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Macpherson et al. Microbiome Res Rep 2024;3:20 https://dx.doi.org/10.20517/mrr.2023.66 Page 5 of 15

[6,7]
digestion, and confer resistance to pathogens . Illustrating this, bacterial strains isolated from the gut of
the bark beetle (Dendroctonus rhizophagus) have been shown to hydrolyze pectin, cellulose, xylan, starch,
lipids, and esters - thereby allowing the host organism to derive nutrition from otherwise indigestible
[31]
substrates . By increasing metabolic capacity and diversity, the insect microbiome thus allows the
exploitation of a wider variety of food sources. Further, bacterial metabolites isolated from the gut of the
fruit fly (Drosophila melanogaster) have been shown to increase immune gene expression of pathways that
promote the activity of Relish/NF-κB - a family of pleiotropic transcription factors that are highly conserved
across Animalia [32,33] . As innate immunity is greatly conserved across flies and mammals, understanding the
microbial modulation of these pathways in insects can also enrich our comprehension of immune signaling
in diverse animal systems. Altogether, the simple growth requirements and efficiency of insect cell lines, as
well as the applicability of many conserved cellular systems across insects and vertebrates, position insect
cell culture as a powerful model system for studying host-microbe interactions relevant to a very wide range
of host species.

Despite their applicability, a significant constraint in the use of insect cell lines for microbiome research is
the limited availability of continuous cell lines derived from relevant insect gut tissue. Gut cell lines have
been established for only nine insect species, and many of the most heavily studied insects, such as
[10]
A. mellifera and D. melanogaster, are not represented . This paucity of gut-relevant cell lines restricts the
range of host-microbe dynamics that can be studied in vitro, while also limiting the study of tissue-specific
interactions. Cell lines derived from embryos or non-gut tissue, for example, may not capture the full
complexity of in vivo gut environments, including physical, chemical, and microbial interactions.

An additional factor to consider when investigating the microbiome using cell lines is how closely a cell line
approximates the gut environment. Primary cell lines, derived from isolated tissue, retain most of their
in vivo functionality and can include gut ultrastructure, but only survive for short periods in cell culture .
[34]
When considering immortalized cell lines, very little has been characterized in detail, whether derived from
humans or insects . The Caco-2 cell line derived from human tissue, which will be discussed in detail in
[35]
this review, is one of the only cell lines characterized enough to determine a relative approximation of gut
tissue conditions (i.e., polarized monolayer, forms tight junctions, expresses many receptors and enzymes
associated with gut tissues) [36,37] .

Recently, insect cell lines derived from various tissues have been subjected to detailed genome or
transcriptome profiling to gain insights into characteristics important for recombinant protein
expression [38-40] . However, when considering insect gut cell lines, genotypic and phenotypic characterization
is lacking.

The RP-HzGUT-AW1 cell line, derived from Lepidoptera member Helicoverpa zea, is one of the first insect
cell lines to be characterized via transcriptomics for expression of insect intestinal epithelial cell gene and
[41]
intestinal stem cell markers and, therefore, is the most characterized in gut approximation .
RP-HzGUT-AW1 exhibits some, but not all, gene markers approximated for intestinal stem cell markers
and differentiated epithelial cells, suggesting the cell line consists of progenitor cells, which form smooth
[41]
muscle junctions between cells . A mechanism to differentiate the progenitor cells further was not
discussed . Going forward, initial characterization of gut cell lines should be performed at the
[41]
transcriptomic level so that the troubleshooting and development of other gut features, such as recreating
structural organization and peritrophic membrane formation, can be attempted. Advanced bio-mimetic
technologies such as organ-on-chip and organoid models have yet to be adapted to insect tissues, although
they could be explored to address these issues.