HUMAN PRACTICES
Implementation
In our project, PHFA, the primary component of the scaffolds for cell development, has vast application potential in biodegradable tissue engineering materials in addition to having a certain degree of supporting strength. PHFA was employed as the primary biological scaffold material to create growth sites for satellite cells produced from chicken muscle using the expertise of tissue engineering. To make cell scaffold materials to achieve a larger specific surface area for cell attachment growth, expansion and shrinkage of flexibly, auxiliary tissue texture and microstructure, maintain muscle tissue structure and 3D simulation cell adhesion in vivo environmental factors such as demand and achieved more than can be used in the production of edible, porosity, the large specific surface area of the microsphere material requirements, We remain focused on the production of PHFA, our scaffold material, using synthetic biology methods. Meanwhile, we also use microspheres to replace the 3D printing technology to prepare the scaffold system. In this way, the replacement scaffold system has stronger physical strength and lower potential capital investment, which can facilitate the industrial promotion of cell-cultured artificial meat.
Figure1:Images of microspheres and laboratory
Currently, many startups are still using animal-derived scaffolds such as gelatin in pilot-scale cultured meat development, but effective non-animal scaffolds that can support 3D-print meat tissue have not yet been established. Some startups claim they have developed new non-animal scaffolds for cultured meat production. For example, Matrix Meats has created electro-spun-nanofiber scaffolds that can be customized to meet the needs of different types of cultured meat development. However, limited data are available on the performance and cost-effectiveness of these scaffolds in the industrial production of cultured meat.
There is currently little scientific research on commercially available scaffolds for cultured meat production, and thus we give further exploration and optimization of methods and biomaterials for the establishment of suitable scaffolds and large-scale cultured meat production, as well as new technologies to enhance the desirable properties of scaffolds, such as porosity and ligand availability.
In adipose tissue engineering, synthetic scaffolds made from PLA, polyglycolic acid (PGA), and PLGA are commonly used, but the further surface functionalization (changes in solubility and cell adhesion sites) required for scaffolds is one of the issues that lead to expensive costs and may limit their industrial application. In muscle tissue engineering, where the most successful materials are of animal origin, natural extracellular matrices, such as collagen or fibronectin, have unique advantages as scaffolds due to their natural properties for cell attachment and growth. And because of variability, safety, and ethical issues, non-animal scaffolds for tissue engineering are being actively developed.
Instead, we developed a microbial-based platform that uses PHFA as a scaffold material. We used a whole-cell synthesis scheme for PHFA, which allows the entire process from glucose to the product polyhydroxy fatty acid ester (PHFA) to be completed within a single cell. The system provides high yield and a high degree of product polymerization, ensuring stable cellular metabolic flow and avoiding problems such as energy flow imbalance, growth arrest, and accumulation of toxic intermediates. It is suitable for continuous synthesis in industrial production.
The dynamically regulated production of PHFA material and the microsphere fabrication technique by double emulsion volatilization method possess desirable features such as low cost, scalability, and tunability. And the scaffold can be a component of food products and eventually present in cultured meat products. PHFA is now widely used in tissue engineering, and its hydrolyzed products are still food-safe and degradable materials. And the gene pathway of the desired material for cell adhesion and growth can be further fused and added to the engineered bacteria through synthetic biology technology using E. coli, allowing the production of PHFA while reducing the cost of microsphere surface modification. This technology makes it possible to satisfy muscle cell culture while utilizing fat to cross-link into the meat. While culturing meat-related cells in bioreactors, synthetic scaffolds also need to be evaluated and optimized to establish effective scaffolds for cultured meat production.
The goal of cultured meat is to create a piece of meat that is highly similar in nutrition, appearance, texture, and flavor to real muscle tissue. However, traditional cell culture methods only yield a thin layer of cells that is almost invisible. Stents originated in the field of medical tissue engineering and are used to provide an optimal microenvironment for tissue growth and regeneration. We shifted from last year's 3D printing technology to microsphere fabrication, which not only greatly reduces costs, but also enables a 3D porous network that mimics the structure and function of the extracellular matrix, which plays a role in cell adhesion and growth, further facilitating industrial production. The porous structure allows the input of gases and nutrients and the output of metabolic wastes, promoting the maintenance of cellular metabolism. It should have a large specific surface area for cell adhesion and growth, flexible contraction and relaxation properties, and good cell affinity and cytocompatibility. Future production of cultured meat may involve the co-culture of multiple cell types to form structured tissues similar to real meat. This will require new scaffolds to support the differentiation of multiple cell types and allow for the spatial heterogeneity of the final product. These scaffolds allow for precise fine-tuning of biomaterial properties such as stiffness and biochemical properties. Meanwhile, the digestibility, edibility, safety, economy, and scalability of PHFA as raw material microspheres should be available for cell culture meat.
Figure2:Polarization microscope image of microspheres
For expansion cultures, cells can be aggregated in stirred bioreactors (e.g., stirred tanks or swinging platform bioreactors [wavy bioreactors]) or grown on microcarriers. While stirred tank bioreactors (STBs) are widely used for animal cell culture in therapeutic development and can support cell proliferation in cultured meat production. STBs typically consist of a cylindrical culture vessel that is mixed by a central impeller and provides a homogeneous and well-mixed environment to provide media and oxygen for cell growth. Stirred tank bioreactors offer the advantage of long-term sterile and homogeneous conditions and a continuous supply of oxygen and nutrients, but impeller-mediated mixing can produce high shear stress on cells. Therefore, some companies are exploring rocking platform bioreactors with wave-like fluid motion to reduce shear stress.
Another type of bioreactor is the perfusion reactor, which has advantages in providing nutrients and draining metabolites. Simeonta et al. designed and developed a perfusion bioreactor using collagen sponges as a scaffold for long-term culture of adult muscle cells and achieved increased cell viability, increased cell density, and uniformity of cell distribution throughout the 3D scaffold. Common types of bioreactors that may be suitable for cultured meat production are shown in the figure.
Figure3:Common types of bioreactors for cultured meat production
Adapting bioreactors for industrial cultured meat production requires considerable optimization to produce cultured meat efficiently and at a very low expense.
In addition, the differentiation and ripening of meat products cannot simulate a 3D structure that resembles the texture, flavor, and nutritional benefits of real meat.
In last year’s design, cells are mixed with scaffold biomaterials and layered together to form meat tissue which 3D printing was used in our project. which has shown great prospects for tissue engineering and regenerative medicine applications. However, 3D printing for cultured meat development may require a simpler and less costly technique than 3D printing in tissue engineering because cultured meat does not require a vascular system as complex as natural tissue and the technique is less suited to the scaling required for cultured meat. This year, we have completed the phased success of the laboratory. In the future, for the large-scale production of cultured meat, we have chosen the co-culture technology of microsphere cells and introduced the reactor.
Microcarriers provide an adhesion surface for cell growth in suspension and are used for stem cell culture and tissue engineering. our project uses the lower-cost microsphere technology, and studies have already successfully expanded bovine myogenic cells on suspended microcarriers. Microcarriers combined with stirred tank bioreactors have also been shown to scale up human MSCs, while our microspheres have a unique porous microstructure designed to optimize gas and nutrient transport, and can also be added to the culture medium for inter-microsphere cell transfer for scale-up production.
As the volume of the stirred tank bioreactor increases, the shear stress on the cells also increases. Minimizing shear while providing uniform perfusion is critical for large-scale cell expansion. Microspheres can be used for high-density walled cell culture to minimize shear stress in large bioreactors while the cell-microsphere system tolerates external shear forces allowing for vessel flexibility. The microsphere cell system can withstand the physical strength of a mechanically stirred bioreactor, presenting great potential for industrialization.
Finally, in this area, we have also explored the use of continuous fermenter culture, and we have now achieved the goal of keeping cells alive in a bioreactor originally designed for bacteria, which namely shows potential for industrialization. The future bioprocess developed for culturing meat may be a closed, continuous, and automated system with monitoring to reduce operational costs and contamination risks.
First, the transformed E. coli is inoculated in small fermenters (small tanks) for fermentation and cultivation as a seed solution. Then it is transferred to a large fermenter (large tank) where the culture is expanded and accumulated to produce the target product PHFA. When the amount of PHFA is accumulated in sufficient quantity, the fermentation broth is transferred to the next device for cell wall fragmentation, and the cell wall and cell membrane are disrupted to some extent by physical crushing and high-pressure homogenizer to maximize the release of the intracellular product PHFA into the liquid phase.
The coating, surface modification, and size of the microspheres can be tailored to the cell and bioreactor type, making the microsphere scaffold for use in liquid phase media easier for cell carriage. We use the introduction of collagen and RGD to the scaffold surface to enhance cell attachment and mimic ECM to create a better environment for cell proliferation, differentiation, and maturation. Due to the unique strength of the microsphere scaffold, the microsphere-cell system can be placed in shake flasks together with a liquid culture medium. By regulating the concentration of serum, we are able to induce cell proliferation followed by differentiation and maturation. Cell culture is started in a bioreactor, by cell expansion, maintaining cell proliferation at exponential growth while preventing differentiation. The cells are then attached to microspheres and transferred to a perfusion bioreactor for tissue maturation as well as cross-linking. The bioreactor for cultured meat production has limited shear stress and a homogeneous environment that supports large-scale cell growth at exponential rates with low energy and consumable requirements. The bioreactor is automated through real-time monitoring to support continuous nutrient supply and waste removal, meeting the high efficiency of the bioreactor.
Even though a lot of experiments need to be conducted to optimize this system, we still collect some valuable data for supporting the better development of this field.
Figure4:Images of microspheres
Figure5:Our design of cultured meat’s industrialized production
Cultured meat as a raw material to replace meat, in the process into different products and simulating different meat tastes and seasoning are different. Under the ToC model, cultured meat brands need to deal with various pressures such as product innovation, precision marketing, and channel segmentation. In contrast, the ToB model is more suitable for our team development.
Portraits of customers to the major traditional meat products reprocessing enterprises as the target customers. Meat processing enterprises are inseparable from the daily life and dietary needs of the people. Due to the influence of traditional eating habits, sales channels, and meat sales radius, catering forms with large meat consumption, such as pigs, cattle, and sheep, are complementary to each other in seasons, and the sales of meat products will increase significantly during holidays. In addition, meat processing enterprises sell a new generation of healthy meat products whose fat composition is mainly unsaturated fatty acids for the production of downstream reprocessed meat products. Traditional meat processing enterprises take meat processing in the middle stream as the core and extend it to the upstream breeding end and the end of the downstream sales respectively. At present, our main business is mainly located in the middle and downstream of the industrial chain.
(1) Meat factory
According to the statistics of "Digital Business", the total revenue of the 13 listed meat products enterprises in 2019 was 165.481 billion yuan, compared with 134.058 billion yuan in the same period last year, an increase of 31.423 billion yuan, a year-on-year increase of 23.44 percent. The average revenue was 12.729 billion yuan.
(2) leisure food raw materials
Data from iMedia Research show that in 2021, the size of China's prefabricated wet market is 345.9 billion yuan, with a year-on-year growth of 19.8%. It is expected that China's prefabricated wet market will maintain a high growth rate in the future, and the size of the prefabricated wet market will reach 107.2 billion yuan in 2026.
(3) Catering enterprises
The cooperation between the artificial meat company and the mature restaurant chain can accelerate the polishing of the product, accelerate the research and development of the taste form of the product, and the production of industrial products. In the case of Beyond Meat, the restaurant segment (To B) had overtaken the original retail segment (ToC) in its Q2 2019 earnings report, just over two years later. The 2019 Q2 results showed that the food service business grew 486% year on year.
There is no obvious cycle phenomenon in the demand for meat products, and the total consumption and per capita consumption of meat show an increasing trend year by year. This proves that the demand gap for meat food continues to exist, the domestic market demand space is vast, and the ToB end has a large development space and a huge market scale. The company's cooperation with meat processing enterprises and large catering brands is the most likely way to achieve scale effects in a short time.
At present, our country’s cultured meat industry is in the early stage of development, the related listed company the man-made meat business has not formed a revenue scale, most of them are in the stage of the market frontier. In our country, cultured meat industry listed companies include the following several
Figure6:Summary of listed companies in China's cultured meat industry in 2021
Figure7:Basic information and revenue performance of listed companies in China's cultured meat industry in 2021 (unit: 100 million yuan)