PhD Scientific Days 2023

Budapest, 22-23 June 2023

Molecular Sciences - Posters K

Microplate-based in vitro assays to investigate the main functions of fibroblasts

Apor Veres-Székely 1,2, Domonkos Pap 1,2, Beáta Szebeni 1,2, Csenge Szász 1, Péter Bokrossy 1, Eszer Lévai1, Attila J. Szabó 1,2, and Ádám Vannay 1,2
1 Pediatric Center, MTA Center of Excellence, Semmelweis University, Budapest
2 ELKH-SE, Pediatrics and Nephrology Research Group, Budapest

Text of the abstract

Introduction: Fibroblasts play a central role in fibroproliferative diseases associated with excessive deposition of extracellular matrix (ECM). Investigation of different properties of fibroblasts, including their migration, proliferation and ECM production is unavoidable both in basic research and in preclinical drug development.
Aims: In the present study we aimed to summarise microplate-based in vitro assays developed or optimized by our research group to examine the main fibroblast functions.
Methods: We established a new Transient Agarose Spot (TAS) assay to investigate cell migration. This method is based on the transitional exclusion of the cells by agarose droplets, placed in the middle of cell-culture plate wells. The dynamic of gap closure, detected by microscopy and graphical analysis indicates the migration capacity of the examined cells. The fibroblast proliferation can be determined by a colorimetric assay using thiazolyl blue tetrazolium bromide dye (MTT), staining viable cells attached to the bottom of cell culture plate. The extent of cell death can be estimated based on the lactate dehydrogenase (LDH) enzyme activity of the cell supernatant. ECM production of fibroblasts can be determined by SiriusRed. Currently we are working on a complex method where the de novo synthetized, immature and mature collagens can be quantified parallelly.
Results, conclusion: During the last few years we optimized several methods to investigate the migration, proliferation and ECM production of fibroblasts. These functional assays are useful tools to reveal the unknown effect of various cytokines (basic research), to screen the efficacy of antifibrotic drug candidates (drug development) or to examine therapeutic success on individual cell cultures (translational research).
Fundings: K-142728, ELKH-POC-2022-024, STIA-KFI-2021, TKP2021-EGA-24, ÚNKP-22-4-II-SE-12, ÚNKP-22-5-SE-17, János Bolyai Research Scholarship, Faculty Excellence Merit Award.