A Novel Culture System Shows that Stem Cells Can be Grown in 3D and Under Physiologic Pulsatile Conditions for Tissue Engineering of Vascular Grafts

Abstract

Currently available vascular grafts have been limited by variable patency rates, material availability, and immunological rejection. The creation of a tissue-engineered vascular graft (TEVG) from autologous stem cells would potentially overcome these limitations. As a first step in creating a completely autologous TEVG, our objective was to develop a novel system for culturing undifferentiated mouse embryonic stem cells (mESC) in a three-dimensional (3D) configuration and under physiological pulsatile flow and pressure conditions. A bioreactor was created to provide pulsatile conditions to a specially modified four-well Labtek Chamber-Slide culture system. Undifferentiated mESC were either suspended in a 3D Matrigel matrix or suspended only in cell-culture media within the culture system. Pulsatile conditions were applied to the suspended cells and visualized by video microscopy. Undifferentiated mESC were successfully embedded in a 3D Matrigel matrix and could withstand physiological pulsatile conditions. Video microscopy demonstrated that the mESC in the 3D matrix were constrained to the wells of the culture system, moved in unison with the applied flows, and were not washed downstream; this was in contrast to the mESC suspended in media alone. Undifferentiated mESC can be grown in 3D and under pulsatile conditions. We will use these results to study the effects of long-term pulsatile conditions on the differentiation of mESC into endothelial cells, smooth muscle cells, and fibroblast cells with the long-term goal of creating a completely autologous TEVG.