Label‐Free Cell Migration Assay Using Magnetic Exclusion

Abstract

biophysical cues. [1,2] Signaling molecules regulate cell migration under normal physiological conditions by controlling cell adhesion and cytoskeleton organization. [1,3,4] Conversely, pathological conditions such as metastatic cancer, vascular disease, and chronic inflammation are associated with unregulated cell migration, contributing to disease progression. [4,5] The wound healing assay is a commonly used in vitro method to study cell migration, cell interactions, and the underlying mechanisms. [6][7][8][9][10] The conventional "scratch" assay creates a cellfree area or scratch in a cell monolayer using a pipette tip or a pin tool. [10,11] Cells from intact surrounding regions migrate and repopulate the cell-free region over time. The migration rate is measured by observing the decrease in the cellfree area at regular intervals. While the scratch assay is simple and inexpensive, it requires 24-48 h to form a cell monolayer. [11] Moreover, by physically scratching the cell layer with a pipette tip or microneedle, cell-free regions of inconsistent shapes and sizes are created. [10,12,13] This process also damages the underlying plastic surface or extracellular matrix (ECM) substrate, which can significantly affect cell migration. [10,12,13] The electric cell-substrate impedance sensing (ECIS) technique, on the other hand, produces consistent cell-free regions without mechanically disrupting the cell layer. [10,12] In ECIS, a confluent cell layer is formed over a small gold film electrode placed at the bottom of a tissue culture well. Electric pulses are applied to induce cell death at the electrode surface, which creates a well-defined region devoid of cells. [14] However, dead cell fragments can remain on top of the electrode and interfere with cell migration. [12,15] Furthermore, the electric pulses can potentially injure the cells surrounding the electrode, thereby affecting their migration. [10] In contrast to the scratch assay and ECIS, the physical exclusion technique creates cell-free areas of reproducible sizes and shapes with minimal cellular damage. [12,16] This method employs physical barriers, such as plastic inserts or silicone stoppers, to prevent cells from settling in a predefined area. However, it requires 12-20 h to form confluent cellular regions around the barrier. [16,17] Commercial inserts are also expensive and unsuitable for multiple uses. [16] Magnetic levitation is another in vitro technique used to form cell clusters in a migration assay. [18] This method is based on the cellular uptake of biopolymer-encapsulated magnetic In vitro wound healing assays are widely used to investigate cell migration during various physiologic and pathologic processes. However, traditional scratch-based assays produce cell-free areas that are not reproducible, whereas the alternate insert-based exclusion method is expensive and timeconsuming. Here, a rapid, label-free, insert-free magnetic exclusion technique, where magnetic fields are used to create cell-free areas is described. Suspensions of diamagnet...

Programing Cell Assembly via Ink-Free, Label-Free Magneto-Archimedes Based Strategy

“…For former research generating cellular patterns on substrates, it is still hard to prepare patterns with micrometer size. The throughput of cell assembly is also limited as only a few assemblies were able to prepare at the same time . In our system, we were about to prepare a hundred microscale cell assemblies in one square centimeter in batches (Figure A).…”

“…In most of the abovementioned research, magnets were used to generate a power source for cell magnetic levitation, the patterns being generated by self-assembly of cells. Puri et al 21 generated a controllable cell exclusion area by using magnetic levitation. However, limited by the size of permanent magnets, only a few simple constructs have been fabricated using the Mag-Arch strategy.…”

“…The throughput of cell assembly is also limited as only a few assemblies were able to prepare at the same time. 21 In our system, we were about to prepare a hundred microscale cell assemblies in one square centimeter in batches (Figure 2A). The assembled endothelial cells not only showed great cell viability (99.98 ± 0.01%, Figure 2B), but also maintained vital biological properties for endothelium such as stretchy cell skeleton formation, proper proliferation, ECM secretion, and tight-conjunction between cells, as shown by the expression of F-actin, K i -67 (Figure 2C), collagen type I, and ZO-1 markers (Figure 2D) in immunofluorescence staining.…”

Biophysical and Biochemical Regulation of Cell Dynamics in Magnetically Assembled Cellular Structures

“…We have previously demonstrated that Gadavist exposure has insignificant effects on HBEC3 KT viability . The impact of Gadavist exposure on primary fibroblast metabolism, an indicator of viability, is investigated using a resazurin-based assay (Presto Blue HS).…”

“…Since cell receptor complexes for OSM and IL6 have a common glycoprotein 130 (gp130) signal transduction chain, we stimulate HBEC3 KT with IL6 to determine whether this cytokine has similar effects on the cell dynamics as OSM . EGF is used as a positive control, as we have previously demonstrated enhanced HBEC3 KT migration due to EGF (0.8 ng mL –1 ) exposure . The annular aggregates are assembled on tissue culture-treated (TCT) and ECM-coated surfaces within 3 h to assess the impact of surfaces on cell-free area closures.…”