PhD Scientific Days 2023

Molecular Sciences II.

Optimization of a microplate-based functional assay to investigate collagen production in vitro

Text of the abstract

Introduction: Increased extracellular matrix (ECM) production of fibroblasts is a key process of tissue fibrosis. The major components of ECM are fibrillar collagens, which are routinely investigated by Sirius Red staining of histological sections. Although there is a great demand both in basic research and preclinical drug development, there is no gold standard method to quantify collagen production in a near high-throughput manner in vitro.
Aims: The aim of this study was to develop and optimize a simple, cost-effective, Sirius Red-based staining method to determine the collagen production of fibroblasts in vitro.
Methods: The collagen production of NRK-49F rat kidney fibroblasts, grown on 96-well tissue culture plates was determined by Sirius Red staining. After aspiring the supernatants, cells were washed, fixed and stained under acidic conditions. The unbound dye was removed by multiple washing steps, then collagen-associated one was eluted by alkaline solution, determining thereby the intracellular and cell-attached forms of collagens. Parallelly, cell supernatants were incubated with acidic Sirius Red solution, the precipitations formed by collagen-dye complexes were centrifuged and washed, then the stain was eluted by alkaline solution. By this step, extracellular, secreted amount of collagens can be measured. The absorbance of eluted dye solutions was recorded at 540 nm with a microplate reader. During the optimization steps, we investigated the optimal dye and acid concentrations, and the effect of various amount of FBS and ascorbic acid of culture medium on the collagen production of fibroblasts stimulated with transforming growth factor ß1 (TGF-ß1).
Results, conclusion: We established a simple, cost-effective, colorimetric, microplate-based method to investigate the collagen production of fibroblasts. Our assay combines two series of staining steps in order to measure both intracellular and secreted collagens in the same samples, earning higher biological relevance. This technique could serve as a useful tool in fibrosis-related basic research and the preclinical screening of antifibrotic drugs, as well.
Fundings: K-142728; TKP2021-EGA-24, STIA-KFI-2021; ELKH-POC-2022-024; ÚNKP-22-4-II-SE-12, ÚNKP-22-5-SE-17; János Bolyai Research Scholarship; Faculty Excellence Merit Award.