A COMPREHENSIVE GUIDE TO CELLULAR AGRICULTURE
Review Paper: Optimizing Bioreactors to Maximize Cultured Meat Production
In our modern world, climate change remains one of the most pervasive threats to the future of humanity. The agriculture industry is a major contributor to this problem: 70% of global freshwater and 50% of the world’s habitable land is used for agriculture. Globally, agricultural-related activities contribute to 24% of the world’s greenhouse gases.
While countries are approaching Day Zero because they don’t have enough water, we are wasting precious resources to produce our food. Here’s the problem: our systems are inefficient. Animals were not designed to be our food or produce maximum outputs of muscle tissue. Their main purpose in life is to survive and reproduce.
However, we are now primarily raising cows, pigs, and chickens with the intention of feeding over 7 billion people and using vast amounts of resources to produce meat. 99% of animals used for food are factory farmed and considered to be industrial products rather than sentient beings.
As the world’s population continues to increase, the demand for food and water will skyrocket and will cause detrimental effects on the wellbeing of our planet. With an anticipated 70% increase in global meat demand by 2050, we will have insufficient planetary resources to keep up with this inefficient system.
We need another solution to our food crisis. One that allows us to stop harming any animals or funding a process that releases so many greenhouse gases.
Abstract
Lab-grown meat (also called cultured meat or in-vitro meat) aims to replicate conventionally produced meat through tissue engineering and cell cultures. The technique of growing tissue with in-vitro methods (cells in Petri-dishes) has been possible for more than 100 years. Yet, the first lab-grown hamburger was only produced in 2013 by a scientific team at the University of Maastricht. At the time, this original burger was incredibly expensive, costing a whopping $300,000 to produce.
However, two years later, the same team was already able to reduce the cost to $11.36, showing the possibilities of reducing costs by scaling production. Yet, despite large advancements being made in cellular agriculture, a commercial production system has not yet developed.
Cultured meat requires stem cells to be placed in very specific environments so that they can proliferate and form muscle tissues. These environments are often maintained by a machine called a bioreactor.
However, scaling bioreactor production remains one major barrier that prevents cellular agriculture to be commercially viable on the international market. Cultured meat is anticipated to be far more efficient than conventional meat and will guarantee the absence of contaminants and antibiotic use during cultivation.
Bioreactors need to be improved so that production quantities are maximized and the optimal conversion of nutrients and resources is ensured. In particular, existing configurations or novel adaptations of any of these must be scaled to a higher capacity. This paper aims to look at potential solutions to improve the efficiency of bioreactors through the integration of automated sensing mechanisms and media recycling mechanisms.
Outline of Paper
— 3.1 Traditional Lab-Based Methods
— 3.2 Bioprocessing
— 3.3 Environmental Considerations
— 3.4 Objectives
4. Environmental Considerations
— 5.1 Sheer Stress
6. Different Ways to Grow Batches of Meat
— 6.1 Batch
— 6.2 Fed-batch
— 6.3 Continuous
— 6.4 Perfusion
— 7.1 Stirred tank bioreactors
— 7.2 Single-use bioreactors
— 7.3 Hollow fiber bioreactors
— 7.4 Airlift bioreactors
9. Automation
— 9.1 Sterilization
How Does Cultured Meat Work?
In order to start the cultured meat process, we need to take muscle stem cells from an animal through a biopsy (taking a sample of cells harmlessly). We then give the cells a type of growth media that has all the growth factors that tell them to arrange themselves in a certain way. We then replicate the right environments for them to grow in with a bioreactor so that they can grow and become muscle tissue.
Several companies have been successful with this process. The first cultivated meat product was sold in Singapore in December, 2020, with several other cultivated meat prototypes taste-tested, including duck, chicken, salmon, yellowtail, shrimp, pork sausage, foie gras, fish maw, fat, beef meatballs, and beef hamburgers amongst others.
Although cellular agriculture products have been proven to be desirable among consumers, it is not ready to dominate the international market. Moving from bench-scale methods to pilot-scale and commercial-scale production involves overcoming significant technical challenges in cell biology and optimizing production.
The Production Problem
We can approach lab-grown meat in different ways. We can grow individual muscle proteins or use complex engineering to co-culture muscle tissue containing muscle, fat, blood vessels, nerves, fibrous tissue, and immune cells.
In both methods, tissue-engineering techniques typically involve using a biomaterial scaffold that supports cells in a three-dimensional organization, lead to the assembly of a tissue that is anticipated to resemble meat in its sensory and nutritional qualities as closely as possible.
For cultured meat to become a viable alternative to traditional meat, production has to be scalable and economical. The specifics of scaling depend on the final intended product and the number of divisions that the stem cell can sustain.
For example, a minced product like burgers will be easier to scale than a full-thickness meat product like steak because the muscle fibers are less complex.
The production of cultured meat relies on cells growing exponentially, and one of the major challenges is when cells stop dividing due to the Hayflick limit.
Introduction to Bioreactors
Although cultivated meat products for taste testing have been produced using standard cell culture dishes and stacked flasks, growing cultivated meat at scale will require the use of bioreactors in volumes up to or beyond several thousands of liters.
Traditional Lab-Based Methods
Traditional expansion methods are ineffective for a couple of reasons:
- It requires many different teams and a complex manual operation, which takes time to organize.
- There are small variations between each batch of lab-grown meat, rendering the final product inconsistent. Consumers will not get the same taste and texture every time even if the product came from the same original team.
- People are not going to be as efficient as machines, especially as the demand for cultured meat grows. The amount of labour and time spent culturing products will be overwhelming.
Bioprocessing
Using bioreactors to manage bioprocesses will be optimal because they are more efficient, cost-effective, and take up less space. We will get more products for the same manual input and each burger will have a consistently high quality.
This fundamental switch in how cells are grown brings in additional considerations such as gas exchange, heat transfer, shear stress, mixing, and foaming. When designing bioreactors, we need to consider all steps of cellular agriculture as shown in the diagram below.
Objectives
- The ultimate goal of bioreactor development is to increase the percentage of nutrients in the medium that is converted to edible animal tissue (known as the medium conversion ratio). The best measure of success is whether the conversion ratio is equal to traditional livestock meat production. Additionally, cell density (cell number per ml medium) and medium use can be optimized using recycling techniques.
- Another goal is to scale up cell production to achieve cost-effectiveness. The labor-intensive parts of the process will need to be automated to reduce cost and the risk of microbial contamination.
Environmental Considerations
The key variables affecting a cultivated meat bioprocess are temperature, oxygen, carbon dioxide, pH, glucose, biomass, and metabolites.
Temperature
The temperature of a cultivated meat bioprocess will depend on the species of interest. Most mammalian cells are grown around 37ºC while insects, fish, and other marine creature cells may be grown at significantly lower temperatures.
Recording and maintaining accurate temperatures during the process is crucial for many parameters. This is typically done through the use of platinum resistance thermometers that contain a stable, non-reactive piece of platinum wire. The electrical resistance of the wire increases linearly with temperature and thus temperature can be inferred by passing current through the wire.
Oxygen
Oxygen must be continually delivered in the form of dissolved oxygen to cells in order to meet metabolic demands. This is referred to as the oxygen utilization rate. Meeting a specific cell line’s oxygen utilization rate requires considerations regarding mixing speed, bubble size, temperature, flow rate, and general properties of the cell culture medium.
Species that grow at lower temperatures, such as fish, may require differing oxygen requirements, as oxygen solubility increases at lower temperatures. Without proper oxygenation (typically 30–40% of air saturation) or with over-oxygenation, cell growth and viability can be rapidly negatively affected.
Carbon dioxide
Carbon dioxide and pH are intimately linked in a cultivated meat bioprocess. As a bioprocess scales, an increasing number and density of cells respire, leading to an increasing concentration of dissolved CO2 in the bioreactor.
In order to decrease CO2 concentrations, surface aeration, or agitation can be used; however, each becomes more difficult with scale due to surface-to-volume limitations and shear stress considerations. CO2 is typically measured using a Severinghaus electrode, which contains a CO2-permeable membrane and electrode that records the resulting pH change within a bicarbonate solution as CO2 is absorbed.
pH
The pH of the cell culture medium must be tightly controlled for optimal cell health. Together with oxygen, the pH can yield information about cell growth rate and metabolism.
The pH in a bioreactor is typically monitored through optical sensors. The standard red-pink color associated with cell culture is caused by the dissolved pH indicator phenol-red, which adjusts color under certain pH thresholds.
Similar indicators can be used in immobilized substrates attached to optical fibers for more practical pH monitoring. Ion-sensitive field-effect transistors can record pH by measuring current changes caused by ion (H+) concentration changes.
It is important to note that bioreactors are the second-largest component of GHG emissions in cellular agriculture, with the first being growing media (primarily because of amino acids). Using clean energy and maximizing the efficiency of bioreactor machinery should also be considered to resolve this problem.
Optimizations
There are many important variables that can affect a bioprocess when scaling. These include efficiency of mass transfer, avoidance of inhomogeneities, heat dissipation, impeller shape and speed, dissolved oxygen, and reactor geometry, amongst others. Each can be affected differently depending on the cell type, scale, and intended downstream use. Adjustments for these variables are likely to be adapted from existing platforms from the cell therapy and biologics fields or assisted by novel engineering and custom-tailored to a specific bioprocess, species, and/or cell type. Thus, the most generalizable optimizations or considerations are discussed below.
Shear stress
Shear stress is the mechanical force induced by the friction of liquid on a cell’s surface. Animal cells are generally more susceptible to shear stress than their bacteria because they do not have a cell wall.
In a bioreactor, shear stress can be caused by the liquid’s turbulence created by the general motion needed to keep the cells in suspension. Larger volumes of cell cultures generally imply stronger shear forces. Shear stress can impact cell viability and differentiation, so they must it must be prevented by installing flow breakers, making cell adaptations, or adding of poloxamers to the culture medium.
The presence of bubbles and their rupturing can also lead to shear stress due to differences. In cells grown in suspension, smaller bubbles (< 1 mm diameter) can lead to higher shear stress.
One way to solve this would be to grow cells on microcarriers (discussed later). This opens up the possibility that an air-lift reactor that relies on the controlled bubbling of gasses could be used for animal cell growth if microcarriers are used. The growth of cells in aggregates or on microcarriers can independently influence the subjected shear forces on cells.
Different Ways to Grow Batches of Meat
Bioreactors grow cells in batches to scale production. There are a couple of ways to grow batches of cultured meat, but all of them have their benefits and drawbacks.
Batch
In batch culture, a vessel is filled with a fixed volume of media and cells are grown to their maximum density before being harvested or transferred to a larger vessel.
Fed-Batch
In fed-batch culture, cells grown in a vessel are fed fresh medium from an in-line, independent feed vessel at variable rates in order to maximize properties such as exponential cell growth or cell densities.
Continuous
In continuous culture, cells are grown in a vessel and new medium is added via an in-line feed vessel at an optimized flow rate. The product, cells, and medium are simultaneously collected in an independent collection vessel at the same or alternative rate.
Perfusion
Perfusion culture is a subset of continuous culture where the cells are retained via a substrate or collection method. It allows the medium to be recycled and high cell densities in a smaller space.
Types of Bioreactors
There are many different bioreactor designs to choose from based on how the medium is mixed and whether the cells are grown in suspension or adhered to a solid surface.
Stirred tank bioreactors
The industry standard for bioreactors is agitated stirred tank reactors, in which cells are grown either in suspension or attached to microcarriers suspended in the agitated medium.
Stirred tank reactors are more sterilized and produce less bubbling than air-lift reactors at the same scale (more on those later). In general, stirred tank reactors permit the growth of cells in suspension with mechanical stirring while maintaining high mass transfer of oxygen. Suspension growth can also occur with attachment-dependent cells through the use of microcarriers
High cell densities can be achieved in stirred tank bioreactors, but scalability is limited because of the high nutrients, waste, pH, and dissolved oxygen gradients created in the bioreactor.
Stirred tank bioreactor vessels have been used for animal cell production in volumes up to 20,000 L. However, these volumes may not be sufficient for cultivated meat, which must achieve a production volume that is much higher than biomedicine.
Single-Use Bioreactors
Single-use bioreactors are cheaper at smaller scales and allow for fast product changeover times. They have the advantage of not requiring heated sterilization, which saves scientists from worrying about time, cross-contamination, water, energy, and sensor costs.
Single-use bioreactors may be a favorable option when considering scale-out methods of production, but they are not economically incentivized because of a batch of cultivated meat products has a lower value. Purchasing a new single-use bioreactor for each batch of meat would become incredibly expensive.
Hollow fiber reactors
A type of single-use bioreactor, hollow fiber reactors, allow cell growth on the outer surface of microfibres or are suspended in the space between them, while nutrients diffuse to the cells from the fiber lumen, which reduces shear stresses.
Hollow fiber bioreactors also provide a tremendous amount of surface area in a small volume. Cells grow on and around the cartridge fibers at densities of greater than 1 x 108 per mL.
The hollow fibers inside the cartridge are small tube-like filters approximately 200 microns in diameter. These fibers are sealed into the cartridge shell so that cell culture medium pumped through the end of the cartridge will flow through the inside of the fiber while the cells are grown on the outside of the fiber.
Nutrients are delivered from the bottom layer of cells to the top, which allows cultures to be maintained for many months of continuous production.
Airlift Bioreactors
Air-lift reactors also permit suspension growth and become advantageous at very large scales (>20,000L), as their mixing does not involve moving parts.
They offer a few benefits, namely inhomogeneity, less shear stress, fewer nutrient or oxygen gradients, and lower power requirements to perform the mixing.
Fun fact: the largest bioreactor ever built was an airlift reactor that held 1,500,000L for microbial cell growth. This airlift reactor design was recently scaled down to 300,000L. We can theoretically feed 75,000 people annually with one airlift reactor!
Scale-out vs. scale-up
To address all the challenges in scaling up cultured meat production, scaling out approach may yield a faster route to getting lab-grown meat to consumers. In this case, production would take place at smaller scales so that meat production requirements are more similar to a restaurant or butcher shop rather than a farm or a large slaughterhouse.
These scales have been estimated to require more manageable bioreactor sizes of 100–1000L volumes to reach local demand. This will make it much simpler to design a facility, and will demand lower energy, land, and capital expenditure. Scaling out may be one way we can buy time and money before large scale-up operations are explored.
Some companies are even working on developing in-home kits because these strategies may give a better short-term path to commercialized cultured meat products. However, production volumes at these scales will likely fail to meet the large demands for global meat consumption so co-existing systems of local scale-out and large scale-up approaches may therefore be required to meet these demands over time.
Automation
Automation can facilitate the prevention of contamination and help with regulatory barriers.
Fun fact: automation is just starting to gain traction in the cell therapy and regenerative medicine fields, which uses similar processes as cultured meat.
Cultivated meat manufacturing fundamentally involves orders of magnitude higher numbers of cells and thus may require significant engineering customization, especially in regards to cell scaling, quality control, harvesting, and product formulation.
Automation can help bring down the production costs of these processes. If a large effort has been dedicated to developing preventative controls and contamination monitoring methods, it will decrease production time, cost of decontamination, and ensure adherence to regulatory and safety guidelines
Sterilization
The most common forms of contamination in lab-grown meat are from adventitious agents such as bacteria, fungi, viruses, or cell cross-contamination.
Different components of the bioreactor are susceptible to entry of various contaminants and need methods to maintain sterility. Various membranes or filters can be used to capture potential contaminant in gas or fluid tubes. Gas filters are commonly made of hydrophobic membranes such as PTFE to prevent aqueous aerosols from entering our cultures.
Pulsed electric fields, which cause cellular permeabilization, may be adapted for the prevention of bacteria and bacterial spore contamination of cell culture media. Additionally, high-temperature short-time sterilization also known as flash pasteurization can be used to inactivate viruses but similarly poses some threat to heat-sensitive ingredients.
In stirred tank reactors, thermal sterilization via steam is the most commonly used method. It is often performed after cleaning of vessels or process components (equipment is washed with high-pressure water jets, rinsed in alkaline and acidic solutions, and dried). Steam sterilization can be performed directly at inlets and outlets, in an empty vessel, on medium within a vessel, or on medium flowing through a continuous process line.
Steam sterilization is dependent on many variables including time, temperature, moisture, direct steam contact, air removal, and drying. High temperatures are usually required (≥121ºC). If contamination is detected, an investigation into the source is performed (faulty filters, micro-cracks, improper sealing) and components are cleaned and re-sterilized.
Conclusion
The main issues in our food systems can be solved with the help of cellular agriculture, but the production of lab-grown meat has to be optimized before this can happen. Bioreactors need to be scaled up to hold more volumes of cell cultures in order for cultured meat to reach the international market.
In the future, with the collaboration of talented engineers, scientists, and business leaders, the problems in our agricultural industry can be disrupted. We can change the world — one cell at a time.
Key takeaways
- The agricultural industry is extremely problematic. 70% of global freshwater and 50% of the world’s habitable land is used for agriculture. Globally, agricultural-related activities contribute to 24% of the world’s greenhouse gases.
- Bioreactors must be scaled up in order to meet global demands for meat. This will require them to hold more volumes of cell cultures, while still meeting safety regulations.
- Different types of bioreactors have their own challenges and benefits. The current status quo of the industry relies on stirred tank reactors that have a maximum volume production 20,000 L.
- Addressing the challenges for cellular agriculture will require an interdisciplinary collaboration in tissue engineering, bioprocessing engineering, and chemical engineering
- Scalability of every step of the design process needs to be considered. Bioreactors must work in contingency with cell lines, scaffolds, and culture media to ensure efficiency on all scales.
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 who’s super interested in the future of biotech. I spend my time reading up on emerging technologies and training 10X mindsets.
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