Observations of cell size dynamics under osmotic stress

Abstract

Cultured mammalian cells [e.g., murine hybridomas, Chinese hamster ovary (CHO) cells] used to produce therapeutic and diagnostic proteins often exhibit increased specific productivity under osmotic stress. This increase in specific productivity is accompanied by a number of physiological changes, including cell size variation. Investigating the cell size variation of hyperosmotically stressed cultures may reveal, in part, the basis for increased specific productivity as well as an understanding of some of the cellular defense responses that occur under hyperosmotic conditions. The regulation of cell volume is a critical function maintained in animal cells. Although these cells are highly permeable to water, they are significantly less permeable to ionic solutes. Appropriate cellwater content is actively maintained in these cells by regulation of ion and osmolyte balances. Transport appropriate to extracellular conditions, leading to accrual or release of these species, is activated in response to acute cell volume changes. Osmotically induced regulatory volume increases (RVI) and regulatory volume decreases (RVD) are known to occur under a variety of conditions. We observed the time evolution of size variation in populations of two CHO cell lines under hyperosmotic conditions. Observations were made using multiple instruments, multiple cell lines, and multiple cell culture conditions. Size variation of CHO A1 was gauged by flow cytometry using an LSRII 1 flow cytometer while CHO B0 cells were quantified using a Cedex 1 cell analyzer. Hyperosmotic stress had a dose-dependent effect on the regulatory control of cell volume. Stressed cultures of CHO cells grown in suspension exhibited a shift in mean cell diameter. This shift in mean was not due to a change in the whole population, but rather to the emergence of distinct subpopulations of cells with larger cell diameters than those in the bulk of the population. ' MONOCLONAL antibodies (mAbs) play an important role in the modern medical, biological and biotechnological landscape. Not only do they occupy a large and growing segment of new biopharmaceuticals but also they are a lab workhorse providing much of the power for in vitro diagnostics and other essential lab techniques. As demand for these molecules continues to grow, our ability to produce them effectively and efficiently becomes critically important.Many researchers and companies are exploring approaches for increasing production and more broadly reducing costs, which for therapeutic mAbs, can range from 300-3,000 US$ per gram (1). These increases have typically been achieved by optimization of cell culture conditions or optimization of the cell lines themselves. Success in these regimes is determined by increased cell density, increasing cell longevity, and by increasing specific productivity.

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