Trout Kidney Extracellular Matrix as a Noncytotoxic Scaffold for Promoting Mesenchymal Stem Cell Growth and Specialization.

PURPOSE: The mesonephric kidney of fish retains its capacity for nephrogenesis and regeneration throughout the organism's lifetime. It is hypothesized that the extracellular matrix (ECM) of trout plays a pivotal role in supporting the proliferation and differentiation of resident cells. The ECM is a complex three-dimensional network of macromolecules secreted by cells during early development and is essential for regulating key cellular functions, including adhesion, migration, proliferation, differentiation, and survival. This study aimed to evaluate the effects of decellularized trout kidney ECM on the growth and differentiation of mouse renal tubular epithelial cells in vitro.

METHOD: In this study, trout kidneys were decellularized and bleached using a combination of sodium dodecyl sulfate (SDS), Triton X-100, and a bleaching buffer. The effectiveness of decellularization was evaluated using quantitative assays and histological staining. The resulting ECM was then coated onto culture dishes at concentrations of 100 and 150 μg/cm2. To assess potential cytotoxicity, an indirect extraction assay was performed. Subsequently, human bone marrow-derived mesenchymal stem cells (hMSCs) were seeded onto the decellularized ECM. Cell viability and proliferation were evaluated using the MTS assay [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] and the CFSE (carboxyfluorescein succinimidyl ester) assay, respectively, and results were compared to an uncoated control group. Finally, gene expression analysis of hMSCs was conducted using quantitative reverse transcription polymerase chain reaction (qRT-PCR).

FINDINGS: The decellularization of fish renal ECM was successfully achieved. Quantitative and qualitative analyses confirmed a significant reduction in DNA content in the decellularized tissue compared to native samples, indicating effective removal of cellular material. At the same time, key ECM components such as collagen and sulfated glycosaminoglycans (sGAGs) were preserved to a substantial extent. Specifically, collagen content decreased from 5.736 μg/mg in native tissue to 4.284 μg/mg in the decellularized ECM, while sGAG content showed a slight decrease from 0.9855 μg/mg to 0.9400 μg/mg, reflecting minimal degradation during processing. Cytotoxicity assessment revealed no toxic effects associated with the decellularized matrices. Furthermore, a significant increase in the proliferation of human mesenchymal stem cells (hMSCs) was observed on day 7 compared to the uncoated control group, suggesting the bioactivity and compatibility of the decellularized trout kidney ECM.