1. Introduction

In the beginning of the twentieth century, a new area of research began to develop. With the cultivation of tissues (explants) followed by the cultivation of primary cells and later by the development of immortalized, permanent cell lines Cell Culture / Cell Biology became a discipline of its own. These early works already included the use of invertebrates, and even Hymenoptera, when muscle explants from Vespa were cultivated to perform polarization optical experiments with reflected light (Pfeiffer, 1941). Since then reams of vertebrate and invertebrate permanent cell lines have been developed, and many of them are now commercially available from different cell culture collections. There also has been, and continues to be, a growing body of published work on the development and use of hymenopteran tissue and cell cultures (Giauffret, 1971; Kaatz et al., 1985; Greany, 1986; Ferkovich et al., 1994; Rocher et al., 2004).

The lack of immortalized cell lines especially for honey bees, Apis mellifera L., continues, however, to be a major limiting factor of many studies trying to examine physiology and disease. Current studies which use bee cell cultures have thus relied on primary cultures or on non-permanent cell lines of low passage number (Lynn, 2001; Bergem et al., 2006; Barbara et al., 2008; Chan et al., 2010; Hunter, 2010; Poppinga et al., 2012). One of the main drawbacks of such primary cell cultures and non-permanent cell lines is that they are usually produced within the laboratory of origin and are thus not made available for widespread use by other researchers. They might also present problems with reproducibility. Even so, such bee cell cultures are and will be useful for e.g., examining bee cell physiology, host cell-pathogen interactions, or the effects of various chemicals on gene and protein expression in bee cells using modern technology and approaches like genomics or transcriptomics.

In contrast to these cell culture approaches, immortalized, permanent cell lines like those established from many non-hymenopteran insects  (mainly Lepidoptera and Diptera) or vertebrates (mainly mammals) have several important advantages. They provide an excellent system to study cellular events, such as gene expression, DNA replication, pathogen interactions and more. They also provide a reproducible system which can be shared and replicated in many laboratories. Thus, as long as a permanent honey bee cell line is not available, insect cell cultures other than Apis mellifera should be screened for their suitability to examine aspects of honey bee biology and pathology. The usefulness of such an approach has been proven recently, when the first heterologous cell culture model for a honey bee pathogen has been reported (Gisder et al., 2011). The cell line IPL-LD65Y, a permanent lepidopteran cell line established from the gypsy moth, Lymantria dispar, was shown to be susceptible to infection by honey bee pathogenic microsporidia (N. apis and N. ceranae) and to support the entire life cycle of Nosema spp. in cell culture providing a new model of microsporidiosis (Troemel, 2011).

To further cell culture based experiments in bee research, we here present protocols for both the isolation and cultivation of non-permanent honey bee cells and the cultivation of permanent insect cell lines proven to be suitable for honey bee research, most of them established from Lepidoptera. We hope that these protocols will foster progress in the development of techniques for the isolation and cultivation of permanent honey bee cell lines.