Super7 passaging method to improve Chinese hamster ovary cell fed-batch performance.

Chinese hamster ovary (CHO) cells are widely used to manufacture biopharmaceuticals, most of all monoclonal antibodies (mAbs). Some CHO cell lines exhibit production instability, where the productivity of the cells decreases as a function of time in culture. To counter this, we designed a passaging strategy that, rather than maximizing the time spent in log-growth phase, mimics the first 7 days of a fed-batch production process. Cultures passaged using this method had lower net growth rates and were more oxidative throughout 6 weeks of passaging. Fed-batch cultures inoculated by cells passaged using this method had increased net growth rates, oxidative metabolism, and volumetric productivity compared to cells passaged using a conventional strategy. Cells from unstable cell lines passaged by this new method produced 80%-160% more mAbs per unit volume than cells passaged by a conventional method. This new method, named Super7, provides the ability to mitigate the impact of production instability in CHO-K1 cell lines without a need for further cell line creation, genetic engineering, or medium development.

CD28 costimulation is essential for human T regulatory expansion and function.

The costimulatory requirements required for peripheral blood T regulatory cells (Tregs) are unclear. Using cell-based artificial APCs we found that CD28 but not ICOS, OX40, 4-1BB, CD27, or CD40 ligand costimulation maintained high levels of Foxp3 expression and in vitro suppressive function. Only CD28 costimulation in the presence of rapamycin consistently generated Tregs that consistently suppressed xenogeneic graft-vs-host disease in immunodeficient mice. Restimulation of Tregs after 8-12 days of culture with CD28 costimulation in the presence of rapamycin resulted in >1000-fold expansion of Tregs in <3 wk. Next, we determined whether other costimulatory pathways could augment the replicative potential of CD28-costimulated Tregs. We observed that while OX40 costimulation augmented the proliferative capacity of CD28-costimulated Tregs, Foxp3 expression and suppressive function were diminished. These studies indicate that the costimulatory requirements for expanding Tregs differ from those for T effector cells and, furthermore, they extend findings from mouse Tregs to demonstrate that human postthymic Tregs require CD28 costimulation to expand and maintain potent suppressive function in vivo.

Reduced mitochondrial membrane potential and metabolism correspond to acute chloroform toxicity of in vitro hepatocytes.

Chloroform is a non-genotoxic compound that is present in drinking water and ambient air as a result of water chlorination but whose carcinogenic mechanism in humans is unknown. Chloroform targets the liver in humans, where cytochrome P-450-dependent biotransformation to phosgene results in mitochondrial damage and cell death. The purpose of this study is to investigate the relationship between cell death, loss of mitochondrial membrane potential (MMP) and reduction of metabolic rates for in vitro cultured mouse hepatocytes after acute exposure to two doses of chloroform. Immediately following a 2-h exposure, culture viabilities were 70% and 54% for concentrations of 7.0 and 8.8 mM, respectively, in contrast with 90.0% for controls. Interestingly, the viabilities of these cultures decreased further, to 6% and 12%, respectively, over the next 24-h period despite being placed in fresh, chloroform-free medium. Measurement of MMP for viable cells at the end of the exposure revealed a decrease in Rhodamine 123 uptake, which indicates a loss of MMP. Additionally, glucose consumption and lactate production rates were reduced during the 6-h period following the exposure. These results support the hypothesis that a subpopulation of cells at the end of an acute exposure may be activated for apoptosis, suggesting a role for apoptosis markers during risk assessment for chloroform.

96-well plate assay for sublethal metabolic activity.

A 96-well plate assay has been devised for estimation of sublethal metabolic activity for compounds administered to in vitro cell cultures during 6- and 24-h exposures. This screen combines a resazurin reduction assay with lactate production and glucose consumption rate assays to assess effects of compounds on both culture viability and metabolic inhibition. In this article, the assay is demonstrated by determining the extent to which five glycolysis inhibitors, namely, phloretin, 2-deoxyglucose, iodoacetate, fluoride, and oxamate, induce metabolic inhibition without cell death for in vitro fibroblast cell cultures. During 6-h exposures, iodoacetate was found to be the most potent inhibitor of glycolysis, whereas iodoacetate and phloretin were most cytolethal. 2-Deoxyglucose had the largest sublethal metabolic range, spanning just over two orders of magnitude in concentrations between its IC(50) values for cytolethality and metabolic inhibition. This method provides a simple and inexpensive way to determine global metabolic and cytolethal effects of compounds for in vitro cell culture systems. It should be of use directly for large-scale screening and ranking of compounds during drug discovery and development, in conjunction with or following some more direct measure of therapeutic efficacy of a prospective drug candidate. Moreover, firsthand determination of the concentrations over which a compound has lethal, sublethal metabolic, or no such effects should serve as a cost-effective and time-saving first step in a given research study, preceding more expensive, lengthy, and/or detailed in vitro methods.

Determination of carbon dioxide production rates for mammalian cells in 24-well plates.

In this report, we describe a method for the quantitative determination of carbon dioxide production rates of mammalian cells. Custom-made, reusable, optically clear plugs are used to seal the wells of a 24-well plate. These plugs prevent the loss of CO2 produced by the mammalian cells cultured in bicarbonate-free medium. Measurements of pH, total liquid phase CO2, and viable cell density are used to estimate the average CO2 production rate during a 6-h incubation period. Using this method, four chemicals well-characterized in regards to toxicity, 2,4-dinitrophenol, antimycin A, rotenone, and cyanide, were found to elicit significant changes in CO2 production for given concentrations within 6 h, without inducing a decline in culture viability. Over longer exposure times, similar concentrations caused growth inhibition but not cell death. An assay based on metabolic change corresponding to growth inhibition that is more sensitive than traditional measures of cell death is a feasible complement to existing methods in drug discovery and toxicity testing.

Toxic concentrations of exogenously supplied methylglyoxal in hybridoma cell culture.

Concentrations at which methylglyoxal, a by-product of cellular metabolism, can be toxic to hybridoma cell cultures were determined using exogenously supplied doses. Trypan blue cell counts of 6-well cultures incubated for 24 h with various methylglyoxal concentrations revealed inhibition of cell growth at 300 muM and higher, with a median inhibitory concentration of 490+/-20 muM. The primary mode of death was apoptosis, as assessed by chromatin condensation, and the effects of methylglyoxal were observed to be complete by approximately eight hours. Yet, the impact of methylglyoxal was a function of the rate of dosing; stepwise addition of MG during the first 6 h of incubation inhibited growth but caused much less cell death than a comparable bolus dose. Inhibition of cellular metabolism by MG was found to coincide with inhibition of cell growth, with a comparable median inhibitory concentration of 360+/-20 muM. The effects on viable cell density and metabolism were both linear at doses approaching zero, with lowest observable effect levels of 54 and 77 muM, respectively. These results provide quantitative estimates for concentrations of methylglyoxal that may be inhibitory to biopharmaceutical-producing cell lines.

Metabolic screening of mammalian cell cultures using well-plates.

For use in a broad spectrum of cell culture applications, we have devised a novel method, termed High-Throughput Metabolic Screening (HTMS), with which to more rapidly screen the overall activity of major metabolic pathways of mammalian cells. This current protocol uses adaptations of theoretical and experimental techniques from metabolic and cell culture engineering. First, HTMS makes use of a simplified metabolic network for metabolic flux analysis. Despite its simplicity, the network is capable of generating flux distributions and ATP production rates that are comparable to a more detailed network. Second, HTMS makes use of microtiter well-plate technology and adaptations of well-known enzymatic assays to increase precision and throughput for cell culture experiments. Multireplicate, multiparallel cultures in the sub-milliliter scale yield very precise metabolic rates using common laboratory equipment and at a fraction of the cost and time of traditional experiments with T-flasks, spinner flasks, or bioreactor systems. The simplicity of the network and the well-plate assays synergistically comprise a new, extremely useful, broadly applicable, and relatively inexpensive way to probe cell cultures for metabolic effects, screen drugs and toxins, optimize media, and support the development of bioprocesses. The simplified network and cell culture and analytical assays are also useful for undergraduate, graduate, and professional training.

24-well plate spectrophotometric assay for preliminary screening of metabolic activity.

A 90-min assay using 24-well plates was developed for screening overall metabolic effects of toxic chemicals by measuring extracellular acidification rate of mammalian cells. During the 90-min test, the pH of each well of a 24-well plate is monitored by measuring the absorbance of phenol red using a spectrophotometric plate reader. The acidification rate is then calculated from the change in pH divided by the time interval and cell density, which is also estimated non-invasively by spectroscopy. The assay was verified by testing the effects of five well-characterized chemical toxins on fibroblast cell cultures. As expected, the responses of the fibroblasts were dependent on the dose and type of toxin. They also corresponded with the established mechanisms of the toxins and with measured lactate production rates. The set-up is simple, inexpensive, and amenable to being automated. The method is easy to perform and rapid. Uses include screening compounds for gross metabolic effects in mammalian cell lines, determining preliminary metabolic dose-response curves for guiding further research, and designing and optimizing media for in vitro systems utilizing cell cultures.