2.3. Isolation and cultivation of non-permanent cell lines
Recently, considerable progress has been made in the development of techniques for the isolation and cultivation of non-permanent honey bee cell lines. These cell lines have some advantages over primary cells because they can be passaged at least once and, therefore, can be cultivated for a longer time than primary cells. All life stages, from eggs to adult, and various tissues, appear to be capable of producing primary cell cultures, with isolations from bee brains (Goldberg et al., 1999), antennae (Barbara et al., 2008) embryos (Chan et al., 2010), haemolymph (VanSteenkiste 1988; Sorescu et al., 2003) fat bodies (Kaatz et al., 1985; Hunter 2010), with the most successful reports supporting use of 4-9 day old developing larvae (Sorescu et al., 2003; Rocher et al., 2004, Hunter 2010). Even so, bee cell proliferation in culture is generally slow. Addition of foetal calf serum (FCS) at a concentration of about 5–20 %, or various amounts of haemolymph or pollen do not appear to affect cell proliferation or rate of growth. Cells normally do not show any signs of differentiation over time, thus cell passages reported are few, up to 5 times over several months (3-8 months cultivation). Larvae with developing head capsule, white eyes, along with the light brown eye, early stage pupae appeared to produce more active cell cultures with diverse cell types, especially when a special culture medium AmWH5 (Table 1) developed for the establishment of non-permanent cell-lines from honey bee tissues was used (Hunter, 2010). An example of such a non-permanent honey bee cell line established from white-eyed pupae is shown in Fig. 1.
Fig. 1. Primary culture of honey bee, A. mellifera, pupae, white head, 17d post explanted. AmWH5 medium (Hunter, WB, USDA,ARS 2011.).