Why Insect Cells May Be the Future of Cellular Agriculture
In the emerging field of Cellular Agriculture, one of the most frequently asked questions is ‘when will we see lab-grown products on the shelves?’
There are factors that contribute to cell ag products not being available on the market, the two biggest being scaling and pricing.
Since the field is still relatively new, the process of growing cells is time and labor-intensive for small yield.
One promising new area of research, Ph.D. candidate Natalie Rubio has been looking to the using insect cells muscle cells as opposed to mammalian cells.
While it may seem kinda weird to eat insect meat, I have an article talking all about it here.
They seem to be much easier to grow and harder to kill, which may make scaling easier and lower production costs.
Let's take a look at the results of these experiments where they put the insects to the test to see just how good these cells are!
In these experiments, they used fruit fly adult muscle progenitor-like cells for the insect cells. This means that these cells have the capacity to differentiate into muscle cells.
For the mammalian cells mouse myoblast cells, which is an embryonic muscle progenitor cell. These are also cells that basically only differentiate into muscle cells.
For the media (AKA cell food) they used two kinds; one that contained FBS (fetal bovine serum) which an animal-derived supplement for media, and then a media that was FBS free.
The Race to Serum-Free
This experiment was to see how cells could adapt to serum-free media and if they could form in monolayers.
Serum or fetal bovine serum (FBS) expensive, animal-derived supplement that cells grow really well with. Monolayers mean the cells grow in, nice single-cell thick sheets in and not in weird clumps.
To start off the experiment, one group of cells was given 100% the serum-free media and the other was gradually weened onto higher concentrations of the serum-free media. The control was cells given media with serum in it.
At the beginning of the in serum free-media race, the immediately adapted cells proliferated about as fast as the control. However, after about 48 hours, their progress slowed and they started becoming a little deformed.
The gradually adapted cells, on the other hand, had a consistent proliferation rate that was comparable to the control over the course of the 2 weeklong experiment.
The adapted insect cells exhibited ‘diauxic growth’, meaning they grew in two different phases. The first phase was while they adhered to the surface they had been initially growing on, and the second phase was after they starting growing in ‘suspension’, meaning growing after detaching from the surface.
The cells stay attached to the surface until they ran out of the room. Once they were a monolayer of cells (a layer one cell thick) over the entire surface they were put suspension.
But the cells started growing so much in suspension that they started growing in clumps. Even after changing the cells to a different type of surface they still were getting all clumpy.
They had to be transitioned to shaker flasks and treated with dextran. Dextran sulfate reduces the clumps but the cells are still floating in suspension, they do not have to adhere to the surface in a monolayer, which is good because at mass production cells will have to grow in suspension. This is very difficult with mammalian cells.
TLDR;
- Cells that were immediately adapted did well for two days but then stopped growing and started deforming
- Cells that were gradually adapted did just as well as the controls and grew very well. They didn't grow adhered to a surface but did grow with little clumping in suspension after behind treated with dextran sulfate, which is known to help de-clump cells.
Tough cells
In the next experiment, it was insect vs mammalian cells, they wanted to see how long the cells could stay alive with just one day worth of media.
Both cell types were given 5ml of media and were left for 25 days.
Over the course of the experiment, the number of live mammalian cells decreased and by the end of the 25 days, most of the cells had died and detached. But the insect cells not ONLY were alive but CONTINUED TO GROW.
While the mouse cells detached because they were dead, the insect cells detached because they ran out of room to grow on the surface so they began to grow in suspension.
By the end of the 25 days, there were 3.5x more viable insect cells than mammalian. Point one to the fruit flies!
Hormones
In this round of experimentation, the insect cells were treated to two different kinds of hormones that naturally occur in insects, one that is a juvenile hormone, and the other was a molting
hormone.
When the cells were treated with the juvenile hormone, one with a smaller dose and one with a dose twice as big as the first. The smaller dose had the largest effect on the cells, which means the larger dose might be somewhat toxic to the cells.
Compared to the control (which were untreated cells), the cells treated with a smaller dose were higher in number and showed slight elongation, a sign that is a precursor to the cell differentiating (turning into a muscle cell) but was a little shorter than the control cells. This is a sign that the hormone might inhibit differentiation.
When the insect cells were treated with the molting hormone, they proliferated slightly more, but nothing significant.
TLDR;
- cells treated with small doses of juvenile insect hormone caused slight increases in proliferation rates
Getting these cells swole!
In order to get the myoblasts (elongated cells) to contract, they ere treated with high concentrations of extracellular potassium. Contracting is important to keep muscle healthy, like working out.
After the potassium treatment, 95% of the cells were A-OK (but just a little bit shorter) and the myoblasts were able to shorten by 31.6% on average when they contracted.
The cells that were still round (pre-myoblast cells) only contracted by an average of 1.6 percent, this varied by around 16%.
TLDR;
- Potassium was effective in making cells be able to shorten more during contractions
Nutritional value
Insect cells are more nutrient-dense then mammalian cells. But just to see, they gave cells iron-fortified media to see if they could increase iron levels and compared it to a control.
Even though it showed way more iron, it indicated that there is a limit to the amount of mineral a cell can absorb.
TLDR;
- Enriching media with nutrients can raise the amount of nutrients present in the cells, but the cells can only absorb so much
Scaffold
Now its time to see if we can make these insects more edible. In this test, chitosan surfacing is used to grow the cells on. Chitosan is a material made from mushroom, which means you don’t have to worry about putting it in your mouth.
After the first day, the plastic surface had a higher total cell population than the chitosan, but the difference in the number of cells that adhered to each surface was not a ‘statistically significant amount’.
This may be because after the cells run out of space quicker on the plastic surface, and detach from the surface,.To see what effect the coating on the surface had another surface was coated with gelatin.
There was improved cell adhesion with gelatin on the plastic surface BUT caused the cells to get clumpy. This didn’t happen with the gelatin-coated chitosan.
There wasn’t a big difference between the plastic surface and the three different chitosan films that varied in concentration (1%, 2%, and 4% concentration)
By the fifth day, the adherent cells growing on plastic had doubled in numbers.
The chitosan films grew to a lesser extent, but importantly the cells were still adherent and alive to create a 3d surface for the cells to grow on (as the meat you eat usually isn’t one cell thin), chitosan sponges with micropores were made by using directional freezing.
The cells were then seeded in the sponges and in both tests formed a monolayer that covered most of the sponge.
After adding 20- -hydroxyecdysone, which is a hormone known to cause increased differentiation, some cells differentiated into myocytes. Those myocytes were pretty similar in length to the myocytes on the plastic, not a statistically significant amount.
Even though the cells were looking pretty good, the sponges themselves were super fragile and had pores that didn’t keep their shape.
The 1% chitosan sponges just turned into sheets and dissolved,. The other two were more durable, and 4% was the stiffest, twice as stiff as the 2%.
To see how the chitosan would react while cooking, well, they cooked it. The chitosan begins to degrade at 325 degrees celsius
TLDR;
- Insect cells grow well on chitosan, which is a mushroom derivative. Sponges made of 2% and 4% chitosan held up best. The chitosan also seems to react well to being cooked and doesn't begin to degrade until 325 degrees celsius.
Takeaways
In the experiments, insect cells showed that they were more durable and was able to even grow in a starvation setting, as opposed to the immediately declining mammalian cells. They were also able to transition to inexpensive serum-free media and go from adherent growth to growth in suspension, which is great for mass production.
Insect cells seem to be a promising cell source for future cellular agriculture products and may help cellular agriculture products be produced simpler, faster, more nutritious, and cheaper.
Works referenced:
In Vitro Insect Muscle for Tissue Engineering Applications
Huge thanks to Natalie Rubio, who helped me understand all the science and answered all my questions!
