The high throughput fabrication of monodispersed microgel building blocks for microporous annealed particle scaffolds provides enhanced control of scaffold porosity. This can impact pore size and subsequent tissue integration outcomes. This protocol uses a high throughput microfluidic approach to generate large volumes of monodispersed microgels which cannot be achieved with other methods such as flow focusing microfluidics, batch emulsion, and electrospraying.
The microgels generated can be formed into microporous annealed particle scaffolds which are advantageous for regenerative medicine applications due to the cell scale porosity that allows for fast tissue and growth. Begin by preparing one glass slide per PDMS microfluidic device. Use tape, filtered air, or isopropyl alcohol washes to remove any dust from the slide.
Place a glass slide in a PDMS device design slide up next to each other on a 96-well plate lid and place it in a plasma cleaner. Close the door and airflow valve and turn on the vacuum pump. Let it run for at least 30 seconds, then turn it off.
Connect the oxygen tank gas tube to the airflow valve. Let the plasma chamber fill with oxygen for 30 seconds, then turn off the oxygen and close the airflow valve. Turn the vacuum pump on and set the radio frequency level to high.
Wait until the chamber turns a violet pink color and allow 30 seconds to pass. When the timer goes off, turn off the plasma and vacuum, then slowly open the airflow valve to release the vacuum. Remove the tray from the plasma cleaner.
Gently flip the PDMS device onto the glass slide to bond them. As bonding happens, observe the slight difference in the transparency of the PDMS. For the best results, store the bonded devices at 60 degrees Celsius until immediately before use.
Prepare the surface treatment by diluting PFOCTS and Novec oil and use one milliliter for three to four devices. Transfer the volume to a one milliliter syringe and attach a 25 gauge needle. Cut a 10 to 12 centimeter piece of Tygon tubing per device.
Cut a piece of PEEK tubing approximately one inch in length. Insert a couple of millimeters of the PEEK tubing into the end of the Tygon tubing to prevent the needle from puncturing the Tygon tubing inlets. Take the devices out of the heated chamber and insert the non-PEEK end of the Tygon tubing into the aqueous inlet hole.
Insert the needle of the surface treatment syringe into the PEEK tubing and cover the oil chamber outlet hole. Inject the treatment slowly and ensure it fills the device without bubbles. Wait for the aqueous chambers to fill first, followed by the smaller channels and then the oil chamber.
Remove the Tygon tubing from the device. Once the device has been filled, let it rest for 10 minutes at room temperature. Fill a five milliliter syringe with oil only and attach a 25 gauge needle.
Aspirate the surface treatment out of the device through the inlets and outlet. Insert Tygon tubing into the aqueous inlet. Insert the syringe with oil into the PEEK tubing and flush each device with oil.
Aspirate the oil out of the device. Repeat the oil flush twice and remove the Tygon tubing. Insert the non-PEEK end of the Tygon tubing into the inlets of the microfluidic devices.
Insert the remaining piece of Tygon tubing with no PEEK tubing on the end into the microfluidic device outlet. Add at least three milliliters of oil to a five milliliter plastic syringe and attach it to a 25 gauge needle. Carefully insert the needle into the PEEK tubing on one of the Tygon inlets.
Gently flush the tubing and device with oil. Collect the oil from the outlet in a conical tube. Repeat the the oil flush on the other Tygon inlet.
Set the syringe pumps to the desired flow rates. Connect the syringe containing the surfactant to the oil inlet via a 25 gauge needle and gently dispense enough oil to prime the tubing and the oil channel of the microfluidic device. Once the device and oil inlets have been set up, add 0.5 milliliters of oil to a new five milliliter syringe which will contain the gel precursor.
Use this small amount of oil to help flush the precursor solution through the microfluidic device. In a conical tube, combine 1.5 milliliters of the PEG backbone solution and 1.5 milliliters of the cross-linker solution. Vortex for 30 seconds and quickly transfer the combined gel precursor solution to the five milliliter syringe.
Connect the syringe with the gel precursor solution to the aqueous inlet via a 25 gauge needle. Gently dispense enough solution to prime the tubing and the aqueous channel. Clamp the syringes onto the syringe pumps and press run.
Look for particles of uniform size from the channels. Collect the microgels from the outlet in a conical tube. Once gelation is complete, use a pipette to carefully remove the oil phase from the bottom of the tube.
Deposit this into an appropriate waste container for fluorinated waste. Add more oil into the microgel collection tube. Mix by gently inverting the collection tube.
Let the collection tube settle for five minutes to allow the phases to separate. Look for the oil phase at the bottom and the aqueous microgel phase at the top. Repeat the oil washes at least two times more.
Add more oil with the gel as demonstrated earlier, then add PBS to the gel. Invert to mix several times. To separate the layers, centrifuge the tube at 2, 000 RCF for about 30 seconds.
Look for the oil phase at the bottom of the tube, gel in the middle and PBS on top. Remove the oil phase with a pipette and discard it in a waste container. Repeat oil and PBS washes twice.
Look for the gel to transition from opaque to clear by the final wash. Remove all oil. Do not remove PBS from the conical tube.
In a chemical fume hood, use a glass pipette to add hexanes to the tube at an equal volume to the PBS. Vortex the conical tube for 30 seconds or until it is mixed thoroughly. Centrifuge at 4, 696 RCF for five minutes.
After separation, look for hexanes in the top layer, PBS in the middle and gel at the bottom. Remove the hexane layer and discard it in a container for organic waste. Aspirate the PBS.
Repeat hexane in PBS washes at least twice or until the gel appears nearly translucent. Wash the gel with PBS once more to remove any remaining hexane residue. Centrifuge at 4, 696 RCF for five minutes and aspirate the PBS layer.
About 67-75%of the 20 kilodalton PEG maleimide was modified with methacrylamide functional groups to ensure a high annealing efficiency. Percent modification was determined by analyzing 1H NMR spectra peaks. The onset of gelation provided insight into the duration of microfluidic microgel generation.
It is recommended to choose gel precursor pH that can initiate gelation between 30 minutes and two hours. After purification and swelling, the microgels had a uniform size and polydispersity index between 1.00 and 1.02 defined as a monodispersed population. After annealing, the microgels formed a porous scaffold visualized with two-photon microscopy.
Correctly bonding the PDMS device to the glass slide and correctly surface treating the device is crucial for the best microfluidic device performance. This technique allows for quick production of uniform microgel building blocks to form map scaffolds which can be used for a variety of in vivo regenerative applications including wound healing.
