Bacterial Decellularization: Non-Chemical Production of Effective Plant Tissue Bio-Scaffolds

Bio-scaffolds for the cellular agriculture field require to be simple, with low associated costs. Here, a method is described to generate decellularized leaf scaffolds utilizing a simple bacterial process, yielding scaffolds with the capacity to sustain myoblast attachment, growth, and differentiation. To best develop a minimal cost decellularization process, we aimed to design the key steps of the method to be as "low-tech" as possible and not use chemical or thermal processing to produce the cellular scaffold. Decellularized leaves (DCL) from the black walnut tree (Juglans nigra) were successfully produced employing a domestic fish tank with a biological filtration system that supports an active aquatic nitrogen-fixing bacterial population, typically within 3-5 days. Following decellularization, the DCL were devoid of any pulp material as confirmed by scanning electron microscopy (SEM). DCL produced in this way are an effective cellular scaffold, and the C2C12 myoblast cell line was shown to attach, proliferate and differentiate on DCL and maintain viability up to 3 weeks post-seeding. Differentiated cellular material grew extensively over the DCL veins and larger differentiated cellular structures extended between individual DCL veins. The data presented provide a proof of concept for an inexpensive, simple, and chemical-free method for leaf decellularization, which supports myoblast attachment, growth, and differentiation. The technology provides clear applications for the cellular agriculture field where cost reduction, scalability, and simplification of established laboratory processes, such as bio scaffold production, is a key factor.