Engineering a Protein Biomaterial: 2D and 3D Cell Culture Applications of Caf1

The environment of mammalian cells is as important in vitro as in the body, allowing their growth and adherence, and shaping their behaviour. In their natural environment, the extracellular matrix (ECM) contains all the signals, mechanical and biochemical, which the cells need in order to function correctly, and the challenge of any biomaterial is to recreate these signals effectively.

The choice of available materials is vast, but in most cases is complicated by either the complexity of conjugating bioactive signals to an inert material, or else using a highly bioactive but poorly defined material that is often derived from an animal. Protein materials have received little attention on account of their problems with low stability and a tendency to aggregate, however by using protein engineering tools we can overcome these disadvantages, and instead benefit from the flexibility that protein structure and chemistry can deliver.

In this work, we demonstrate how the bacterial protein, Caf1 - which is non-bioactive, highly stable and free from animal material – can be engineered to support the growth of various cell types, including induced pluripotent stem cells (iPSCs). We then show how Caf1 can be used in the differentiation of stem cells down both heart and bone lineages, and how it may function in a wound healing context. Finally, we describe how the innate capacity of Caf1 to refold and self-assemble following thermal denaturation allows both increased biological complexity and new material possibilities, providing an easy route for 3D cell culture.