Development of BIOnanotechnologies based on Extracellular Vesicles, applied in early diagnostic, prognosis and therapy of atherosclerotic disease (BIOVEA) – 83PCCDI/2018
Duration: 31 March 2018 – 31 December 2020
Funding: 5.287.500 RON
Coordinator: Institute of Biology and Cellular Pathology „Nicolae Simionescu“ Bucharest
National Institute of Research-Development in Pathology and Biomedical Sciences „Victor Babeş” Bucharest
Carol Davila” University of Medicine and Pharmacy Bucharest
Clinical County Emergency Hospital „Pius Brînzeu” Timișoara – OncoGen Centre for Cellular and Gene Therapies
Specific objectives: (1) identification of extracellular vesicles (VEC) as specific biomarkers for atherosclerotic disease; (2) development of early diagnostic and prognosis VEC-based technology; (3)identification of an optimal source for VEC generation; (4) development of VEC-based therapeutic nanotechnologies in atherosclerotic disease.
Implementing these new investigation models (in vitro and in vivo), methods and transferrable technologies into clinical practice, for diagnostic and treatment purposes, will directly impact the health state of population.
The project plans isolation of mesenchymal stem cells from bone marrow (MSCs) and adipose tissue (ADSCs) of healthy hamsters. Harvesting of biological samples will be performed under general anesthesia of experiment animal, followed by adherence procedures to the culture flasks of cells from the 2 selected sources. Characterization of stem cells will include: optical microscopy (morphologic aspects), flow cytometry (stem cells specific markers: CD90, CD73, CD29, and CD105), RT-PCR for genes of interest, assessment of plasticity – differentiation towards adipocytes, osteoblasts and chondrocytes; cytokines profile will be generated by ELISA method, while the adherence ability to different cytokine and chemokine substrates will be tested by flow chamber. In vitro generation of VEC implies successive centrifugations and ultra-centrifugations of cell culture supernatant. In the last phase of this project the quantitative and qualitative assessment of VEC will be performed using flow cytometry procedures (based on surface markers – CD9, CD63, CD81), RT-PCR, and electron microscopy. MSC and ADSC-derived VEC will be compared and classified based on markers expression. VEC effect will be tested in vitro on other types of murine cells (endothelial cells), quantified through proliferation (xCELLigence) and viability assays. The final result will be a panel of surface markers, collections of MSCs and ADSCs morphological and functional characteristics, as well as VEC obtained from these cellular types. This will lead to the identification of the optimal source of cells generating VEC further to be used for their therapeutic potential in coronary atherosclerotic disease.
Researchers at OncoGen are involved in establishing a cellular source for in vitro generation of extracellular vesicles, wihth a therapeutic potential in the coronary atherosclerotic disease.