A protocol for screening cells based on deformability using parallel microfiltration

Abstract

Cell mechanical phenotype, or 'mechanotype', is emerging as a valuable biomarker for the physiological or diseased state of cells. Established techniques such as micropipette aspiration and atomic force microscopy provide quantitative insights into the viscoelastic properties of cells. However, to acquire data across a larger number of samples within a reasonable timescale requires higher throughput measurements. The recent development of fluidic-based mechanotyping methods enable data acquisition at rates of up to 2000 cells/second 1,2 , but require image-based analysis that hinders simultaneous measurements across an array of cell samples. Here we present a protocol for measuring the ability of cells to filter through micron-scale pores using a simple and scalable technique called parallel microfiltration (PMF). Cell filtration depends on the ability of cells to deform through micron-scale pores, and can thus provide a measure of cell deformability. The PMF method can be used for screening of multiple cell samples in parallel based on cell filtration. The entire protocol, from preparation of cell suspensions to readout of filtration, can be completed within an hour. Here we demonstrate the applicability of PMF to measure human promyelocytic leukemia (HL-60) cells, as well as human ovarian (OVCAR3 and OVCAR5) and breast cancer (MDA-MB-231) cells. The method can easily be adapted for use with other mammalian cell types.

DYT1 Dystonia Patient-Derived Fibroblasts Have Increased Deformability and Susceptibility to Damage by Mechanical Forces

“…To begin to determine if DYT1 dystonia is associated with altered cellular deformability, we performed our previously described PMF assay (Qi et al, 2015; Gill et al, 2017) on NIH3T3 fibroblasts with impaired torsinA function. In PMF, cell suspensions are filtered through porous membranes on the timescale of seconds by applying a defined magnitude of air pressure.…”

“…The number and size distribution of cells in each well were quantified using an automated cell counter (TC20, BioRad Laboratories, Hercules, CA, United States). Finally, a defined magnitude of air pressure which was monitored using a 0–100 kPa pressure gauge (Noshok Inc., Berea, OH, United States), was applied to the 96-well plate outfitted with a custom pressure chamber for 40 or 50 s (Qi et al, 2015; Gill et al, 2017). To quantify retention volumes following filtration, we measured the absorbance at 560 nm of the phenol red-containing cell medium using a plate reader (Infinite M1000, Tecan Group Ltd., Männedorf, Switzerland).…”

β-adrenergic signaling modulates cancer cell mechanotype through a RhoA-ROCK-myosin II axis

“…To measure whole cell deformability, we used parallel microfiltration (PMF) (27,28). Cells were trypsinized with 0.25% trypsin-EDTA and cells in suspension were counted using an automated cell counter (TC20, Bio-Rad) and resuspended in medium to a density of 5×10 5 cells/ml.…”

Stress hormone signaling through β‐adrenergic receptors regulates macrophage mechanotype and function

“…To measure the deformability of cells, which is indicated by their ability to filter through 5 mm pores of a membrane with applied pressure, we used parallel microfiltration (PMF) as described in our previous studies (29,30). Briefly, cells were treated with drugs for 24 h, and we prepared a suspension of single cells by trypsin treatment for 3 min followed by scraping to harvest additional cells that remain adhered.…”

“…S1E). To determine effects of b-AR activation on both monocyte and macrophage physical properties, we treated cells with isoproterenol and measured their ability to deform through micron-scale pores using PMF (29,30). In this assay, air pressure is applied to drive a suspension of cells through 5 mm diameter pores over a relatively short timescale of 30 s, and the volume of fluid retained above the membrane is determined; the retention volume depends on the number of pores that have been occluded by cells, which is largely determined by the physical properties of cells, including cell size and deformability (29).…”