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When switching back and forth between different dish configurations, it is important to maintain a constant inoculum density (as measured in cells/mm2 of dish surface) and a similar amount of medium/cell. The latter may have to vary, however, since meniscus formation in small wells (e.g., 24-, 48-, or 96-well plate wells) may require adding proportionately more medium/well in order to keep the cells in the center submerged. Table 2.1 lists some commonly used dish sizes, their surface area, and the amount of medium commonly added per well.
Originally, all cell and tissue culture was carried out in glass plates or vessels. This method is extremely labor intensive, since it requires that all plates be carefully washed and resterilized after use. The availability of cheap disposable sterile plasticware has made culture of cells on glass a special-purpose exception rather than the rule. In some cases it may be necessary to have cells grown on glass. The manipulation involved in fixing, staining, and viewing cells for imunohistochemistry, in situ mRNA analysis, or scanning electron microscopy, for example, are best carried out on cells attached to a glass slide or coverslip. There are several ways to do this. Sterile "chamber slides" containing 1, 2,4, 8, or 16 chambers per slide are commercially available. This glass (or plastic) slide has a plastic superstructure attached by a silicon gasket that divides the slide into the requisite number of sections and contains the cells and medium. This is protected by a plastic cover. The cells are plated onto the glass surface in the usual medium and placed in the incubator. When the cells have reached their desired state of confluence, the slides are removed and the cells fixed and/or stained for observation. The same cells can be placed in all chambers and each well stained individually, or different cells can be placed in each well and the entire slide stained at one time. The plastic grid can be removed from the slide and the slide can be handled like a normal glass slide for staining, mounting, and observation using an upright microscope and short working distance objectives. This method has the advantage of flexibility and convenience; however, some cells will not stick well to these surfaces.
An alternate method is to grow cells directly on coverslips placed in a plastic tissue culture dish. Several square coverslips can be placed in a 60- or 100-mm plastic tissue culture dish or round coverslips can be individually placed into the wells of 24-well multiwell plates. The coverslips must be sterilized. It is best to clean them thoroughly to remove any chemicals and coatings left on by the manufacturer. This may be done by soaking a num-
ber of coverslips for 2 hr in acetone, rinsing and soaking overnight in ethanol, and after a second ethanol rinse flaming to sterilize (or let coverslips drain dry). Each can then be placed in a dish with a sterile forceps. The glass surface can be coated with attachment factors and the cells cultured as usual. The coverslips and attached cells can then be fixed and stained and mounted directly on a slide for viewing.
It is easiest to handle No. 2 thickness coverslips, which do not break as easily as the thinner ones. However, these may be too thick for the optimal working distance of some objectives.
The shape and size of cells in vivo will depend on their environment, what they are attached to, the hormonal signals they are receiving, and their function. Thus, epithelial cells may form a single cell layer attached to a basement membrane with thick, highly polarized cells. Peritubular cells may also be attached in a single layer but may be very thin, acting more as a physical barrier than a protein or mucopolysaccharide-secreting factory. Interstitial cells such as those in the ovary and testis may be almost round in cross-section and may occur in connected clumps in the tissue. In the artificial environment of in vitro culture systems, most cells will be rounded when grown in suspension and flattened when grown on a surface such as tissue culture plastic. Some cells, such as pancreatic b cells or testicular Leydig cells, may show increased function when they are allowed to maintain a rounded shape; other cell types may need to attach and spread to survive (Folkman and Moscona, 1978; Gospodarowicz et al., 1978).
Basement Membrane and Attachment Factors
Plastic or glass surfaces can be coated with a purified attachment protein such as fibronectin, laminin, or collagen. This is discussed more completely in Chapter 8 on serum-free culture. Basement membrane can also be laid down on the culture dish by one cell type, the cells removed sterilely, and a second cell type cultured on this cell-produced ECM (Gospodarowicz et al., 1980, 1982; Mather et al., 1984). This is less defined and more laborious than using purified protein components, but might well support a cell's growth or function better. A procedure for producing plates coated with cell-produced ECM is outlined later in this chapter. In addition, a cell can be selected to produce the tissue-type-specific ECM that would be most appropriate for the cell to be grown in vitro, thus providing a more physiologically relevant culture model. For example, a stromal cell line may be used to produce ECM to grow the epithelial cell type normally found adjacent to that stromal cell.