Deformability-induced lift force in spiral microchannels for cell separation

1Heriot-Watt University, School of Engineering and Physical Science, Department of Biological Chemistry, Biophysics and Bioengineering Edinburgh Campus, Edinburgh, EH14 4AS, Scotland 2 Centre for Innovation Competence – Humoral Immune Reactions in Cardiovascular Diseases, University of Greifswald, Fleischmannstr. 42, 17489 Greifswald, Germany and: Deutsches Zentrum für Herz-Kreislaufforschung, Partner Site Greifswald, Fleischmannstr. 42, 17489 Greifswald, Germany 4Biomedical Engineering Division, James Watt School of Engineering, University of Glasgow, G12 8LT


Real-time fluorescence and deformability cytometry
While there are many available well-established technologies for assessing cell mechanotype such as Atomic Force Microscopy (AFM) 15 , micropipette aspiration 16 , magnetic tweezers and optical stretchers 17 , these methods suffer from low-throughput 18 where is the circularity defined as being the projected cell area and the cell perimeter Deformation (D) in the channel is independently measured from the initial cell shape and therefore any treatment-induced morphological changes to shape. Consequently, when possible, a differential deformation DD parameter has been introduced 21 .

Summary of Triplicate results
The hydrodynamic behaviour of cells was assessed in terms of lateral equilibrium position (measured as a distance from the particle centre to the outer wall [µm]) obtained at the end of the spiral channel by monitoring the ROI, by high-speed microscopic imaging. For one replica of one condition at one flow rate we obtained at least 10000 events. As an example, we provide SFig. 5 showing a single image extracted from a video recorded for soft cells at flow rate corresponding to Re=119 in the spiral channel with 360 × 60 µm 2 cross-section. All of the raw files can be accessed upon a request.

SFig. 5
An exemplary image extracted from a video recorded for soft cells at flow rate corresponding to Re=119 in the spiral channel with 360 × 60 µm 2 cross-section. in comparison to the statistical summary of the lateral equilibrium position (expressed as distance from the pouter wall [µm]) reported as median (represented as the symbol) and the interquartile range (indicated by the short vertical lines). Vertical dotted lines indicate four sections of the channel corresponding to four outlets of the channel (0-90 µm-outlet A, etc.).

Design I: Hydrodynamic behaviour of cells of cellular deformability model
Hydrodynamic behaviour of cells (10000 per condition) of five different deformabilities (soft max, soft half-max, soft, stiff half-max and stiff) (A) in comparison to reference 15 µm beads in design I spiral microchannele with 360 × 60 µm 2 cross-section at five different flow rates corresponding to Re=79, 119, 158, 198 and 237 The lateral equilibrium positions were measured as a distance from the outer wall (µm) at the end of the spiral channel and there were generated by image analysis. Here, it is reported as median (represented as the symbols) and the interquartile range (indicated by the short vertical lines). Vertical dotted lines indicate four sections of the channel corresponding to four outlets of the channel (0-90. µm-outlet A, 90-180 µm-outlet B, etc.). Events belonging to a given section have the highest probability of being captured within the corresponding outlet and tables showing statistical summary (mean and standard deviation from the mean (SD), median, 25 th (Qi) and 75 th (Q3) percentile as well as minimal (min) and maximal (max) measured value) of latera equilibrium positions obtained for at least 10000 events.

Summary of flow cytometric viability assay.
On the top-an exemplary scatter plot showing gating strategy for live cell (green, negative for both Alexa Fluor 488-annexin V and propidium iodide (PI) fluorescence), apoptotic cells (orange, annexin V-positive and PI-negative) and necrotic (red, annexin V-positive and PI-positive). Summary of flow cytometric assessment of the presence of live, apoptotic and necrotic Jurkat cells before (stained control and after processing (stained test).
Design I spiral channel with 360 × 60 µm cross-section at highest applied flow rate (Re=237) for three replicas.