3D Hypoxia Microenvironment Created by a Single Cell Layer-by-Layer Assembly Spheroid Demonstrates the Role of miR-382 and Cell Autophagy in Renal Fibrosis

Abstract

Compared with conventional 2D cell culture systems, 3D cell culture systems can better mimic the sophisticated internal environment. Hyaluronic acid (HA) is a fundamental element of the extracellular matrix and can be modified in many ways to alter its properties. Herein, in order to develop a 3D hypoxia culture system, we firstly synthesized HA-EDA with HA and ethylene-diamine (EDA) and then wrapped single renal mesangial cell layer-by-layer (LBL) with cationic HA-EDA and anionic HA. Unexpectedly, these wrapped cells formed a 3D hypoxia multi-cellular spheroid after being treated with trypsin. Based on the developed 3D hypoxia microenvironment, we found miR-382 might play a vital role in renal fibrosis. Amazingly, cell autophagy was detected both in 3D spheroid and 3DM cells (cells migrated from the 3D spheroid). However, different from the 3D spheroid, the 3DM cells displayed alleviated accumulation of TGF-β1 and Fn, which demonstrated the renoprotective role of autophagy in renal fibrosis. The high ratio of CD24+ cells in 3DM cells revealed that cells surviving through the autophagic process could acquire some characterization of progenitor cells via dedifferentiation. The developed 3D multi-cellular spheroid highlighted the role of hypoxia and cell autophagy in renal fibrosis.