The mammalian central nervous system is developmentally derived from neuroepithelial cells in the neural plate. These neuroepithelial cells grow and differentiate in response to signals from their surrounding environment. Many of those signals have been well characterized and others remain to be discovered. In cell culture, a conditioned medium, a feeder cell layer, or a tissue extract has been used as supplement in addition to those factors well characterized for maintaining the multipotent status of neural progenitor cells. To date, there have been many types of neural progenitor cells established in culture from various stages of development and from different regions of the nervous system of various species. This chapter will provide a brief introduction to those cultures and a detailed method for culturing rat neural epithelial cells at embryonic stage E9 and characterizing them in vitro and in vivo.

Temperature reduction in CHO cell batch culture may be beneficial in the production of recombinant protein and in maintenance of viability. The effects on cell cycle, apoptosis and nucleotide pools were studied in cultures initiated at 37°C and temperature shifted to 30 °C after 48 hours. In control cultures maintained at 37 °C, viable cells continued to proliferate until the termination of the culture, however, temperature reduction caused a rapid decrease in the percent of cells in S phase and accumulation of cells in G-1. This was accompanied by a concurrent reduction in U ratio (UTO/UDP-GNAc), previously shown to be a sensitive indicator of growth rate. Culture viability was extended following temperature shift, as a result of delayed onset of apoptosis, however, once initiated, the rate and manner of cell death was similar to that observed at 37 °C. All nucleotide pools were similarly degraded at the time of apoptotic cell death. Temperature reduction to 30 °C did not decrease the energy charge of the cells, however, the overall rate of metabolism was reduced. The latter may be sufficient to extend culture viability via a reduction in toxic metabolites and/or limitation of nutrient deprivation. However, the possibility remains that the benefits of temperature reduction in terms of both viability and productivity are more directly associated with cultures spending extended time in G-1.

1.Introduction. 2.Setting up a Cell Culture Lab. 3.The Physical Environment. 4.Media. 5.Standard Cell Culture Techniques. 6.Looking at Cells. 7.Contamination, How to Avoid It, Recognize It, and Get Rid of It When It's There. 8.Special Considerations for Serum Free Culture-Established Cell Lines. 9.Primary Cultures. 10.Establishing a Cell Line. 11.Special Growth Conditions. 12.Cell Culture for Commercial Settings. Glossary. Appendices: Equipment List. List of Cell Lines and Where to Get Them. Preparation of Bovine Pituitary Extract. Index.

Complex nutrient mixtures, which are usually called "media," are almost always supplemented with serum, with another complex biological fluid (e.g., milk, embryo extracts, and plasma), or with a defined mixture of hormones and growth factors. The choice of medium and supplements can have a major impact on the growth, function, and even phenotypic and genetic stability of cells in vitro. This choice thus becomes an important part of developing a useful and meaningful in vitro model system. This chapter defines the various roles that the medium plays in supporting cell function and outlines a method for selecting and optimizing medium in growing the cell of choice.

This chapter discusses the simultaneous measurement of cell cycle and apoptotic cell death. Many assays of apoptosis and/or cell cycle use flow cytometry. The chapter describes two different assays to measure apoptosis and cell cycle simultaneously using flow cytometry. The first involves the use of terminal transferase (the TUNEL assay) together with propidium iodide for the identification of cell cycle, while the second uses fluorescently labeled annexin V, together with propidium iodide as an indicator of cell-membrane integrity, and additionally Hoechst 33342 for the determination of cell cycle. The terminal transferase assay is performed using fixed cells, and it is, therefore, useful in the analysis of samples collected over time. The annexin V assay is performed using unfixed cells; thus, it provides information regarding membrane integrity.