(440e) Investigating Glyphosate Toxicity through the Development of Gut Organoids and 3D Printed Thin Films

Glyphosate (N-Phosphonomethylglycine), the most widely utilized herbicide in modern agriculture, has prompted concerns regarding its environmental impact and potential health implications to non-target organisms, particularly humans. Traditional toxicological methods provide controversial information on glyphosate toxicity and may not accurately reflect human responses, as 2D in-vitro models lack human physiological complexity. This research aims to develop a 3D micro-engineered organ equivalent (MOE) of the human small intestine (gut organoid), aiming to mimic the in-vivo environment more effectively than traditional 2D cell cultures. This gut organoid will be used to study the toxicity of glyphosate incorporated in a 3D-printed thin film matrix. CaCO2 and nHDF cells were cultured in modified Collagen I solution at different concentrations (0.5 and 2 mg/ml) and cell ratios (1:1, 1:10, and CaCO2 only, respectively) to develop gut organoids. Immunostaining analyses (IHC) were performed on Days 7 and 11 to evaluate epithelial (ZO-1) and stromal (vimentin) markers. To develop the 3D printed film, a 20-gram water-based ink was formulated using Hydroxypropyl Methylcellulose (HPMC) as base polymer, glyphosate, and other functional excipients (glycerin and polyvinylpyrrolidone). Rheological analysis was performed to characterize the ink properties, including flow/viscosity, amplitude sweeps, and thixotropy tests. Subsequently, 3D films were successfully printed from a custom design using a CELLINK BIOX printer using a print pressure of 60 kPa and a print speed of 10 mm/s. The gut organoid development suggests that the 2 mg/ml collagen concentration with both cell types produces more favorable epithelial morphology than the 0.5 mg/ml concentration. The presence of fibroblasts is essential for the formation of the epithelial layer could be deduced from the organoid formation. The non-uniform shapes of the organoids which posed challenges for preparation and sectioning, were further tuned through 3d printed well plates with structures to support the spherical formation of the organoids during crosslinking. The overall study shows that a gut organoid was fabricated, and 3D-printed glyphosate films were successfully printed. These platforms will allow us to conduct in vitro toxicological evaluation of glyphosate at concentrations relevant to human physiology. Future works to be concluded before the conference include the dissolution profile of the glyphosate films, as well as the permeability of the gut organoids using 1 mg/ml FITC Dextran fluorescein-isothiocyanate (FITC)-labeled Dextran 4 kDa. The impact of various relevant glyphosate concentrations exposure to the gut organoids will be evaluated through Live/Dead cytotoxicity, ATP assays, and disruption of the epithelial barrier.