Improving reliability of preclinical drug/toxicant screening assays is urgently needed. Nowadays, standard tests are based on cell monolayer (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 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-MSC) demonstrate excellent potential. The aim of this work is to create a 3D organotypic tissue in order to perform an in vitro test to screen potential toxic/drug molecules, employing a continuous, stable, non tumorigenic rat DP-MSC, 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’. Appropriate protocols to induce specific differentiation were performed in accord with literature. Results demonstrated that MUR are able to create 3D 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. 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.
Developing an Organotypic in vitro 3D Toxicological Test by a Continuous, Stable, non-Tumourogeic Mesenchymal Progenitors Cell Line Derived from the Rat Dental Pulp
BERTA, Giovanni Nicolao;DI SCIPIO, FEDERICA;SPRIO, ANDREA ELIO;SALAMONE, PAOLINA;
2010-01-01
Abstract
Improving reliability of preclinical drug/toxicant screening assays is urgently needed. Nowadays, standard tests are based on cell monolayer (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 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-MSC) demonstrate excellent potential. The aim of this work is to create a 3D organotypic tissue in order to perform an in vitro test to screen potential toxic/drug molecules, employing a continuous, stable, non tumorigenic rat DP-MSC, 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’. Appropriate protocols to induce specific differentiation were performed in accord with literature. Results demonstrated that MUR are able to create 3D 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. 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.
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