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Applications utilizing cells encapsulated in small picoliter size drops have often been limited by the inability to control the number of cells per drop. This protocol demonstrates a method to control cell encapsulation by combining two distinct microfluidic phenomena, fluid dynamic cell ordering and drop generation at a flow focusing micro nozzle. In an upstream channel, a sufficiently high flow rate of aqueous solution with suspended cells or particles is provided to cause the formation of trains with equal spacing in the direction of flow downstream.

A flow focusing drop generation nozzle is used to form aqueous drops at kilohertz rates in a surfactant stabilized admissible oil carrier fluid. The ordered cell particle trains are then combined with drop generation by tuning the aqueous and oil flow rates to provide drop generation rates that are synchronized with the arrival of longitudinally ordered cells or particles at the drop generation nozzle, while particles are used as cell surrogates. To demonstrate this protocol flow rates must be sufficiently small as to limit fluid.

Sheer stresses on biological cells, the overlapping of the cell ordering drop generation and cell viability. Aqueous flow rate constraints provides an ideal operational regime for controlled encapsulation of single and multiple cells. Analysis of video microscopy data show that both single and double cell or particle encapsulation efficiencies outperform random encapsulation efficiencies and greatly reduce the number of drops, which do not contain the desired number of cells or particles.

Confinement of cells and drops limits the dilution of cell secretions, highlight cell heterogeneity and also controls intercellular signals when compared to a bulk suspension. Efficient means for encapsulating these cells into drops provides a valuable tool for biological researchers To begin this procedure. Design a microchannel pattern as shown in this figure in AutoCAD software.

The long channel section represents the aqueous flow ordering channel with a width of 27 microns. The enlarged nozzle schematic shows equal channel widths of 27 microns for the aqueous ordering channel and the oil channel, followed by the nozzle contraction of 22 microns and sudden expansion to a wider 61 micron channel. The device height is 52 microns.

Both aqueous and oil inlets have large debris filters with gaps on the order of the ordering channel width as illustrated by this enlarged schematic of the oil inlet.Shown. Here is a true image of the ordering channel and nozzle injected with dye. Employ a third party manufacturer to print a high resolution transparency mask on Mylar film or quartz where channels are transparent on a dark background.

Subsequently create a silicon and SU eight photoresist master for replica molding. Tape the master mold onto the bottom of a Petri dish for PDMS replica molding. After replica molding is complete and the device outline has been cut out, use a 0.75 millimeter outer diameter biopsy punch to punch fluidic ports in the three round regions shown in this figure a dear scotch tape to the pattern side of the PDMS and peel to remove any dust after plasma bonding.

The PDMS to a clean glass microscope slide. Place the entire device into an oven and warm the oven up gradually to 120 degrees Celsius. Leave the device in the 120 degrees Celsius oven overnight to complete bonding and to return the PDMS to its original hydrophobic state.

An alternative method for making the glass surface of the channel hydrophobic is to inject a coating such as aquae into the fluidic ports and then purge with air. Using a one milliliter syringe and a syringe needle draw in several hundred microliters of air, followed by a tiny amount of aquil enough to fill only the metal syringe Tip carefully, but firmly inject the aqua followed by the purging air into the fluidic ports. Without breaking the PDMS to glass bond aggressively.

Repeat the air purge on all inlet and outlook ports while wiping off any excess aqua in order to avoid any deposits that may clog the channels upon drying. Controlling the cell concentration is essential to achieving the proper number of cells to the proper number of drops. This has two challenging components.

The first is estimating the proper concentration beforehand, and the second is maintaining that concentration during the experiment For the particular device used in this study, eight to 15 micron cells or particles should adequately order for controlled encapsulation. In this demonstration, 9.9 micron polys diary microspheres will be used as cell surrogates to prepare the aqueous particle suspension concentration to achieve ideal audit encapsulation, start with a microsphere stock concentration of 1%solids by weight using previous data for full ordering. As a guide, increase the concentration to 1.5%solids by gently centrifusion one milliliter of the stock sample, removing 250 microliters of supinate liquid and Resus suspending the particles by vortex mixing or gentler mixing.

When using cells, both cells and polystyrene particles have a specific gravity greater than one, although not shown in this video. For long-term experiments lasting on the order of many minutes to hours, the solution may be buoyancy matched by adding a solute such as calcium chloride for particles or opti prep for cells. After the cell or particle suspension is ready, prepare a 10 milliliter sample of the continuous fluorocarbon oil phase in a 15 milliliter centrifuge tube vortex.

Mix the surfactant and fluorocarbon oil to approximately 2.5%weight in weight. Here we obtain a 2.4 weight in weight mixture, which is acceptable to set up the micro encapsulation experiment. First, turn on the power for the inverted optical microscope and high speed camera.

Focus on the microchannel layer and inspect the channels for clogs and any debris that could become dislodged. Select a channel free of large debris or obvious clogs. Cut three lengths of translucent tigon PVC tubing for the aqueous inlet oil inlet and emulsion outlet to minimize dead volume.

Cut just enough tubing to reach from the syringe pumps to the microscope. Stage cut tubing ends at a 45 degree angle To facilitate insertion into the fluidic ports, use tweezers to press fit the tube ends into the fluidic ports. Then press fit a 30 gauge blunt tip stainless steel syringe needle into the free end of the aqueous inlet tube.

Repeat for the oil inlet tube. Move the device and attach tubing to the microscope stage using a 20 times objective align and focus on the device nozzle. Manually adjust the microscope for Kohler illumination to provide optimal illumination at the focal plane.

Using the high speed camera software crop, the field of view around the nozzle to allow for higher frame rates and then set the frame rate, exposure time, and other camera settings as required for optimal recording. Next, fill a one milliliter syringe with the previously prepared well mixed aqueous phase solution. Fill a three milliliter syringe with the oil phase solution.

Tilt one of the filled syringes vertically and flick to move air bubbles to the syringe outlet. Slowly depress the plunger long enough to push the air to the syringe tip holding the syringe vertically. Connect the syringe to the respective syringe needle already attached to the device.

Depress the plunger to force the air through the syringe needle dead volume until fluid is pushed through the tubing almost to the device securely mount the syringe to a syringe pump and engage the plunger block. Repeat connections for the second syringe and mount it to a second syringe pump power on each syringe pump and program each pump. Using the manufacturer's protocols, set the initial flow rates to 50 microliters per minute for the oil phase and five microliters per minute.

For the aqueous phase start, the pumps wait for each fluid to enter the device and fill the channels pushing out remaining dead air. This may take several minutes using the initial flow rates. Observe the formation of drops at the nozzle.

Slowly increase the aqueous flow rate to observe ordering of particles in the long aqueous solution channel. As the flow rate increases do not increase the flow rate to the point where jetting of the aqueous fluid stream is triggered at the nozzle as shown in this example, using a different drop generation nozzle note, the unsteady flow and inconsistent drop is here. If the particle concentration is too low to provide alternating trains with relatively few missing particles and the sample was not buoyancy matched, physically tilt the syringe pump towards the syringe outlet to provide gradual settling of particles towards the syringe outlets.

Once adequate ordering occurs, adjust the oil flow rate to tune the generation frequency and size of drops. Iteratively adjust both flow rates to achieve desired encapsulation rates and drop volumes. Once stable ordered encapsulation is confirmed, move the outlet tubing from the waste reservoir into a collection reservoir for future use.

Single and double particle or cell encapsulation can be achieved at high efficiency. Utilizing this protocol shown here is a representative result of single particle encapsulation with an oil flow rate of 60 microliters per minute and an aqueous flow rate of nine microliters per minute. Lambda, the average number of particles for drop is 0.95.

Particle spacing in the direction of flow is about 17 to 18 microns for fully ordered alternating particles. The drop generation rate in this example is 6.1 kilohertz with an average drop size of 24.4 picoliters. The histogram compares the drop encapsulation particle efficiency of order of single particle encapsulation with plus.

On statistics, the random encapsulation for a sample size of 517 drops, the average fraction of drops containing one particle is 79.5%as opposed to a predicted random encapsulation fraction of 36.7%The fraction of particles that end up in the correctly encapsulated drops was observed to be 83.7%for a sample size of 491 particles, while the random encapsulation fraction was 37.3%Double particle encapsulation is achieved simply by reducing the oil flow rate to 30 microliters per minute while maintaining the aqueous flow rate at nine microliters per minute. Here lambda, the average number of particles per drop is 1.8. Similar to single particle encapsulation.

Particle spacing in the direction of flow is about 17 to 18 microns for fully ordered alternating particles. The larger drops have an average drop size of 39.8 picoliters and have formed at a rate of 3.8 kilohertz compared to random encapsulation. For a sample size of 383 drops, the average fraction of ordered encapsulation drops containing two particles is 71.5%as opposed to a predicted random encapsulation fraction of 26.8%The fraction of particles that tended up in the correctly encapsulated drops was observed to be 79.5%for a sample size of 689 particles, while the random encapsulation fraction was 33.4%Taken.

Together, these results show that both single and double particle encapsulation efficiencies outperform random encapsulation efficiencies by over a factor of two and greatly reduce the number of drops, which do not contain the desired number of cells or particles. The importance of proper particle or cell concentrations for high encapsulation efficiency is illustrated in this experimental run. While the oil and aqueous flow rates are the same as in the double particle encapsulation run shown previously, the average number of cells per drop lambda is decreased to 1.57.

Consequently, full ordering does not occur, and thus holes in the trains emerge leaving sun drops with fewer than anticipated particles. This histogram shows the decreased efficiency for two particle encapsulation due to the lower value of Lambda. For a sample size of 324 drops, the average fraction of drops containing two particles was 55.9%with nearly as many single particle drops as double particle drops.

This indicates that Lambda should be equal or close to the number of desired cells per drop to maximize correctly encapsulated particles or cells with the exact value of lambda chosen, according to whether fewer or more cells per drop is tolerable. In this demonstration, we exploit buoyancy mismatches to enable real-time control of the concentration during the experiment. However, for longer term experiments, it may be advisable to buoyancy match for more consistent results following the encapsulation of cells into aqueous drops.

Time dependent experiments may be performed in a centrifuge tube or under a microscope by re-injecting the drops into microfluidic arrays.