Engineering Murine Adipocytes and Skeletal Muscle Cells in Meat-like Constructs Using Self-Assembled Layer-by-Layer Biofabrication: A Platform for Development of Cultivated Meat

Abstract

Global meat consumption has been growing on a per capita basis over the past 20 years resulting in ever-increasing devotion of resources in the form of arable land and potable water to animal husbandry which is unsustainable and inefficient. One approach to meet this insatiable demand is to use biofabrication methods used in tissue engineering in order to make skeletal muscle tissue-like constructs known as cultivated meat to be used as a food source. Here, we demonstrate the use of a scaffold-free biofabrication method that forms cell sheets composed of murine adipocytes and skeletal muscle cells and assembles these sheets in parallel to create a 3D meat-like construct without the use of any exogenous materials. This layer-by-layer self-assembly and stacking process is fast (4 days of culture to form sheets and few hours for assembly) and scalable (stable sheets with diameters >3 cm are formed). Tissues formed with only muscle cells were equivalent to lean meat with comparable protein and fat contents (lean beef had 1.5 and 0.9 times protein and fat, respectively, as our constructs) and incorporating adipocyte cells in different ratios to myoblasts and/or treatment with different media cocktails resulted in a 5% (low fat meat) to 35% (high fat meat) increase in the fat content. Not only such constructs can be used as cultivated meat, they can also be used as skeletal muscle models.

Scaffolding Biomaterials for 3D Cultivated Meat: Prospects and Challenges

“…[120,188] However, developing novel bioreactors and automated assembly methods will be necessary for this technique to apply to scaled CM production. [120] For all of these approaches, it will be critical for bioreactor designs to simultaneously take into account cost considerations and the necessity of reliably maintaining sterile operating conditions. [419] Methods for producing muscle fiber alignment and spatial heterogeneity, including acoustic cell patterning, bioprinting, lithography, embossing, micromolding, and incorporation of growth factors into scaffolds in defined patterns, require further exploration.…”

“…Furthermore, the use of 2D cell sheets is an interesting concept with the advantage that no exogenous scaffold material is introduced into the construct. [ 120 , 188 ] However, developing novel bioreactors and automated assembly methods will be necessary for this technique to apply to scaled CM production. [ 120 ] For all of these approaches, it will be critical for bioreactor designs to simultaneously take into account cost considerations and the necessity of reliably maintaining sterile operating conditions.…”

“…This method has also recently been explored as a method for cultivating meat from a combination of muscle cells and adipocytes. [ 120 ] Expected challenges when attempting to use cell sheet‐based methods for CM include the space needed to grow sufficient numbers of cells in 2D culture as well as the labor‐intensive nature of stacking multiple sheets. These challenges could potentially be addressed by novel bioreactor geometries and automated methods for tissue assembly.…”

“…Several studies have reported the successful 2D coculture of myogenic and adipogenic cells from agriculturally relevant species, [ 429 , 430 , 431 , 432 ] while others have reported small‐scale 3D cultures using human or mouse cells. [ 120 , 428 , 433 ] In addition, the company Ants Innovate is developing a scaffolding system to support the growth of marbled meat containing muscle cells and fat. They have filed a patent on their biomaterial system, [ 434 ] and the founders have recently published an article using a cellulose sponge for organoid culture.…”

Reprocessing seafood waste: challenge to develop aquatic clean meat from fish cells

“…In recent years, the technology of 3D cell culture has been developed and applied to a cell sheet 1 . Although these technologies have been applied in the medical field 2 – 4 , they are expected to be applied in other fields, such as the food industry 5 , 6 . Our aim is to explore applying this technology to produce marine food resources.…”