mammary gland targeted expression of the transgene has been employed. Using production genes driven by regulatory sequences of milk protein genes a number of valuable therapeutic proteins have been produced in the milk of transgenic bioreactors, ranging from rabbits to dairy cattle. Unlike bacterial fermentors, the mammary gland of transgenic bioreactors appear to carry out proper postsynthetic modifications of human proteins required for full biological activity. In comparison with mammalian cell bioreactors, transgenic livestock with mammary gland targeted expression seems to be able to produce valuable human therapeutic proteins at very low cost. Although not one transgenically produced therapeutic protein is yet on the market, the first such proteins have recently entered or even completed clinical trials required for their approval.

Growth kinetics, nutrient uptake, and expression of the Alcaligenes eutrophus poly(beta-hydroxybutyrate) synthesis pathway in transgenic maize cell suspension cultures.

Transgenic suspension cultures of Black Mexican Sweet maize (Zea mays L.) expressing the Alcaligenes eutrophus genes encoding enzymes of the pathway for biosynthesis of the biodegradable polymer poly(beta-hydroxybutyrate) (PHB) were established as a tool for investigating metabolic regulation of the PHB pathway in plant cells. Cultures were grown in a 2 L modified mammalian cell bioreactor and in shake flasks. Biomass doubling times for transgenic bioreactor cultures (3.42 +/- 0.76 days) were significantly higher than those for untransformed cultures (2.01 +/- 0.33 days). Transgenic expression of the bacterial enzymes beta-ketothiolase (0.140 units/mg protein) and acetoacetyl-CoA reductase (0.636 units/mg protein) was detected by enzyme assays and immunoblots. However, over the first 2 years of cultivation, reductase activity decreased to 0.120 units/mg proteins. Furthermore, the PHB synthase gene, although initially present, was not detectable after 1.5 years of cultivation in suspension culture. These facts suggest that transgenic expression of PHB pathway genes in plant cells may not be stable. A hydroxybutyrate derivative was detected via gas chromatography even after 4 years of cultivation. Although the method used to prepare samples for gas chromatography cannot directly distinguish among PHB polymer, hydroxybutyryl-CoA (HB-CoA), and hydroxybutyric acid, solvent washing experiments indicated that most or all of the signal was non-polymeric, presumably H-CoA. The synthesis of HB-CoA appeared to be linked to substrate growth limitation, with HB-CoA accumulation increasing dramatically and cell growth ceasing upon depletion of ammonium. This suggests that the PHB synthesis pathway in plants is subject to regulatory mechanisms similar to those in prokaryotic cells.

Evaluation and applications of optical cell density probes in mammalian cell bioreactors

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.

Scaleup of spinfilter perfusion bioreactor for mammalian cell retention

A spinning cylindrical filter, known as a spinfilter, permits the mammalian cell bioreactor operation at high perfusion rates leading to very high cell densities (10(7) mL(-1)). Filter screens with openings (25 mum) slightly larger than the average cell size have been used to retain single cells in suspension over a long period of operation without clogging. We have previously shown why it is necessary to optimize the rotational speed of the spinfilter in order to achieve efficient cell retention and avoid potential screen clogging. Effects of bulk mixing and perfusion rate on screen fouling and cell retention were also investigated. Based on this analysis, in this article, we suggest strategies for scaleup of spinfilters. Experimental data from 12- and 175-L (working volume) bioreactors is shown in support of the scaleup analysis.

Fluctuations in Continuous Mammalian Cell Bioreactors with Retention

Continuous flow bioreactors with cell retention have been increasingly used for the cultivation of mammalian cells. The potential advantages of such bioreactors are high cell concentrations and volumetric productivities. In many reported cases, these systems have shown fluctuations in cell concentrations of various frequency and magnitude. To analyze the dynamics of the fluctuations, a model-based approach is followed. Simulations showed that large fluctuations in biomass resulted in response to fluctuations in the retention ratio when the system is operated at high dilution rate and high cell retention. The dependence of cell concentration fluctuations on variations in dilution rate and retention ratio was established by a cross-correlation statistical analysis on available experimental data. The slower dynamics and the fluctuation propensity of retention systems suggest that continuous culture without retention is more convenient for kinetic studies. In all likelihood, continuous culture with retention can be stabilized by controlling both the retention ratio and the dilution rate.

Performance of mammalian cell culture bioreactor with a new impeller design

To improve the oxygen transfer in a mammalian cell bioreactor, a new type of impeller consisting of a double-screen concentric cylindrical cage impeller (annular cage impeller in short) was designed and its mass transfer rate evaluated. This new impeller design increases the specific screen area, and the convective mass transfer rate through the annular cage was significantly increased. The oxygen transfer rates with the new impeller and the commercially available cell-lift impeller (CelliGen by New Brunswick Scientific Co.) were evaluated and their performance compared at various rates of aeration and agitation. The results showed that with the new impeller, the oxygen transfer rate was increased by 19% in water and 21% in cell-free culture medium supplemented with 10% horse serum, the total hybridoma cell concentration was increased to 3.4 x 10(7) cells/mL, and the IgG(1) subtype monoclonal antibody (MAb) product concentration was also increased to 512 mg/L in perfusion culture of murine hybridoma cell line 62'D3. These improvements in oxygen transfer rate, cell concentration, and MAb product concentration are all very significant. The mass transfer resistance in the cell-lift impeller system was found to be mainly due to the surface area of the single-screen cage impeller. The new annular cage impeller not only provided the increased surface area for convective oxygen transfer but also protected cells from hydrodynamic shear damage, thereby achieving a significant bioprocess improvement in terms of higher viable cell concentration, higher product concentration, and higher oxygen transfer rate in the mammalian cell bioreactor system.

A comparison of three different mammalian cell bioreactors for the production of monoclonal antibodies

Three different commercially available stirred tank reactors for mammalian cell culturing were compared for the ability to support hybridoma cell growth and monoclonal antibody production in batch mode operation. Despite quite similar vessel geometries differences were found both in growth and production profiles in the systems. These differences can possibly be related to the different aeration modes used in the bioreactors, and the levels of shear stress created by stirrer and agitator in the tanks.

Evaluation of a hollow fiber oxygenator for use in bubble—free mammalian cell bioreactors

AbstractThe oxygen transfer capabilities of a hollow fiber oxygenator for intended use in bubble‐free aerated bioreactors have been evaluated experimentally. The oxygenator (480 mL), which is the main component of an artificial heart/lung unit used routinely in cardiopulmonary bypass procedures, was assessed for use in a recycle stream to determine if the oxygen requirements in bubble‐free hybridoma cell bioreactors could be supported on a large scale. Oxygen transfer to simulated medium in a 15 L bioreactor was found to depend primarily on the liquid recirculation rate (1 to 5 L/min) and was not seriously affected by the air flowrate in the oxygenator (0.5 to 15 L/min). Based on the experimentally determined mass transfer coefficient across the membrane, it was found that the oxygenator could support a 100 L fermentor at an average cell density of 2xl06 cells/mL with a specific uptake rate of 4.8 mgO2/(109 cells ‐ h).Furthermore, if oxygen enriched air were to be used in the oxygenator, a single unit could support cell densities beyond 8xl06cells/mL in a 100 L working volume fermentor.

Comparison of an electrochemiluminescent homogenous immunoassay and an ELISA for monitoring productivity in mammalian cell bioreactors

An Enzyme Linked Immuno Sorbent Assays (ELISA) and an Electro-chemiluminescent Immuno Assay (CIA) are compared for the purpose of monitoring product formation in mammalian cell bioreactors. The ELISA had a relative standard deviation of 10%, compared to 8% for the CIA . The CIA was found to be a fast and accurate alternative to the ELISA. The use of more than one immuno assay format was also shown to provide additional insight into process performance.

Design and Scaleup of an Anchorage‐dependent Mammalian Cell Bioreactor

Design and Scaleup of an Anchorage‐dependent Mammalian Cell Bioreactor

The Influence of Shear Stress on Cultured Vascular Endothelial Cells: The Stress Response of an Anchorage‐Dependent Mammalian Cell

AbstractThe influence of hydrodynamic forces on anchorage‐dependent mammalian cells is of interest both in biomedical research and in the development of scaled‐up mammalian cell bioreactor systems. Our own studies, reviewed here, have demonstrated that, in response to a fluid‐imposed laminar shear stress, cultured vascular endothelial cells undergo a dramatic alteration in structure and function. From these and related studies, it may be possible to obtain some insight into the general response of anchorage‐dependent mammalian cells to hydrodynamic forces. An unanswered question is: how does such a cell recognize and then transduce a fluid mechanic signal?

Analyses of Bioreactor Performance by Nuclear Magnetic Resonance Spectroscopy

There are many commercially and scientifically relevant products that must be produced in mammalian cell systems. Growth and maintenance systems (bioreactors) for mammalian cells are not well characterized, however, since these cells are more fastidious and sensitive than are microorganisms. Optimization of mammalian cell bioreactors can be facilitated by nondestructive monitoring of cellular physiology and metabolism in situ. Nuclear Magnetic Resonance (NMR) spectroscopy is ideal for this application. NMR can monitor a variety of parameters of intra and extracellular metabolites that contain nuclei with net magnetic moments. Such analyses can be used to determine energization, levels of 31P and 1Hcontaining metabolites, selected enzyme kinetics, compartmentalized ion activities, metabolic fates of 3H, 2H, 13C, 15N or 19Flabeled tracers, O2 tension, compartmentalized redox potential, membrane potential, cell number and cell volume. These can all be monitored with reasonable time resolution with minimum perturbation of the sample.