This Exponential Technology Is Going To Change Everything.
By: Sayyida Hashim
Tags: Biotechnology, Innovation, Meat, Technology, Cellular Agriculture

How do you make ACELLULAR products from CELL cultures?

Let’s take the example of milk (without cows).

Here’s a question: what makes milk, milk?

Chances are you’re probably looking at me like I’m crazy. And no, I’m not.

Here’s a better way of wording it: what gives milk its nutrition and taste?

Answer: Whey and Casein.

With that, we know that whey and casein are the heroes behind this incredible fluid, but how do we replicate them? Luckily, it happens so that our cells and proteins have a language → genetic code, which every living thing can understand. Essentially what happens is that we pass on this genetic code, which we obtain in a non-invasive procedure or maybe from an online database, to another host (eg. yeast). This will be our starter culture. As our yeast friend follows instructions, he unknowingly brews up some of these animal proteins!

..and Voila! You’ve got the main components that makes milk, well, milk!

See, that wasn’t so complicated, was it?

Now let’s get back to Roan’s story to understand how we can obtain cellular products from cell cultures. To let him free while making the process more efficient, we decided to produce lab-grown meat. The question is, how the hell do we even make meat without an animal?

Creating meat — without animals.

Step 1: Understanding what ‘meat’ is.

Meat is mainly the muscle tissue of an animal. Animal muscle is roughly 75% water, 20% protein and 5% fat, carbohydrates and assorted proteins.

Muscles are made of cells called myofibers, which are made up of myofibrils. It contains two main proteins — actin and myosin.

All this probably seems too confusing because of the similar names, but don’t worry! Here is a diagram that sums up everything:

Muscle Structure

Here is what one myofibril looks like:

A closer look of myofibril

Step 2: Let’s gather some cells!

Cells are fascinating — they come in every little shape and size but what type do we use?

Well, out of all the peculiar ‘lil cells, we use the myosatellite stem cell which are cells that can give rise to skeletal muscle cells. We find these little critters under the basal layer of the muscle where they chill till their time comes.

They are relatively easy to obtain and all you’d need is a teeny-tiny biopsy from Roan (who will need a local anaesthetic for that but don’t worry, it won’t hurt.)

But this certainly won’t be enough; skeletal muscle is composed of connective tissue, stromal tissue, muscle fibers and various other stem cell populations. Myosatellite cells reside in muscle fibers and so, it is a necessity to disassociate these for our lab-grown goodies.

Skeletal Muscle Fibres — this is where you find myosatellite cells! | Source

To do this, we first physically separate them after which proteases, enzymes which catalyze [accelerate] proteolysis [breakdown of proteins], such as trypsin, pronase and dispase, purify this. Then, we filter it to obtain the mononucleated cell population, which consists of stromal cells, myosatellite cells, somatic cells and blood cells. To further separate the myosatellite cell from these (yes, there are a LOT of separation to get them!), we have various approaches on the basis of:

Physical features [density gradient centrifugation]

features [density gradient centrifugation] Biological features [preplating, cytochalasin B treatment]

features [preplating, cytochalasin B treatment] Molecular features [fluorescence‐activated cell sorting, magnetic-activated cell sorting]

…and Tada! We have some myosatellite cells!

The only problem is that these being multipotent stem cells, stem cells which can only differentiate into limited cell types, abide by the Hayflick limit 😓

The Hayflick limit occurs due to the degradation of telomeres which are caps at the end of chromosomes that help preserve DNA.

The Hayflick limit restricts the cell from dividing unlimitedly, which would serve as a problem while expanding operations to a larger scale.

Of course, we could use iPSCs/ Embryonic stem cells (Pluripotent and Totipotent respectively) for this as they don’t have to abide by the Hayflick limit but they do tend to go a bit wild in the differentiation part.

Whatever cell we decide to use though, our aim will be to help them proliferate (multiply) as much as they can + differentiate and mature.

Immortalizing?

We know that multipotent stem cells have to abide by a Hayflick limit, that restricts it from dividing boundlessly. After maximum cell division occurs, the cell enters a state of senescence and cannot divide anymore.

What can we do about this?

While we could rely on mutations in-vitro that allow the cell to bypass cellular checkpoints, this could have unpredictable consequences on cell biology, which could also possibly make it useless.

As of now, research is still underway but various other methods promise immortality or at least prolonged Hayflick limit. Some of them are:

Adding genes+ stopping the expression of cell cycle genes

Targeting proliferative pathways with small molecular compounds.

Inhibiting various proteins such as STAT3, p38 etc…

Mimicking an injured/regenerative state via growth in the presence of cytokines

After these cells are collected, Roan’s involvement is over and he can go back to doing cow things. YAY!

Because of this limited involvement, we could pick the healthiest, happiest cow there is for our meat (and take into consideration additional factors for maximum proliferation like sex and age)!

Step 3: Creating Muscle 💪

Now, we have a bunch of purified, immortalized myosatellite cells. What next?

Where + how do these cells grow?

The cells after proliferation go on a little journey to turn into muscle. Here is a pictorial representation of it (made by yours truly 😉):

This process is known as myogenesis | Image by meeeee :)

Basically, the activated myosatellite cells will turn into myoblasts via a gene regulation shift which involves the up-regulation of MYOD and MYF5 & the down-regulation of PAX7. During this, these transcription factors change gene expression to turn these myoblasts into myocytes. These myocytes then go around and look for friends of the same kind to fuse with so that they can form myotubes. These myotubes together form tasty muscle! 😋

Several strategies have been used by scientists to form myotubes in a way that allows them to be packed together into tasty meat, some of which include:

seeding cells around a gelatin ring promoting muscle fusion.

ring promoting muscle fusion. using plastic microcarriers to increase surface area for cells to bind on to and hence harvesting more cells in a smaller space.

While all these genetic stuff are happening, the cell undergoes structural changes too with the help of actin, myosin, calcium, ATP hydrolysis and other proteins to form characteristics for sarcomeric organization.

Of course, you can’t just expect these cells to differentiate and do everything on its own. We’d need to replicate Roan’s body environment for this.

But, firstly, what even is Roan’s body environment?

Temperature : 37 degree Celsius

: 37 degree Celsius CO2 level: 5%, to mimic the natural blood CO2 levels.

5%, to mimic the natural blood CO2 levels. The cells usually grow in a dense protein jungle known as Extracellular Matrix or ECM that give it structure.

From this, we know that we’ll have to put a check on all these parameters while having a way to clean waste products in the process. What we currently use for this is a cell culture medium, which is basically a bath of nutrients so that our cells can do everything as it does inside Roan.

Some important stuff for the cells to grow (constituting the cell culture media) are:

Basal media: Composed of everything you’d expect a cell to need; amino acids, carbohydrates, lipids, vitamins, salts and trace minerals.

SERUM SERUM SERUM : Biology is complex enough without having to literally grow meat in the labs and so the cells will need to satisfy some additional requirements for it to grow nicely. This is provided by a serum (which is something like a super-powered cocktail 🍸). Our current choice of serum isn’t the most eco-friendly or ethical one as it is derived from animals; Fetal Bovine Serum (FBS) . FBS is a by-product of the dairy industry and so, it would really contradict the key aim of having lab-grown products. Moreover, FBS comes with a hefty price 💰 tag, costing $318 (USD)/200 mL. Yikes!

: Biology is complex enough without having to literally grow meat in the labs and so the cells will need to satisfy some additional requirements for it to grow nicely. This is provided by a (which is something like a super-powered cocktail 🍸). Our current choice of serum isn’t the most eco-friendly or ethical one as it is derived from animals; . FBS is a by-product of the dairy industry and so, it would really contradict the key aim of having lab-grown products. Moreover, FBS comes with a hefty price 💰 tag, costing $318 (USD)/200 mL. Yikes! Cell Signalling Molecules → help cells communicate with each other!

These cells are kept in an incubator that satisfies all the temperature + CO2 requirements previously mentioned to really give it the cow-body-feel.

Now these cells can swim around and do what they need to do!

Based on factors like the number of cells and their metabolic rate, we replace this medium periodically (every 24–72 hours) to remove cellular waste.

Step 4: Polishing it all up to suit the both of us!

Okay, here’s a question: would you eat a beef burger which is one cell line thick?

Of course not! Unfortunately, this is exactly where we are headed if we stop right here.

You see, these cells love to stick on to their surfaces. They just cling on there which wouldn't be the best if you want to make something like steak.

how cells (red) usually grow in surfaces (blue) — self-made :D

So to bring in some depth, we use something called a scaffold which makes it more 3D and meat-like. This replicates the ECM in which cells grow.

Since cells love to stick on to surfaces, we can create some surfaces similar to the structure we want to attain and it’ll just blindly stick on to it! That’s genius, am I right?

creating more surfaces! self-made :)

Which unfortunately also means…

NO NUTRIENTS FOR SOME CELLS!

How is that, you ask? Well, cells get their nutrients from the growth medium through diffusion. This is a problem because diffusion is very picky and only works up till a certain point. This certain point isn’t very high to rely on diffusion — 200 microns, about the width of the human hair.

If we continue to rely on diffusion, we’ll be stuck with 200 microns wide cultured Big Macs. Ermm… no thanks.

We still don’t have an answer to this. yet.

What is currently in the works (artificial circulatory system)

Your scrumptious Big Macs are ready to serve!

And that’s it! You’ve got some fresh lab-grown Big Macs, ready to serve 🍔

It’s the exact same thing as the regular Big Mac, except it uses 99% less land, 98% less water, releases 96% fewer emissions and you get to see Roan’s happy face as he runs around doing cow things.