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

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

Visualization 4: Lensfree diffractive tomography for the imaging of 3D cell cultures

ROI1 of the crop 1 (3D view flyover) Originally published in Biomedical Optics Express on 01 March 2016 (boe-7-3-949

Visualization 6: Lensfree diffractive tomography for the imaging of 3D cell cultures

ROI1 of the crop 2 (3D view flyover) Originally published in Biomedical Optics Express on 01 March 2016 (boe-7-3-949

Visualization 2: Lensfree diffractive tomography for the imaging of 3D cell cultures

3D view of the full reconstructed volume (3D view flyover) Originally published in Biomedical Optics Express on 01 March 2016 (boe-7-3-949

Visualization 3: Lensfree diffractive tomography for the imaging of 3D cell cultures

Crop 1 of the reconstructed volume (3D view flyover) Originally published in Biomedical Optics Express on 01 March 2016 (boe-7-3-949

Selective individual primary cell capture using locally bio-functionalized micropores.

BACKGROUND: Solid-state micropores have been widely employed for 6 decades to recognize and size flowing unlabeled cells. However, the resistive-pulse technique presents limitations when the cells to be differentiated have overlapping dimension ranges such as B and T lymphocytes. An alternative approach would be to specifically capture cells by solid-state micropores. Here, the inner wall of 15-µm pores made in 10 µm-thick silicon membranes was covered with antibodies specific to cell surface proteins of B or T lymphocytes. The selective trapping of individual unlabeled cells in a bio-functionalized micropore makes them recognizable just using optical microscopy. METHODOLOGY/PRINCIPAL FINDINGS: We locally deposited oligodeoxynucleotide (ODN) and ODN-conjugated antibody probes on the inner wall of the micropores by forming thin films of polypyrrole-ODN copolymers using contactless electro-functionalization. The trapping capabilities of the bio-functionalized micropores were validated using optical microscopy and the resistive-pulse technique by selectively capturing polystyrene microbeads coated with complementary ODN. B or T lymphocytes from a mouse splenocyte suspension were specifically immobilized on micropore walls functionalized with complementary ODN-conjugated antibodies targeting cell surface proteins. CONCLUSIONS/SIGNIFICANCE: The results showed that locally bio-functionalized micropores can isolate target cells from a suspension during their translocation throughout the pore, including among cells of similar dimensions in complex mixtures