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Characterisation of growth enhancing factor production in different phases of in vitro fish macrophage development
1Department of Biological Sciences, University of Alberta, Edmonton, Canada.
Abstract:
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.
Insights
Macrophage growth factor (MGF) activity in culture supernatants (CCM) peaks during the proliferative phase of goldfish kidney macrophage (IVDKM) development. Optimizing MGF collection from IVDKM cultures enhances purification and aids antimicrobial studies.
Area of Science:
- Immunology
- Cell Biology
- Aquatic Animal Health
Background:
- Macrophage growth factors (MGF) are released by goldfish macrophages.
- Understanding macrophage proliferation is crucial for immunological studies.
Purpose of the Study:
- To characterize the developmental phases of in vitro derived kidney macrophages (IVDKM).
- To determine the optimal timing for collecting macrophage-conditioned culture medium (CCM) for MGF purification and functional studies.
Main Methods:
- Morphological and flow cytometric analysis to define IVDKM developmental phases (lag, proliferative, senescence).
- Assay of CCM from different IVDKM developmental phases for growth-inducing capabilities.
- Correlation of CCM composition with IVDKM growth and senescence.
Main Results:
- IVDKM growth exhibits distinct lag, proliferative, and senescence phases.
- Maximal MGF activity is found in CCM from the proliferative phase.
- CCM from the senescence phase shows reduced macrophage growth induction.
- A link exists between CCM composition, IVDKM growth, and entry into senescence.
Conclusions:
- Collecting IVDKM CCM during the proliferative phase maximizes MGF activity and minimizes inhibitory factors.
- This optimized collection strategy aids MGF purification and the study of macrophage antimicrobial mechanisms.