Entomoculture

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Entomoculture is the subfield of cellular agriculture which specifically deals with the production of insect tissue in vitro. [1] It draws on principles more generally used in tissue engineering and has scientific similarities to Baculovirus Expression Vectors or soft robotics. [2] The field has mainly been proposed because of its potential technical advantages over mammalian cells in generating cultivated meat. [1] The name of the field was coined by Natalie Rubio at Tufts University. [1]

Contents

Process

Entomoculture functions along the same principles as cellular agriculture in general. First, embryonic cells are derived from an insect. Embryonic stem cells 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. [1] These stem cells are then immersed into a culture medium 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 factors. Such additives are frequently produced by recombinant protein production—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. The bioreactor replicates the environmental characteristics that would otherwise be emulated in vivo, including temperature and osmolarity, to promote cell differentiation into muscle tissue. [3]

Founder cells and fusion-competent myoblasts connect to form multinucleated muscle fibres. Smooth muscle tissue.jpg
Founder cells and fusion-competent myoblasts connect to form multinucleated muscle fibres.

In the bioreactor, stem cells undergo myogenesis—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. [4]

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. [4]

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. [1]

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. [1]

Culture conditions. As mammalian cells digest and metabolize glucose, they produce byproducts such as lactic acid 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. [5] As a result, insect cultures can have their media replaced at intervals as long as 90 days. [5] This is compounded by the fact that insect cells do not deplete added nutrients as fast as mammalian cells. They consume triglycerides, 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. [1]

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. [1]

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 (FBS) into the culture media. [1]

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. [1] [6]

Suspension cultures. When mammalian muscle cells grow in vivo, a fundamental part of their proliferation relies on their attachment to the extracellular matrix (ECM). To replicate this relationship, mammalian cells are usually cultured in adherent monolayers — 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 microcarriers, 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 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. [1]

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Tissue engineering Biomedical engineering discipline

Tissue engineering is a biomedical engineering discipline that uses a combination of cells, engineering, materials methods, and suitable biochemical and physicochemical factors to restore, maintain, improve, or replace different types of biological tissues. Tissue engineering often involves the use of cells placed on tissue scaffolds in the formation of new viable tissue for a medical purpose but is not limited to applications involving cells and tissue scaffolds. While it was once categorized as a sub-field of biomaterials, having grown in scope and importance it can be considered as a field of its own.

Cultured meat Animal flesh product that has never been part of a living animal

Cultured meat is a meat produced by in vitro cell cultures of animal cells. It is a form of cellular agriculture, with such agricultural methods being explored in the context of increased consumer demand for protein.

Chinese hamster ovary cell

Chinese hamster ovary (CHO) cells are an epithelial cell line derived from the ovary of the Chinese hamster, often used in biological and medical research and commercially in the production of recombinant therapeutic proteins. They have found wide use in studies of genetics, toxicity screening, nutrition and gene expression, particularly to express recombinant proteins. CHO cells are the most commonly used mammalian hosts for industrial production of recombinant protein therapeutics.

G<sub>0</sub> phase Quiescent stage of the cell cycle in which the cell does not divide

The G0 phase describes a cellular state outside of the replicative cell cycle. Classically, cells were thought to enter G0 primarily due to environmental factors, like nutrient deprivation, that limited the resources necessary for proliferation. Thus it was thought of as a resting phase. G0 is now known to take different forms and occur for multiple reasons. For example, most adult neuronal cells, among the most metabolically active cells in the body, are fully differentiated and reside in a terminal G0 phase. Neurons reside in this state, not because of stochastic or limited nutrient supply, but as a part of their developmental program.

Bioreactor Device or system that supports a biologically active environment

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 process is carried out which involves organisms or biochemically active substances derived from such organisms. This process can either be aerobic or anaerobic. These bioreactors are commonly cylindrical, ranging in size from litres to cubic metres, and are often made of stainless steel. It may also refer to a device or system designed to grow cells or tissues in the context of cell culture. These devices are being developed for use in tissue engineering or biochemical/bioprocess engineering.

Cell culture Process by which cells are grown under controlled conditions

Cell culture is the process by which cells are grown under controlled conditions, generally outside their natural environment. After the cells of interest have been isolated from living tissue, they can subsequently be maintained under carefully controlled conditions. These conditions vary for each cell type, but generally consist of a suitable vessel with a substrate or medium that supplies the essential nutrients (amino acids, carbohydrates, vitamins, minerals), growth factors, hormones, and gases (CO2, O2), and regulates the physio-chemical environment (pH buffer, osmotic pressure, temperature). Most cells require a surface or an artificial substrate to form an adherent culture as a monolayer (one single-cell thick), whereas others can be grown free floating in a medium as a suspension culture. The lifespan of most cells is genetically determined, but some cell culturing cells have been “transformed” into immortal cells which will reproduce indefinitely if the optimal conditions are provided.

Growth medium Solid, liquid or gel used to grow microorganisms or cells

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 types of media are used for growing different types of cells.

Matrigel is the trade name for the solubilized basement membrane matrix secreted by Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells produced by Corning Life Sciences. Matrigel resembles the laminin/collagen IV-rich basement membrane extracellular environment found in many tissues and is used by cell biologists as a substrate for culturing cells.

Industrial fermentation is the intentional use of fermentation in manufacturing products useful to humans. In addition to the mass production of fermented foods and drinks, industrial fermentation has widespread applications in chemical industry. Commodity chemicals, such as acetic acid, citric acid, and ethanol are made by fermentation. Moreover, nearly all commercially produced industrial enzymes, such as lipase, invertase and rennet, are made by fermentation with genetically modified microbes. In some cases, production of biomass itself is the objective, as is the case for single-cell proteins, baker's yeast, and starter cultures for lactic acid bacteria used in cheesemaking.

Fed-batch culture is, in the broadest sense, defined as an operational technique in biotechnological processes where one or more nutrients (substrates) are fed (supplied) to the bioreactor during cultivation and in which the product(s) remain in the bioreactor until the end of the run. An alternative description of the method is that of a culture in which "a base medium supports initial cell culture and a feed medium is added to prevent nutrient depletion". It is also a type of semi-batch culture. In some cases, all the nutrients are fed into the bioreactor. The advantage of the fed-batch culture is that one can control concentration of fed-substrate in the culture liquid at arbitrarily desired levels.

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 therefore expansion capacity can be multiplied several times over. It provides a straightforward way to scale up culture systems for industrial production of cell or protein-based therapies, or for research purposes.

Plant tissue culture is a collection of techniques used to maintain or grow plant cells, tissues or organs under sterile conditions on a nutrient culture medium of known composition. It is widely used to produce clones of a plant in a method known as micropropagation. Different techniques in plant tissue culture may offer certain advantages over traditional methods of propagation, including:

A chemically defined medium is a growth medium suitable for the in vitro cell culture of human or animal cells in which all of the chemical components are known. Standard cell culture media commonly consist of a basal medium supplemented with animal serum as a source of nutrients and other ill-defined factors. The technical disadvantages to using serum include its undefined nature, batch-to-batch variability in composition, and the risk of contamination.

Moss bioreactor

A moss bioreactor is a photobioreactor used for the cultivation and propagation of mosses. It is usually used in molecular farming for the production of recombinant protein using transgenic moss. In environmental science moss bioreactors are used to multiply peat mosses e.g. by the Mossclone consortium to monitor air pollution.

A 3D cell culture is an artificially created environment in which biological cells are permitted to grow or interact with their surroundings in all three dimensions. Unlike 2D environments, a 3D cell culture allows cells in vitro to grow in all directions, similar to how they would in vivo. These three-dimensional cultures are usually grown in bioreactors, small capsules in which the cells can grow into spheroids, or 3D cell colonies. Approximately 300 spheroids are usually cultured per bioreactor.

The in vivo bioreactor is a tissue engineering paradigm that uses bioreactor methodology to grow neotissue in vivo that augments or replaces malfunctioning native tissue. Tissue engineering principles are used to construct a confined, artificial bioreactor space in vivo that hosts a tissue scaffold and key biomolecules necessary for neotissue growth. Said space often requires inoculation with pluripotent or specific stem cells to encourage initial growth, and access to a blood source. A blood source allows for recruitment of stem cells from the body alongside nutrient delivery for continual growth. This delivery of cells and nutrients to the bioreactor eventually results in the formation of a neotissue product. 

A Hollow fiber bioreactor is a 3 dimensional cell culturing system based on hollow fibers, which are small, semi-permeable capillary membranes arranged in parallel array with a typical molecular weight cut-off (MWCO) range of 10-30 kDa. These hollow fiber membranes are often bundled and housed within tubular polycarbonate shells to create hollow fiber bioreactor cartridges. Within the cartridges, which are also fitted with inlet and outlet ports, are two compartments: the intracapillary (IC) space within the hollow fibers, and the extracapillary (EC) space surrounding the hollow fibers.

Cellular agriculture Production of agriculture products from cell cultures

Cellular agriculture focuses on the production of agriculture 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, fats, and tissues that would otherwise come from traditional agriculture. Most of the industry is focused on animal products such as meat, milk, and eggs, produced in cell culture rather than raising and slaughtering farmed livestock which is associated with substantial global problems of detrimental environmental impacts, animal welfare, food security and human health. Cellular agriculture is field of the biobased economy. The most well known cellular agriculture concept is cultured meat.

In vitro spermatogenesis is the process of creating male gametes (spermatozoa) outside of the body in a culture system. The process could be useful for fertility preservation, infertility treatment and may further develop the understanding of spermatogenesis at the cellular and molecular level. 

Cell suspension Type of cell culture

A cell suspension or suspension culture is a type of cell culture in which single cells or small aggregates of cells are allowed to function and multiply in an agitated growth medium, thus forming a suspension. Suspension culture is one of the two classical types of cell culture, the other being adherent culture. The history of suspension cell culture closely aligns with the history of cell culture overall, but differs in maintenance methods and commercial applications. The cells themselves can either be derived from homogenized tissue or from another type of culture. Suspension cell culture is commonly used to culture nonadhesive cell lines, plant cells, and insect cells. While some cell lines are cultured in suspension, the majority of commercially available mammalian cell lines are adherent. Suspension cell cultures need to be agitated and may require specialized equipment or flasks. These cultures, like all cell cultures, need to be maintained with nutrient containing media and cultured in a specific cell density range to avoid cell death.

References

  1. 1 2 3 4 5 6 7 8 9 10 11 Rubio, Natalie R.; Fish, Kyle D.; Trimmer, Barry A.; Kaplan, David L. (2019-04-17). "Possibilities for Engineered Insect Tissue as a Food Source". Frontiers in Sustainable Food Systems. 3. doi: 10.3389/fsufs.2019.00024 . ISSN   2571-581X. S2CID   116877741.
  2. ↑ Ikonomou, L.; Schneider, Y.-J.; Agathos, S. N. (2003). "Insect cell culture for industrial production of recombinant proteins". Applied Microbiology and Biotechnology. 62 (1): 1–20. doi:10.1007/s00253-003-1223-9. ISSN   0175-7598. PMID   12733003. S2CID   23929531.
  3. ↑ Datar, Isha; Betti, Mirko (2010). "Possibilities for an in vitro meat production system". Innovative Food Science & Emerging Technologies. 11: 14–18. doi:10.1016/j.ifset.2009.10.007.
  4. 1 2 Gunage, Rajesh D.; Dhanyasi, Nagaraju; Reichert, Heinrich; VijayRaghavan, K. (2017). "Drosophila adult muscle development and regeneration". Seminars in Cell & Developmental Biology. 72: 56–66. doi: 10.1016/j.semcdb.2017.11.017 . ISSN   1096-3634. PMID   29146144.
  5. 1 2 Rubio, Natalie R.; Fish, Kyle D.; Trimmer, Barry A.; Kaplan, David L. (2019-02-11). "In Vitro Insect Muscle for Tissue Engineering Applications". ACS Biomaterials Science & Engineering. 5 (2): 1071–1082. doi:10.1021/acsbiomaterials.8b01261. PMID   33405797. S2CID   92606475.
  6. ↑ Ikonomou, L.; Bastin, G.; Schneider, Y. J.; Agathos, S. N. (2001). "Design of an efficient medium for insect cell growth and recombinant protein production". In Vitro Cellular & Developmental Biology. Animal. 37 (9): 549–559. doi:10.1290/1071-2690(2001)037<0549:doaemf>2.0.co;2. ISSN   1071-2690. PMID   11710429.
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