Angela Jacobi

Stem cell mechanics, determined predominantly by the cell’s cytoskeleton, plays an important role in different biological processes such as stem cell differentiation or migration. Several methods to measure mechanical properties of cells are currently available, but most of them are limited in the ability to screen large heterogeneous populations in a robust and efficient manner—a feature required for successful translational applications. With real-time fluorescence and deformability cytometry (RT-FDC), mechanical properties of cells in suspension can be screened continuously at rates of up to 1,000 cells/s—similar to conventional flow cytometers—which makes it a suitable method not only for basic research but also for a clinical setting. In parallel to mechanical characterization, RT-FDC allows to measure specific molecular markers using standard fluorescence labeling. In this chapter, we provide a detailed protocol for the characterization of hematopoietic stem and progenitor cells (HSPCs) in heterogeneous mobilized peripheral blood using RT-FDC and present a specific morpho-rheological fingerprint of HSPCs that allows to distinguish them from all other blood cell types.

Cell mechanical measurements are gaining increasing interest in biological and biomedical studies. However, there are no standardized calibration particles available that permit the cross-comparison of different measurement techniques operating at different stresses and time-scales. Here we present the rational design, production, and comprehensive characterization of poly-acylamide (PAAm) microgel beads mimicking biological cells. We produced mono-disperse beads at rates of 20 – 60 kHz by means of a microfluidic droplet generator, where the pre-gel composition was adjusted to tune the beads’ elasticity in the range of cell and tissue relevant mechanical properties. We verified bead homogeneity by optical diffraction tomography and Brillouin microscopy. Consistent elastic behavior of microgel beads at different shear rates was confirmed by AFM-enabled nanoindentation and real-time deformability cytometry (RT-DC). The remaining inherent variability in elastic modulus was rationalized using polymer theory and effectively reduced by sorting based on forward-scattering using conventional flow cytometry. Our results show that PAAm microgel beads can be standardized as mechanical probes, to serve not only for validation and calibration of cell mechanical measurements, but also as cell-scale stress sensors. Significance Statement: Often vastly different cell mechanical properties are reported even for the same cell type when employing different measurement techniques. This discrepancy shows the urgent need for standardized calibration particles to cross-compare and validate techniques. Microgel beads can serve this purpose, but they have to fulfil specific requirements such as homogeneity, sizes and elasticities in the range of the cells, and they have to provide comparable results independent of the method applied. Here we demonstrate the standardized production of polyacrylamide microgel beads with all the features an elastic cell-mimic should have. These can not only be used as method calibration particles, but can also serve as cell-scale sensors to quantify normal and shear stresses exerted by other cells and inside tissues, enabling many new applications.

Cell mechanical characterization has recently approached the throughput of conventional flow cytometers. However, this very sensitive, label-free approach still lacks the specificity of molecular markers. Here we combine real-time 1D-imaging fluorescence and deformability cytometry (RT-FDC) to merge the two worlds in one instrument — opening many new research avenues. We demonstrate its utility using sub-cellular fluorescence localization to identify mitotic cells and test for their mechanical changes in an RNAi screen.