Physiological alterations of GS-CHO cells in response to adenosine monophosphate treatment.

Growth-arrested strategies (e.g. hypothermia and hyperosmolarity) have been widely employed to enhance cell-specific productivity (qP) in mammalian cell culture bioprocess. In addition to enhanced qP, alterations in cell physiology, such as cell size and cell cycle phase, have also attracted extensive attention under growth-arrested conditions. However, to date, very few reports on associations between physiological changes in growth-inhibiting approaches have been published. In this study, we explored associations between the physiological changes of GS-CHO cells in response to adenosine monophosphate (AMP) treatment. In dose response studies, AMP treatment resulted in suppressed proliferation, accumulated S-phase cells, increased cell size and enhanced qP. Subsequently, six GS-CHO clones exhibited the physiological alterations in varying degrees when treated with 7 mM AMP. But more importantly, a significant positive correlation between total intracellular protein content and mean electronic volume, an indicator of cell size (P < 0.01) was found, indicating that total intracellular protein was the determining factor in increasing cell size in this growth-arrested strategy. Besides, our results provide additional evidence that treatment with growth-arrested agents may increase cell size; the agent per se did not cause the increased productivity.

Measuring dissolved oxygen to track erythroid differentiation of hematopoietic progenitor cells in culture.

As stem cell technologies move from the developmental to the commercial stage strategies must be developed to monitor culture operations. These will ensure consistency of differentiation programs and maintenance of optimum cell viability during production runs. Due to the sensitivity of stem cells to their environment, and their variability in response to external stimuli, accurate monitoring of in vitro conditions will be crucial for effective large-scale culturing of therapeutic stem cells. Here we describe a simple method to monitor the expansion and maturation of adult human haematopoietic stem/progenitor cells into red blood cells in vitro by measuring the oxygen consumption rate of cultures. Cell cultures followed a characteristic pattern of oxygen consumption that is reflective of in vivo erythroid maturation. This method could be easily developed as an online system to map erythroid differentiation and maturation of cultured cells as effectively as the more time consuming process of flow cytometric analysis of surface marker expression patterns.

Defining viability in mammalian cell cultures.

A large number of assays are available to monitor viability in mammalian cell cultures with most defining loss of viability as a loss of plasma membrane integrity, a characteristic of necrotic cell death. However, the majority of cultured cells die by apoptosis and early apoptotic cells, although non-viable, maintain an intact plasma membrane and are thus ignored. Here we measure the viability of cultures of a number of common mammalian cell lines by assays that measure membrane integrity (a measure of necrotic cell death) and assays that measure apoptotic cells, and show that discrepancies in the measurement of culture viability have a significant impact on the calculation of cell culture parameters and lead to skewed experimental data.

Analysis of an artificially selected GS-NS0 variant with increased resistance to apoptosis.

Heterogeneity in gene expression and phenotypic traits is an inherent feature within the "clonal" cell lines used for biopharmaceutical production. This feature can allow the selection of cell lines with improved phenotypes and adaptation to growth under preferential conditions to improve productivity or provide a platform to study the molecular basis of improved characteristics. A repeated process of extended batch culture of a recombinant antibody producing GS-NS0 myeloma cell line generated a stable variant cell line displaying increased resistance to both environmental stresses and chemical apoptosis inducers, and resulted in extended culture viability and increased antibody production. An interesting feature of the variant cell line was an altered metabolic state with consumption of lactate as the culture progressed. The variant cell line also showed altered expression of proteins associated with autophagy suggesting a role for this process in extending cell survival in culture. Targeted transcriptomic analysis was carried out on the parental and variant cell lines using a qRT-PCR array containing a panel of apoptosis-associated genes to elucidate both the predominant apoptotic pathways in the NS0 cell line with batch culture progression, and the altered gene expression contributing to increased survival in the variant line. Results indicated a balance between pro- and anti-apoptotic signaling is triggered with the onset of cell death in the NS0 cell line. Pro-survival pathways such as NFκB signaling and the unfolded protein response were implicated along with death receptor, endoplasmic reticulum stress and p53 mediated apoptotic pathways. The identification of altered gene expression in the variant cell line also provides several potential targets for cell engineering strategies to create improved cell lines for production.

Selection methods for high-producing mammalian cell lines.

The selection of high-producing mammalian cell lines represents a bottleneck in process development for the production of biopharmaceuticals. Traditional methods are time consuming (development times often exceed six months) and significantly limited by the number of clones that can be feasibly screened. The market for therapeutic proteins is set to double by 2010, so there is an increasing need to develop methods for the selection of mammalian cell lines stably expressing recombinant products at high levels in an efficient, cost-effective and high-throughput manner. Alternatives include higher throughput methods based on flow-cytometric screening and recently developed automated systems for the selection of high-producing cell lines.