STSM at Karolinska Institute - Report from Sónia Siquenique

Through this STSM, I aimed at gaining epertise in working with human intestinal organoids, to further enrich the Home Institution's expertise in developing more physiological relevant biomimetic in vitro intestinal models.

With regard to the accomplished practical work, initially, human-derived intestinal organoids were maintained in culture for 7 days (Figure 1) and subsequently passaged or differentiated into apical-out configuration (Figure 2), allowing me to become familiar with key aspects of their handling, differentiation, and maintenance.

Figure 1. Human jejunal organoids in culture for 7 days (Magnification: 4x).

Figure 2. Differentiated human jejunal organoids into apical-out configuration (Magnification: 40x). Another major goal of this STSM was to evaluate the intestinal permeability of exenatide-based delivery systems using this 3D intestinal model. Permeability studies were conducted by testing 1 µM of exenatide released from delivery systems, using human intestinal organoids differentiated into the apical-out configuration, after 24 hours.

In parallel, I acquired hands-on training in the development of monolayers derived from intestinal organoids—another key objective of this STSM. Organoids were enzymatically dissociated into single cells and seeded with different cells densities onto inserts (24-well plate format) coated with a Matrigel/Collagen I mixture (1:1). Media was changed every other day for 5 days and TEER values were measured to ensure the formation of a complete monolayer (Figure 3).

Figure 3. Monolayers-derived from human intestinal organoids seeded at different cell densities.

As an alternative approach to evaluate the efficacy of the delivery systems in the absence of human pancreatic islets, a bioluminescence resonance energy transfer (BRET)-based assay was employed to monitor the activation of the GLP-1 receptor (GLP-1R). This method enabled us to assess the functional engagement of the receptor by the released recombinant exenatide, providing valuable insights into the bioactivity of the delivery systems.

In summary, a new collaboration was established between the Home (i3S, Porto, Portugal) and Host Institution by my STSM focusing on the development of advanced in vitro intestinal models to be used as an alternative to animal models for drug testing. This transfer of knowledge contributes to the reduction of animal experiments used for drug testing and nanomedicines, in accordance with the goals of working group 1 of the CA IMPROVE. I have received specific training in culture of human intestinal organoids and in the establishment of monolayers-derived form intestinal organoids that will be applied at my Home Institution to improve the reproducibility and physiological relevance of the in vitro intestinal model.

Beyond scientific outcomes, the STSM contributed significantly to my personal and professional development. The opportunity to work in a new research environment abroad enriched my doctoral training, in line with the goals of working group 4 of the CA IMPROVE.