Multiplexed End-point Microfluidic Chemotaxis Assay using Centrifugal Alignment

A fundamental challenge to multiplexing microfluidic chemotaxis assays at scale is the requirement for time-lapse imaging to continuously track migrating cells. Drug testing and drug screening applications require the ability to perform hundreds of experiments in parallel, which is not feasible for assays that require continuous imaging. To address this limitation, end-point chemotaxis assays have been developed using fluid flow to align cells in traps or sieves prior to cell migration. However, these methods require precisely controlled fluid flow to transport cells to the correct location without undesirable mechanical stress, which introduce significant set up time and design complexity. Here, we describe a microfluidic device that eliminates the need for precise flow control by using centrifugation to align cells at a common starting point. A chemoattractant gradient is then formed using passive diffusion prior to chemotaxis in an incubated environment. This approach provides a simple and scalable approach to multiplexed chemotaxis assays. Centrifugal alignment is also insensitive to cell geometry, enabling this approach to be compatible with primary cell samples that are often heterogeneous. We demonstrate the capability of this approach by assessing chemotaxis of primary neutrophils in response to an fMLP (N-formyl-met-leu-phe) gradient. Our results show that cell alignment by centrifugation offers a potential avenue to develop scalable end-point multiplexed microfluidic chemotaxis assays.

Introduction measure of chemotactic capability. While this approach is simple to perform, it often produces 48 inconsistent results because the chemical gradient experienced by each cell across the membrane 49 interface can vary with time, location, and cell density 9 . Transwell assays also require a large 50 number of cells (10 5 -10 6 ) and large volumes of chemoattractant solutions, limiting the number 51 and types of experiments that could be performed, especially on primary cell samples obtained 52 from patients 11 . 53 Microfluidic approaches have been used to develop improved chemotaxis assays that 54 provide more reliable chemical gradients, as well as that reduce the requirements for cells and 55 reagents [12][13][14][15][16][17][18][19][20] . These devices typically involve generating chemoattractant gradients through 56 passive diffusion or through active perfusion. Cells are then introduced into this gradient in order 57 to observe their migration via time-lapse microscopy. This approach has a limited capacity to 58 perform multiple chemotaxis assays in parallel because of the need to continuously track cell 59 migration in each assay using microscopy. While multiplexing these experiments could be achieved using automated microscopy, this approach is not scalable for high throughput 61 applications such as drug testing and drug screening 21 . 62 To address the need for scalable multiplexed chemotaxis assays, several approaches have  Here, we developed a scalable end-point chemotaxis assay that uses centrifugal force to 76 align cell samples to a common starting point in order to provide a multiplexed end-point 77 chemotaxis assay without the need for continuous microscopy. Individual microfluidic devices are 78 arrayed in a rotational symmetric manner on a glass slide substrate. Unlike trap-or sieve-based 79 alignment, this approach obviates the need for carefully controlled fluid flow by aligning cells via 80 centrifugation against a barrier feature. We validated this approach by assessing the migration of 81 primary neutrophils along an fMLP gradient in order to demonstrate its potential as a simple, 82 scalable, and multiplexed chemotaxis assay requiring minimal equipment.

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The microfluidic device for end-point chemotaxis assays comprise of two reservoirs 86 connected by a thin microchannel that limits diffusion between the reservoirs (Figure 1A & 1C). 87 The microfluidic channel contains a barrier feature that enables centrifugal alignment of the cell 88 sample at a common starting point before starting each chemotaxis assay ( Figure 1B). The barrier 89 feature includes a small degree of concavity designed to trap cells against movement created by 90 tangential forces (perpendicular to channel) resulting from angular acceleration caused by starting  the reservoir past the barrier feature and generally excluded from the assay. In order to prevent fluid leakage during the alignment process, the outlets were sealed using acrylic tape. After 106 pipetting the cell sample, the chemoattractant was pipetted into the source reservoir.

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Chemoattractant molecules were diffused from the source reservoir into the sink reservoir through 108 the gradient channel to establish a gradient ( Figure 1B). The device was then placed inside an 109 incubator to allow chemotaxis to take place at stable temperature and gas conditions. Finally, after 110 an appropriate amount of time, the device is imaged to determine the resulting cell positions.

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To study gradient formation in our microfluidic device, as well as the impact of the barrier 113 feature, we first developed finite element models of the gradient with and without a barrier feature. 114 Our model showed that the barrier feature caused the gradient profile to flatten near the barrier,  Figure 2D). The correlation coefficient between gradient strengths in 124 these two scenarios is 0.96, suggesting that the difference is a simple scale factor error.

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To ensure the gradient profile formed using FITC-Dextran is representative of the gradient 126 profile formed using fMLP, we further tested gradient formation using Rhodamine B, which has a To align cells against the barrier feature, the microfluidic device is rotated using a spinner 133 to produce a centrifugal force ( ! ) towards the center of rotation: where " is the density of neutrophils 28 and # is the density of water, V is the volume of a 136 neutrophil, is the angular velocity, l is the distance from the axis of rotation. For small particles 137 moving in a liquid at low Reynolds number, they need to overcome the Stokes drag force ( % ): If cells could migrate consistently at ! and cells were evenly distributed in channels at the 149 beginning, the number of collected cells at barriers (N) could be calculated by: Where W is the width of migration channel, t is the centrifugal spinning time, % is the cell 152 distribution density, k is a constant.

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To experimentally confirm our model, we loaded the microfluidic device with human concavity. We then measured the value of k by fitting Equation (4) to data points from 0 to 1500 160 RPM and found a high-quality fit ( $ > 0.98, Figure 3E). Based on these results, we selected 161 1500 RPM for 1 min as the condition for cell alignment throughout our study.

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To validate neutrophil chemotaxis in the microfluidic device, we performed chemotaxis 164 assay in gradients of fMLP using human neutrophils. Neutrophils were isolated from whole blood 165 using a commercial magnetic immunoselection kit and resuspended in a cell migration media.  In devices with fMLP gradient, most of the cells were observed to migrate away from the barrier 192 ( Figure 5A-C). Varying the fMLP gradient, we found that neutrophils appear to require a threshold fMLP gradient to activate chemotaxis since migration was observed only when the fMLP 194 gradient was greater or equal to 25 nM (Figure 5D-F, p < 0.005). There also appears to be a 195 decrease in migration distance from 25 nM to 100 nM, but this trend was not statistically 196 significant (p > 0.05). Together, these data present support the use of centrifugal cell alignment as 197 a rapid and convenient multiplexed end-point cell migration assay. 198

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In this study, we investigated an approach to develop a multiplexed end-point chemotaxis   ≤10 and ≥25 nM fMLP were statistically significant (p < 0.005).