Abstract: A method of forming cell spheroids cultured in a serum-free manner on nanoscale coatings of hyaluronic acid with high molecular weight is disclosed. Corneal stromal cells are cultured on the hyaluronic acid coatings with nanotopography in the serum-free manner. Experimental results show that the cells cultured on the hyaluronic acid coatings (1.1-1.7 nm) increase cell-cell interaction, and when the cells form three-dimensional spheroids, they have higher biological synthetic capabilities and can secret more extracellular matrix, which has potential applications in corneal stromal tissue reconstruction.