Entomoculture is the subfield of
cellular agriculture
Cellular agriculture focuses on the production of agricultural products from cell cultures using a combination of biotechnology, tissue engineering, molecular biology, and synthetic biology to create and design new methods of producing proteins, f ...
which specifically deals with the production of insect tissue ''in vitro''.
It draws on principles more generally used in
tissue engineering
Tissue engineering is a biomedical engineering discipline that uses a combination of Cell (biology), cells, engineering, Materials science, materials methods, and suitable biochemistry, biochemical and physicochemical factors to restore, maintai ...
and has scientific similarities to Baculovirus Expression Vectors or
soft robotics
Soft robotics is a subfield of robotics that concerns the design, control, and fabrication of robots composed of compliant materials, instead of rigid links.
In contrast to rigid-bodied robots built from metals, ceramics and hard plastics, the ...
. The field has mainly been proposed because of its potential technical advantages over mammalian cells in generating cultivated meat.
The name of the field was coined by Natalie Rubio at
Tufts University
Tufts University is a private research university on the border of Medford and Somerville, Massachusetts. It was founded in 1852 as Tufts College by Christian universalists who sought to provide a nonsectarian institution of higher learning. ...
.
Process
Entomoculture functions along the same principles as cellular agriculture in general. First, embryonic cells are derived from an insect. Embryonic
stem cells
In multicellular organisms, stem cells are undifferentiated or partially differentiated cells that can differentiate into various types of cells and proliferate indefinitely to produce more of the same stem cell. They are the earliest type o ...
are totipotent cells, meaning they retain the capacity to differentiate into any or all of the different kinds of specialized cells. These cells can either be taken from primary cultures (directly from the animal) or from cryopreserved secondary cultures.
These stem cells are then immersed into a
culture medium
A growth medium or culture medium is a solid, liquid, or semi-solid designed to support the growth of a population of microorganisms or cells via the process of cell proliferation or small plants like the moss ''Physcomitrella patens''. Different ...
so that they can proliferate. Culture media consist of basal media, which is a composition of the various nutrients essential to cell growth. This mixture diffuses into the cell and once it consumes enough, it divides and the population multiplies. To optimize growth, this culture media is generally supplemented with other proteins and
growth factor
A growth factor is a naturally occurring substance capable of stimulating cell proliferation, wound healing, and occasionally cellular differentiation. Usually it is a secreted protein or a steroid hormone. Growth factors are important for regu ...
s. Such additives are frequently produced by
recombinant protein production
Protein production is the biotechnological process of generating a specific protein. It is typically achieved by the manipulation of gene expression in an organism such that it expresses large amounts of a recombinant gene. This includes the tran ...
—translating the respective genes into bacteria that are then fermented to produce several copies of the protein. The proliferated number of stem cells can then be seeded onto a scaffold to initiate a larger composition or can be placed directly into a
bioreactor
A bioreactor refers to any manufactured device or system that supports a biologically active environment. In one case, a bioreactor is a vessel in which a chemical reaction, chemical process is carried out which involves organisms or biochemistry, ...
. The bioreactor replicates the environmental characteristics that would otherwise be emulated ''in vivo'', including temperature and osmolarity, to promote cell differentiation into muscle tissue.
![Smooth muscle tissue](https://upload.wikimedia.org/wikipedia/commons/9/9a/Smooth_muscle_tissue.jpg)
In the bioreactor, stem cells undergo
myogenesis
Myogenesis is the formation of skeletal muscular tissue, particularly during embryonic development.
Muscle fibers generally form through the fusion of precursor myoblasts into multinucleated fibers called ''myotubes''. In the early development o ...
—the differentiation into muscle tissue. This is a complex process involving the formation of founder cells and fusion-competent myoblasts. About 4–25 of the fusion-competent myoblasts then fuse with one founder cell to create multinucleated myofibers, which collectively become larval muscle.
When muscle tissue is developed ''in vivo'', the larval muscle is destroyed during metamorphosis. A similar process transpires involving the adult muscle precursors, which use the old larval muscle as a template to create the mature muscle. However this process is not of significant relevance in ''in vitro'' cultivation, as development is stopped prior to metamorphosis.
Comparison to mammalian cell culture
In terms of cultured meat, entomoculture has mainly been proposed due to its potential advantages over mammalian cell culture. Such advantages can be ascribed to the differences in biology between the two cell types that enable insect cells to tolerate conditions more favourable for industrial production.
Temperature. Mammalian cells developing ''in vivo'' are incubated at 37 degrees Celsius. Simulating such a warm climate in a bioreactor requires energy inputs which thus increase production costs. Insect tissue can be grown to scale at room temperature or colder with little to no hindrance in cell development.
Culture conditions. As mammalian cells digest and metabolize glucose, they produce byproducts such as
lactic acid
Lactic acid is an organic acid. It has a molecular formula . It is white in the solid state and it is miscible with water. When in the dissolved state, it forms a colorless solution. Production includes both artificial synthesis as well as natu ...
which accumulate and acidify the cell's environment. The ability that cells have to uptake nutrients depends on the pH of the environment—it must be within a certain window for optimal growth. Lactic acid accumulation leads to inferior growth conditions for the cell. As such, the environment must be "rebalanced"—which is typically accomplished by replacing the entire culture medium as frequently as every 2–3 days. However, the saturated culture media may still contain viable nutrients, which makes the practise wasteful and expensive. Insect cells in part circumvent lactate production but are also tolerant to more acidic environments. When insect cell growth was compared at a pH of 5.5, 6.5 and 7.5, negligible difference was noted.
As a result, insect cultures can have their media replaced at intervals as long as 90 days.
This is compounded by the fact that insect cells do not deplete added nutrients as fast as mammalian cells. They consume
triglyceride
A triglyceride (TG, triacylglycerol, TAG, or triacylglyceride) is an ester derived from glycerol and three fatty acids (from ''tri-'' and ''glyceride'').
Triglycerides are the main constituents of body fat in humans and other vertebrates, as w ...
s, glucose and proteins at a slower rate, suggesting that they have more efficient metabolic pathways. Additionally, insect cell cultures are typically contaminated with lipid cells called trophocytes or vitellophages, which are precursors to insect egg yolk cells. These cells are a natural source of fat that other insect cells can consume.
Osmolarity. Mammalian cells also require a relatively precise measure of carbon dioxide and oxygen to grow — as such, cultures are usually supplemented with an extra 5% carbon dioxide. Insects can go without this supplement.
Serum-free culture media. Culture media is an instrumental part of cellular agriculture and generating cultured tissue because it is effectively what allows scientists to begin with a relatively small sample of animal stem cells and end up with enough to constitute an entire tissue. To proliferate, a cell does not only require essential nutrients and macromolecules but also growth factors. When mammalian cells grow ''in vivo'', these growth factors are supplied by the animal's blood. To replicate this, the culture medium usually consists of a basal mixture supplemented with extra growth factors. The basal medium makes up the bulk of the culture and contains most of the nutrients while the growth factors are added in trace amounts. As a result, the natural starting point is combining
Fetal Bovine Serum
Fetal bovine serum (FBS) is derived from the blood drawn from a bovine fetus via a closed system of collection at the slaughterhouse. Fetal bovine serum is the most widely used serum-supplement for the in vitro cell culture of eukaryotic cells. Th ...
(FBS) into the culture media.
FBS is somewhat controversial because it comes from the blood of a dairy cow fetus. The two issues with this is that it is a) reliant on animals, hence defeating the goal of cellular agriculture and b) expensive because it is so inaccessible. Additionally, from a scientific perspective, FBS is chemically undefined, meaning that its composition varies between animals. For the sake of research consistency, this is not desirable. The ideal culture medium is one that is simple, can stimulate proliferation, is unreliant on animals, is accessible and is cheap. However, because mammalian cells rely on a complex array of growth factors, finding a culture medium that satisfies all five of these criteria is an ongoing challenge. Insect cells on the other hand, are biologically simpler organisms than mammals. They contain a fluid called hemolymph rather than blood, so they do not rely on all the same growth factors as mammalian cells. Instead, insect cell medium typically uses a basal medium (such as Eagle's Medium, Grace's Insect Medium or Schneider's Drosophila Medium), which is supplemented with plant based additives such as yeastolate, primatone RL, hydrolysates, pluronic lipids and peptides.
Suspension cultures. When mammalian muscle cells grow ''in vivo'', a fundamental part of their proliferation relies on their attachment to the
extracellular matrix
In biology, the extracellular matrix (ECM), also called intercellular matrix, is a three-dimensional network consisting of extracellular macromolecules and minerals, such as collagen, enzymes, glycoproteins and hydroxyapatite that provide stru ...
(ECM). To replicate this relationship, mammalian cells are usually cultured in adherent
monolayer A monolayer is a single, closely packed layer of atoms, molecules, or cells. In some cases it is referred to as a self-assembled monolayer. Monolayers of layered crystals like graphene and molybdenum disulfide are generally called 2D materials.
...
s — cultures where the cells grow on a substrate in layers only one cell thick. This necessitates using bioreactors with a lot of surface area that, when scaled up to the industrial level, is unfeasible. The alternative is to use
microcarrier
A microcarrier is a support matrix that allows for the growth of adherent cells in bioreactors. Instead of on a flat surface, cells are cultured on the surface of spherical microcarriers so that each particle carries several hundred cells, and the ...
s, which are small pieces of material that float in the culture medium to increase the overall surface area the cells can attach to. This also introduces the need to
vascularize. When mammalian cells are grown in adherent cultures, the cells not in direct contact with culture medium stop growing, forming
necrotic
Necrosis () is a form of cell injury which results in the premature death of cells in living tissue by autolysis. Necrosis is caused by factors external to the cell or tissue, such as infection, or trauma which result in the unregulated dige ...
centres. Unlike mammalian cells, insect cells are also able to grow unattached to anything — or in
suspension cultures. This means that bioreactors do not need a large surface area, and can instead be produced in much more practical shapes.
References
{{Reflist
Agriculture
Cellular agriculture