[Abstract unavailable

The incidence of apoptotic and necrotic cell death was compared in CHO, SF9 insect cells and murine plasmacytoma (J558L) and hybridoma (TB/C3) cells during in vitro cultivation in batch cultures. Acridine orange staining and fluorescence microscopy enabled the visualization of a classic morphological feature of apoptotic cell, the presence of condensed and/or fragmented chromatin. DNA gel electrophoresis was employed to show an additional characteristic of the process, the endonuclease-mediated fragmentation of DNA into multiples of 180 base pairs. The levels of apoptosis at the end of batch cultures of plasmacytoma and hybridoma cell lines were found to be 60% and 90% of total dead cells, respectively. However, employing the above-mentioned techniques, the biochemical and morphological features of apoptosis were not found in CHO and SF9 insect cells. Some factors affecting the induction of apoptosis during the batch culture of the hybridoma and plasmacytoma cell lines were identified. The most effective inducer was found to be glutamine limitation, followed by (in order of importance) serum limitation, glucose limitation, and ammonia toxicity. Blockage of the cell cycle of the plasmacytoma and hybridoma cells using thymidine resulted in the induction of apoptosis. This has important implications for the development of cell culture processes that minimize cell division and thereby increase specific productivity.

Chinese hamster ovary cells have been engineered to inducibly over-express the p21(CIP1) cyclin-dependent kinase inhibitor, to achieve cell cycle arrest and increase cell productivity. In p21(CIP1)-arrested cells production of antibody from a stably integrated lgG4 gene, was enhanced approximately fourfold. The underlying physiological basis for enhanced productivity was investigated by measuring a range of cellular and metabolic parameters. Interestingly, the average cell volume of arrested cells was approximately fourfold greater than that of proliferating cells. This was accompanied by significant increases in mitochondrial mass, mitochondrial activity, and ribosomal protein S6 levels. Our results suggest that p21(CIP1)-induced cell cycle arrest uncouples cell growth from cell-cycle progression, and provides new insight into how improved productivity can be achieved in a cell line commonly used for large-scale production of pharmaceutical proteins.

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The question is addressed as to whether cells which are subject to high-energy dissipation rates in agitated bioreactors show an apoptotic response. Murine hybridoma cells in batch culture were agitated in bench-scale (1-L) bioreactors without gas sparging. At an energy dissipation rate of 1.5 W m(-3) there was no apparent damage. At 320 W m(-3) cell viability declined, and increasing proportions of the dead cells displayed the morphological features of apoptosis, but necrosis also remained as a significant mechanism of death. When cells were subjected to the intensive energy dissipation rate of 1870 W m(-3) in a bioreactor without gas headspace, the cell number dropped by 50% within 2 h and a subpopulation of smaller-sized cells emerged. This excluded trypan blue but showed some apoptotic characteristics such as reduced and condensed DNA content and low F-actin content. The incidence of apoptotic activity was further demonstrated by the appearance of numerous apoptotic bodies. Analysis of the cell cycles of both small and normal size populations indicated that greater proportions of S and G2 cells had become apoptotic and there was evidence of preferential survival of G1 cells. It is suggested that two mechanisms of cell death are apparent in hydrodynamically stressful situations, but their relative expression depends on the energy dissipation rate.

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Using mechanical cell properties measured by micromanipulation, and a model of cell distortion in laminar flow fields, a method has been developed for predicting disruption of animal cells by laminar shear stresses. Predictions of the model were compared with measured losses of cell number and viability of TB/C3 murine hybridomas sheared in a cone and plate viscometer at shear rates up to 3950 s(-1), and shear stresses up to 600 Nm(-2), achieved by enhancement of viscosity with dextran. In all cases, the experimental, results and predictions were within 30%. Such excellent agreement suggests it might be possible to use micromanipulation measurements of animal cell mechanical properties to predict cell damage in more complex flow fields, such as those in bioreactors.

Here, we have studied two parameters critical to process control in mammalian cell culture; dissolved oxygen (dO 2 ) and pH, measured with fluorescent sensors thus allowing the study of the metabolic state of cells in culture without removing or damaging cells during cultivation. Two cell lines, namely, NS0 and CHO were batch-grown in 24-well plates at different serum concentrations with the sensors implemented in the bottom of each well. The data showed a good relationship between the dO 2 and pH data obtained from fluorescent probes and the growth and death characteristics of cells. The method has provided a high throughput on-line multi-parametric analysis of mammalian cell cellular activity.