Importance of endogenous extracellular matrix in biomechanical properties of human skin model.

The physical and mechanical properties of cells modulate their behavior such proliferation rate, migration and extracellular matrix remodeling. In order to study cell behavior in a tissue-like environment in vitro, it is of utmost importance to develop biologically and physically relevant 3D cell models. Here, we characterized the physical properties of a single cell type growing in configurations of increasing complexity. From one human skin biopsy, primary dermal fibroblasts were isolated and seeded to give monolayer (2D model), spheroid (3D model poor in extracellular matrix) and tissue-engineered cell sheet (3D model rich in endogenous extracellular matrix). Living native human dermis tissue was used as a gold standard. Nanomechanical and viscoelastic properties at the cell scale were measured by atomic force microscopy (AFM) while biphoton microscopy allowed collagen detection by second harmonic generation and scanning electron microscopy helped in model morphological characterization. In all models, fibroblasts presented a similar typical elongated cell shape, with a cytoskeleton well-arranged along the long axis of the cell. However, elastic moduli of the tissue-engineered cell sheet and native dermis tissue were similar and statistically lower than monolayer and spheroid models. We successfully carried out AFM force measurements on 3D models such as spheroids and tissue-engineered cell sheets, as well as on living native human tissue. We demonstrated that a tissue-engineered dermal model recapitulates the mechanical properties of human native dermal tissue unlike the classically used monolayer and spheroid models. Furthermore, we give statistical evidence to indicate a correlation between cell mechanical properties and the presence of collagens in the models studied.

Gene Electrotransfer in 3D Reconstructed Human Dermal Tissue.

Gene electrotransfer into the skin is of particular interest for the development of medical applications including DNA vaccination, cancer treatment, wound healing or treatment of local skin disorders. However, such clinical applications are currently limited due to poor understanding of the mechanisms governing DNA electrotransfer within human tissue. Nowadays, most studies are carried out in rodent models but rodent skin varies from human skin in terms of cell composition and architecture. We used a tissue-engineering approach to study gene electrotransfer mechanisms in a human tissue context. Primary human dermal fibroblasts were cultured according to the self-assembly method to produce 3D reconstructed human dermal tissue. In this study, we showed that cells of the reconstructed cutaneous tissue were efficiently electropermeabilized by applying millisecond electric pulses, without affecting their viability. A reporter gene was successfully electrotransferred into this human tissue and gene expression was detected for up to 48h. Interestingly, the transfected cells were solely located on the upper surface of the tissue, where they were in close contact with plasmid DNA solution. Furthermore, we report evidences that electrotransfection success depends on plasmid mobility within tissue- rich in collagens, but not on cell proliferation status. In conclusion, in addition to proposing a reliable alternative to animal experiments, tissue engineering produces valid biological tool for the in vitro study of gene electrotransfer mechanisms in human tissue.

Calcium Electroporation: Evidence for Differential Effects in Normal and Malignant Cell Lines, Evaluated in a 3D Spheroid Model.

BACKGROUND: Calcium electroporation describes the use of high voltage electric pulses to introduce supraphysiological calcium concentrations into cells. This promising method is currently in clinical trial as an anti-cancer treatment. One very important issue is the relation between tumor cell kill efficacy-and normal cell sensitivity. METHODS: Using a 3D spheroid cell culture model we have tested the effect of calcium electroporation and electrochemotherapy using bleomycin on three different human cancer cell lines: a colorectal adenocarcinoma (HT29), a bladder transitional cell carcinoma (SW780), and a breast adenocarcinoma (MDA-MB231), as well as on primary normal human dermal fibroblasts (HDF-n). RESULTS: The results showed a clear reduction in spheroid size in all three cancer cell spheroids three days after treatment with respectively calcium electroporation (p<0.0001) or electrochemotherapy using bleomycin (p<0.0001). Strikingly, the size of normal fibroblast spheroids was neither affected after calcium electroporation nor electrochemotherapy using bleomycin, indicating that calcium electroporation, like electrochemotherapy, will have limited adverse effects on the surrounding normal tissue when treating with calcium electroporation. The intracellular ATP level, which has previously been shown to be depleted after calcium electroporation, was measured in the spheroids after treatment. The results showed a dramatic decrease in the intracellular ATP level (p<0.01) in all four spheroid types-malignant as well as normal. CONCLUSION: In conclusion, calcium electroporation seems to be more effective in inducing cell death in cancer cell spheroids than in a normal fibroblast spheroid, even though intracellular ATP level is depleted in all spheroid types after treatment. These results may indicate an important therapeutic window for this therapy; although further studies are needed in vivo and in patients to investigate the effect of calcium electroporation on surrounding normal tissue when treating tumors.

Efficient In Vitro Electropermeabilization of Reconstructed Human Dermal Tissue.

DNA electrotransfer is a successful technic for gene delivery. However, its use in clinical applications is limited since little is known about the mechanisms governing DNA electrotransfer in the complex environment occurring in a tissue. The objectives of this work were to investigate the role of the extracellular matrix (ECM) in that process. Tumor ECM composition was shown to modulate in vivo gene electrotransfer efficiency. In order to assess the effects of ECM composition and organization, as well as intercellular junctions and communication, in normal tissue response to electric pulses, we developed an innovative three-dimensional (3D) reconstructed human connective tissue model. 3D human dermal tissue was reconstructed in vitro by a tissue engineering approach and was representative of in vivo cell organization since cell-cell contacts were present as well as complex ECM. This human cell model presented multiple layers of primary dermal fibroblasts embedded in a native, collagen-rich ECM. This dermal tissue could become a useful tool to study skin DNA electrotransfer mechanisms. As proof of the concept, we show here that the cells within this standardized 3D tissue can be efficiently electropermeabilized by milliseconds electric pulses. We believe that a better comprehension of gene electrotransfer in such a model tissue would help improve electrogene therapy approaches such as the systemic delivery of therapeutic proteins and DNA vaccination.