A simple device for planting replicate mammalian cell cultures

Summary The design and use of a unit for planting uniform inocula for replicate cultures are described. Its design permits continuous gassing of suspensions of mammalian cells with humidified CO₂, thus stabilizing the pH (±<0.05 pH unit) of culture media buffered with sodium bicarbonate. The unit can be readily modified to deliver different volumes; identical samples can be dispensed simply and rapidly, with minimal cell damage and chance of microbial contamination. Quantitative data regarding sample uniformity and growth subsequent to planting with this unit are presented.     

Regulating apoptosis in mammalian cell cultures

Cell culture technology has become a widely accepted method used to derive therapeutic and diagnostic protein products. Mammalian cells adapted to grow in bioreactors now play an integral role in the development of these biologicals. A major limiting factor determining the output efficiency of mammalian cell cultures however, is apoptosis or programmed cell death. Methods to delay apoptosis and increase the longevity of cell cultures can lead to more economical processes. Researchers have shown that both genetic and chemical strategies to block apoptotic signals can increase cell culture productivity. Here, we discuss various strategies which have been implemented to improve cellular viabilities and productivities in batch cultures.     

A method for the rapid establishment of normal adult mammalian colonic epithelial cell cultures

Summary Normal colonic epithelial cell cultures of mammalian origin are required to facilitate the study of both normal cellular functions as well as pathogenesis of certain (human) colonic diseases. To date, little information is available regarding the growth requirements of colonic epithelial cells in culture of eitehr animal or human origin. Such data would enable the development of a long-term culture system for these cells. In this study, we present methodology that results in the establishment of homogeneous cultures of adult rabbit colonic epithelial reproducibly, quickly, and in quantity. The epithelial nature of the cultures is unambiguously established by intermediate filament typing using antikeratin antibodies. Such culutres can now be used for a variety of functional studies as well as to investigate the growth requirements of colonic epithelial in culture. This work was supported by the Blinder Foundation for Crohn’s Disease Research, Harbor UCLA IBD Center (AM 36200) and grant AM 27806 from the National Institutes of Health, Bethesda, MD.     

Why do human hepatitis viruses replicate so poorly in cell cultures?

The five viruses which classically cause hepatitis in man represent diverse families of viruses and share in common only a striking hepatotropism and substantial restrictions to replication in conventional cell cultures. Hepatitis A virus is unique among these viruses in that it is amenable to propagation in cell culture, but replication of this virus is much slower and less efficient than replication of other picornaviruses. This probably reflects less efficient cap-independent viral translation, as well as restrictions at other points in the replication cycle. We speculate that the significantly restricted replication of hepatitis viruses in cell culture reflects evolutionary forces controlling their transmission and propagation through human populations.     

A method for growing stationary plant suspension cell cultures

Summary Stationary culture of plant cell suspensions has been achieved. Slurries, produced when small amounts of agar (0.1–0.4%) were added to culture media, were used to suspend plant cells. Growth proceeded more slowly than in standard shake culture, but cells remained viable for months of culture. This method of growing plant cells in stationary culture should be useful for general applications including long-term cell culture, shipment of cultures, and physiological, molecular biological, and pathological studies. Mention of a trademark, proprietary product, or vendor does not constitute a guarantee or warranty of the product by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products or vendors that may also be suitable. Editor’s Statement This procedure for growing stationary suspension cultures in an agar slurry should be useful for shipping suspensions and for long-term maintenance of little used or back-up cultures.     

Fluorometric determination of DNA in mammalian cell cultures by PicoGreen

An intercalating fluorochrome, PicoGreen, was assessed for its ability to determine the concentration of DNA in clarified mammalian cell culture broths containing monoclonal antibodies. Fluorescent signal suppression was ameliorated by sample dilution or by performing the assay above the pI of secreted IgG. The source of fluorescence in clarified culture broth was validated by incubation with RNase A and DNase I. At least 91.8% of fluorescence was attributable to nucleic acid and pre-digestion with RNase A was shown to be a requirement for successful quantification of DNA in such samples.     

In situ pH maintenance for mammalian cell cultures in shake flasks and tissue culture flasks

pH in animal cell cultures decreases due to production of metabolites like lactate. pH control via measurement and base addition is not easily possible in small‐scale culture formats like tissue‐culture flasks and shake flasks. A hydrogel‐based system is reported for in situ pH maintenance without pH measurement in such formats, and is demonstrated to maintain pH between 6.8 and 7.2 for a suspension CHO cell line in CD CHO medium and between 7.3 and 7.5 for adherent A549 cells in DMEM:F12 containing 10% FBS. This system for pH maintenance, along with our previous report of hydrogels for controlled nutrient delivery in shake flasks can allow shake flasks to better mimic bioreactor‐based fed batch operation for initial screening during cell line and process development for recombinant protein production in mammalian cells. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

A simple apparatus for measuring cell settling velocity

Accurate cell settling velocity determination is critical for perfusion culture using a gravity settler for cell retention. We have developed a simple apparatus (a settling column) for measuring settling velocity and have validated the procedure with 15-μm polystyrene particles with known physical properties. The measured settling velocity of the polystyrene particles is within 4% of the value obtained using the traditional Stokes' law approach. The settling velocities of three hybridoma cell lines were measured, resulting in up to twofold variation among cell lines, and the values decreased as the cell culture aged. The settling velocities of the nonviable cells were 33-50% less than the corresponding viable cells. The significant variation of settling velocities among cell populations and growth phases confirms the necessity of routine measurement of this property during long-term perfusion culture.     

Transtubular bioreactor: A perfusion device for mammalian cell cultivation

Summary A transtubular mammalian cell perfusion chamber has been developed that allows separate delivery and control of medium and gas flow to the cells. The hydrodynamic and mass transfer characteristics of the reactor are studied experimentally for both gas and liquid components. Feasibility and potential of the transtubular reactor are demonstrated by the growth of a hybridoma cell line.     

Production of recombinant h-AT III with mammalian cell cultures using capillary electrophoresis for product monitoring

The importance of mammalian cell cultures for biotechnological production processes is steadily increasing, despite the high demands of these organisms on their culture conditions. Efforts towards a more efficient bioprocess generally concentrate on maximizing the culture's life time, the cell number, and the product concentration. Here recombinant BHK 21 c13 cells are used to produce rh-AT III, an anticoagulant of high therapeutic value. The influence of the process mode (batch, repeated batch, continuous perfusion) and the process temperature (30°C vs. 37°C) on the above mentioned parameters is investigated. It is possible to increase the length of the culture from 140 h (batch) to more than 500 h (continuous perfusion culture), while concomitantly increasing the cell density from 0.72 10⁶/ml (batch) to 2.27 10⁶/ml (repeated batch) and 2.87 10⁶/ml (continuous perfusion culture). The accumulation of toxic metabolites, such as lactate, can be curtailed by reducing the bioreactor temperature from 37°C to 30°C during the later part of the exponential growth phase. Fast and reliable product monitoring became essential during process optimization. Capillary zone electrophoresis (CZE) in uncoated fused silica capillaries was studied for that purpose and compared to the standard ELISA. Under optimized conditions an AT III quantification could be done within 2 min with CZE. The detection limit was 5 g/ml. A relative standard deviation of less than 0.9% was calculated. The detection limit could be lowered by one order of magnitude by using a two dimensional system, where an liquid chromatographic (LC) system is coupled to the CZE. Concomitantly the resolution is improved. The two-dimensional analysis required 5 min. Membrane adsorbers (MA) were used as stationary phase in the LC-system, to allow the application of high flow rates (5–10 ml/min). The correlation between the LC-CZE analysis and the standard AT III-ELISA was excellent, with r²: 0.965. Using the assay for at line product monitoring, it is shown, that the process temperature is of no consequence for the productivity whereas the process mode strongly influences this parameter.     

Computer programs for modeling mammalian cell batch and fed-batch cultures using logistic equations

A MATLAB^® toolbox was developed for applying the logistic modeling approach to mammalian cell batch and fed-batch cultures. The programs in the toolbox encompass sensitivity analyses and simulations of the logistic equations in addition to cell specific rate estimation. The toolbox was first used to generate time courses of the sensitivity equations for characterizing the relationship between the logistic variable and the model parameters. Subsequently, the toolbox was used to describe CHO cell data from batch and fed-batch mammalian cell cultures. Cell density, product, glucose, lactate, glutamine, and ammonia data were analyzed for the batch culture while fed-batch analysis included cell density and product concentration. In all instances, experimental data were well described by the logistic equations and the resulting specific rate profiles were representative of the underlying cell physiology. The 6-variable batch culture data set was also used to compare the logistic specific rates with those from polynomial fitting and discrete derivative methods. The polynomial specific rates grossly misrepresented cell behavior in the initial and final stages of culture while those based on discrete derivatives had high variability due to computational artifacts. The utility of logistic specific rates to guide process development activities was demonstrated using specific protein productivity versus growth rate trajectories for the 3 cultures examined in this study. Overall, the computer programs developed in this study enable rapid and robust analysis of data from mammalian cell batch and fed-batch cultures which can help process development and metabolic flux estimation.     

A simple method for examining organotypic CNS cultures with nomarski optics

Summary This paper describes a method for examination of living organotypic cultures of CNS with Nomarski differential interference-contrast optics. Cultures grown in Maximow assemblies. which promote the best differentiation of the tissue but are optically faulty, are transferred for Nomarski observation to a simple sandwich chamber which combines the optical perfection of the usual sandwich chamber with the flexibility and safeguarding of sterility characteristic of the Maximow assembly. Thus cultures can be transferred repeatedly between their maintenance and observation chambers. In the resulting microscopic images, it is posible to visualize delicate unmyelinated fibers, myelinated cell bodies and other features which are normally impossible to demonstrate in living cultures as well as to improve the images of other structures such as large neuronal perikarya and myelinated axons. This work was supported in part by NIH Grant NS 11425     

Gene expression profiling for mechanistic understanding of cellular aggregation in mammalian cell perfusion cultures

Aggregation of baby hamster kidney (BHK) cells cultivated in perfusion mode for manufacturing recombinant proteins was characterized. The potential impact of cultivation time on cell aggregation for an aggregating culture (cell line A) was studied by comparing expression profiles of 84 genes in the extracellular adhesion molecules (ECM) pathway by qRT‐PCR from 9 and 25 day shake flask samples and 80 and 94 day bioreactor samples. Significant up‐regulation of THBS2 (4.4‐ to 6.9‐fold) was seen in both the 25 day shake flask and 80 and 94 day bioreactor samples compared to the 9 day shake flask while NCAM1 was down‐regulated 5.1‐ to 8.9‐fold in the 80 and 94 day bioreactor samples. Subsequent comparisons were made between cell line A and a non‐aggregating culture (cell line B). A 65 day perfusion bioreactor sample from cell line B served as the control for 80 and 94 day samples from four different perfusion bioreactors for cell line A. Of the 84 genes in the ECM pathway, four (COL1A1, COL4A1, THBS2, and VCAN) were consistently up‐regulated in cell line A while two (NCAM1 and THBS1) were consistently down‐regulated. The magnitudes of differential gene expression were much higher when cell lines were compared (4.1‐ to 44.6‐fold) than when early and late cell line B samples were compared (4.4‐ to 6.9‐fold) indicating greater variability between aggregating and non‐aggregating cell lines. Based on the differential gene expression results, two mechanistic models were proposed for aggregation of BHK cells in perfusion cultures. Biotechnol. Bioeng. 2013; 110: 483–490. © 2012 Wiley Periodicals, Inc.     

Cytotoxicity of two proteins isolated from Bacillus thuringiensis var. israelensis crystals to insect and mammalian cell lines

Abstract Two toxic proteins (proteins A and B) were isolated from Bacillus thuringiensis var. israelensis crystals using ion-exchange chromatography. A cell cytotoxicity assay based on the uptake of [³H]uridine by tissue culture cells following exposure to the toxic proteins, was used to measure the viability of the cells. Proteins A and B were cytotoxic to Aedes aegypti and MA104 cells. Protein B was also cytotoxic to Spodoptera frugiperda cells.     

Spectroscopic methods and their applicability for high‐throughput characterization of mammalian cell cultures in automated cell culture systems

The number and use of automated cell culture systems for mammalian cell culture are steadily increasing. Automated cell culture systems require miniaturized analytics with a high throughput to obtain as much information as possible from single experiments. Standard analytics commonly used for conventional bioreactor samples cannot handle the high throughput and the low sample volumes. Spectroscopic methods provide a means of meeting this analytical requirement and afford fast and direct access to process information. In the first part of this review, UV/VIS, fluorescence, Raman, near‐infrared, and mid‐infrared spectroscopy are presented. In the second part of the review, these spectroscopic methods are evaluated in terms of their applicability in the new field of mammalian cell culture processes in automated cell culture systems. Unlike standard bioreactors, these automated systems have special requirements that apply to the use of spectroscopic methods. Therefore, they are compared with regard to cell culture automation, throughput, and required sample volume.     

A simple HPLC technique for accurate monitoring of mammalian cell metabolism

We have developed a simple and accurate isocratic HPLC method, without any prederivatisation, for the determination of glucose, lactate, glutamine, glutamate, pyrrolidone carboxylic acid and alanine in samples from mammalian cell cultures. The method has been successfully validated with enzyme analysis for each of the compounds. Quantification of pyrrolidone carboxylic acid makes the correction for glutamine decrease due to chemical decomposition very simple and accurate, and avoids some possibly erroneous calculations.     

Dianthramide glucosides from tissue cell cultures of Delphinium staphisagria L

Tissue cell cultures of Delphinium staphisagria L. produced three dianthramide glucosides N-(2'-beta-glucopyranosylsalicyl)-5-hydroxyanthranilic acid methyl ester, N-(2'-beta-glucopyranosyl-5'-methoxysalicyl)-5-hydroxyanthranilic acid methyl ester and N-(2'-beta-glucopyranosyl-5'-hydroxysalicyl)-5-hydroxy-6-methoxyanthranilic acid methyl ester, together with known methyl esters of N-salicylanthranilic acid and N-(2'-beta-glucopyranosyl-5'-hydroxysalicyl)-5-hydroxyanthranilic acid. Structures of the glucosides were established by MS, 1-D and 2-D NMR techniques.