Controlling Particle Fraction in Microporous Annealed Particle Scaffolds for 3D Cell Culture

Granular materials, such as MAP scaffolds, are porous injectable materials made from interlinked micro-size hydrogel particles. Controlling particle fraction is critical to material properties and cellular responses. Variable particle fractions in MAP scaffolds result in a range of scaffold properties and cell responses, but this technique allows users to define the final particle fraction in the MAP scaffolds they create.

MAP scaffolds can be used to promote the repair and regeneration of complex wounds, as well to deliver drugs. Particle fraction, for example, affects the rate and degree of cellular infiltration and regeneration. This method can be applied across all classes of granular hydrogels.

Now that we can make MAP scaffolds with user-controlled particle fractions, we can reproducibly study the bioactivity of these materials and investigate unique cell responses, such as confinement. Setting up the microfluidic microgel generation requires time and patience as you learn the techniques. It's best to have multiple devices prepared and at the ready in case you run into issues with the setup.

Begin with microfluidic production of Hyaluronic Acid, or HA, and Norbornene, or NB, microgels. Prior to drying the microgels, weigh a cryo-safe screw-cap tube. Then in a sterile hood, transfer the washed and purified microgels to a cryo-safe screw-cap tube using a positive displacement pipette.

Add 70%ethanol to the purified microgels and mix well. Centrifuge the tube for five minutes at 5, 000 g. Aspirate the supernatant, and add 70%ethanol.

Mix well and incubate overnight at four degrees Celsius. The next day, briefly centrifuge to ensure the microgels are at the bottom of the tube. Add liquid nitrogen to a cryogenic container, and place the tube of microgels to flash freeze.

After five to 10 minutes, remove the tube of microgels with forceps. Quickly remove the cap, and cover the tube with lab-grade tissue. Secure the tissue with a rubber band, and transfer it to a lyophilization container, or chamber.

Load the sample on the lyophilizer following the manufacturer's instructions, and lyophilize at 0.066 Torr and minus 63 degrees Celsius. Weigh the lyophilized microgels from the tube before storing them at room temperature. Mix polydimethylsiloxane, or PDMS elastomer base with the curing agent at a 10 to one ratio by mass.

Pour the PDMS mixture into a large plastic Petri dish and degas in the desiccator for 30 minutes, or until all the bubbles have disappeared. Once all the bubbles have disappeared, carefully place the 3D-printed mold into the PDMS to minimize the formation of new bubbles. Place in the oven at 60 degrees Celsius for two hours to cure the PDMS.

After curing, use a knife, or razor blade to gently trace around the perimeter of the culture device, and carefully remove the mold. Use a four-millimeter biopsy punch to remove any PDMS from the bottom of the wells. Cut the devices to fit on a glass cover slip.

Use tape to remove dust from the bottom side of the culture device. Place the clean glass cover slip and culture device on a hot plate at 135 degrees Celsius for 15 minutes to remove moisture. In a fume hood, use a corona plasma gun high on the glass cover slip and the bottom side of the device for 30 seconds.

Then quickly bond the treated surfaces together. Gently apply pressure to ensure a good seal between the culture device and glass cover slip. Place the device in a 60 degree Celsius oven overnight to secure the bond.

The following day, autoclave the device to sterilize before use in vitro. Next, prepare the Microporous Annealed Particle, or MAP scaffold components, based on the desired particle fraction for cell culture in MAP scaffolds. Weigh lyophilized microgels to determine the mass of microgels.

Then reconstitute the microgels in 84%of the final MAP volume of cell media based on the chosen weight percent MAP. Allow the microgels to swell for 20 minutes. Dissolve the Hyaluronic Acid, or HA-tetrazine, and cell media in 16%of the final MAP volume.

Once cells have reached the desired confluency, lift and count the cells. Transfer 10, 000 cells per microliter MAP to a new tube. Centrifuge the cells, and aspirate the supernatant from the pellet without aspirating the cells.

Add microgels and cross-linker to the pellet using a displacement pipette. Mix well, and seed 10 microliters per well. Pipette in a circular motion to evenly distribute the mixture in the well.

Allow the microgels to anneal at 37 degrees Celsius for 25 minutes before adding cell media to fill the wells. Maintain the 3D cultures at 37 degrees Celsius, and change media as needed. To avoid aspirating the scaffold, stabilize the pipette tip along the ridge of the upper well.

At the desired time points, fix samples by replacing media with 50 microliters of 4%paraformaldehyde per well for 30 minutes at room temperature. Wash the samples three times with 50 microliters of PBS, or preferred buffer, and proceed for immunofluorescence, or fluorescence staining using 50 microliters per well as the working volume. Microfluidic devices with a flow-focusing region were shown to produce HA-NB microgels of either 50, or 100 microns in diameter.

The medium for lyophilizing the microgels was optimized to minimize cryogel formation by using 70%ethanol. However, other media, such as isopropyl alcohol, water, and acetonitrile can be used interchangeably to facilitate cryogel formation. To facilitate bio-orthogonal interlinking of HA-NB microgels a linear HA-tetrazine cross-linker was synthesized.

Proton NMR spectroscopy shows a successful modification of 11%of HA repeat units with a tetrazine pendant group. The dried lyophilized microgel product was rehydrated with special volumes and annealed to achieve different weight percent formulations of microgels in MAP scaffolds. These weight percentages of microgels in MAP scaffolds corresponded to unique particle fractions.

Cell culture in MAP scaffolds is shown here. Cells in 3D culture in MAP scaffolds were fixed on day five, stained and imaged on a confocal microscope. Single Z-slices show differences in cell growth and scaffolds comprising different weight percent MAP.

Select your lyophilization media appropriately depending on whether you want cryogels, or not. Do not skip incubation steps, so that your microgels have sufficient time to swell in the media. Standard techniques such as microscopy, flow cytometry, RNA sequencing, and more can be used to assess cell responses within these granular materials with defined porosities.