マイクロ流体プローブとのライブの細胞層の急速なサブトラクティブパターニング

The primary objective of the method presented in this video article is to rapidly generate spatially defined cell patterns to facilitate in-situ spatiotemporal analysis of cell-to-cell interactions. The presented strategy leverages the microfluidic probe technology. The microfabricated probe head localizes nanoliter volumes of biochemicals on biological substrates to pattern cell monolayers.

The main advantage of an MFP-based patterning technique is that it is nearly instantaneous, owing to local lysis, allowing real-time modification of the patterns to be generated. We first had the idea for this method when we noticed that other methods to generate cell patterns require long, multi-step protocols before actually seeing the success of the cell patterns. To run stable experiments with a hydrodynamic flow confinement on a cell surface, the MFP parameters need to be optimized.

For this purpose, visual demonstration is critical. Aditya Kashyap and Julien Cors will be demonstrating this procedure. The operational modules of the platform comprise motorized syringes, motorized stages, and a controller.

The MFP is connected to a motorized Z-stage to control the gap distance between the head and the substrate, and the substrate holder is attached to the X and Y motorized stages constituting the scanning system. The MFP head has fluidic vias to connect to the pumping station, mounting holes to mount the head onto the Z-stage, and channels that exit the polished apex. The apex is set coplanar to the cell culture substrate.

To prepare the pumping station and fluid-handling apparatus, clean syringes and syringe plungers by sonication and 0.5%bleach solution in ultrapure water prior to and post live-cell experiments. Rinse them thoroughly with water. Fill the syringes with water by immersing their tips completely in a water bath and aspirating using the plunger.

Purge the liquid while within the water bath with the syringe shaft contacting the seal at the exit of the syringe. Continue to aspirate and purge until no air bubbles are seen in the syringe columns. Connect the filled syringes to the syringe pumps using lower-lock connectors.

Use the switch valve mounted on the pumps to direct the liquid from the syringe to one of two capillaries leading either to the MFP head or to the liquid reservoirs. Purge both capillaries with water from the syringes at a flow rate of about 10 to 50 microliters per minute, depending on the size of the syringe, until about 10 microliters of water are left in the syringes. Clean the MFP head by sonication using glassware detergent for standard cleaning, or 0.5%bleach for stringent cleaning, for five minutes.

Purge the channels with water by immersing the apex in water and applying a vacuum to the vias. Inspect the channels under a stereo microscope for potential obstructions, and repeat the previous step, if necessary. Next, mount the cleaned head on the head holder and screw the connector onto the head.

Insert the purged capillaries into an appropriate microfluidic connector adapter which interfaces with the channel vias in the head. Screw the head holder to the Z-stage, which is used for the control of gap distance between the head and the cell monolayer, before performing end-point calibration as described in the text protocol. Obtain a crude zero-gap distance by bringing the MFP head over a chamber slide without cells and slowly descending in five-micron steps.

Upon probe contact with the substrate, Newton's rings should be observed. This is a crude estimate. An accurate position is to be obtained after adjust coplanarity of the probe apex to the substrate.

When formed, ensure that the Newton's rings are symmetric. To ensure coplanarity of the probe apex, adjust the tilt of the head using a goniometer at the interface of the head and the Z-stage. Move the MFP head 20 microns away from the substrate and adjust the tilt using the goniometer.

Repeat the descent, zeroing, and tilt adjustment until the Newton's rings are symmetric on contact. With the tilt adjusted, set the Z position, which produces symmetric Newton's rings, at zero. Next, prepare the processing liquid for the inner injection channel and the extraction buffer solution required for the outer injection, as described in the text protocol.

Using the drain line connect to the syringe pumps, purge the remaining water and aspirate the degassed solutions into the injection syringes at 40 microliters per minute until the syringes are full. This ensures bubble-free filling of the syringes, connectors, and capillaries. To prepare the cell monolayers on chamber slides, expend cells in T-flasks using standard cell-culture protocols.

On reaching cell confluence in the culture flasks, trypsinize and collect cells. Seed 0.2 million cells per square centimeter in each of the two-chamber slides for patterning. Culture the cells over 48 hours using the cells in one of the chambers as a control for cell growth and viability.

On reaching cell confluence on the chamber slides, incubate the cells for 45 minutes with 500 microliters of cell-tracker dye solution at a 10 micromolar concentration prepared in serum-free medium. This is done for cell visualization during patterning. Wash the labeled cells with PBS by gently flushing each chamber using a pipette.

Subsequently, culture the cells in serum-supplemented medium for the patterning experiments. The hydrodynamic flow confinement localizes liquids using simultaneous injection and aspiration of a processing liquid. In the hierarchical confinement, multiple processing liquids are confined, with the outer HFC shielding the sample from the inner-processing HFC.

Move the MFP over the cell monolayer to a gap distance of 50 microns from the glass slide. This gap distance ensures contact of the hierarchical HFC and also accounts for monolayer surface and thickness variation. Injection sodium hydroxide at six or eight microliters per minute through I-two.

Evaluate other flow rates using the flow rules in the text protocol. Modulate the size of the inner HFC by changing the ratio of injection flow rates QI-two and QI-one using the injection syringes. For example, use a QI-one between 1.3 microliters per minute and four microliters per minute, with the QI-two fixed at eight microliters per minute, to result in a sodium hydroxide footprint of 150 to 300 cells.

Inject complete medium from the outer-most apertures on the MFP head at a flow rate of 20 microliters per minute to account for evaporation of the medium and aspiration during the operation of the hHFC. To pattern the cell monolayers, set the stage software to scan the probe head over the cell monolayer in user-defined patterns at a scan velocity of 10 microns per second and a gap distance of 50 microns. With the nested hHFC in operation and in contact with the monolayer, scan the MFP with the trajectory of the desired pattern to effect patterned cell removal.

For a coculture after the first cell-type removal, seed a different cell line to fill the gaps, as before. Prepare the sampling station for downstream analysis of the lysate. Shown here is an example of a sampling station, which comprises a three-D printed eight-strip PCR holder.

Wipe the tube holder with 70%ethanol or other surface decontaminants, based on the stringency desired for the application. Use a magnetic clip on the tube holder to attach it onto the substrate holder. After preparing the sampling station, position the MFP head 100 microns from the monolayer.

Begin operating the hHFC with 50 millimolar sodium hydroxide solution as the processing liquid for the inner injection channel with a flow rate of one microliter per minute for QI-one, six microliters per minute for QI-two, minus-seven microliters per minute for QA-one, and minus-17.5 microliters per minute for QA-two. Once the flow confinement has stabilized, descend the probe head to a gap distance of 50 microns to perform sodium-hydroxide-based local lysis at the chosen subpopulation of cells with the hHFC. Once lysis of the subpopulation is complete, direct the head towards the tubes in the sampling station.

Eject the collected lysate into the PCR tubes directly. After processing the lysate, as described in the text protocol, load the lysate into a standard qPCR workflow as set by the supplier of the instrument. By controlling the ratio of the injection liquids in the hierarchical HFC, the size of the footprint in contact with the cell surface is modulated.

The flow rates were chosen, such that chemical effect, rather than shear, is the dominant mechanism for cell lysis. This was corroborated by determining the shear using computational fluid dynamics. Shown here is the generation of a grid of cell islands by scanning trajectory programming with the MFP.

Modulation of footprint size using the injection liquid ratio allows control of the resolution of cell removal. This method can be used for the sequential coculture of multiple cell populations, therefore generating complex patterns of cell-to-cell interactions. The use of sodium hydroxide as a processing liquid makes the lysate compatible with PCR-based analysis of the cells.

Here, the DNA content was quantified from five and one footprint, lysed using the described method. Once mastered, this technique can be performed on cells in less than 30 minutes. While attempting this procedure, it's important to remember to make sure that the channels and the capillaries are free of air bubbles, as this may adversely affect the HFC.

Don't forget that working with sodium hydroxide, a corrosive chemical solution, can be extremely hazardous, and precautions such as safety glasses and lab coats should always be taken while performing this procedure. Following this procedure, we can locally analyze cells by immunostaining or DNA-RNA sequencing by MFP-mediated lysis in order to answer additional questions, like which proteins are over-expressed due to geometric confinement of cells. After its development, this technique can pave the way for researchers in the field of molecular biology to explore inter-and extracellular signaling during tissue generation and degeneration.

After watching this video, you should have a good understanding on how to subtractively pattern cell monolayers set up in MFP for local processing and make complex geometries of cocultures of cells.