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Page 6 of 15 Macpherson et al. Microbiome Res Rep 2024;3:20 https://dx.doi.org/10.20517/mrr.2023.66

The lack of basic insect gut cell line characterization is a major hurdle in the development of biologically
relevant cell lines that respond to stimuli (e.g., microbial metabolites, chemical treatments of interest) in a
manner that is predictable and comparable to in vivo conditions. Thus, while current insect cell cultures
offer unique insights, their limited representation of genuine, characterized gut tissues can constrain the
depth and overall breadth of host-microbe interaction studies. Here, we highlight lessons learned from
human gut-derived cell lines and describe several of the latest techniques that show promise for enabling
targeted derivation of insect gut cell lines.

Caco-2 and the microbiome: an exemplar approach
Cell culture techniques have been effectively leveraged to gain insight into mammalian epithelial cell-
microbial relationships over the past several decades. The human cell line model of the gut epithelium most
widely used for this purpose is likely the Caco-2 line, derived by Fogh in 1977 from a human colon
adenocarcinoma biopsy. Despite their derivatization from the colon, these cells behave most similarly to
[42]
small intestine epithelial tissue, both biochemically and morphologically . Demonstrating their broad-
spectrum application, Caco-2 cells have been used in thousands of studies to illuminate the pathogenesis of
deleterious microbes, probiotic formulations, and immune-microbe interactions [43-45] . For example, Caco-2
cells have been employed to confirm both the invasiveness of, and the cytokine response to Fusobacterium
nucleatum - a bacterium associated with colorectal cancer - in a relevant intestinal cell model [45,46] .
Additionally, this cell line has been used to evaluate the potential beneficial properties of the yeast
[44]
Kluyveromyces marxianus, strain B0299 , where it was observed that the yeast dampened the release of
pro-inflammatory cytokines by cells treated with lipopolysaccharide, an inflammatory stimulus, which
ultimately led to the yeast being considered as a therapeutically useful probiotic .
[44]

A further aspect that extends the applicability of the Caco-2 cell model to biomedical science is that the cells
can be co-cultured with other cell types and incorporated into several advanced biomimetic models. For
instance, the co-culture of Caco-2 and Raji B cells, a lymphoblast-like cell line, facilitates the production of a
model of specialized microfold epithelial cells, which overlay the gut-associated lymphoid tissues . This
[47]
model can subsequently be utilized to study the transport of microbial-derived micro- and nano-
compounds across this epithelial barrier cell type [47,48] . Caco-2 cells have also been integrated into organ-on-
chip models, which are micro-biomimetic systems that incorporate multiple cell types and provide a
[49]
framework for cellular growth . This model system more closely represents in vivo tissue, improving the
efficacy of in vitro studies.

Considering the extensive insights gained from using mammalian cell lines such as Caco-2, similar
methodologies should be applied to insect models. However, since spontaneously immortalized tissue is
rare to identify in insects under natural circumstances, the pivotal challenge of deriving gut-specific cell
types lies primarily in the isolation procedure rather than the general maintenance steps. The tailored
methodologies that show the most promise for isolating gut-derived cell lines able to grow continuously
(i.e., which exhibit immortalization) under laboratory conditions are discussed below.

PRODUCING IMMORTALIZED INSECT GUT CELL LINES
Cell types relevant to insect gut cell line development
Contrasting the human gut epithelium, the insect gut epithelium is relatively simple, consisting of two
major cell types: columnar epithelial cells and endocrine cells [Figure 2] . Additionally, stem cells, goblet
[50]
cells, and copper cells (responsible for acidification of the digestive tract) are often present [Figure 2] [50,51] .
Columnar epithelial cells closely resemble the stereotypical enterocytes found in humans and function
similarly. We suggest that columnar epithelial cells, as the most prevalent cell type in the insect gut