Real Architecture for <scp>3D</scp> Tissue ( <scp>RAFT</scp> ): Mechanical Properties and Ability to Support the <scp>3D</scp> Culture of Porcine Corneal Endothelial Cells

A suitable tissue-engineered equivalent is necessary to support cultured cells for cell therapy transplantation, thereby alleviating the increasing demand for donor tissue. A compressed collagen I hydrogel, Real Architecture For 3D Tissue (RAFT), was introduced to substitute native corneal stroma for supporting endothelial cell growth as an extracellular cellular matrix (ECM) tissue equivalent. Here, the RAFT's mechanical properties and transparency were optimized to tailor for designing a corneal endothelium transplantation graft. Meanwhile, the gene and protein expression of ZO-1, Na/K ATPase, and N-Cadherin were used to investigate the impact of mechanical properties on cell behavior. The results showed that increasing the collagen concentration and reducing the initial loading volume could generate a stiffer, thinner, and more transparent RAFT. Staining results showed that porcine corneal endothelial cells (PCECs) remained alive, forming a high cell density monolayer with ZO-1 and Na/K ATPase expressions on various stiffnesses of RAFTs. Gene and protein expression results showed that PCECs could grow on various stiffnesses of RAFTs (elastic modulus ranged from 1.17 ± 0.11 to 2.08 ± 0.14 MPa), expressing ZO-1, Na/K ATPase, and N-Cadherin. In short, RAFTs fabricated with 0.4 of 5 mg/mL collagen I shared similar optical and mechanical properties to the native cornea, with a thickness of 73.67 ± 1.70 μm, a stiffness of 0.40 ± 0.03 MPa, an elastic modulus of 1.17 ± 0.07 MPa, and light transmittance of 60.71% ± 1.17%. This is an ideal tissue-engineered cell carrier suitable for developing a cell-seeded RAFT graft for cell therapy.