Isolation and primary culture of Galleria mellonella hemocytes for infection studies

Abstract

Galleria mellonella larvae are increasingly used to study the mechanisms of virulence of microbial pathogens and to assess the efficacy of antimicrobials. The G. mellonella model can faithfully reproduce many aspects of microbial disease which are seen in mammals, and therefore allows a reduction in the use of mammals. The model is now being widely used by researchers in universities, research institutes and industry. An attraction of the model is the interaction between pathogen and host. Hemocytes are specialised phagocytic cells which resemble neutrophils in mammals and play a major role in the response of the larvae to infection. However, the detailed interactions of hemocytes with pathogens is poorly understood, and is complicated by the presence of different sub-populations of cells. We report here a method for the isolation of hemocytes from Galleria mellonella. A needle-stick injury of larvae, before harvesting, markedly increased the recovery of hemocytes in the hemolymph. The majority of the hemocytes recovered were granulocyte-like cells. The hemocytes survived for at least 7 days in culture at either 28°C or 37°C. Pre-treatment of larvae with antibiotics did not enhance the survival of the cultured hemocytes. Our studies highlight the importance of including sham injected, rather than un-injected, controls when the G. mellonella model is used to test antimicrobial compounds. Our method will now allow investigations of the interactions of microbial pathogens with insect hemocytes enhancing the value of G. mellonella as an alternative model to replace the use of mammals, and for studies on hemocyte biology.

A Straightforward Method for the Isolation and Cultivation of Galleria mellonella Hemocytes

“…Surprisingly, our results after following the literature [ 14 , 15 , 16 ] showed that almost all of the hemocytes isolated directly from larvae ( Figure 2 A) were nonviable after 24 h of isolation ( Figure 2 B) and completely dead after 6 days (see Figure 2 C). Clearly, these apparently easy protocols described for the cultivation of G. mellonella hemocytes had some limitations, as a similar survival rate was expected during isolation and incubation.…”

“…The literature contains different protocols for G. mellonella hemocyte isolation and use [ 14 , 15 , 16 ]. As a summary of such protocols, in our laboratory, we obtained hemocytes from larvae of approximately 250 mg.…”

Morphological and Ultrastructural Characterization of Hemocytes in an Insect Model, the Hematophagous Dipetalogaster maxima (Hemiptera: Reduviidae)

“…For TEM assays, hemolymph collected from 10 insects was pooled and centrifuged at 500× g for 10 min at room temperature [ 31 , 32 ] to enrich the preparation with hemocytes without inducing visible cell damage. The supernatant was discarded and the pellet was suspended and fixed in Karnovsky mixture containing 4% formaldehyde and 2% glutaraldehyde in 0.1 M cacodylate buffer for 2 h and processed as previously described [ 33 ].…”

Methodological Approaches To Assess Innate Immunity and Innate Memory in Marine Invertebrates and Humans

“…Trypan blue vital dye is a useful procedure for estimating alterations in membrane permeability associated with cell death in mammals (Louis and Siegel, 2011). The same procedure is often adopted for the evaluation of immune cell viability in different invertebrate species (Rocha et al, 2014;Senior and Titball, 2020). The main drawbacks of this technique are the toxicity of trypan blue, which limits the possibility of using it to few minutes after staining, and the high number of false negatives (e.g., lack of detection of cells already engaged into the apoptotic pathway as they still have intact membranes).…”