Engineering substantia propria (or stroma of cornea) that mimics the function and anatomy of natural tissue is vital for in vitro modelling and in vivo regeneration. There are, however, few examples of bioengineered biomimetic corneal stroma. Here we describe the construction of an orthogonally oriented 3D corneal stroma model (3D-CSM) using pure electro-compacted collagen (EC). EC films comprise aligned collagen fibrils and support primary human corneal stromal cells (hCSCs). Cell-laden constructs are analogous to the anatomical structure of native human cornea. The hCSCs are guided by the topographical cues provided by the aligned collagen fibrils of the EC films. Importantly,…