Cellular agriculture: meat without slaughter

By Clarie Lo

Around 12000 years ago, the human civilisation stopped foraging for food, and troops of hunter-gatherers settled as farmers. The raising, slaughtering and downstream processing of livestock has never stopped in order to meet the ever-rising demand for meat. As the food industry blossoms, the availability of arable land begins to shrink, and problems like foodborne diseases and antibiotic resistance start coming to the surface.

In 1913, Alexis Carrel and Albert Ebeling decided to culture small fragments of embryonic chick muscle tissues on a slide, which miraculously stayed alive and actively proliferated for an entire year.1 These “immortal cells” gave birth to a new mode of agriculture: cellular agriculture.  

In order to cultivate meat, muscle stem cells need to be harvested as the initial cell line. They are adult stem cells found within skeletal muscle tissues, and their self-renewal is induced upon injuries to replenish the supply of mature cells via cell division and differentiation. To access this valuable resource, an animal is fixated, followed by a biopsy to extract muscle samples under anaesthesia. Then, scissors and meat mincer are used for physical dissociation, followed by enzymatic dissociation with protease, often trypsin.

After the removal of debris with filtration and centrifugation, the stem cells need to be isolated from the remaining sample. This could be achieved in two ways: Fluorescence Activated Cell Sorting (FACS) or Magnetic Activated Cell Sorting (MACS). Both techniques are based on the principle of labelling different cell types with different antibodies. FACS makes use of fluorescently tagged antibodies, while MACS attaches magnetic beads to them.2  

Stem cells will subsequently be cryopreserved, a process through which their structures are kept intact at a low temperature. Meanwhile, immortalisation is also carried out to overcome the Hayflick limit of cell division, which describes the doubling potential of stem cells. For human diploid cells to overcome their division capacity of around 40 to 60 rounds,3 telomerase expression is to prevent the shortening of telomeres, a hallmark of cell senescence.

When the production of cultivated meat is set in motion, the muscle stem cells are taken out of the cell bank and placed into a culture medium. This solution of nutrients not only feeds cells for in-vitro growth and maturation, but also plays a key role in maintaining the pH and osmotic pressure of the environment that cells are stimulated to proliferate and differentiate in. Therefore, a suitable culture medium is usually composed of the following ingredients: a carbon-based energy source like glucose, amino acids for protein synthesis, salts to maintain osmolarity, vitamins as enzyme cofactors, and water.

On top of the basal medium, scientists have also been adding foetal bovine serum, which accounts for 5 – 20% of the final medium content. The serum is harvested by collecting blood of mature foetuses found in slaughtered cows. For every 100 livestock processed by the abattoir, 8 of them are pregnant females, so there is a fixed supply of this additional component. The serum will then be coagulated, centrifugated and screened for microbial contaminants.

Despite its ability to secure long-term cell viability, the extraction of foetal bovine serum is not animal-free. The culture medium is also susceptible to batch-to-batch variability, as the serum content varies between individual cows. This stimulated the formulation of Essential 8. This is a chemically defined, albumin-free solution containing only essential chemicals like FGF2 and TGFB1.4 Some companies even replaced these relatively expensive growth factors with co-cultures of “feeder cells”, which supplements the stem cells with extracellular secretions.5

Up until this point, the cells are still suspended within the liquid in an unstructured way. In order to form our familiar piece of meat, scaffolds are required to define their overall 3D structures. These scaffolds mimic the extracellular matrix to provide support to cells and act as geometric cues to induce appropriate differentiations. There are three major types of scaffolds: edible, degradable before human consumption, and reusable, which can naturally detach during downstream processing. They could be made from animal proteins produced by fermentation like collagen, fibronectin and laminin, or chitin in fungi and cellulose in plants. Polymers such as PLGA and PCL are non-edible but reusable synthetic alternatives. Optimisation is required for desirable seeding efficiency and degradation rate.

Scaffold fabrication can be achieved using extrusion bioprinting. The printable material, known as bioink, is a bulk of water-rich molecules named hydrogels. The scaffold cells, polymers, and linker molecules are packaged within it. By computing its placement, a strand is pushed out of the printing chamber through a nozzle as thin as a 400-micron-thick human fingernail.6 Scaffolds are also designed specifically for myocytes and adipocytes. The former uses an aligned scaffold to construct myotubes with interspersed microbeads of the latter. This allows the creation of a marbled piece of meat, retaining the juiciness provided by intramuscular fat.7

Just like any manufacturing industry, when cellular agriculture is taken out of a laboratory context, it has to occur in automated and controlled chambers in order to reduce manual costs of large-scale production. These chambers are called bioreactors. Single use plastic bioreactors are very popular in the present due to their fast turnaround time and low energy cost, as heated sterilisation is not required. However, companies have been shifting to bioreactors that are made of stainless steel, which can avoid unnecessary plastic waste while being less expensive in the long run because they are not discarded after each round of production.

Apart from their constituent materials, bioreactors also differ in their system designs. Agitated bioreactors carry out the convective mixing of culture medium and cells as one integral batch. Hence, a stirring paddle is often included as an essential module. In contrast, perfusion bioreactors create a steady flow of medium over a fixated cell population. For example, cultured cells can be arranged into a hollow fibre membrane using a porous straw as the scaffold. This allows the passage of medium even amid high cell density.8

From the first lab-grown burger in 2013 in Netherlands, to the regulatory approval for cultivated chicken by the Singaporean government in 2020, flavour tests have been returning with increasingly positive results. Scientists are no longer satisfied by securing the accurate flavour, texture and tenderness. The concept of enhanced meat with boosted nutritional profile emerges. For example, instead of pills, Andrew Stout from Tufts University successfully induced the phytonutrient production of vitamin precursors for elderlies to consume.9 Some biotechnology companies even expanded their stem cell library into non-farmable exotic animals, like Vow Food who cooked dumplings with kangaroo meat.

As of now, cellular agriculture is yet to become the mainstream method of food production. However, there is always the prospect of doing so, just like how our ancestors dropped their bows and spears.

References:

1.         Ebeling AH. The permanent life of connective tissue outside of the organism. Journal of Experimental Medicine. 1913 Mar 1;17(3):273–85.

2.         Pan J, Wan J. Methodological comparison of FACS and MACS isolation of enriched microglia and astrocytes from mouse brain. Journal of Immunological Methods. 2020 Nov;486:112834.

3.         Hayflick L. The limited in vitro lifetime of human diploid cell strains. Experimental Cell Research. 1965 Mar;37(3):614–36.

4.         Desai N, Rambhia P, Gishto A. Human embryonic stem cell cultivation: historical perspective and evolution of xeno-free culture systems. Reprod Biol Endocrinol. 2015;13(1):9.

5.         Llames S, García-Pérez E, Meana Á, Larcher F, del Río M. Feeder Layer Cell Actions and Applications. Tissue Engineering Part B: Reviews. 2015 Aug;21(4):345–53.

6.         Zhang YS, Haghiashtiani G, Hübscher T, Kelly DJ, Lee JM, Lutolf M, et al. 3D extrusion bioprinting. Nat Rev Methods Primers. 2021 Dec;1(1):75.

7.         Rowat A. Methods and compositions for cell culture on heterogeneous scaffolds. WO2020219755A1.

8.         Fenge C, Klein C, Heuer C, Siegel U, Fraune E. Agitation, aeration and perfusion modules for cell culture bioreactors. Cytotechnology. 1993;11(3):233–44.

9.         Stout AJ, Mirliani AB, Soule-Albridge EL, Cohen JM, Kaplan DL. Engineering carotenoid production in mammalian cells for nutritionally enhanced cell-cultured foods. Metabolic Engineering. 2020 Nov;62:126–37.

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