Rebuilding the World’s fisheries while feeding the world
Cellular Aquaculture & Cultured Fish: The Alternative Protein Guide Part 2
A fish named Frank walks into a bar… wait fish can’t walk. Let me try again.
Two cells walk into a bar… oh, that doesn’t work either. Hold on.
I walk into a bar… shoot, that’s illegal, I’m 15.
Fine. No bar.
So, there’s me, Frank the fish, and Frank the fish’s cells in the form of sushi… Frank gets to enjoy being alive, and I get to enjoy Frank’s yummy muscle tissue at the same time. Now that’s what I call a fun time.
And this *impossibility* is possible with a FANTASTIC technology called tissue engineering (aka cultured fish). I’ll be giving a technical overview of cultured fish; a technology near and dear to my heart (literally though, I’m currently on a boat in Amsterdam, the ocean is 2 seconds away).
Here are some pre-requisites you should know:
- Cultured fish is grown from stem cells (pre-muscle tissue cells) in a controlled environment (a replication of the cell’s original animal’s interior).
- The controlled environment is made up of a protein & nutrient-rich soup (called a growth medium), it also has these biological support structures (scaffolds).
- All of this happens in a bioreactor tank — which delivers nutrients and removes waste products.
Before you get skeptical about a 15-year-old delivering content… 👋I’m Isabella; I speak at conferences all over the like Microsoft Ready, C2, World Summit AI and Elevate, I’m currently working with Ira Van Eelen, daughter of the inventor of cultured meat and I’m a nerd for all things tech 🤓.
How DEEP is the water… (Why we need Cultured Fish)
(Yes, all these titles are going to be fish-related puns. Sorry not sorry!)
- Food Scarcity: (supply=decreasing; demand= increasing) → 90% of fisheries are depleted/at max capacity, and 1/3 of fish stocks are depleting faster than they can repair. Fish demand is increasing by 30% in the next decade.
- Food health: the US mislabels 33% of seafood. Often, mislabeled fish are species that tend to be higher in pollutants (e.g., mercury).
- Food allergies: Fish and shellfish cause >90% of food allergic reactions in the U.S.
- Food price: 3B people rely on seafood as their primary source of calories, with less supply, seafood gets expensive.
So here’s the deal: we can make the same biological fish in labs, without the oceans, without the toxins, and even without the allergens. It’s more ethical, sustainable, healthier and potentially cheaper.
We could produce fish anywhere around the world! Landlocked countries with high poverty rates can have access to mercury-free omega-3 fats.
Fun fact: The alternative protein industry is reaching a market value of 140 Billion in the next 10 years.
Let's learn about this fish muscle tissue 😁👇
Fishy Muscle Tissue
Our friendly swimmers contain 3 types of muscle tissue. Different ratios of the tissues = different species of fish.
There’s red muscle tissue which contains many blood vessels, capillaries & mitochondria. This type of muscle has an abundance of slow-twitch fibers which LOVE oxygen.
They perform “aerobic metabolism”; i.e., turn oxygen into energy (ATP). Red muscle is the marathoner; it can work for extended amounts of time at a good pace (you wouldn't sprint a marathon)!
Moreover, the red muscle has more myoglobin which carries oxygen to the muscle fibers.
White muscle is the opposite; it has fast-twitch fibers which don’t need much oxygen (they’re powered by sugar through a process called glycolysis). Their anaerobic metabolism lets them make a lot of energy (ATP) but only for short periods of time, and they have less myoglobin (oxygen-carrying molecule). They’re the sprinters of the bunch.
Pink muscle is a combo of red & white.
To create cultured fish, we combine the muscle types in different ratios.
Remember, different fish = different “patterns” of red, white or pink tissue.
Fish have an abundance of “white” muscle tissue (whether they’re marathoners or sprinters). Where they differ is in the amount of red muscle tissue. The more red muscle in a fish means they’re more of a long-distance swimmer. Fish with less red (i.e. mostly white tissue) are the sprinters.
Note: salmon’s redness is unique. They’re reddish not because of their muscular activity (salmon = sprinters = mostly white muscle ), but because of their diet. When salmon eat clam fish they consume astaxanthin (let’s call it fun-pigment-related-to-carotene in carrots).
Fun-pigment-related-to-carotene in carrots brings out that colour 🙌.
Culturing meat & culturing fish is not the same task
Fish’s muscles (red, white, pink) are different than muscle tissue of land animals. For example, raw fish; it’s yummy (unless you’re one of *those* anti-sushi people). However, raw chicken? Pork? Lamb? No way.
Land animals have firmer muscles because they have to support themselves from a thing called “gravity”… heard of it? I hear the guy that pioneered physics was pretty important in its discovery!!
Jokes aside, gravity makes land muscles un-snackable when they’re raw; but when they’re cooked, the connective tissue is softened and those juicy flavours flow!
Whereas fish’s muscles aren’t fighting gravity. They float all the time. (The buoyancy force in the water neutralizes gravity).
If my dog were an animal, she’d definitely be a fish. Oh, wait… How does that work?! I’m confusing myself, moving on.
So here’s what we know:
- Fish give rise to many health and economic problems but fear not! Cultured fish is here 😉
- fish has 3 different muscle tissues (red, white, pink), and different ratios of these muscles = different species of fish
- Fish muscle is completely different than land animal’s muscles because fish live in water, making the engineering task different than cultured meat.
Note: Each muscle (red, white, pink) has different oxygen requirements. E.g., red muscle relies on aerobic metabolism (oxygen to produce energy); therefore, it has higher oxygen needs. But, white muscle can survive with very little oxygen. We need to consider these oxygen variations when designing our tissue engineering approach (explanation coming).
You have to SEA this cultured fish process
Cultured fish begins with a cell 🦠 that has the potential to be red, white & pink muscle. There’s 32 000 different types of fish we can choose cells from.
Here’s the framework for creating any cellular bite of your choice🍣.
When a cell loves itself very very much…
Phase 1: Proliferation.
As a food fan, I’m going to use recipes. Here’s our recipe for phase 1 of cell culturing:
- Get a starting sample of cells (from a fish)
- Add a growth-medium to make these cells proliferate
The goal of this phase is to increase the # of cells by a lot. With a large sampling of cells, we can then do what I call “finessing their biology” 😎(aka phase 2).
Best FISHES to create muscle tissue
… getting, cause, wishes?
Phase 2: Tissue perfusion (fancy word for creating muscle tissue).
We take the pile of cells and make them differentiate. Differentiating cells means letting them “mature.” For example, muscle cells mature through a process called myogenesis; demonstrated below.
Myogenesis is like a startup-accelerator.
However, instead of taking a potential billion $ idea and turning it into a billion-dollar company with the right resources and mentorship; myogenesis takes potential muscle tissue cells and gives them resources (growth medium + nutrients) and mentorship (scaffolds aka support structures).
potential cells + resources + mentorship = harnessing their potential + lunch
The last “stage” of this process is creating myofibers. Our goal in phase 2 is to make many of these fibers.
Note: myofibers = many myotubes. The myofibers are made of many small fibers call myofibrils
Myofibrils contain the proteins actin (pink) and myosin (blue).
This magical structure is what we enjoy to eat.
- Actin = cell movements
- Myosin = responsible for converting energy in the cell
These proteins help muscles move (contract) with the energy they create.
Before we continue, here’s some terminology :
Scaffolds: Helpful structure that “supports” cell growth
Myo-fibers: building blocks of muscle tissues (many fibers = muscle); think of these fibers as the “bricks,” and tissue as the “house”; the bricks build the house.
Extra Cellular Matrix: The body’s natural scaffold; inside the body ECMs are like “cacoons” & homes for cells, so they can grow up and become awesome 😎
To create this actin/myosin contraption (phase 2), we follow this recipe:
- Use scaffolds to form 3D structures
- Use scaffolds to promote differentiation of cells
A scaffold might look something like this:
- Get a starting sample of cells (from a fish)
- Add the nutritious medium (*magic*) to make these cells proliferate
- Use scaffolds to form 3D structures
- Use scaffolds to promote differentiation of cells
ANDDDDD, that’s the BLUEprint to culturing swimmers(get it, ’cause water’s blue?? I don’t think this one sunk in… sorry I’ll stop).
How the industry cod use development
To create cod(or any fish, really)the focus is on improving:
- Cell lines ( the “starting stones” of this technology)
- Cell medium (the resources to feed the cells )
- Scaffolding/structural advancement (“cellular mentorship”)
- Bio-reactor tanks
 Whale, what are the Cells we need?
Studying fish in labs is new. Only 2 databases have studied fish cell lines.
Cell lines are cells that have been cloned many times in a liquid medium. The medium promotes cell growth + keeps cells alive.
The 2 studies studied 100,000 cell lines.
🛑Before we move on, let me take you through some cell culture fundamentals.
Cells have a limit to the # of times they can duplicate. This number is around 30, and it’s also called the “Hayflick limit.”
Immortalized cells (aka tumour cells) are Hayflick limit rebels 😎. They can generate infinite cells (with the advantage of never running out of these cells). Generally, immortalized cells have a mutation to do this. The disadvantage is they don’t behave like “normal” cells.
Primary cells are the cells derived directly from the sample (i.e., tissue).
Once these cells start hanging out in the culture medium (a nutritious soup that supports their growth), they become dominated by fibroblasts; a type of cell that creates the ECM or Extra-cellular matrix (mom of the cell to support them).
primary cells + medium → cell lines → [add mutation] immortalized cells / [no mutation] normal cells → fibroblasts produce its own support (ECM)
Now you’re a culture insider ;)
Of the 100,000 cell lines, not 12, not 367, but 0 were myoblastic (cells that become muscle tissue).
9 samples had primary cells from muscle, but none differentiated into muscle tissue.
So, ya. There has been minimal research on cell lines of fish, which, obviously makes it an area of development… this whole technology relies on cells… especially primary cells from a muscle that differentiate into muscle tissue.
I wrote an in-depth article about cultured meat, where I describe the possible stem cells types, I’ll link the article at the bottom of this one in case you want to learn more after you’re done reading this.
I’ll briefly cover two potential primary cells for cultured fish:
- Myosatellite Stem Cells are multipotent, meaning they can become most types of muscle tissue, but nothing more. Since we’re not creating hearts or lungs, these cells are acceptable for cultured fish (and meat in general). Pros: It’s easy to turn them into muscle tissue; cons: they can only proliferate ~30 times.
- Induced Pluripotent Stem Cells(iPS) are pluripotent meaning they can become almost every cell in the body (except placental cells). Pros: they can proliferate indefinitely (one cell can feed the world!!!); cons: they’re hard to control (i.e., more challenging to turn into muscle tissue)
Takeaway: using appropriate primary fish cells, we can create fish flesh 🥰. With 32,000 fish species out there, we sure have a lot to explore!! Now that’s a large school of fish.
Designing the next Ocean
Cultured meat’s medium is a significant challenge for growing cells; in this case, growing seafood. But it is essential because it gives cells the nutrients and factors they need to grow.
The medium is the cell’s environment. We want to trick the cells into thinking they’re still in their original environment (Frank’s body). If we trick them into thinking they’re still in Frank, they’ll grow like normal.
The catch is, right now our medium is slightly expensive and contains animal ingredients (AKA cultured fish police aren’t going to be happy)…
The medium typically has FBS (fetal bovine serum) or FCS (fetal calf serum). These ingredients help encourage cell proliferation.
Cell culturing is the only place where multiply and divide mean the same thing (cell proliferation)… it’s THAT cool 😎
There are 2 types of media we need.
- Basal Medium = the “nutrients.”
- Growth Medium = encouraging differentiation, attaching to support structures; all activities that have to do with growing/maturing.
Yes, I know what you’re thinking; this is correlated with the 2 phases of cell culturing. Maybe I was wrong at first, but you’re sure thinking about that now. (phase 1 = proliferation, phase 2 = tissue perfusion (differentiation)).
The culture medium’s requirements:
- Meet oxygen demands of the cells (varies depending on the type of muscle: red, whi — oh you know the drill!)
- Not made with FBS/FCS: these animal serums are *currently* vital to proliferation. I.e., when we try to grow cells without them, proliferation rates plummet. The media, however, needs to be plant-based (because of ethics & company values). The standard in cultured meat world is companies won’t use animal products.
- Fish cells have a higher buffering capacity, which makes them less sensitive to changes in pH — however, we still need to ensure the pH levels are “good,” the medium would monitor + (have the ingredients to) maintain these levels.
- Fats are nutritionally relevant in seafood: omega-3 long-chain polyunsaturated fatty acids (PUFAs), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)are 3 very long words that you probably skimmed, and I’ll never be able to spell, but the implication is the media should be fatty acid-rich.
- Proper Osmolality (# of dissolved particles in the fluid), (should be as close as possible the osmolality of the original animal).
Cultured fish’s medium needs monitoring. There are so many variables (pH, oxygen, etc.) within the cells that have a profound impact on growth, so, creating built-in sensors is part of medium development😍.
Fish are hypoxic (survives with small amounts of oxygen)… which makes growing these cells a more manageable task because the monitoring of oxygen and other factors is less strict.
We have barely started the research into plant-based, acceptable, scalable medium, but I’d argue it’s one of the most important hurdles to lab-grown fish.
We haven’t designed this magical medium for our dear fish cells🐟🧫 yet. Right now, the use of FBS and FCS is crucial to cell proliferation; when we remove these ingredients, the cells get all bratty and stop making babies.
“It is likely that animal-based media are used merely because of a lack of well-researched options.” Aka let’s get cracking on this research folks!
A SoFISHticated Scaffold
Put short & concisely; scaffolds are the cacoons of the cells. They support them.
To use an analogy: a house is the muscle tissue, the myofibers are the bricks, and the scaffolds are the glue that keeps the bricks together.
I’ll link that in-depth article here & at the bottom, which also goes deeper into the science behind scaffolds.
Note: scaffolds are a porous material.
The Scaffold types we’re exploring for cultured fish are:
- Cellulose Skelton (plants)
- Insect and crustacean exoskeletons
- Chitosan (algae, fungi, yeast)
Scaffolding is still an open research area in tissue engineering; we’ve BARELY started fiddling with its possibilities.
However, fish’s structure is more straightforward than other meat’s, which, makes creating scaffolds easier. Yay Frank!
When designing scaffold systems, we need can either have:
- Biodegradable scaffolds (that the cells eventually break down to create their natural cacoon systems with the fibroblasts we nerded over earlier).
- OR — Integrated into the final product
Scaffolds need to:
- Be a 3D support structure can guide differentiation & shape the fish product.
- Have the right proteins so that the cells adhere to the scaffold. Example: in biological organisms proteins such as elastins, collagens, fibronectin, and laminin do this with the ECMS; our scaffolds likely need these proteins. (Alternatively, the media can include these proteins).
- Include fish glycosaminoglycans (GLY)(chains of sugars & proteins). Glycosylation is vital to attaching cells to the scaffold — we need fish GLY because fish protein glycosylation processes are different than mammals. Think of scaffolds being your parents, and GLYs being your cool aunts. You get into a fight with your parents, but your cool aunt convinces you to stop being a stubborn teenager and listen to them. Yay! However, if it was a stranger trying to be the “GLY,” you probably wouldn’t listen. TL;DR: cells need a trusted GLY.
- The right porosity (we don’t have a porosity # yet, and it varies from species, but it is an important tribute of scaffolds)
Note: GLY is not a scientific name; it is me being unable to spell.
- We need to use the right proteins to facilitate the correct biological reactions (e.g. GLYs) in cells, so they adhere to scaffolds.
- The scaffolds need to be successful at raising their cells to become c̶o̶o̶l̶ ̶c̶a̶t̶s̶ muscle tissue.
Any FIN is possible, even Bioreactors
Here’s the recipe thus far:
Cell lines + Medium (or Media) + Scaffolds = well… not cultured fish yet, more like a cellular stew.
To make it all come together, we need bioreactors. We might need 2 (one for each phase in the process):
- Stirred tank bioreactors = those that encourage multiplying/dividing
- Tissue Perfusion bioreactor = scaffold & differentiate the cells to create tissues
Note:  has been built to scale because of industries like pharma; however,  only exists in mini-versions for labs, and needs some engineering cleverness for the #scale-up.
It varies from company to company if they want to combine the reactors or make the phases very distinct 🤷♀️. But the reactors have 2 processes.
These bioreactors are closed systems which deliver the relevant resources and remove waste products.
Cells are whiney and demanding because they have lots of desires. For example, when there’s too much lactic acid in the tank, they start protesting (dying).
SO as a result, we need to be on the ball with monitoring the conditions for them. Back to the analogy of a house, we’re the cleaning/weather folks that keep the sun shining and bricks dust free. Happy cells = clean house & sunshine.
Things to monitor in the tanks:
- Lactic acid build-up (a waste product of metabolism): this causes changes in pH which impacts protein productions and oh no… not GLY too 😥
- Oxygen to keep those cells breathing
- Temperature (but fish are cool… literally… they can live in cooler temperatures saving cultured fish factories $ on energy). It just can’t be too chilly. Lower culturing temperature = fewer energy costs😍.
- CO2 build-up
Fish cells are cool because they’re less picky than most cells (beef cells I’m looking at you), so mass production is easier to achieve.
However, the MAJOR challenge is scaling these systems. We haven’t designed mass production for cultured food… aka it’s “easier” but not “easy”.
We’re imagining is a beer-brewery type situation:
Summary of the problems
Before I get into the summary, I’m going to tell you an *industry secret*.
Cultured meat isn’t expensive. I know what you’re thinking… what about that $330,000 burger?! (For context: the world’s first cultured meat “proof of concept” was grown by Mosa Meat and presented in August 2013). There was a huge fuss because yelp would rate it at least 10 dollar signs.
But cultured meat is a spinoff of the medical industry. 3 PhDs were working on bringing this brand-new concept to life for 3 years. So yes, it took a lot of money. They used medical supplies (culture medium, machines, etc.) which are not built to scale and also 10 dollar signs on yelp.
It can cost 200,000 to engineer a heart valve: an existing medical technology. I don’t see any New York Times pieces complaining about that amount of $$$$$$! Cultured meat was a new medical research technology that was run by 3 expensive professors, fancy medical materials and no automation, so 300K is a pretty good deal.
Now, with a handful of companies, more advanced machinery + automation, the price is under $10 for that same hamburger.
Bringing cultured meat to fruition isn’t much of a cost battle — it’s a scale and regulation party. Onwards to the summary of challenges😍👇
Cell Lines → With 6 companies (and 🤞more upcoming) we’re starting to understand fish cells more. In 2017 we made the world’s first cultured fish patty, and we’re now culturing shrimp. Proof of concepts are coming!!
Medium → One of the main things driving these companies is cruelty-free food. ∴They're trying to get away from FBS and make plant-based media.
FBS comes from a fetal cow’s blood.
Beef consumption = ⬆️.
Cattle-raising technique’s improvement= ⬆️. We produce FBS when female cows get accidentally sent to the slaughterhouse; however:
Improving techniques =fewer mistakes = less pregnant cows getting slaughtered.
∴ FBS Supply = ⬇️.
According to BioSera( a leading supplier of animal culture), “the present world supply of FBS is short and will continue decreasing.” Companies need to focus on medium production once they’re ready to scale. (Currently, they buy their media from supplies like BioSera).
With lots of pending cultured meat/fish companies in the world, FBS is getting expensive + there isn’t enough supply to grow all these products!
No company exists that is creating media for food cells… but, the next billion-dollar company is producing -based media for food😉😎🌱. (PS. companies like JUST are creating a plant repository of ingredients + their chemical qualities; becoming a useful asset for building 🌿media).
Scaffolds → Luckily, this is a prominent area of research that scientists are working on… designing the scaffold might not be hard (it’s only a porous & economically feasible material).
However, hooking the cells onto the scaffold & encouraging growth is the challenge. This lies in the ingredients in the media — ex, fish glycosaminoglycans might be important for this.
Development of scaffolds & tissue-perfusion growth media could be intertwined.
Bio-reactors → We’ve done some research on the first 3 challenges (cell lines, media, scaffolds), and the science works (we can make lab-grown fish/meat).
Most companies are planning to release their products in ~5–10 years which leaves them time to research the fundamentals of delivering the nutrients to the cells in a scalable way.
Cultured meat teams are starting to diversify: adding chefs (for flavour improvement), engineers with scaling experience (but no medical background), and people that know how to scale 😍.
Beyond these scientific hurdles (which need the right engineers to solve) we’re dealing with government regulations (TL;DR: no one knows how to handle regulations for cultured meat products because it’s “new” food) and there’s additionally some conflicted consumers.
Schools of Fish
Leading companies in the space that can really SEA [the future of] food.
Wild Type which raised 3.5M is working on salmon (yes, they’ll include the fun-pigment-related-to-carotene).
Finless-foods made the first lab-grown fish burger in 2017, they’ve raised 3.5M. They’re also considering using immortal-cells (but don’t worry, they’re not tumours!) This could make production + scaling of cultured fish easier.
Shiok is YC’s first cell-based meat company focuses on shrimp and raised 4.6M.
Only 6 companies?! 32 000 types of fish?
We need more people working on seafood that tastes yummy… we wouldn’t want anything that tastes funny… 😋
We’re in the most technically advanced point in history. We have self-driving cars, smart furniture and people trying to get to MARS.
As a techie, I can’t wait to see the food tech revolution unfold. What a fine period to be alive… and WOW what a great time to be a fish. 👋See you soon…
If you want to learn more about the technical details of stem cells for clean meat, read this blog.
If you’d like to learn more about using longer words to describe cultured fish, I’d recommend this paper.