Preparation of Extracellular Matrix Paper and Construction of Multi-Layered 3D Tissue Model

Construction of organized three-dimensional (3D) tissue with extracellular matrix (ECM) and multiple types of cells is important for tissue engineering to enable tissue function and enhance cellular function. However, the concentration of ECM and the thickness of the 3D tissue have been limited in previous methods due to a lack of permeability to nutrients and oxygen. Besides, it is difficult to use matured natural ECM as a cell scaffold without chemical modification due to its insolubility. In this article, we focus on multi-layered structure, which is commonly found in living tissue such as skin, blood vessels, and other organs. Here, we describe the preparation of a paper-like scaffold (ECM paper) from micro-fibered natural ECM and the construction of 3D multi-layered tissue composed of cell layers and ECM layers by stacking cell-seeded ECM papers. The thickness and components of the ECM layers are easily controllable by changing the composition of the ECM papers, and the fibrous structure of ECM paper shows high permeability and permits cell migration. Additionally, the ECM microfiber, which is physically defiberized from natural ECM, has a high ECM concentration equal to that of living tissue and high stability under physiological conditions. Therefore, this set of protocols enables construction of multi-layered 3D tissue composed of precisely controlled ECM layers and cell layers that may contribute to the assembly of tissue models. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Preparation of extracellular matrix paper Basic Protocol 2: Evaluation of cellular function of cells on extracellular matrix paper Basic Protocol 3: Construction of multi-layered 3D tissue.