Optimization of in vitro bioassays to investigate fibroblast functions
Szász Csenge
KÁROLY RÁCZ CONSERVATIVE MEDICINE PROGRAM
Dr. Fekete Andrea
Semmelweis Egyetem I. sz. Gyermekgyógyászati Klinika Koós Aurél terem
2026-06-15 14:00:00
Gyermekkori betegségek klinikuma, élettana és prevenciója
Dr. Szabó Attila
Dr. Veres-Székely Apor
Dr. Cseprekál Orsolya
Dr. Pongrácz Judit Erzsébet
Dr. Liliom Károly
Dr. Cserhalmi Marcell
Dr. Varga Ágnes
Fibrotic processes become activated in several acute tissue injuries and chronic diseases, resulting in organ failure over time and contributing significantly to death cases worldwide. Similarly, cancer is another leading cause of mortality in most developed regions. Moreover, fibroblast and cancer cells share similar functional characteristics of proliferation, collective migration and production of extracellular matrix elements. In order to alleviate the medical burden imposed by these diseases, we aimed to develop an efficient screening system for the investigation of new therapeutic options.
Following induction with different growth factors playing central role in fibrosis and cancer progression, the effect of kinase inhibitors on proliferation, a crucial phenomenon in the course of these diseases was examined with the well-known enzyme activity-based MTT assay with optimized conditions for our experimental setup. To distinguish between proliferation inhibition and cytotoxicity, it was paired with LDH test.
To overcome the disadvantages of the current gold standard assay for collective cell migration, the scratch assay, we developed a novel migration test called TAS assay. Instead of creating gap in an already formed monolayer of cells, TAS assay applies agarose spot as a removable physical barrier prior to cell seeding to avoid the modifying effect of physical damage on cell migration and ensure a uniform, small, easily traceable initial gap, resulting in a cost-effective, precise migration assay. The implementation of cell staining even allowed the transfer of the detection and evaluation into a microplate reader, resulting in a highly automated test and a significant shortening of assay time.
Finally, to address the challenges of modelling and measuring collagen production in vitro, we established a Sirius Red collagen stain-based assay. Since the non-polymerized soluble collagens in the supernatant represent an important fraction of total collagens and are usually discarded, the aim was to optimize the staining procedure for their labelling as well. Furthermore, the cell culture conditions were also adjusted to minimize the effect of interfering factors and achieve maximal collagen production.
The resulting near-high throughput in vitro screening system consists of cost-effective, simple, relatively fast assays that enable the simultaneous measurement of the main fibroblast and cancer cell functions to obtain comprehensive understanding about the effect of the tested compounds.
