倒胶体晶体聚（乙二醇）脚手架制作：肝脏组织工程三维细胞培养平台

The overall goal of this procedure is to fabricate PEG-based inverted colloidal crystal scaffolds with an extracellular matrix coding for liver tissue engineering applications. Liver tissue engineering is an exciting field for tissue engineering and regenerative medicine. We re creating 3D engineering scaffold that allow liver cell to maintain their function for a long time.

The implications of this technique extends toward drug testing as liver cells cultured with this system may retain their function longer and better response to drug. Though this method can provide insight into tissue engineering, it can also be applied to other systems, such as those using stem cells. Demonstrating the procedure will be Hitomi, a graduate student from my laboratory.

To prepare the micro-bead molds, first cut the tips from 0.2 milliliter boil-proof microcentrifuge tubes at the 40 microliter level. Use waterproof glue to adhere the tops of the cut tubes to glass microscope cover slips and then glue around the molds. Next, replace the water in a vial of polystyrene microspheres with 70%ethanol.

Place the sphere solution in an ultrasonic bath and sonicate the mixture at 30 watts for three minutes to loosen the aggregated spheres. Once the microspheres have been prepared, pipette 100 microliters of ethanol into each of the molds. Then, cut four millimeters off of the top of a 200 microliter pipette tip and use it to pipette 25 microliters of the polystyrene microspheres into the mold twice.

Place the molds on a rocking shaker and set it to 120 RPM. Let the samples sit on the shaker overnight. The next day, place the molds under a microscope and check that the spheres are ordered hexagonally.

Let the ethanol evaporate at room temperature for two nights. At this time, carefully place the mold and bead complex into a 130 degrees Celsius oven for six hours to anneal the polystyrene beads and form the negative of the 3D scaffold. First, synthesize polyethylene glycol diacrylate macromers using established protocols as described in these two references.

Next, prepare a 50%solution of PEGDA in deionized water and centrifuge the mixture until it is completely dissolved. For extracellular matrix conjugated scaffolds add an addition 10%of a krill oil PEG NHS into the 50%PEGDA solution. Then, prepare a 20%stock solution of the photo-initiator in 70%ethanol.

Add 50 microliters of the photo-initiator solution for each milliliter of the PEGDA mixture. Then, vortex the mixture for one minute. Next, peel away the molds from the glass slides, push the lattices out carefully using a spatula and place them into separate 1.5 milliliter centrifuge tubes.

Pipette 300 microliters of the PEGDA solution into each tube, and then centrifuge the tubes to allow the PEGDA solution to infiltrate into the polystyrene lattice. Next, remove the lattice from the tube using tweezers and carefully blot excess PEGDA solution from the surface. Place the lattice on paraffin film covered glass with the flat, circular surface facing up.

Then, expose the scaffold to 365 nanometer ultraviolet light for five minutes using a UV spot lamp. Place around ten PEGDA polymerized crystal lattices into a new vial and add 20 milliliters of THF. Shake the vials on an orbital shaker at 300 RPM for six hours.

Remove the vial every one to two hours and exchange the polymer-containing solvent with fresh THF. After the last rinse, add water to the used THF solution to check if the PS spheres have completely dissolved. If the solution is white, perform additional rinses until the solution runs transparent.

To sterilize the scaffolds, prepare a 50 milliliter microcentrifuge tube with two milliliters of 70%ethanol per scaffolds and place the scaffolds in the tube using a spatula. Allow the scaffolds to soak in ethanol for one hour. After one hour, carefully pour the ethanol out and replace it with an equal volume of sterile PBS.

Then, centrifuge the scaffolds to remove any bubbles. Store the centrifuge scaffolds at four degrees Celsius and remove them every one to two hours to replace the solution with fresh PBS. After a few changes in PBS, remove the scaffolds to be coated in collagen and place them in a tube containing one milliliter of collagen type one per scaffold to be coated.

Next, centrifuge the tube as before, and then, shake the scaffolds at 400 RPM on an orbital shaker for 30 minutes. Store the scaffolds overnight at four degrees Celsius. Culture hepatocarcinoma cells in 100 millimeter cell culture dishes with 10 milliliters of culture medium.

Change the medium every three days until the cell density reaches 75 to 80%of the culture surface. When the cells are ready, carefully place the scaffolds with the flat surface facing upward into a 24-well plate. Then, rinse each scaffold with two milliliters of PBS.

Aspirate the PBS and add two milliliters of growth medium to the scaffolds. After 30 minutes, remove the medium and allow the scaffold to dry for one hour. Next, harvest the hepatocarcinoma cells using the trypsin digestion method.

After pelleting the cells, remove the supernatant and re-suspend the pellet so that there are one million cells per 25 microliters. Slowly pipette 25 microliters of the cell suspension directly on top of each scaffold. Then, cover the plate and place it in the incubator overnight.

Next, transfer the scaffolds carefully into a new 24-well plate and pipette two milliliters of medium into each well before returning the plate to the incubator. A scanning electron microscope of the scaffold is shown here. It is made up of a hexagonal closest packed arrangement of pores that average 100 to 105 microns in diameter which forms interconnects with the neighboring pores that are mostly between 35 and 40 microns in diameter.

When seeded with hepatocarcinoma cells, the cells grown and proliferate on the scaffolds that are pre-coated in collagen type one but do not expand in number on the bare scaffolds. In addition, the hepatocarinoma cells on the collagen-coated scaffolds showed greater cell function in a concentration dependent manner. On day 14, the hepatocarcinoma cells cultured in the 400 microgram per milliliter coated ICC scaffolds secreted more than three times the amount of albumin as those cultured in the bare scaffold.

While attempting this procedure, it is important to remember to make sure the polystyrene spheres in the lattice are arranged properly. Poor arrangement will lead to poor inter-connectivity which can adversely affect cell activity. After watching this video, you should have a good understanding of how to fabricate celciated PEG-based inverted colloidal crystal scaffolds with a coating.

Don't forget that working with tetrahydrofuran can be hazardous and precautions such as operating in a fume hood and wearing gloves and goggles should always be taken while performing this procedure.