206 Evaluation of two in vitro models as a possible platform for research on mare endometrosis

Currently, there are no trusted models to study endometrosis; thus, an in vitro system allowing evaluation of potential antifibrotic drugs is crucial. This work aimed to assess two different models, using mare endometrial fibroblasts and endometrial explants, to which a cocktail of cytokines were added to induce the fibrotic phenotype. Three endometrial fibroblast lines previously isolated and characterized in the laboratory were used. Fibroblasts were cultured until reaching 90% confluency, then transferred to an induction medium (DMEM-HG plus with 1% FBS, 1 mM sodium pyruvate, 2 mM l-glutamine, 1× AAM) for 18 h at 38°C and 5% CO₂. After this time, a fibrotic phenotype induction medium (DMEM-HG plus 1× AAM and 10 ng mL⁻¹ each of TGFβ, TNFα, IL1b β, and IL6) was incubated for 24 h at 38°C in 5% CO₂. RNA/protein was extracted to evaluate the relative expression of genes related to fibrosis (αSMA, COL1A2, COL3A1, CTGF, MMP2, MMP9, TIMP 1, and TIMP2) and αSMA protein by western blot and MMP9 by zymography. For the explant model, five uteri from the slaughterhouse were collected, and only those coming from healthy reproductive tracts were processed into pieces of approximately 30 mg and 3 mm. Fifteen explants per uteri were washed several times in 1× PBS solution supplemented with 2× AAM and placed individually, using a 12-well plate, in 1 mL of DMEM-HG supplemented with 0.1% (wt/vol) BSA, 1× AAM, for 1 h at 38°C, 5% CO₂. After 1 h, the culture medium was replaced with the fibrotic phenotype induction medium and incubated for another 24 h. After this time, RNA/protein was extracted to evaluate the relative expression of genes related to fibrosis (αSMA, COL1A2, COL3A1, CTGF, MMP2, MMP9, TIMP 1, and TIMP2) and αSMA protein by western blot and MMP9 by zymography. Uninduced fibroblasts and explants were the negative controls. Statistical analysis used unpaired t-tests, with significance defined by P < 0.05. Zymographs and western blots were analyzed using the ImageJ program. In the fibroblast model, an increase was observed in the relative expression of the genes αSMA, COL1A2, COL3A1, MMP2, and MMP9 in the induced fibroblasts (P < 0.05), and no differences were observed in CTGF expression. There was an increase in αSMA protein in the western blot level after induction. The zymography showed an increase in the secretion of MMP9 with enzymatic activity in the culture supernatant. In the explant model, there were no statistical differences in the expression of the αSMA gene compared with the control. In contrast, increases in the relative expression levels of the COL3A1, MMP2, and MMP9 genes were detected (P < 0.05). At the protein level, there was a significant increase in αSMA expression in the western blot, and the MMP9 activity was corroborated by zymography, with a slight increase in protein activity in extracts of the explants treated with the induction cocktail. The potential of the fibroblast and explants as in vitro biological models of endometriosis was tested in this study. The models were challenging and need improvement, yet they could be of use in future research for the treatment of endometrosis. Of note is that explant culture retained the three-dimensional structure of the original tissue with epithelial and stromal interaction.

Funding for this study was provided by Fondecyt 121039.