Optimization of Culture Media and Cell Ratios for 3D In Vitro Skeletal Muscle Tissues with Endothelial Cells.

A major challenge of engineering larger macroscale tissues in vitro is the limited diffusion of nutrients and oxygen to the interior. For skeletal muscle, this limitation results in millimeter scale outcomes to avoid necrosis. One method to address this constraint may be to vascularize in vitro-grown muscle tissue, to support nutrient (culture media) flow into the interior of the structure. In this exploratory study, we examine culture conditions that enable myogenic development and endothelial cell survival within tissue engineered 3D muscles. Myoblasts (C2C12s), endothelial cells (HUVECs), and endothelial support cells (C3H 10T1/2s) were seeded into Matrigel-fibrin hydrogels and cast into 3D printed frames to form 3D in vitro skeletal muscle tissues. Our preliminary results suggest that the simultaneous optimization of culture media formulation and cell concentrations is necessary for 3D cultured muscles to exhibit robust myosin heavy chain expression and GFP expression from GFP-transfected endothelial cells. The ability to form differentiated 3D muscles containing endothelial cells is a key step toward achieving vascularized 3D muscle tissues, which have potential use as tissue for implantation in a medical setting, as well as for future foods such as cultivated meats.

Aggregating in vitro-grown adipocytes to produce macroscale cell-cultured fat tissue with tunable lipid compositions for food applications.

We present a method of producing bulk cell-cultured fat tissue for food applications. Mass transport limitations (nutrients, oxygen, waste diffusion) of macroscale 3D tissue culture are circumvented by initially culturing murine or porcine adipocytes in 2D, after which bulk fat tissue is produced by mechanically harvesting and aggregating the lipid-filled adipocytes into 3D constructs using alginate or transglutaminase binders. The 3D fat tissues were visually similar to fat tissue harvested from animals, with matching textures based on uniaxial compression tests. The mechanical properties of cultured fat tissues were based on binder choice and concentration, and changes in the fatty acid compositions of cellular triacylglyceride and phospholipids were observed after lipid supplementation (soybean oil) during in vitro culture. This approach of aggregating individual adipocytes into a bulk 3D tissue provides a scalable and versatile strategy to produce cultured fat tissue for food-related applications, thereby addressing a key obstacle in cultivated meat production.

Perspectives on scaling production of adipose tissue for food applications.

With rising global demand for food proteins and significant environmental impact associated with conventional animal agriculture, it is important to develop sustainable alternatives to supplement existing meat production. Since fat is an important contributor to meat flavor, recapitulating this component in meat alternatives such as plant based and cell cultured meats is important. Here, we discuss the topic of cell cultured or tissue engineered fat, growing adipocytes in vitro that could imbue meat alternatives with the complex flavor and aromas of animal meat. We outline potential paths for the large scale production of in vitro cultured fat, including adipogenic precursors during cell proliferation, methods to adipogenically differentiate cells at scale, as well as strategies for converting differentiated adipocytes into 3D cultured fat tissues. We showcase the maturation of knowledge and technology behind cell sourcing and scaled proliferation, while also highlighting that adipogenic differentiation and 3D adipose tissue formation at scale need further research. We also provide some potential solutions for achieving adipose cell differentiation and tissue formation at scale based on contemporary research and the state of the field.