A COMPREHENSIVE GUIDE TO CELLULAR AGRICULTURE

Proposal: Using 4D Scaffolding to Improve the Texture of Cultured Meat

To many environmentalists, cellular agriculture is nothing short of a miracle. This sensational technology has become the poster child for the future of food production, allowing meat-eaters to eat their favourite foods without comprising the well-being of our planet.

Many industry experts explain cellular agriculture as a way to produce animal products without the animal. Cellular agriculture creates a win-win situation for everybody: consumers get to eat the meat they love, animals don’t have to be slaughtered, and the agricultural industry emits 99% fewer greenhouse gasses. Who would hate a claim like that?

But in order for that to happen, we have a major barrier to overcome. One that is a must-have for consumers (especially my fellow food-lovers 😍). We need to nail the texture and taste of cultured meat. To grow meat in a lab that tastes similar to what we get in-vivo in a living animal, we need to examine one of the most important aspects of cellular agriculture — scaffolds.

The Current Process

Growing meat in a lab is no easy feat. There are many different factors to consider before a muscle stem cell can become a steak, and they can be broken down into 4 main steps:

How cells differentiate into myofibers (muscle tissue).
  1. Getting the cells. Multipotent Stem Cells (cells that have the potential to become almost any type of specialized cell) are extracted from an animal through a painless biopsy. They are then given the right gene factors and developed into a cell line.
  2. These stem cells are then immersed in a culture medium (which I like to compare to a magic potion). The culture media gives the cells all the essential nutrients, carbohydrates, proteins, and fats they need to grow and multiply.
  3. The stem cells are “seeded” into a scaffold (type of mold that gives them a structure to grow onto). Current scaffolding methods are static and can be either edible or inedible.
  4. All of the cell cultures are then put in a bioreactor which keeps the cells in constant motion and introduces the cells to growth factors. These growth factors encourage them to differentiate into the many different kinds of specialized cells we find in meat.

The purpose of this process is to replicate the environment that cells are in when they are actually growing into muscle tissues in a living animal (whether it be a cow, pig, chicken, or fish).

In the animal, stem cells attach to the network of extracellular matrix (ECM) proteins through receptors located in the muscle fibers. I like to think of the extracellular matrix is like the instruction book for cells — it is a 3-dimensional mesh of glycoproteins, collagen, and enzymes that transmits signals telling cells how they should arrange themselves.

This needs to be replicated in our petri dish so that our cells are arranged in a way that we would naturally see them, especially in structured meat like steak for example.

A key component to achieving this through cellular agriculture includes using a scaffold. Scaffolds are the glue that holds everything together in meat. Without having our cells seeded into a scaffold, they would grow sporadically into a bath of meat mush.

Scaffolds serve as a support network that cells expand and differentiate onto. Over time, when provided with appropriate signals, the cells multiply in number and produce new tissue that takes on the shape of the scaffold as the scaffold degrades.

The Scaffolding Situation

There are many considerations when finding the right scaffold for the job.

A: Collagen hydrogel B: Collagen Sponge C: Collagen hydrogel scaffold D: Stained collagen hydrogel scaffold Source: Research Gate

These include:

  • 🔎 Porosity — the number of pores/openings in the scaffold diffuse gas and nutrients to the innermost layers of cells which mitigates cell death from lack of contact with the growth medium
  • 💎 Crystallinity determines the rigidness of the final shape, promotes thermal stability, and water retention in the cells
  • 🍃 Vascularization — vascular tissue found in plants can be used to help to transport fluids to the cells. It can help with cell alignment and facilitate gas and nutrient exchange.
  • 🧪️ Biochemical properties — determines the cell adhesion through chemical bonding. The necessary chemical cues must be produced to encourage cell differentiation.
  • 🍔 Edibility — scaffolds that are not removed from the muscle tissue must be edible to ensure consumer safety

Scaffolds have to be made from porous materials that allow oxygen and nutrient flow and waste product removal to maintain the cells’ metabolic functions. Current scaffolds are chosen to do so with popular options being chitin and chitosan, decellularized plant tissue, and collagen hydrogels.

However, even if static scaffolds can get us pretty close to the texture and taste of regular meat, they’re still not perfect because they cannot fully replicate the dynamic interactions that the cells have with proteins in the cow.

4D Scaffolding

How cells can be seeded into a 4D scaffold

There is a solution to overcome the major drawbacks of static scaffolds: using 4D printing to create a dynamic scaffold that will better emulate the in vivo environment inside an animal.

Four-dimensional (4D) bioprinting is a recently developed technology based on 3D bioprinting. It has a unique ability to transform shape upon response to intrinsic or external stimuli. This may help more accurately mimic the dynamics of the native tissues.

The 4D bioprinting strategy is based on the integration of stimuli-responsive biomaterials within the 3D bioprinting technology. The fabrication of biologically active constructs that can alter their shapes upon desired stimulation, such as immersion in water, to achieve prescribed functionality.

The selection of materials that comprise the bioink is crucial for successful bioprinting with live cells. Naturally derived polymers, such as polysaccharides, offer the advantage of effectively mimicking the biological nature of the native ECM environment.

Alginate used to create a 4D scaffold

Alginate (polymer found in brown seaweed) and hyaluronic acid are two of the most frequently used biopolymers for bioprinting/biofabrication due to their capability to support cell survival and differentiation in vitro, low toxicity, and relatively low cost. Gelatin is another option that can be used because of its high collagen content.

Regardless of which of these materials used to base the hydrogels, they need to be programmed or induced to undergo multiple controllable shape changes over time. Hydrogel scaffolds should swell when additional water is added to them and absorbed, changing the shape that the muscle cells are growing on. The amount of swelling can be tuned by changing aspects of the hydrogel material.

We can actually control the timing and the extent of shape change that occurs. This helps mimic the continuous different shape changes that developing tissues undergo and can support a higher cell density — meaning that more cells can be placed per scaffold (which allows for more meat).

This proposed 4D biofabrication process does not pose any negative effect on the viability of the printed cells, and the self-folded hydrogel-based tubes support similar cell survival as 3D scaffolding processes. Consequently, the presented 4D biofabrication strategy allows the production of dynamically reconfigurable structures with tunable functionality and responsiveness, governed by the selection of suitable materials and cells.

Why is this a good idea?

The 4D scaffold will exhibit changes in physical or chemical properties to stimuli such as heat, moisture, light, magnetic field or pH. By creating a scaffold that mimics the movement of muscles in animals, we can better replicate the cell-cell interactions in a cow and better allow the muscle stem cells to assemble into the muscle tissues in a lab. We’ll be able to create lab-grown meat that tastes the same as what we are used to eating in no time.

How do we go about doing this?

An overview of the 4D printing process
  1. In the first step, methacrylate alginate or hyaluronic acid polymer solutions can be printed onto glass surfaces in two-dimensional (2D) rectangular shapes.
  2. The solutions can then be loaded with a photo-initiating system for green light crosslinking (forming covalent bonds that hold portions of several polymer chains).
  3. Next, the printed polymer films are crosslinked with green light and mildly dried. After immersion of the crosslinked films in water, FBS, or another cell culture media in the third step, they can be folded into tubes or another shape depending on the final product. The shape transformation of the films occurs in a matter of seconds.
  4. If the printed solution contains live cells, the cells can be evenly distributed throughout the walls of the tube after folding.

The swelling behavior of the photo-crosslinked hydrogels depends on the composition of the aqueous media. Both polymers swell strongly (15–30 times) in pure water. We can control the amount of swelling by adding a relatively high concentration of monovalent ions like Na+, phosphate-buffered saline (PBS) buffer. This has the potential of decreasing the swelling ratio of both polymers down to 6.

Conclusion

There are still some scalability issues that need to be resolved before cellular agriculture becomes commercially viable on a global scale. The unshakeable truth is that lab-grown meat has to taste as good — or even better — than traditional meat for there to be a large market for it. Cultured meat companies need to find a way to consistently produce lab-grown meat in high quantities while maintaining the integrity of structured meat.

The proposed 4D fabrication strategy allows large-scale production of the self-folded tubes and scaffolds since there are virtually no limitations in the number of printed films. It is versatile and can have different polymers be integrated into this approach depending on the needs of a specific product.

The future is bright. In recent years, cellular agriculture has rightfully stepped into the limelight as a contender for being the future of meat production due to ethical and environmental reasons. The cultured meat industry has made leaps in progress and is growing exponentially. At a time where climate change remains one of the biggest threats to the future of humanity, every bit of effort will count towards making a better world.

Hey there! Thanks for making it to the end of the article. Before you click out…

My name is Kimberly Liang (just call me Kim ☺️) and I’m a 16-year-old innovator/business enthusiast/musician who’s super interested in the future of biotech. I spend my time reading up on emerging technologies and training 10X mindsets.

If you want to find me:

Here’s my LinkedIn and Twitter. If you want to stay updated in my journey, sign up for my monthly newsletter!

Until next time 👋

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Kimberly Liang

Kimberly Liang

I’m a 16-year-old innovator with huge ambitions to change the world. I research emerging technologies and neoteric mindsets to maximize my impact.