Decreasing the cost of YPR Culture Medium for Entomoculture

A culture medium is a substance containing all the essential nutrients like carbohydrates, fats, proteins, salts, and vitamins which a cell needs to grow. As the culture medium diffuses into the cell, the cell will grow, divide and the cell line will “proliferate”.

Culture medium is an instrumental part of cellular agriculture and generating cultured tissue because it’s effectively what allows us to take a relatively small sample of animal stem cells and end up with an enough to constitute an entire cut of meat. These stem cells would then be put into a bioreactor where they are exposed to a whole bunch of environmental cues which encourage them to differentiate into the specialized kinds of cells we get in meat.

In order to supply all the essential nutrients to the cell, the culture medium usually consists of a basal mixture supplemented with extra additives.

Generally, these additives are what complicate things. With mammalian cells, when their tissue is grown in vivo (i.e. in their body), extra growth factors are supplied by their own blood. And so, in order to conveniently replicate this, cellular agriculture leveraged Fetal Bovine Serum (FBS) which, as it sounds, is the blood of a cow fetus. The two issues with this is that it is a) reliant on animals, hence defeating the goal of cellular agriculture and b) expensive because it is so inaccessible.

There is no other substance that we can easily obtain that can singlehandedly provide all these growth factors, so we turn to making them all individually using recombinant protein production. But, at the moment, this too is crazy expensive.

This naturally leads us to one of the biggest challenges facing cellular agriculture: finding the ideal culture medium which is one that is simple, can stimulate proliferation, is unreliant on animals, is accessible and is cheap.

But, this isn’t so much the case for entomoculture — cellular agriculture which deals specifically with insects. Insects naturally contain a substance called haemolymph rather than blood and so don’t even rely on the same growth factors as mammalian cells in the first place. As a result, they can be adapted to serum free culture mediums which don’t contain animal byproducts. At this point, the biggest challenge remains making these culture media cheap.

Yeastolate-Primatone (YPR) is an entomoculture media developed by Ikonomou et al in 2001 through a series of fractional factorial experiments on ingredients referenced in pre-existing formulations. It was shown to stimulate the proliferation of Sf-9 and High Five cells, prolong the stationary phases of these two lines and support recombinant protein production post baculovirus infection.

While the specific composition of YPR is publicly available, it is not sold commercially. But, it’s raw ingredients cost about $28.88 per litre. While this may seem significant, it’s much cheaper than many other culture media (particularly those used for mammalian cells). The Essential 8 culture media, for instance, costs $376.80 per litre.

Still, this puts the starting cost of our meat at well over $28 per kilogram, which isn’t exactly competitive in a grocery store. To put it in perspective, a kilogram of ground beef costs $11.25 CAD — the raw production costs are about $10.00 CAD.

So, in this article, we’re going to go over 5 potential ways to reduce the cost of YPR media. This analysis is based on the Good Food Institute’s An analysis of culture medium costs and production volumes for cultivated meat which did a similar thing for the Essential 8 culture medium.

The point of these solutions are to be realistic — meaning they can be done with out some sort of major technological advancement — and also based on conservative estimates — meaning they’re on the cautious side.

So, what makes YPR this expensive?

YPR is made up of 6 different ingredients which are

YPR = IPL-41 + Glucose + Glutamine + Yeastolate Ultrafiltrate + Primatone RL + Pluronic F68.

• IPL-41: 51 ingredient basal medium which comprises the bulk of the culture medium and supplies most of the nutrients.
• Glucose: A sugar
• Glutamine: An amino acid
• Yeastolate Ultrafiltrate + Primatone RL + Pluronic F68: Additives which supply extra peptides, amino acids, lipids, vitamins and nucleosides.

As we can see

• 40.9% of the cost is contributed by yeastolate ultrafiltrate
• 39.1% is from glucose
• 10.7% is from Primatone RL

As such, any measures we take to decrease the cost of culture media will focus primarily on these 3 ingredients.

We can also take a closer look into each of the 51 components of the basal medium and figure out that the leading expenses in it are

• maltose (26.8%)
• glutamine (36.2%)
• sucrose (7.6%)

So, anything we do with in the basal medium should focus on these ingredients.

What changes can be made?

If we want to decrease the cost of the culture medium, we should start by trying to cut the cost associated with yeastolate ultrafiltrate, glucose, and primatone RL. This can done either by reducing the amounts of the aforementioned ingredients, replacing them with cheaper alternatives or sourcing them at a reduced price.

Scenario A: Yeastolate ultrafiltrate is added to the culture medium at 4% rather than 6%.

Yeastolate is considered to be the most important supplement to insect cell cultures as it provides peptides, amino acids, carbohydrates, nucleosides and vitamins. So at first, this might seem counterintuitive — wouldn’t this just be unproductive for cell growth?

Many existing insect culture formulations add yeastolate to the media at 4%, suggesting that it is possible. But, in order not to significantly stunt cell growth during the process, we could do this by

• adapting the cell line over a few generations to tolerate lower concentrations.
• engineering the yeastolate to be more “potent” or more effective at interacting with the cells thus decreasing the amount the cell line needs.

Here, the cost associated with the yeastolate would drop to $157,600 per 20,000 L.

Scenario B: Yeastolate ultrafiltrate is replaced with yeast extract.

Yeast extract is effectively the food grade version of the pharmaceutical grade yeastolate ultrafiltrate. Yeastolate ultrafiltrate was used primarily because the YPR culture medium was originally invented for baculovirus infection

But since cellular agriculture is about producing food, food grade should do just fine. Sigma Aldrich sells yeast extract for $68.00 per 250 g. As such the cost associated with the yeast extract is $32,640 per 20,000 L.

Scenario C. Scale the production of carbohydrates to the level of D-glucose.

In the IPL-41 basal medium, D-glucose is produced at $0.0008 per gram. On the other hand, maltose is produced at $0.359 per gram, sucrose at $0.062 per gram and the non-basal media glucose at $1.13.

Firstly, since the glucose in the basal medium is identical to the non-basal medium glucose, the two should be produced at the same price leading to a total non-basal media glucose cost of $168.

D-glucose has a molecular complexity of 151 and maltose and sucrose have complexities of 342 and 395, respectively. This suggests that maltose and sucrose would be harder to synthesize, however when scaled to the level of d-glucose, they should be able to be produced at a price proportional to their molecular complexity. For maltose, this would lead to a production price of $0.0018 per gram and $36 for the entire 20,000 L. For sucrose, this would lead to a production price of $0.002 per gram and $66 for the entire 20,000 L.

Now, glucose is widely recognized as the most important carbohydrate for insect culture, with maltose and fructose coming in as secondary sources after glucose depletion.

On the other hand, sucrose has been suggested to only be consumed by cells post baculovirus infection which does not happen in the context of cellular agriculture, implying that it is not necessary for our purposes. As such, we will eliminate it from the formulation. Now the cost of the basal medium is $17,589.86 for 20,000 L.

Scenario D. Scale the production of glutamine.

Glutamine is an amino acid that is, while not essential, recognized to be important for the proliferation of certain insect lines including High Five Cells which this media was originally designed for.

Glutamine has a molecular complexity of 146 in comparison to the molecular complexity of glutamic acid which is 145. This implies that the two should be able to be produced for the same price. As we are currently able to obtain glutamic acid at $0.03 per gram, we should be able to produce the requisite 20,000 grams of glutamine in the basal medium for $600. The additional glutamine supplementation could be produced at $2100 for 20,000 L.

Scenario E: Primatone RL is replaced with soy hydrolysate.

Now the main cost driver in the culture medium is primatone RL. Primatone RL is a main provider of oligopeptides, iron, salts, and amino acids to cell lines.

Plant hydrolysates are common medium supplements which fulfil the same purpose. Soy hydrolysate, or HySoy is one such example which can be obtained at $113.00 per kg. By replacing primatone RL with HySoy, the corresponding cost for 20,000 L would be $11,300.

Compounding Scenarios B, C, D and E result in the total cost of the culture medium to be $3.08 per litre!

Other Possibilities

In terms of further scenarios, we could also consider eliminating the non essential and non important amino acids. In addition to the nine essential amino acids, glutamine, cystine and tyrosine are considered to be important for successful proliferation. This suggests that the other amino acids (glycine, proline, arginine, asparagine, aspartic acid, serine and alanine) could be eliminated.

However, this could place some strain on the cells. Making this kind of decision could also really depend on on the cell kind and species, and so further testing would have to be done to figure out how important each amino acid is.

How much would production cost?

Now comes the question of how much producing a specific amount of meat — let’s say a kilogram — would actually cost. This depends on how much culture medium those cells require.

Step 1: Number of Cells per Bioreactor.

The growth configuration we’re going to be focusing on is the batch process. This entails cells being grown in batches which are all inoculated at one time for use. This is different (and less efficient) than the continuous or semi-continuous process which is more of a perpetual letting cells grow and collecting them. The reason we’re going with the less efficient process is to ensure that the estimates are not optimistic.

We’ll consider a 27,000 L bioreactor which is at the upper bound of size feasibility. While bigger bioreactors definitely exist, animal cells aren’t typically able to withstand the amount of pressure present in those models.

Cells proliferate best in what is called a seed train bioreactor, so we’ll start them off here. Once we want them to differentiate and structure onto the scaffold, we’ll put them into a perfusion bioreactor.

In a seed train bioreactor, minimum cell density requirements are considered to be 200,000 cells/ml — anything much lower will trigger cell apoptosis. Maximum cell density is 7 x 10⁷ cells/mL (this is the biggest source of discrepancy, as the maximum cell density for insect cells has yet to be definitively decided for cellular agriculture). So, in the proliferation stage, we’ll

• start the cells at minimum density
• have them proliferate until they reach maximum density
• put them into a larger bioreactor at minimum density
• repeat until we get to a bioreactor of 27,000 L.

Expanding the culture from the minimum to the maximum density will take a 350 fold increase. We’ll have our largest seed train bioreactor be 27,000 L, the middle one be 77 L and the smallest be 0.2 L.

Step 2: Amount of Time

Expanding the culture from the minimum to the maximum density will take a 350 fold increase, which is less than 8 doublings. High Five cells are reported to have a doubling time of 24 hours, meaning that each bioreactor stage will take 8days. Together, the three bioreactor stages will take 24 days.

After, we want to seed our cells onto a scaffold so that they mature in a certain configuration which takes an additional 10 days, bringing the total time to 34 days or about 5 weeks.

Step 3: Total Yield per Batch

We’re going to consider the average cell volume to be 3375 micrometers³ (this is a rough estimate as different kinds of cells in different species will be larger or smaller). A cubic meter of ground meat weighs about 881 kg which includes some fat.

This puts our total at

yield = (density of meat) x (unit conversion) x (avg. volume per cell) x (cell density) x (size of largest reactor)
= (881 kg/m³) x (10^-18 um/m³) x (3.375 x 10³ um/cell) x (7 x 10⁷ cells/ml) x (2.7 x 10⁷ ml/batch)
= 5,620 kg.

Now, this all has to be put into the perfusion bioreactor for differentiation. So, we’ll double it to account for the necessary “void space” in and around the cells. This puts our reactor’s spatial requirements at about 11, 240 L.

Step 4: Required amount of Culture Medium

We’re going to calculate a couple different possibilities for how much culture medium we require.

The minimum amount we require is enough to fill the largest bioreactor which is 27,000 L. This assumes that the culture medium is never replaced.

The maximum amount we require is if we change the culture medium every 6 (once per stage)

0.2 + 77+ 27,000 + 11,240 = 38,318 L

Assumptions & Oversimplifications

Now, there are a whole bunch of assumptions and oversimplifications that we’ve made throughout this process. While the assumptions can all be justified, the main oversimplifications we made which would lead to some discrepancy with the actual final result is that

• some cells will die and senesce
• some cells that will not adhere to the scaffold, and so will not be part of the finished product
• some cells won’t differentiate.
• we need to use different ingredients for different cell lines
• medium cost takes into account labour and preparation, not only the raw ingredients
• high cell density requires different medium conditions as low cell density
• other costs that go into a finished meat product in general.

And given that we didn’t even factor in things like the energy required to run the bioreactor and the labour, this would not accurately represent the actual production costs. A little under 15 dollars per kilogram is better than 200, but is still falls short of the $10 mark represented by current commercial ground meat.

What would it take to be competitive?

In terms of lowering the cost of cultured meat production, besides lowering the cost of the culture medium, the other thing we can do is produce more cells per batch (more biomass for a given amount of culture medium means less cost per a given quantity).

As mentioned above, one thing that has yet to be definitively decided is the max density that insect cells can be cultured at. And, indeed, out of all the variables that go into calculating yield per batch (density of meat, size of the bioreactor, size of the cell and cell density), the only one that can be changed is the cell density. So, what would we need our cell density to be in order to get the cost per kilogram down to $10.00 CAD?

Well, this would mean that

our required kg/batch = (cost per litre) * (litres used) / (cost per kg) = (3.08) * (27,000) / 10 = 8,305 kg

implying that

Max cell density = (kg/batch) / [(bioreactor size) * (cell size) * (conversion unit) * (weight)]

= 8,305 / [(2.7 x 10^ 7 mL/batch) x (3.375 x 10³ um³/cell) x (10 ^-18 um/mL) x (881 kg/m³)]

= 1.03 x 10⁸ cells/mL.

So, we would need to be able to culture our cells at 1.03 x 10⁸. Which, is close to 1.5 times the current density.

Overall, the YPR culture media analysis completed here is optimistic that cultured insect meat could be grown at a price that is competitive in the modern market.

Further examination could be done into the amino acids included in the formulation in order to decrease the cost. Additionally, developments of higher density cultures would imply less cost per unit of meat.