Ex Vivo Meat Production
Wild Type is a San Francisco-based cell-based meat company. Last year, the company raised a $3.5M seed round to develop cell-based fish and foie gras. This patent application is their first one that has been made public.
The patent application itself reads like a description of their entire technology platform. There are a broad range of topics covered, from media development to bioreactor design. Given the breadth of topics covered, it’s clear that not everything is meant to be novel technology. Instead, it seems like Wild Type is going for an “omnibus patent application” strategy –using a broad patent application to reserve an early priority date, buying them time to figure out which parts of their technology are the most promising. Presumably, later they will file continuations of this patent application that will focus in on a narrower aspect of their technology.
Instead of listing out everything that’s covered in the patent application, I’ll instead focus on the parts that are the most interesting to me.
The first type of product that Wild Type discusses is fish meat containing a mixture of mature muscle and fat cells. Wild Type enumerates many of the possible cell types that can be used to make cell-based fish, as well as the process of turning each cell type into mature muscle and fat. I think this list could be useful for new companies trying to understand their options for creating cell-based fish.
Embryonic stem cells (ESCs): ESCs are totipotent, meaning that they can differentiate into any cell type. They can also proliferate indefinitely without any modification. By starting with these cells, or other pluripotent cells, Wild Type can make both muscle and fat from the same batch of proliferated cells.
Induced pluripotent stem cells (iPSCs): It’s possible to revert mature fish cells back to pluripotency through exposure to certain reprogramming molecules. The resulting iPSCs can then proliferate indefinitely and eventually differentiate into muscle and fat.
Embryonic germ cells: These are another pluripotent stem cell that can be isolated from early fish embryos.
Fibroblasts: Fibroblasts are found in connective tissue, and are responsible for making the extracellular matrix. These cells can be turned directly into myocytes or adipocytes without first going through an intermediate pluripotent state. This process is called “transdifferentiation” or “direct reprogramming.”
Precursor cells: These are cells which are already committed to either the muscle (myosatellite cells) or fat (adipocyte precursors) lineage, but which are not yet fully differentiated. The difficulty with this path is that the cells won’t naturally proliferate indefinitely. Therefore, they would need to be immortalized, e.g. through spontaneous immortalization. Spontaneous immortalization involves proliferating cells until an individual cell naturally mutates in a way that allows it to proliferate indefinitely. That cell then takes over the culture and serves as the basis for the immortalized cell line. The benefit of this method is that it avoids genetic modification, which could be favorable for regulation or public perception.
The ultimate goal is to end up with mature myocytes and adipocytes that make up the final product. Muscle and fat can either be cultured separately and then combined, or they can be cultured together. In the later case, stem cells would remain pluripotent for the entire proliferation stage, then be differentiate into both muscle and fat at the same time, in whatever proportion makes sense for the end product.
Wild Type indicates that the final muscle tissue will have at least 50% high glycolytic  and anaerobic muscle fibers in order to improve the taste of the end product. As compared to land animal meat, fish generally has a much higher proportion of glycolytic muscle fibers, which contributes to the difference in texture between the two types of meat. However, Wild Type doesn’t indicate how they intend to control the ratio of glycolytic vs oxidative fibers in the end product.
The second product that Wild Type focuses on is cultured foie gras. Foie gras is a promising product for cultured meat in part because of its high price and homogenous texture. On a cellular level, all foie gras is composed of mature hepatocytes that have gone through steatosis–the process of retaining an abnormally high amount of fat within the cell. In the case of animal meat, force feeding a duck a high-fat diet can trigger steatosis in the duck’s liver.
The possible starting cell types are the same as for fish, except in this case the desired goal is a mature hepatocyte. Once a mature hepatocyte is obtained, there are many ways to induce steatosis. The simplest way is culturing the cells in media with a high lipid content. In this scenario, the cell’s natural lipid metabolic pathways will cause it to absorb some of the surrounding lipids. However, some lipids might go to waste. It is also possible to synthetically increase the cell’s ability to absorb lipids by using genetic engineering, which may be less wasteful.
One benefit of growing foie gras via cell culture versus using an animal is that it’s easier to control the types of lipids contained in the cells. Wild Type suggests exposing the cells to high amounts of omega-3 fatty acids, which could make the foie gras healthier than its animal-based counterpart.
In my opinion, the most interesting piece of technology discussed in the patent application is the use of recombinases to make “foodprint-free” cell modifications–genetic modifications that are later removed by enzymes. There is little or no foreign genetic material in the cell by the time it is sold as meat, potentially allowing it to avoid the GMO label.
Recombinases are enzymes that can trigger “genetic recombination.” In practice, this means that they can add, remove or reverse strands of genetic material. Recombinases work by identifying “DNA recognition sites” , which sit on either side of the genetic material that will be operated on. The recognition sites each have a directionality that determines how the recombinase behaves. If the sites are pointed in the opposite direction, the recombinase will reverse the genetic material in between the two sites. If the sites are pointed in the same direction, the recombinase will excise the gene and “glue” the ends together, creating a circular DNA fragment which can be cleaned up by the cell. This fragment is not part of any chromosomes, meaning that it won’t be copied over during the next cell division. This technology usually leaves behind a single DNA recognition site, making it not strictly footprint-free. However, both the original cell and all of its clones will have a minimized genetic footprint.
This allows Wild Type to genetically modify a cell to express a gene of interest, e.g. to prevent differentiation and promote proliferation, then later completely remove the gene from the cell. Figure 15 illustrates a construct that makes this possible. The construct is flanked by two “pLox” regions pointed in the same direction–these are the DNA recognition sites that the recombinase will bind to. In between the pLox regions are the gene of interest, then a tetracycline responsive element (TRE) , then the gene coding for the Cre recombinase. When Wild Type wants to turn off the gene of interest, they expose the cell to doxycycline, which triggers the expression of the recombinase. The recombinase will then excise the gene of interest, the TRE gene, and the Cre gene. Using this technique, Wild Type can get the functionality of genetic modification, with a minimal effect on the genome of the cell. It’s unclear whether Wild Type has further technology that can make this excision completely footprint-free.
Another application of recombinase technology shown in Figure 14 allows Wild Type to switch between proliferation and differentiation. The cell starts out expressing genes which allow the cell to proliferate while maintaining pluripotency. Again, doxycycline induces the expression of the recombinase, but it also induces the expression of MyoD, a growth factor that leads to muscle cell differentiation. The recombinase then completely excises the genes that were maintaining pluripotency and proliferation. Unlike the previous system, this system is not completely footprint free, since the construct coding for MyoD and the recombinase remain inside the cell. However, it might be possible to remove this construct later using a different recombinase system .
The two constructs discussed here induce recombinase expression with doxycycline, but any method of inducing recombinase expression would work. For example, Wild Type discusses a different system using a synthetic Notch receptor . In nature, Notch receptors allow cells to trigger gene expression in neighboring cells through signaling molecules on the surface of each cell. By creating such a system that triggers recombinase expression, Wild Type can excise a gene in an entire population of cells without having to include a media additive like doxycycline.
One of the main benefits of using systems like these is that if the cell is completely footprint free, the meat will not be GMO based on the most recent USDA regulations . Even if the cells are not completely footprint free, it’s possible that regulatory bodies will prefer a lower genetic footprint.
There are a few other interesting pieces of technology that I’ll mention briefly:
Wild Type provided further confirmation that mushroom extract and other plant-based solutions can serve as a cheaper replacement for fetal bovine serum (FBS) . They do experiments where they transition from FBS to shiitake mushroom extract and soybean hydrolysate.
Wild Type discusses the “hanging drop” method of transitioning adherent cells to 3D culture. They place a drop of media on a plate small enough that when the plate is turned upside down, the drop is suspended by its own surface tension. They then culture cells in the drop. The force of gravity pulling down on the cells encourages the cells to form cell aggregates (i.e. clumps of cells), which can then be moved to a 3D suspension culture.
Wild Type identified glucomannan (a polysaccharide found in konjac) as a promising material for edible scaffolding or microcarriers. Glucomannan is abundant and cheap, and has a neutral taste. The concentration of glucomannan in the end product could be a way to adjust the elasticity of the meat. Wild Type experimentally confirmed that duck fibroblasts and fish myosatellite cells can grow attached to glucomannan scaffolds.
Thanks to Alene Anello and Parendi Birdie for comments on drafts of this piece.
 What are glycolytic fibers? Muscles are essentially bundles of many muscle fibers. Muscle fibers are the individual cells in muscle tissues, each containing mitochondria that generate energy for muscle contraction. Certain types of muscle fibers, called “glycolytic” fibers, primarily use anaerobic respiration to generate energy. Muscles with many glycolytic fibers generally are more resistant to fatigue, but cannot generate large amounts of force quickly. On the other hand, oxidative muscle fibers primarily use aerobic respiration for energy. Muscles with many oxidative fibers can generate a lot of energy quickly, but also tire quickly once the local oxygen supply is depleted. All muscles have some proportion of both glycolytic and oxidative fibers, but the ratio varies based on the needs of the particular muscle.
 Wild Type doesn’t specify what type of recombinase is required, but the most common is Cre recombinase with LoxP DNA recognition sites.
 See Memphis Meats’ patent in the first part of the patent analysis series for an explanation for tetracycline-based transduction. In short, the TRE allows Wild Type to trigger transcription of a gene by exposing the cells to the small molecule doxycycline.
 The Cre-Lox recombinase system is the most common system, but there are other similar systems, such as the Flp-FRT system. Although they don’t discuss this, Wild Type can use different system to trigger events at different times throughout the culture.
 Like the one described in https://www.ncbi.nlm.nih.gov/pubmed/26830878.
 Discussed in one of the earliest cell-based meat papers: https://www.sciencedirect.com/science/article/pii/S0094576502000334