The overall goal of this procedure, is to demonstrate a convenient, efficient, and high throughput microfluidic purification technology, to separate free nanoparticles from cells, following cell engineering with the nanoparticles. The major want of this technology is to allow the quick and efficient removal of unbound nanoparticles After the cell laboring procedure, we gather up all our ses-fa-tion. This microfluidic cell purification technology achieves efficient size based separation due to the differential inertial focusing effects of cells and nanoparticles in a spiral micro-device.
It can process up to 10 million cells per mil concentration which is very useful for regenerative medicine, as well as latch volume cell processing. This technology is high desired for cell engineering field, which often using nanoparticle to labor cells, for imaging purpose or function control. Demonstrating the procedure will be Hui Min Tay, a research assistant from my laboratory, as well as Doctor David Yeo, a post doctor research fellow from Professor Xi's laboratory.
Culture mesenchymal stem cells to greater than 80%confluency on a six well plate prior to labeling the cells with the nanoparticles. Similarly culture THP-1 cells to a density of one million cells per milliliter. Next, dissolve one milligram of silica nanoparticles with one milliliter of two hundred micromolar calcium dye solution.
And stir the particles overnight. Also, fabricate PLGA calcium AM nanoparticles using methods described in the reference shown here. Mix either one milligram of the PLGA calcium AM nanoparticles or 150 micrograms of the calcium silica nanoparticles in 01%poly-l-lysine solution in water by pipetting the solution up and down for one minute.
And then let it incubate at room temperature for 15 to 20 minutes. Next centrifuge the nanoparticles. Remove the excess poly-l-lysine supernatant and re-suspend the nanoparticles in one milliliter of the appropriate culture medium for the cell type to be labelled.
Add 1 milligrams of the labelled nanoparticles to one milliliter of medium for every one million cells in culture. Pipette the mixture of cells, nanoparticles, and medium thoroughly for one minute. And then incubate the mixture for approximately 24 hours.
Once labelled, dissociate and harvest the stem cells, and re-suspend them to a concentration of between 100, 000 and 1, 000, 000 cells per milliliter for microfluidic processing. Use labelled THP-1 cells at the same concentration. In order to fabricate the microfluidic spiral device, first prepare a silicone wafer master mold using standard microfabrication techniques.
Then, mix 30 grams of the base PDMS prepolymer and three grams of a curing agent thoroughly in a weighing boat. Degas the mixture under vacuum for 60 minutes to remove any air bubbles. Pour the PDMS mixture onto the master mold, patterned with the spiral channel design, carefully to a height of five to 10 millimeters.
Then degas the mixture for 60 minutes to remove any air bubbles. Repeat this process until all bubbles are eliminated. Next, place the wafer in an oven, and ensure that the wafer is not tilted during curing, to ensure a constant device height.
Cure the PDMS at 80 degrees Celsius for two hours. Once cool, cut out the PDMS spiral device using a scalpel and carefully peel the PDMS slab from the master mold. Then, use a scalpel to trim the edges of the device to ensure a smooth surface for bonding.
Next, use a 1.5 millimeter biopsy punch to create two holes for the inlets, and two holes for the outlets. Wash the device with isopropanol to remove any debris. And dry the device in an 80 degree Celsius oven for five minutes.
Then, use masking tape to clean the bottom surface of the PDMS device, and one side of a glass slide. Carefully place the glass slide and PDMS device into a plasma chamber with the clean surfaces exposed. Switch on the plasma power to maximum, lower the chamber pressure until the chamber turns pink in color, and expose the components for 60 seconds.
Then, remove the slide and PDMS device from the chamber. And bond them together by pressing the plasma exposed surfaces tightly together. Ensure no bubbles are trapped between the two surfaces.
Heat the bonded device on a hot plate set at 80 degrees celsius for two hours to strengthen the bond. Cut two pieces of tubing 15 to 20 centimeters in length for the inlets, and attach a 23 gauge needle on one end of each tube. Then, cut two pieces of the same tubing that are five to 10 centimeters in length for the outlets, and attach them to the outlet holes of the device.
Prior to running the sample, manually prime the device with a syringe containing 70%ethanol until it flows from the outlet tubing. Allow the ethanol to sit in the device for at least 30 seconds to sterilize it. Then, load 30 milliliters of filtered PBS with 1%BSA into a 60 milliliter syringe.
Also, load three milliliters of labelled cells into a three milliliter syringe and check that no air bubbles are trapped in either syringe. Remove any air bubbles by gently ejecting a few drops of liquid out of the nozzle. Connect the inlet syringe tips and tubing to the syringes and secure the syringes to the syringe pumps.
Insert the tubes into their respective inlets of the device, and ensure that there are no bubbles along the tubing. With the fluidics now set up, mount the device onto an inverted phase contrast microscope for real time imaging during the cell sorting process. Secure a small waste beaker and two 15 milliliter tubes close to the device on the microscope stage.
Place the outlet tubing from both of the device's outlets into the waste beaker. Now, set the flow ratio for sample to sheath buffer to one to 10 by setting the flow rate of the sample syringe to 120 microliters per minute and the sheath syringe to 1, 200 microliters per minute. Run the device for one and a half minutes to give the flow rate a chance to stabilize.
Use a high speed camera to confirm the presence of inertially focused cells near the channel inner wall under bright field with phase contrast. Once steady state flow is achieved, and the device is running correctly, position the outlet tubes into different vials to collect separate eluents from the cell outlet and waste outlet. This device is able to correctly sort a labelled suspension of THP-1 monocytic cells from fluorescent silica nanoparticles.
High separation efficiencies were obtained as confirmed by both high speed imaging and flow cytometry analysis. The single step purification strategy enables continuous recovery of a labelled suspension and adherent cells that are suspended in fresh buffer solution without the need for centrifugation. The device is capable of high efficiency separation with both silica and PLGA nanoparticles and can separate up to 10 million cells per milliliter with the same high efficiency.
After watching this video, you should have a good understanding of how to label cells with nanoparticles. And how to purify the labelled cells by removing excess unbound particles using our specialized microfluidic device.
