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Figure 2. Targeted zones to produce insect gut cell lines. Schematic representation of the insect digestion system, which can be broken
down into three regions: the foregut, midgut, and hindgut. The midgut is the primary choice from which to derive cell lines on account of
it playing a key role in shaping the gut microbiome and being the principal location of nutrient absorption, pathogen defense, and the
production of signaling molecules [50] . The midgut has several cell types, including columnar epithelial cells (CC), endocrine cells (EC),
goblet cells (GC), and stem cells (SC) [50] . Only the most relevant of the top five cell types are shown. Columnar epithelial cells are the
most common, followed by endocrine cells and stem cells, making these cells the first selection for cell culture [50] . Created with
BioRender.com [20] .

epithelium, represent a good target for successful cultivation during cell line isolation procedures. It is
important to note that non-target cell types, such as muscle cells, exist within the insect gut as well . As a
[50]
result, cell lines are not always composed of their target cell type and should be verified by, at minimum,
microscopy prior to further experimentation.

Methods to attain immortalized insect gut cell lines
Cell lines are considered immortalized once they no longer possess the ability to respond to cell cycle
controls. These controls, present at the G and G cell cycle checkpoints, limit cellular proliferation and,
1
2
when required, lead to cellular senescence or apoptosis . Bypassing these controls can, on rare occasions,
[52]
[50]
occur naturally in a process known as spontaneous immortalization . Alternatively, this can be achieved
deliberately, for example, through the insertion of proto-oncogenes, pathogen invasion, Clustered Regularly
Interspaced Short Palindromic Repeats (CRISPR)-Cas9, and exposure to carcinogenic chemicals or ionizing
radiation [53-56] . Immortalization is particularly difficult to achieve in mature tissue, such as the gut
epithelium, because of its limited replicative potential and greater likelihood of senescence .
[48]
Notwithstanding these difficulties, many insect cell lines have been successfully produced from mature
tissue. Table 1 lists the currently available insect cell lines that have been immortalized. Several common
techniques for the production of insect gut cell lines are relevant to discuss.

Spontaneous immortalization
Spontaneous immortalization occurs when a cell line is immortalized without exposure to carcinogens or
direct genetic manipulation . Continuous passaging of a primary cell line increases the likelihood of
[50]
[50]
random epigenetic changes or chromosomal rearrangements . Often, a mutation occurs in the p53
pathway [50,65] . Although considered a rare event, spontaneous immortalization has led to the production of
all currently existing insect gut cell lines . Notably, midgut cell lines have been successfully produced in
[10]
this manner, including CT/BCIRL-SfMG1-0611-E8-KZ (Fall armyworm midgut) .
[10]