HUMAN PRACTICES
Proof-of-concept
In the natural state, animal muscle cells grow by attachment and are embedded in the corresponding tissues. To simulate the in vivo environment, muscle cell culture in vitro needs to use an appropriate scaffold system for adhesion support growth, assist in forming cell tissue texture and microstructure, and maintain the three-dimensional structure of muscle tissue[1]. The most ideal scaffold system should have a relatively large specific surface area for cell attachment and growth, which can flexibly shrink and expand, simulate cell adhesion in vivo environment and other factors, and be easily separated from the cultured tissue[2].Therefore, the development of degradable scaffolds as part of muscle tissue is currently the focus of the development of cell cultured meat projects, and the materials used to construct scaffolds have also become the focus of our attention. Inspired by the fat components in traditional cultured meat, we turned our attention to fatty acids, and finally, we focused on PHFA after screening[3]. We have designed a non-natural pathway for PHFA biosynthesis and successfully constructed a system that can complete the polymerization of hydroxyl fatty acids. By optimizing the enzyme activities in the metabolic pathway, we have been able to obtain a certain yield of PHFA products. It can provide raw materials with excellent mechanical properties, safety, non-toxic, and high biocompatibility for the subsequent production of a microsphere scaffold system.
In the microsphere scaffold preparation stage: we have mastered the mature microsphere scaffold preparation technology and developed porous, and large specific surface microsphere scaffold system by adjusting the microsphere state most suitable for cell growth through the rotation speed, PHFA concentration, and ammonium bicarbonate concentration. Not only that, the diameter of the microspheres is small and the specific surface is large, which can be effective for material transportation. Carrying on surface modification, such as adding collagen and RGD can improve the affinity of biological cells, cell tissue surface adhesion rate, and structure stability, conducive to accelerating the growth rate of cells, It has substantially solved the problems of recycling difficulty, high cost, and insufficient stability of 3D printing scaffolds last year. Moreover, the large-scale production of microspheres is easy, which enables us to reduce the cost of large-scale tissue culture of artificial meat, and further provides powerful technical support for the industrial production of cell cultured meat.
In the cell culture stage: Firstly, we verified the non-toxicity of the material. We measured the cell proliferation on the surface of the microsphere by the CCK-8 method, so as to judge the activity of the microsphere material by whether there are viable cells and the number of viable cells. We successfully observed that the cell proliferation on the surface of the microspheres was good, which provided a strong demonstration for the edible of the subsequent artificial meat scaffolds from a certain level. In the future, we hope that the microsphere-cell system will have a richer taste and a more meat-like texture. The quality of taste depends on the differentiation ability of the cells. So we observed and recorded the cell system continuously. Then we found that myogenic cells differentiated significantly in the laboratory culture stage. It indicates that the microsphere-cell system has a good differentiation capacity and has the potential to reach the differentiation goal of the finished artificial meat eventually. We constructed and cultured the entire microsphere-cell system using various innovative approaches such as GRGDSP modified microspheres. In order to test the effectiveness of our innovative culture, we use cytological experimental indicators such as live-dead cell staining for evaluation. The evaluation process also argued the excellent nature of our constructed cell-microsphere system and the rationality of the culture method from a certain perspective. Such advantages enable us to achieve better cell adhesion, growth, and value addition on the microsphere surface. Eventually, our team laid the foundation for further industrialization by cultured meat crude products.