High-throughput mechanophenotyping of multicellular spheroids using a microfluidic micropipette aspiration chip

Cell spheroids are in vitro multicellular model systems that mimic the crowded micro-environment of biological tissues. Their mechanical characterization can provide valuable insights in how single-cell mechanics and cell–cell interactions control tissue mechanics and self-organization. However, most measurement techniques are limited to probing one spheroid at a time, require specialized equipment and are difficult to handle. Here, we developed a microfluidic chip that follows the concept of glass capillary micropipette aspiration in order to quantify the viscoelastic behavior of spheroids in an easy-to-handle, more high-throughput manner. Spheroids are loaded in parallel pockets via a gentle flow, after which spheroid tongues are aspirated into adjacent aspiration channels using hydrostatic pressure. After each experiment, the spheroids are easily removed from the chip by reversing the pressure and new spheroids can be injected. The presence of multiple pockets with a uniform aspiration pressure, combined with the ease to conduct successive experiments, allows for a high throughput of tens of spheroids per day. We demonstrate that the chip provides accurate deformation data when working at different aspiration pressures. Lastly, we measure the viscoelastic properties of spheroids made of different cell lines and show how these are consistent with previous studies using established experimental techniques. In summary, our chip provides a high-throughput way to measure the viscoelastic deformation behavior of cell spheroids, in order to mechanophenotype different tissue types and examine the link between cell-intrinsic properties and overall tissue behavior.

HEK293T spheroids were aspirated using pipettes with a diameter of 65±5 µm, fabricated by pulling borosilicate glass pipettes (Harvard Apparatus, 1 mm OD, 0.5 mm ID) with a laser-based puller (Sutter Instruments Co Mode P-2000) and cutting them with a quartz tile. Cell adhesion to the pipette walls was prevented by incubating the pipettes in 2 mg/mL PolyEthyleneGlycol-PolyLysine (PLL(20)-g[3.5]-PEG(2)/PEG(3.4)-Biotin(20%), SuSos AG, Dubendorf, Switzerland) in MRB80 solution (80 mM piperazine-N,N'-bis(2-ethanesulfonic acid) (Pipes), pH 6.8, 4 mM MgCl 2 , 1 mM EGTA [Sigma]) for 30 minutes. Spheroids were suspended in a CO 2 -equilibrated medium and kept in a custom-made sample chamber. The chamber was constructed by adhering two microscope slides to a custom-made aluminum spacer of 3 mm thickness using vacuum grease (Beckman Coulter). The pipette was introduced into the chamber, aligned with a spheroid and an aspiration pressure was attained by vertically displacing a water reservoir connected to the pipette using a vertical translational stage (LTS300, Thorlabs). Spheroids were aspirated using a pressure ∆P = 5 cmH 2 O and visualized on an inverted microscope (Nikon Eclipse TI) with a 10x air objective. After aspiration, the pressure gradient was removed and the retraction of the tongue was recorded. The creep advancement of the tongue was recorded with an ORCA Flash 4.0 digital camera using a 1s interval for a total of 10 minutes, of which 5 minutes corresponded to aspiration and the next 5 minutes to retraction. Data was obtained for two independent experiments, which were performed at room temperature. As the experimental set-up did not include a heating stage to keep the experimental chambers at the physiological temperature of 37 °C, aspiration of spheroids was only performed in the first hour after they came out of the incubator.
The critical pressure ∆P c to aspirate the spheroids was derived from: AutoCAD screenshots of the full design that was printed on the silicon wafer using soft lithography. (A) First layer, which was printed to a height of ±50 µm, with the design enframed with alignment arrows. For slab 1, the alignment arrows are written by the laserwriter (µMLA, Heidelberg Instruments) as pillars, while for slab 2 an entire rectangular surface is written except for empty wells complementary to the arrows of slab 1. Alignment crosses are written to facilitate the alignment of layer 2 with layer 1 in the next step of the writing process. (B) Final developed design, where the second written layer is indicated in green. (C) Zoomed-in section of layer 1, clarifying the placement of alignment arrows. (D) Zoomed-in section of layer 2 (with the middle part cut out) to demonstrate how the right part of the wafer design is mirrored to the left part. This is necessary to create the correct final design, as PDMS slab 2 is turned over when aligning it with PDMS slab 1. (E) Side view of the final developed design on the silicon wafer, which is used for PDMS casting. Slab 1 contains arrow pillars and slab 2 complementary arrow wells (both indicated in red) to facilitate easy alignment before bonding the two slabs in order to create the final device.

Supplementary Figure 3: Pressure distribution across aspiration channel for a microfluidic device that is misaligned 20 µm along both the x-and y-axis.
A 3D numerical COMSOL simulation of the pressure distribution assuming the device is connected to a 60 cm long straight rectangular channel mimicking the outlet tube. A pressure gradient of 700 Pa is simulated for the condition that all pockets but one are clogged by spheroids. The misalignment has negligible effects on the pressure drop across the aspiration channel in comparison to a perfectly aligned chip (Fig. 3A).  Table S1.)

Supplementary Movies
Movie 1: Microfluidic multi-channel aspiration experiment to determine spheroid mechanics.
A brightfield video of HEK293T spheroids being aspirated into the aspiration channels under an applied hydrostatic pressure of ∆P = 700 Pa for a total duration of 5 minutes. Scale bar is 200 µm.
Movie 2: NIH3T3 cell spheroids move out of pockets during retraction measurement.
A brightfield video of NIH3T3 spheroids being aspirated under a hydrostatic pressure of ∆P = 1500 Pa for the first 10 minutes, after which the pressure gradient is removed and spheroid tongues start retracting. The tongues retract within seconds, making it impossible to record a retraction curve and extract a retraction rate to derive a critical pressure ∆P c . Scale bar is 200 µm.
Movie 3: MCF10A cell spheroid retraction with remaining small aspiration pressure of 100 Pa.
A brightfield video of an MCF10A spheroid being aspirated under a hydrostatic pressure of ∆P = 1500 Pa for the first 10 minutes, after which the pressure gradient is reduced to a small remaining aspiration pressure of 100 Pa and the spheroid tongue starts retracting. The tongue again retracts rapidly, within seconds, thereby ruling out the probability that this fast retraction is caused by a backflow. Scale bar is 50 µm.
A brightfield video of an MCF10A spheroid in a chip that was not coated with pluronic F-127 solution, being aspirated under a hydrostatic pressure of ∆P = 1500 Pa for the first 10 minutes after which the pressure gradient is removed and the spheroid tongue starts retracting. The tongue again rapidly retracts, within seconds, thereby demonstrating that this fast retraction is probably not governed by the surface treatment of the aspiration channels. Scale bar is 50 µm.