Currently available meat products are composed of animal tissues which exist in a wide range of formats, from minced meats to highly structured full cuts. There is now considerable interest in the use of cell culture and tissue engineering in the future of food manufacturing processes, specifically in the production of meat [[1], [2], [3]]. While the emerging industry of cultivated meats continues to evolve, there is a need to create a wide range of structured meat products [4,5]. Although still emerging, the broader goal of cellular agriculture [2,3,6] is to replace products produced by traditional agricultural methods with biotechnological approaches notably through synthetic biology and tissue engineering [2,3]. One specific focal point within the field is the cultivation of mammalian cells in vitro for the preparation of meat-like products [2,7,8]. Although a number of challenges remain to achieve this goal, a significant body of work has begun to address issues such as the large scale production of relevant cell types, creating sustainable and ethically sourced media and developing suitable scaffolds [2,3,7,8]. While exciting, it is important to also recognize that the future potential of these methods are still debated as efforts to address the continued dependence on animal products in traditional cell culture (for example, fetal bovine serum), water/electricity use and any potential health benefits in the final meat products are still ongoing [9,10]. With that said, there remains an intense interest in developing solutions which address these issues, and others, potentially opening up a new future in which foods can be more sustainably created and distributed globally.
Tissue engineering and biomaterial approaches are being intensely examined as a way to utilize mammalian cells and scaffolds to re-create structured consumer ready meat products. In recent years, there has been an increase in studies on the use of plant-derived biomaterials for tissue engineering applications [4,7,[11], [12], [13], [14], [15], [16], [17], [18], [19], [20]]. For example, decellularization of plant tissues can result in cellulose-rich three dimensional (3D) scaffolds both in vitro and in vivo [4,7,[11], [12], [13], [14], [15], [16], [17], [18], [19], [20]]. At this point a multitude of plant tissues have been shown to support the growth of numerous mammalian cell types (myoblast, adipocyte, fibroblast, epithelial, osteoblast, tendon etc) which demonstrates the general utility of plant-derived biomaterials for potential biomedical and food-based tissue engineering applications [4,7,[15], [16], [17], [18], [19], [20], [21]]. A growing body of literature also suggests that plant-derived proteins can be utilized to create scaffolds for tissue engineering and are broadly compatible with mammalian cell culture. Proteins such as soy, zein and camelina, etc have been studied, but of particular interest are gluten proteins derived from wheat, such as gliadin and glutenin [[22], [23], [24]]. These wheat derived proteins can be purified and made into films suitable to culture mammalian cells. For instance, glutenin films have been demonstrated to be an acceptable substrate for osteoblasts [23]. In the same study, a gluten film was shown to support the growth of osteoblasts but with less efficiency due to the cytotoxicity of gliadin [23]. Wheat protein based scaffolds can also be obtained through electrospinning, in which ultrafine fibrous structures can be obtained, creating a polymer melt film of wheat glutenin [24]. Such scaffolds have been shown to support the culture of adipose derived mesenchymal stem cells [23]. The use of naturally derived 3D biomaterials has gained interest due to their potential for use in cellular agriculture applications and the potential for them to become a key element of future meat products [7,20,25].
Notably, bread crumbs are a common ingredient in the production of many meat products [26] and was one of the ingredients in the 2013 demonstration of a cultured beef burger [5,[27], [28], [29]]. While the crumb itself was only utilized as traditional filler and texturizer, here we studied the possibility of utilizing a bread product as a 3D scaffold for in vitro mammalian cell culture. The main ingredient of the bread scaffolds we studied was wheat flour, which is primarily composed of starch (∼70%) but its strength and stability is mainly attributed to its protein content (∼12%), represented by gluten and non-gluten (albumin and globulins) proteins [30]. As described above, such proteins have found potential utility in the creation of biomaterials [22,24]. A requirement for an optimal biomaterial is a structure with high porosity to prevent an anoxic microenvironment. The porous nature of bread results from the presence of a leavening agent, such as yeast or baking powder. The reaction between sodium bicarbonate, the active ingredient in baking powder, and water yields bicarbonate, an anion that decomposes into water and carbon dioxide at ambient temperature and is further favoured when exposed to heat. The carbon dioxide helps the bread rise in addition to leaving behind pores as it exits the crumb. The porous structure of the crumb allows for the migration of cells within the bread which consequently makes it an appealing biomaterial. Moreover, the time required to make the bread is less than an hour, which is highly efficient in comparison to other methods of creating scaffolds for tissue engineering, including plant-based scaffolds. In fact, the methods described here only require a few hours to produce scaffolds, in contrast to the extensive multi-day processes required by traditional plant tissue decellularization approaches [4,7,[11], [12], [13], [14], [15], [16], [17], [18], [19], [20]].
Our objective in this study was to demonstrate that mammalian cells could remain viable and proliferate in vitro within scaffolds made of porous bread crumb. We demonstrate that bread scaffolds remain intact over the course of up to four weeks in cell culture, can be modified to control their mechanical properties and that mammalian cells will proliferate and remain viable within the scaffolds. Importantly, the recipe we utilize relies on sodium bicarbonate, rather than yeast, to create the required porosity, thus avoiding potential contamination of the scaffold with an unwanted cell type. The data presented here supports a simple and highly accessible new method for creating cell culture scaffolding utilizing ancient approaches. We demonstrate below that several cell types can be cultured on these scaffolds and that they remain viable and do not exhibit signs of significant oxidative stress or cytotoxicity. Furthermore, we demonstrate that mouse myoblasts are also capable of differentiating and fusing into multinucleated myotubes on the scaffolds. This demonstrates the possibility of culturing skeletal tissues onto the bread crumb itself. The inherent porosity of the bread crumb along with its simple production method results in a potentially useful biomaterial that can be utilized for in vitro 3D cell culture.
Time will be required to fully elucidate how such a scaffold may be effectively scaled in an industrial cultured meat setting, however the results reveal another novel path forward towards the goal of meat production in an animal free manner. Importantly, the use of bread-derived scaffolds overcomes the processing steps required for decellularizing the plant-derived scaffolds previously described by several groups, including our own. It also overcomes the processing steps involved in purifying plant proteins for use in traditionally engineered biomaterials. The potential for the dual-use of bread crumb as both a scaffold for expanding/differentiating mammalian cells as well as a downstream filler/texturizer in meat products is also of interest. Of course, future work will have to build upon this first proof-of-concept study which establishes the use of bread as scaffolding biomaterial. Optimization of surface chemistry, physical properties, and porosity/structural control are all areas of future development. Regardless, the research presented here establishes a novel approach for developing edible biomaterials that can potentially be employed in the production of laboratory grown meat and other cellular agriculture applications.