Speaker
Description
Introduction
Organoids are miniaturized three-dimensional layered constructs offering unprecedent resemblance with the structural, biological and functional characteristics of organs. These models provide a new framework to study the cellular processes, the physiology and the treatment of pathologies at the organ-level. Beyond in vitro modelling, organoids can offer a new alternative for in vivo applications in regenerative medicine.
The standard procedure to grow organoids relies on inducing the self-assembly of stem cells in weak hydrogels, commonly Basal Membrane Extract (BME). However, BME derives from tumorous mouse which hinders the possibility of clinical translation. Beyond that, the micro-environment it provides to organoids is poorly controlled, lacks of hierarchical structure, and offers a composition radically different from the native extracellular matrix (ECM)1. Meanwhile, 2D and prosthetic materials have largely been developed using native proteins, but for organoids, biomimetic materials remain little approached2.
We herein propose new materials for organoid culture, composed solely of ECM proteins, with a porous structure to promote nutrients and oxygen diffusion. We compare the ability to support organoids formation of i) fibrillar native-like collagenous matrices, ii) materials derived from decellularized extra-cellular matrix (dECM), and iii) the standard BME. Our materials represent a new alternative for organoid culture, that is animal-free, compatible with clinical translation, and mimic closely the physiology of tissues.
Methods
Highly concentrated collagen I solutions are ice-templated to allow ice crystals growth and the subsequent collagen segregation in-between the crystals. The ice crystals are subsequently melted at low temperature to reveal pores, while collagen packing is maintained and its self-assembly into fibrils simultaneously induced3. dECM solutions are prepared from corpus spongiosum (dECM-CS), and structured using the same ice-templating process and topotactic gelation. All materials are characterized to assess their composition (IHC, hydroxyproline assay, electrophoresis), native-like features (TEM, PLOM, DMA), and textural aspects (SEM). Urothelial stem cells isolated from patient tissues are seeded and grown on the materials with appropriate culture medium. The organoid formation and properties are characterized and compared between the materials and the control (confocal microscopy, IHC, TEM, qPCR).
Results
We successfully obtained porous yet dense matrices, in the wet state, with both molecular type I collagen and dECM-CS solutions (Fig.1). Preliminary results of urothelial stem cells culture in our models demonstrate their ability to direct the self-assembly of cells into organoids. Additionally, we were able to differentiate bladder and urethral organoids based on cytokeratins makers. Further results are expected to uncover the materials properties that specifically instruct organoids formation.
Discussion
Such biomaterials recapitulate the structural, biological and functional features of biological tissues. By exploring the physics of ice, innovative collagen self-assembly techniques, and more complex materials deriving from native tissues, we tailor the biological composition, the collagen conformation, and the hierarchical and textural aspects of the materials’ interfaces to direct the self-assembly of stem cells into organoids.
References
1 Kozlowski, M. T., Crook, C. J. & Ku, H. T. Commun Biol 4 (2021).
2 Kretzschmar, K. & Clevers, H. Dev Cell 38, 590-600 (2016).
3 Martinier, I. et al. Biomater Sci 12, 3124-3140 (2024).
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