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Macpherson et al. Microbiome Res Rep 2024;3:20 https://dx.doi.org/10.20517/mrr.2023.66 Page 11 of 15

intestinal Caco-2) were of A. mellifera origin and the detoxification abilities observed were contingent on
[94]
the cell lines employed . The relevance of cross-species studies is thus context-dependent, underscoring
the importance of deriving and employing species-specific cell lines in insect-related research to ensure the
accuracy of results.

Despite progress in the field, a significant knowledge gap remains regarding the impact of specific toxins on
host tissue in isolation as well as in the presence of various resident and foreign microbes. This gap may be
addressed by insect gut cell lines, which in turn could help to advance pest management strategies, and to
shield vulnerable species from the noxious effects of toxin exposure.

Exploring specific symbiont-derived pathogen defense in host environment
The application of cell lines in the investigation of host-pathogen-symbiont interactions will promote a
more comprehensive understanding of symbiont-derived defense and its impact on the host, while avoiding
confounders associated with host models. These confounders include the inherent colonization of the host
by other microbiome members, which could make any interaction studies non-specific, or would require
the use of germ-free hosts (which often are not available). Few studies have used cell line models to look at
such interactions. One study of note used a rotating wall vessel-derived (RWV) 3-D HT29 organotype
model, composed of a human adenocarcinoma cell line with epithelial morphology (HT-29), to investigate
[95]
the interactions between symbiont, human host, and pathogen during infection . The RWV model was
coinfected with commensal bacteria Lactobacillus reuteri and the pathogen Salmonella enterica, in order to
[95]
determine the protective ability of L. reuteri and its impact on the host . The study revealed that L. reuteri
reduced pathogen growth and adhesion to HT-29 cells, leading to L. reuteri being considered a potential
[95]
candidate for probiotic therapy against Salmonella infection . This study highlights the potential of cell
culture to elucidate mechanisms underlying symbiont-derived pathogen defense, as well as the
repercussions for all cells involved.

A wider range of host-specific gut cell lines could improve insect work by creating an accurate environment
to inoculate both pathogen and potential symbiont, resulting in more natural responses to what would
happen within the specific host. With the use of insect gut cell lines, research of this nature could help to
identify safe probiotic candidate bacteria against the most relevant “incurable” insect pathogens, such as
Paenibacillus larvae in honeybees, gain insights on microbiome members important for host defense, and
[96]
the impact of both pathogen and symbiont presence in host tissues .

LIMITATIONS
The key limitation associated with cell culture is its inability to fully recapitulate in vivo organ and tissue
environments . While the simplistic model of cell culture is efficacious in the study of isolated variables, it
[1]
consequently cannot assess the impact of the same variables on complex systems, such as the reproductive
and immune systems. This discrepancy could be addressed in the future through the production of
co-culture and microfluidic systems. Another limitation of cell lines is that they cannot reproduce the
dramatic gut remodeling that many insects undergo between life stages . Additionally, immortalized cells
[50]
tend to behave differently than the primary cells from which they were derived. This is due to changes in
important regulatory genes necessary to prevent senescence. One notable difference between the currently
available immortalized insect midgut cells and primary cells of the same origin is the lack of polarization,
which limits studies on epithelial barrier function . As a result of these limitations, it is important to
[97]
understand cell culture as a tool for preliminary and mechanistic work, which must be validated with
appropriate in vivo studies.