16 Feb Microfluidic analysis of enzymatically dissociated tissues

“Biomedical research relies on identification and isolation of specific cell types using molecular biomarkers and sorting methods such as fluorescence or magnetic-activated cell sorting. Labelling processes potentially alter the cells’ properties and should be avoided, especially when purifying cells for clinical applications. A promising alternative is the label-free identification of cells based on physical properties. Sorting real-time deformability cytometry (soRT-DC) is a microfluidic technique for label-free analysis and sorting of single cells. In soRT-FDC, bright-field images of cells are analyzed by a deep neural net (DNN) to obtain a sorting decision, but sorting was so far only demonstrated for blood cells that show clear morphological differences and are naturally in suspension. Most cells, however, grow in tissues, requiring dissociation before cell sorting which is associated with challenges including changes in morphology, or presence of aggregates. Here, we introduce methods to improve the robustness of analysis and sorting of single cells from nervous tissue and provide DNNs which can distinguish visually similar cells. We employ the DNN for image-based sorting to enrich photoreceptor cells from dissociated retina for transplantation into the mouse eye.”

“Cell preparation, soRT-FDC setup and chip design. (A) Retinae from reporter mice (Nrl-eGFP) or human retinal organoids (Crx-mCherry) are dissociated and resuspended in measurement buffer for soRT-FDC. (B) Sketch of the soRT-FDC setup. Two syringe pumps supply a microfluidic chip with sample and sheath fluid. Lasers excite fluorescence signal which is measured by avalanche photodetectors and the cell is imaged by a high-speed camera. A high-power LED illuminates the cell. Interdigital transducers (IDTs) excite surface acoustic waves, which push selected cells towards the target outlet. (C) Figure shows the 2D-CAD design of the entire sorting chip and zoomed in versions show specific parts. The red rectangles indicate filter assemblies, which consist of a cascade of pillars with decreasing distance. The orange rectangles indicate a unit of several serpentines, which helps to divide aggregates of cells and to increase the spacing between cells. The layout was designed using KLayout 0.25.3.” Reproduced under Creative Commons Attribution 4.0 International License from Herbig, M., Tessmer, K., Nötzel, M. et al. Label-free imaging flow cytometry for analysis and sorting of enzymatically dissociated tissues. Sci Rep 12, 963 (2022).

Figures and the abstract are reproduced from Herbig, M., Tessmer, K., Nötzel, M. et al. Label-free imaging flow cytometry for analysis and sorting of enzymatically dissociated tissues. Sci Rep 12, 963 (2022).https://doi.org/10.1038/s41598-022-05007-2 under Creative Commons Attribution 4.0 International License

Read the original article: Label-free imaging flow cytometry for analysis and sorting of enzymatically dissociated tissues