PROGRAMMA FINALE - ABSTRACTS ONLINE

ABSTRACT

Title
3D-cell-sheets from a continuous, stable, non-tumorigenic, mesenchymal stem cell line derived from rat dental pulp: an innovative tool for toxicological tests.
 
Authors
F. Di Scipio

Doctoral School in Experimental Medicine and Therapy, Experimental and Clinical Pharmacology and Therapy - Cycle XXIV.
 
Department of Clinical and Biological Sciences, University of Turin.
 
Abstract
Improving reliability of preclinical drug/toxicant screening assays is really needed. Nowadays, standard tests are based on cell monolayer, two dimensional (2D)-cell-culture models lacking cell-cell and cell-matrix interactions, and animal models (expensive, ethically controversial and sometimes not predictive of human response). Innovative three dimensional (3D)-cell-based models are coming thanks to the rapid progress in tissue engineering.Unfortunately, the ability to produce bioengineered tissues is function of maintaining differentiated cells in culture for appropriate time.Stem cells obtained from different adult tissues may be able to overcome this problem. In our knowledge,dental pulp mesenchymal stem cells (DP-MSCs) demonstrate excellent potential. The aim of this work was to create a 3D organotypic tissue in order to provide an in vitro test to screen potential toxic/drug molecules, employing a continuous, stable, non tumorigenic rat DP-MSCs, named MUR. For this purpose, MUR were grown until confluence onto thermo-responsive surfaces obtained by poly-N-isopropyl-acrylamide immobilization. MUR-derived monolayer could be easily obtained without using proteolytic digestion by lowering temperature below 20°C for 30’. Multiple cell sheets could be co-layered to fabricate a 3D structure. Appropriate protocols to induce specific differentiations were performed in accord with literature. Results demonstrated that MUR were able to create cell sheets after 4 days of culture. The phenotype assessed by means of immunohistochemistry showed that stem cell features were preserved within the cell sheet, with a positivity for Nkx-2.5, c-kit, c-myc, a-SMA, type-I collagen, CX43 and GATA-4. MUR-derived sheets were able to differentiate to osteoblasts and adipocytes when appropriately stimulated.To validate the use of our method as toxicological test, different drugs with proven side effects on bone and adipose tissue have been employed on differentiated cell sheets. Although cell sheets do not represent normal architecture for many mammalian tissues, our 3D approach should contribute to more easily identify new toxics/drugs and could improve the success rate at early stages of the drug-discovery process.