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Page 2 of 15 Macpherson et al. Microbiome Res Rep 2024;3:20 https://dx.doi.org/10.20517/mrr.2023.66

other animals, plants, and fungi. By breaking down the organism into discrete parts, cell culture models
[1]
offer a high-resolution view of the current subject, free of in vivo confounders . As well, the homogeneity of
cell culture, with a singular cell type and a well-defined genetic profile, allows for exceptional
reproducibility . The application of cell culture has revolutionized in vitro research endeavors, especially in
[1]
genetics, pathology, and microbiology.

In vivo animal models are a commonly used approach to scientific research, serving as valuable tools for
confirming in vitro findings and investigating various biological processes, diseases, and potential
treatments. These models allow researchers to study complex physiological and pathological phenomena in
a controlled environment. Although insect animal models raise less ethical concerns and debate than
vertebrate models, there are several limitations when completing in vivo research. Practical barriers,
encompassing the acquisition and maintenance of these insects, can prove daunting for researchers. Such
issues include obtaining administrative approval, securing sufficient space and equipment for rearing
insects, or possessing the knowledge to supply the environmental conditions and nutritional needs required.
Further, in studies that investigate microbe-host tissue interactions, researchers face the intricate task of
mitigating confounding factors linked to the host. This involves addressing challenges like immunological
response and the potential off-target effects of other microbes within the host. Creating germ-free insect
models, a crucial step to limit these confounding factors, is labour-intensive and may induce detrimental
changes to the form and function of the host. Another notable concern is the overwintering of insects in
temperate or continental climates, which can impact research timelines and reproducibility. Recognizing
these challenges, researchers are exploring alternative models, such as cell lines, for preliminary and
mechanistic studies within controlled environments, offering a potential workaround to some of the
obstacles inherent in using live insect models.

One field where cell culture models have proven to be uniquely powerful is in the study of the microbiome,
which is the community of microorganisms (and their genetic potential, metabolites, structural elements,
and environmental conditions) living within the intestinal tract of the host . Cell culture of human
[2]
epithelial cells, particularly of intestinal origin, has improved our understanding of how microbes (or
[3]
[4]
microbial metabolites) impact intestinal transepithelial permeability , immune signaling , and cellular
proliferation mechanisms relevant to cancer .
[5]
Despite great promise, researchers have not yet adopted this approach concerning the insect microbiome.
Similar to the human gut microbiome, the insect gut microbiome serves as a critical intermediary between
the environment and host health, aiding in digestion, detoxification, and combating pathogens , yet it
[6,7]
remains poorly understood. Crucial to both ecosystem health and global food security, insects are facing
rising threats of habitat destruction, pesticide use, and antimicrobials . Given their importance and
[8,9]
vulnerability, it is essential to understand the microbes so inextricably tied to their health. In order to study
host-gut microbiome interactions in insects, tissue-specific intestinal cell lines must be obtained from insect
hosts. Overall, cell lines can be separated into two major categories: primary and immortalized. Primary cell
lines are obtained directly from insects and maintained under stringent laboratory conditions for short
periods of time before they eventually die out. Immortalized lines are an asset because they allow for
laboratory manipulation without the need for available live insect sources. Of the 1,500 immortalized insect
cell lines openly available, a mere 0.012% represent midgut tissue . Of this fraction, only 9 of the over
[10]
1 million known insect species are represented [10,11] . In this review, we explore the characteristics of insect
cell lines, their use in studying host-microbial interactions, and emerging techniques for the
immortalization of novel insect gut cell lines.