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

A BRIEF HISTORY OF INSECT CELL CULTURE
The roots of insect research at the cellular level can be traced back to 1912, when Glaser and Chapman
examined how wilt disease affected spongy moth (Lymantria dispar) hemocytes - key immune effector cells
of invertebrates that participate in phagocytosis, encapsulation, and clotting systems . In the following
[12]
decades, insect cell culture techniques continued to advance, resulting in the successful establishment of
[13]
multiple primary cell lines . Continuous cell lines, however, remained elusive during this early work, as
[13]
cell lines were unable to survive beyond 3 months . This milestone is generally accepted to have been
finally achieved by Grace (1962) when he derived a continuous cell line from the ovarian tissue of emperor
gum moth (Opodipthera eucalypti) pupae . Since this time, insect cell culture has blossomed along with
[14]
advancements in aseptic techniques and complex media formulations. A notable cell line, Sf-21, which was
produced by Vaughn et al. in 1977, is used in the mass production of baculovirus stocks and recombinant
proteins for human vaccine production to date [15,16] . Insect cells have also been used in infectious disease
research, pesticide screening, and “bio-bots” (small robots incorporating animal biomass), and have the
potential to be used as a food source [13,17,18] . At present, over 1,500 insect cell lines spanning 7 orders of
[11]
taxonomy are cataloged on Cellosaurus , displaying significant progress in the field over 80 years.

THE GROWTH REQUIREMENTS OF INSECT CELL LINES
Insect cell lines exhibit broad adaptability to culture conditions and many lines can be successfully
cultivated in temperatures ranging from 22-34 °C, 0%-5% CO , pH 6.0-6.8, and ambient humidity [15,19] . In
2
contrast, mammalian cells typically require tight adherence to in vivo conditions during incubation,
including a consistent temperature of 35-37 °C, 5% CO , pH 7.0-7.3, and 95% relative humidity [Figure 1] .
[15]
2
Thus, insect cell lines are less fastidious compared to mammalian lines, most notably lacking the
requirement of a CO incubator. Insect cells also grow more rapidly, are less sensitive to changes in pH, and
2
[15]
express significantly more protein than mammalian cells . This versatility may be, at least in part, due to
the exothermic nature of insects which requires them to tolerate a range of ambient temperatures.

Several types of media have been developed that satisfy the nutritional requirements and chemical
conditions needed by insect cells [15,21] . In general, insect cells require essential amino acids, inorganic salts,
sugars, and vitamins . Additionally, media is often supplemented with growth-promoting substances,
[21]
namely insect hemolymph or fetal bovine serum . These conditions are similar to mammalian cells and are
[21]
subject to modification depending on the cell line.

The control of pH in insect cell lines is primarily achieved with a phosphate buffer system, which does not
require a CO incubator to function . Mammalian cells, by contrast, commonly utilize either HEPES [4-(2-
[15]
2
hydroxyethyl)-1-piperazineethanesulfonic acid], a CO -independent buffer system, or a bicarbonate buffer,
2
[22]
which relies on regulated CO conditions to effectively control pH . HEPES is not usually recommended
2
for insect cell culture; however, some literature has reported its use, namely in AmE-711, a spontaneous cell
line derived from embryonic western honey bee (Apis mellifera) tissue [15,19] . Insect cell lines display a
markedly elevated rate of oxygen consumption and a decreased rate of lactate production in comparison to
mammalian cells. This discrepancy may be exaggerated in cell culture, as a large proportion of mammalian
cell lines are derived from cancerous tissue. A well-documented phenomenon, the Warburg effect, is the
observation that cancerous tissue exhibits reduced oxygen consumption and heightened lactate production
compared to healthy tissue [23,24] . Nonetheless, insect cells’ increased gas exchange (and therefore CO
2
[15]
accumulation) necessitates the introduction of base to increase pH . The addition of base increases the
[15]
osmolarity of the media and, in turn, increases the risk of osmotic shock in cell culture . The degree to
which low lactate production ameliorates the acidic effect of heightened CO production in insect cells is
2
unknown and could be a promising area to explore in future studies.