Intestinal organoids – a potential screening tool for advanced drug delivery systems

Introduction

Nano-sized formulations for targeted oral drug delivery show promise in treating local gastrointestinal diseases, offering a patient-friendly alternative to predominantly parenteral treatments1. However, assessing advanced delivery systems requires more comprehensive intestinal models. Bridging the gap between conventional 2D cell-based systems and in vivo models, 3D organoids are able to partially recapitulate a cellular phenotype closer to the normal human intestinal epithelium2. Reversing the polarity of organoids from apical-in ("standard configuration") to an apical-out orientation enables direct access to the apical surface of the epithelium3. Thus apical-out organoids represent a powerful new tool for the precise evaluation of drug delivery system efficacy.

Aim

To establish an in vitro 3D human organoid model derived from stem cell-containing crypts from ileal biopsies and characterize the model in terms of morphology, differentiation and protein expression.

Method

Culture conditions were established to generate 3D organoids by isolating stem cell-containing crypts from human ileal tissue. Organoids were initially derived as apical-in cultures in an extracellular matrix (ECM). To access the apical surface of the epithelium, the polarity of the organoids was reversed by removing the ECM. Apical-in organoids were characterized for structural morphology (size, budding formation, shape) using brightfield and fluorescence microscopy. Mass spectrometry-based proteomics was utilized to evaluate cell composition (differentiation and immaturity marker expression), functionality (tight junction protein expression), and the expression of proteins involved in the cell uptake of drug delivery vehicles and intracellular transport.

Results

Microscopic analysis revealed that the apical membrane of the organoids faced the suspension environment, confirming that the polarity was successfully reversed (Fig 1A). Apical-out organoids maintained consistent morphological structures over a period of eight weeks (eight passages). Proteomic analysis indicated expression of ileum-specific biomarkers and fundamental ADME machinery. Furthermore, expression of regulatory proteins of major endocytic pathways such as clathrin-mediated endocytosis (CLTC), caveolin-mediated endocytosis (CAV1) and macropinocytosis (CTBP1) and markers for intracellular compartments (Rab5a, Rab7, LAMP1) in organoids was comparable to biopsies (Fig 1B).

Conclusion

Apical-out ileal organoids were successfully established with physiology-similar properties and will in the next step be evaluated as a screening tool for advanced drug delivery systems.

Acknowledgement

The authors are grateful to the European Union for financial support (GENEGUT, GA 101057491).

References: 1 Chu, J. N. et al. Nat. Rev. Gastroenterol. Hepatol. 19, 219–238 (2022); 2 Gunti, S. et al. Cancers 13, 874 (2021); 3 Co, J. Y. et al. Cell Rep. 26, 2509-2520.e4 (2019)