A Review of Materials & Methods Used for Cellular Agriculture Scaffolds
One of the claims cellular agriculture loves to make is that it produces animal products without the animal. And what most consumers will infer from this statement is that it will produce animal products exactly as we think of them today. In order for that to happen, we need one of the most important contributions to cellular agriculture — a scaffold.
Let’s take a step back. Culturing tissue in vitro generally consists of 3 main steps.
- Stem cells (undifferentiated cells which have the potential to differentiate into many or all of the different kinds of specialized cells) are taken from an animal or from a secondary “cryopreserved” culture.
- These stem cells are immersed in a culture medium in order to proliferate. A culture medium is a mixture containing all the essential carbohydrates, proteins, fats, vitamins, nutrients and growth factors that a cell needs to grow. As the culture medium is consumed by a cell, it will grow, divide and hence, the population will “proliferate”.
- The stem cells are put into a bioreactor in order to differentiate. A bioreactor is a machine which exposes the stem cells to a variety of environmental cues to encourage them to differentiate into either founder cells or progenitors. These founder cells and progenitors then connect to form multinucleate myofibres — i.e. the muscle.
However, this alone will not allow us to go from a handful of stem cells to a steak, for instance. A steak is an example of a structured meat product — it’s characterized not only by the kinds of cells present but their overall arrangement as well. Our current process does nothing to encourage any specific arrangement; all we’ve done is let a bunch of cells float around randomly. What we’d end up with is an unstructured meat product (like a burger) which is uniform in its composition.
This is where the scaffold comes in. A scaffold is a mold which the cells are attached to in order to achieve a certain shape. The stem cells are “seeded” to the scaffold between steps 2 and 3 so that it is placed in the bioreactor during differentiation.
The scaffold is meant to simulate the Extra-cellular Matrix (ECM): the 3-dimensional mesh of glycoproteins, collagen and enzymes which is responsible for transmitting mechanical and biochemical cues to the cells. These signals dictate how the cells should arrange themselves as well as influences their individual development. So, unsurprisingly, an effective scaffold can’t just be made of any material.
Some characteristics which render a material similar to the ECM (or are just helpful in general) include
- Porosity. Pores are minute openings on the surface of the scaffold. They can be created in order to release the scaffold’s pre-existing cellular components. They also help diffuse gas and nutrients to the innermost layers of adherent cells which prevents against developing a “necrotic centre” (created when cells which are not in direct contact with the culture medium have died due to a lack of nutrients).
- Vascularization. Vascular tissue found in plants contain the organs responsible for internally transporting fluids. It forms natural topographies which is a low cost way of promoting cell alignment by replicating the natural physiological state of myoblasts. It may also help with gas and nutrient exchange.
- Biochemical Properties. A scaffold’s biochemical properties should be similar to those of the ECM. It must facilitate cell adhesion through textural qualities or chemical bonding. Additionally, it must produce the chemical cues which encourage cell differentiation. Alternatively, the material should be able to blend with other substances which have these functional qualities.
- Crystallinity. The degree of a material’s crystallinity dictates qualities such as rigidity. High crystallinity is often due to hydrogen bonding which in turn increases thermal stability, tensile strength (important for maintaining the scaffold’s shape), water retention (important for hydrating the cells) and young’s modulus.
- Degradation. Certain materials degrade into compounds which are beneficial to cells, although on the other hand, sometimes this degradation is irrelevant or detrimental for the cells. Degradation would allow us to remove the scaffold from the finished product so that it is purely animal tissue — thereby increasing its resemblance to in vivo meat. However, the rate at which it is done should be controllable or at timed in such away that it doesn’t disappear in the middle of muscle development.
- Edible. If scaffolds are unable to be removed from the animal tissue, they must be edible to ensure consumer safety. In this way, it would also be beneficial if they are made out of ingredients which are nutritional, for instance, high in protein.
In recent years, a number of academic research groups and companies have emerged in order to figure out what raw materials have the characteristics which would make them suitable scaffolds as well as how best to turn them into scaffolds. The remainder of this article is going to summarize some of the key findings.
Decellularized Plant Tissue
Cellulose is the most abundant polymer in nature and constitutes the exoskeletons of plant leaves. Due to it’s abundance, it can be obtained at a relatively low cost. It is also versatile and biocompatible.
Through a process called “decellularization”, it is coated in an SDS surfractant which creates pores. These pores then release the plant’s cellular components, and it becomes decellularized plant tissue. This material has been extensively studied by academic researchers in the Pelling Group and Gaudette Group at the University of Ottawa and Worchester Polytechnic Institute, respectively.
Through cross-linking (forming covalent bonds between individual polymer chains to hold them together) the plant tissue’s mechanical properties can be changed so that it more closely resembles skeletal muscle tissue. This can also be done by blending the plant tissue with other materials.
On the other hand, decellularized plant tissue typically lacks mammalian biochemical cues, so it need to be coated with other functional proteins in order to compensate. However, C2C12 growth was not shown to change significantly between the bare scaffold and the same scaffold with a coating of collagen or gelatin proteins. But, the seeding efficiency (rate at which cells attach to the scaffold) was improved.
An advantage of decellularized plant tissue is the natural topography afforded by the vasculature in the leaves. This helps replicate the natural physiological state of the myoblasts which promotes cell allignment. The other ways of doing this are usually quite a bit more expensive including 3d printing, soft lithography and photo lithography. Vascularization can also help overcome the 100–200 nm diffusion limit of culture medium into cells which usually produces necrotic centres in muscle conglomerates.
Another way to do this is having a porous scaffold which supports angiogenesis (the development of new blood vessels). While this has been shown to work for Apply Hypanthium, not all plants are nearly as porous.
The alternative to plant cellulose is bacterial cellulose which is typically more pure than plant cellulose as it is free from contaminants like lignin and hemicellulose. Bacterial cellulose has more hydrogen bonding between it’s polymer strands and so it has greater crystallinity. It also has smaller microfibrils which allow it to retain more moisture and have smaller pores. The substance itself can be produced using waste carbohydrates (which may suggest it can be achieved at a reduced cost) and it causes juiciness and chewiness in emulsified meat (which would mean that even if it can’t be taken out of the final product, it will contribute to the texture profile).
Chitosan and Chitin
Chitin is the second most abundant polymer in nature and is found in the exoskeletons of crustaceans and fungi. As cellular agriculture is interested in being unreliant on animals, chitin dervied from fungi is of greater interest. It has mostly been studied by the aforementioned Pelling Group.
Chitosan is derived from chitin in a process known as alkaline deacetylation (substituting out certain amino acid groups). The degree of this process determines the physical and chemical properties of the chitosan.
Chitosan has antibacterial properties, in particular, it has bactericidal effects on planktonic bacteria and biofilms and a bacteria static effects on gram negative bacteria like E.coli. This is important as it neutralizes compounds which are potentially harmful for humans to eat without using antibiotics which many consumers prefer to stay away from.
Chitosan’s resemblance to glycosaminoglycans and internal interactions between glycoproteins and proteoglycans make it highly biocompatible. It can also be easily blended with other polymers in order to select for more bioactive factors.
One potential disadvantage of chitosan is that it degrades in the presence of lysozymes (a naturally occuring enzymes). But, this can be resisted using the deacetylation process. This is not entirely a negative thing, as the byproducts produced through degradation have anti-inflammatory and anti-bacterial properties. It is just important to match the level that cells rely on the matrix for structure with degradation.
Recombinant Collagen
Collagen is a family of proteins which makes up the primary structure of connective tissue. It is typically derived from bovine, porcine and murine sources. As these are all animal sources, cellular agriculture overcomes this through the use of transgenic organisms which are capable of producing the amino acid repeats which make up the collagen.
Collagen naturally exists as Collagen type I and has been produced as porous hydrogels, composites and substrates with topographical cues and biochemical properties. Synthetic kinds of collagen have also been produced through recombinant protein production — Collagen type II and III, tropoelastin and fibronectin. One main challenge with these proteins is that they can not be modified post translation. However, an alternative fibrillar protein has been isolated in microbes which lacks collagen’s biochemical cues but has this kind of gene customizability.
A big focus of recombinant collagen production is yield optimization — how it can be produced most effectively. Plants, in particular, tobacco look like the most promising option, however, bacteria and yeast are also viable alternatives.
Textured Soy Protein
Textured Soy Protein is a soy flour product often used in plant based meat which has been shown to support the growth of bovine cells by the Levenberg Group at the Israel Institute of Technology.
It’s spongy texture enables efficient cell seeding and its porosity encourages oxygen transfer. Additionally, it degrades during cell differentiation into compounds that are beneficial to certain cells.
Mycellium
Mycellium are the roots of mushrooms. Altast Foods Co. is using solid state fermentation in order to grow mushroom tissue on mycelium scaffolds. They then harvest this tissue and use it to create bacon analogs.
Nanomaterials
Nanomaterials are materials which exhibit unique properties at the nanoscale. Biomimetic Solutions — a London based scaffold currently involved in the SoSV incubator — is leveraging nanomaterials in order to create scaffolds.
Nanocellulose Sponges
Cass Materials in Perth, Australia is using a dietary fibre called Nata de Coco (derived from coconuts) to create nanocellulose sponges for their BNC scaffold.
Nata de Coco is biocompatible, has a high porosity, facilitates cell adhesion and is biodegradable.
Immersion Jet Spinning
Immersion Jet Spinning is a method of creating scaffolds by spinning polymers into fibres initially developed by the Parker Group at Harvard University.
iRJS platform uses centrigual force to extrude a polymer solution through an opening in a rotating reservoir. During extrusion, the solution forms a jet which elongates an aligns as it is shot through the air gap. The jet is directed into a vortex-controlled precipitation bath which chemically crosslinks or precipitates polymer nanofibers. By adjusting parameters like air gap, rotation and the solution, we can change the diameter of the resulting fibres.
This method can spin scaffolds out of PPTA, nylon, DNA and nanofiber sheets. A nanofibrous scaffold made this way out of alginate and gelatin was able to support the growth of C2C12 cells. Rabbit and bovine aortic smooth muscle myoblasts were also able to adhered to the gelatin fibres. They formed aggregates on shorter fibres, and aligned tissue on the longer ones.
Electrospinning
A company called Matrix Meats is using electrospinning — a process which uses electric force to turn charged polymers into fibres for scaffolds. Their scaffolds have been shown to allow meat marbling, is compatible with multiple cell lines and is scalable.
Needless to say, there is not one material or method of production that is universally superior to the other possibilities. Each of them have their own helpful attributes as well as weaknesses. It is probable that many of these weaknesses will be overcome by blending different materials with complementary characteristics.
