Growth Factor-Free Pre-vascularization of Cell Sheets for Tissue Engineering.

The therapeutic efficacy of tissue-engineered constructs is often compromised by inadequate inosculation and neo-vascularization. This problem is considered one of the biggest hurdles in the field and finding a solution is currently the focus of a great fraction of the research community. Many of the methodologies designed to address this issue propose the use of endothelial cells and angiogenic growth factors, or combinations of both, to accelerate neo-vascularization after transplantation. However, an adequate solution is still elusive. In this context, we describe a methodology that combines the use of the stromal vascular fraction (SVF) isolated from adipose tissue with low oxygen culture to produce pre-vascularized cell sheets as angiogenic tools for Tissue Engineering. The herein proposed approach takes advantage of the SVF angiogenic nature conferred by adipose stem cells, endothelial progenitors, endothelial and hematopoietic cells, and pericytes and further potentiates it using low oxygen, or hypoxic, culture. Freshly isolated nucleated SVF cells are cultured in hyperconfluent conditions under hypoxia (pO2 = 5 %) for up to 5 days in medium without extrinsic growth factors enabling the generation of contiguous sheets as described by the cell sheet engineering technique. Flow cytometry and immunocytochemistry allow confirming the phenotype of the different cell types composing the cell-sheets as well the organization of the CD31(+) cells in branched and highly complex tube-like structures. Overall, a simple and flexible approach to promote growth factor-free pre-vascularization of cell sheets for tissue engineering (TE) applications is described.