Direct transfection of clonal organoids in Matrigel microbeads: a promising approach toward organoid-based genetic screens

Abstract

Organoid cultures in 3D matrices are relevant models to mimic the complex in vivo environment that supports cell physiological and pathological behaviors. For instance, 3D epithelial organoids recapitulate numerous features of glandular tissues including the development of fully differentiated acini that maintain apico-basal polarity with hollow lumen. Effective genetic engineering in organoids would bring new insights in organogenesis and carcinogenesis. However, direct 3D transfection on already formed organoids remains challenging. One limitation is that organoids are embedded in extracellular matrix and grow into compact structures that hinder transfection using traditional techniques. To address this issue, we developed an innovative approach for transgene expression in 3D organoids by combining single-cell encapsulation in Matrigel microbeads using a microfluidic device and electroporation. We demonstrate that direct electroporation of encapsulated organoids reaches up to 80% of transfection efficiency. Using this technique and a morphological read-out that recapitulate the different stages of tumor development, we further validate the role of p63 and PTEN as key genes in acinar development in breast and prostate tissues. We believe that the combination of controlled organoid generation and efficient 3D transfection developed here opens new perspectives for flow-based high-throughput genetic screening and functional genomic applications.

Gelatin methacryloyl and its hydrogels with an exceptional degree of controllability and batch-to-batch consistency

“…Protein-based hydrogels including collagen, fibrin, and Matrigel ® , as natural extracellular matrices (ECM) analogues, have been widely investigated for various applications, like three-dimensional (3D) culture, tissue engineering, and regenerative medicine, because they are biocompatible and have excellent innate bioactive properties such as enzyme degradation and good control over cellular activities (adhesion, proliferation, migration, and differentiation) 1–4 . Matrigel ® is one of the most popular protein-based hydrogels 5–7 . Matrigel ® is a gelatinous protein mixture derived from Engelbreth-Holm-Swarm mouse sarcoma tumors and contains mainly proteins (laminin, type IV collagen, and entactin) and a small portion of proteoglycans and growth factors 5 .…”

Current Advances in 3D Tissue and Organ Reconstruction

“…In parallel, in our experience, the use of micro-bioreactors significantly enhance the acquisition and maintenance of a high plasticity state, providing a useful platform in stem cell organoid technology for long-term culture of different cell types, ranging from epigenetically erased cells to embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), as well as mesenchymal stem cells (MSCs) [ 65 ]. Interestingly, parallel studies also demonstrated that encapsulation systems can be successfully used to store and cryopreserve organoids without altering cell growth and differentiation ability, as well as to perform post-treatments, such as direct transfection, and post-genetic screen and organoid analysis [ 83 ].…”

Exploiting CRISPR Cas9 in Three-Dimensional Stem Cell Cultures to Model Disease

“…The third method involves direction delivery of Cas9 and sgRNA to the organoids without generating single cells ( Matano et al, 2015 ). Of the three methods, the first two have been employed more widely due to difficulties associated with efficient gene delivery through 3D matrices ( Laperrousaz et al, 2018 ). Since hydrogel matrices can often pose diffusional limitations to highly charged, relatively large non-viral gene delivery vectors, limitations remain in using CRISPR Cas9 to study organoids.…”