Lymphoid cells often grow poorly or die when grown at low density. The reasons are still not well understood, but may relate to requirements for growth factors, and possibly also toxic products from the tissue culture vessels. Choice of the particular batch of FCS may make a big difference.
Although it is rarely necessary on a routine basis, the growth of small numbers of cells is sometimes improved by incubating the tissue culture ware with medium overnight at 37°C, and then replacing it with fresh medium before the cells are added. It is presumed that something toxic in the plastic is being washed out.
These problems can be overcome (at least partly) by culturing together with a slow-growing or nongrowing population of cells. These cells are usually termed 'feeders', implying that they make something needed for growth. If culture conditions are optimal, feeder cells may make little difference. They do no harm however, and may reduce variation between experiments (Fazekas and Scheidegger, 1980). The current concept of the action of feeder cells is that they supply growth factors, particularly IL-6, but they are very likely also to supply others (see Section 8.3.2). If the fusion mixture is plated out at high cell density, added feeders may make little or no difference because the spleen cells used for fusion also act as feeders.
As noted previously, the presence offeeder cells may make a crucial difference between success and failure of production of rat hybrids.
Commonly used feeder cells include thymocytes (typically 106/0.2 ml well; Lernhardt et al., 1978; Oi and Herzenberg, 1980), normal spleen cells (lOVml; Galfre and Milstein, 1981) or peritoneal cells (Hengartner et al., 1978; typically 2 x 104/0.2 ml). Peritoneal cells are harvested by washing out the peritoneal cavity with sterile saline, using a syringe and needle and taking care to avoid puncturing the gut. Roughly half are lymphocytes and half are macrophages. If the mice are from specific pathogen-free colonies, yields will be 3-5 x 106 cells per mouse. 'Conventional' mice will yield up to 10 times as many cells.
Peritoneal cells from rats are collected by anaesthetizing the rat, injecting 20 ml of sterile culture medium into the peritoneal cavity, and gently massaging the abdomen. After 2-5 min, the rat is killed and the medium aspirated. Typically, one may recover about 2 x 107 cells (Ravoet and Bazin, 1990).
The feeder cells do not have to be histocompatible with the hybrids. They do not even need to be from the same species. Rat thymocytes seem to function well for mouse-mouse hybrids.
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