Characterisation of growth enhancing factor production in different phases of in vitro fish macrophage development

We previously described the release of macrophage growth factor(s) (MGF) into culture supernatants (CCM) by a goldfish macrophage cell line (GMCL) and in vitro derived kidney macrophages (IVDKM). In this study, we report that IVDKM growth can be subdivided into three developmental phases, defined using both morphological and flow cytometric characteristics: a lag phase, a proliferative phase, and a senescence phase. Analysis of the growth inducing capabilities of CCM indicated that maximum activity was consistently found in supernatants isolated from IVDKM cultures during the proliferative phase of development. In contrast, CCM from the senescence phase proved to be poor inducers of macrophage growth. Overall, we identify a link between the seeding-CCM composition, the extent of IVDKM growth and the rate of entrance into a senescent state characterised by IVDKM apoptotic cell death. Use of IVDKM CCM obtained at the peak of macrophage growth maximised macrophage growth factor (MGF) activity, and prevented the introduction of negative regulators of IVDKM proliferation, which will contribute significantly to our MGF purification efforts. Furthermore, the collection of IVDKM, prior to their commitment into apoptotic pathways, will prove to be essential in the selection of specific cell subsets for studies of antimicrobial mechanisms of macrophages.