細胞を含んだハイドロゲルの刺激細胞挙動のひずみ勾配チップ

The overall goal of this gradient strain chip is to provide a simple approach to generate non continuous gradient static strains on a 3D hydrogel for the investigation of cell behaviors under a series of strain conditions. This method can help answer key questions in the bioengineering field such as the stimulations of a stem cell differentiation and the regulation of cellular alignment for artificial tissue organ development. The main advantage of this technique is that various strains can be applied on the cell layer and hydrogels in the same micro environment for comparison of cell behavior.

Visual demonstration of this method is critical as the chip preparation steps are difficult to learn since the light should be complete in three to four hours for base of experiment to ensure high cell variability. Weigh 10 grams of gelatin powder and add it to a glass flask with 100 milliliters of Dulbecco's phosphate buffered saline, or DPBS. Put a magnetic stir bar into the flask and place the flask on a stirring hot plate.

Cover the flask with aluminum foil to avoid water evaporation. Set the hot plate temperature to 50 to 60 degrees and the stirrer at 100 RPM for one hour to dissolve the gelatin powder. After the gelatin has dissolved, add eight milliliters of methacrylic anhydride very slowly using a pipette.

Let it react at 60 degrees Celsius for three hours. Then add prewarmed DPBS into the flask to a final volume of 500 milliliters. Allow the solution to mix well for 15 minutes to stop the reaction.

Meanwhile cut a dialysis membrane into several 25 millimeter long tubes. Immerse them in deionized water for 15 minutes. And make a knot to close one end of the dialysis tubes.

Load the appropriate amount of the polymer solution into the dialysis tubes and close the other end. Then place the filled tubes in a five liter plastic beaker with deionized water for a week. Renew the deionized water twice a day and maintain the solution at 40 to 50 degrees Celsius during the dialysis process.

Following dialysis collect the solution from the tubes into a 500 milliliter glass bottle. Pour approximately 450 to 500 milliliters of solution into a 500 milliliter filter cup. And apply a vacuum to force the solution to pass through the filter membrane for sterilization.

Then transfer the sterilized polymer into several 50 milliliter sterilized tubes. Transfer the sterilized polymer into several 50 milliliter sterilized tubes and store them in a minus 80 degrees Celsius freezer for three to five days. Freeze dry the minus 80 degrees Celsius polymer for one week using a freeze drier to form GelMA which can then be stored in a minus 80 degree Celsius freezer.

For chip fabrication first prepare the polymethacrylate or PMMA, played and molded as described in the text protocol. Then prepare the PTMS cover and PTMS plug by properly mixing 30 grams of PTMS elastomer and three grams of PTMS curing agent. Degas the mixture under a vacuum chamber for one hour.

Cast 1.8 to two grams of the mixture into the PMMA mold for the PTMS cover. Also use the appropriate amount to fill each cavity for the PMMA mold for the PTMS plug. In addition cast 10 grams of uncured PTMS mixture in a blank 10 centimeter plastic plate.

Put these molds in a vacuum chamber to degas for 30 minutes. Cure the PTMS for two hours at 80 degrees Celsius. After cooling down the cured PTMS on the mold, detach the PTMS covers and the PTMS plugs from the PMMA molds.

Next punch two holes with diameters of about three millimeters at the ends of the flow channels of the PTMS covers. Cut the PTMS sheet molded from the 10 centimeter plastic plate into many one centimeter by one centimeter cubes. And punch a three millimeter hole in each PTMS cube.

Glue two uncured one centimeter by one centimeter PTMS cubes onto the openings of the PTMS cover to serve as medium reservoirs and to aid in the curing process of the gradient circular hydrogel patterns. Then cure for one hour at 80 degrees Celsius. Next bond the PTMS covers with the two PTMS reservoirs onto a TMSPMA coated slide.

To do so, pretreat the bonding side of the PTMS cover and the TMSPMA coated slide under an oxygen plasma machine for 90 seconds of oxygen plasma treatment. Contact the plasma treated surface of the PTMS covers and the TMSPMA slide and press them closely for permanent bonding through the formation of the silicon oxygen silicon bond. Print a photomask by printing the established layout on a transparent film.

Then cut the printed photomask to 25 millimeters by 37.5 millimeters in size. Weigh 25 milligrams of freeze dried GelMA into 0.3 milliliters of prewarmed cell culture medium in a 1.5 milliliter black microcentrifuge tube. Put the microcentrifuge tube on a laboratory stirrer hot plate until the GelMA dissolves in the medium.

Add 50 milligrams of photoinitiator to one milliliters of DPBS in a microcentrifuge tube. And place it in an 80 degree Celsius oven for 15 minutes or until the photoinitiator has dissolved. Add 25 microliters of the 10%photoinitiator to the microcentrifuge tube.

Pipette several times to mix well. Next count three million NIH3T3 cells using an automated cell counter. After centrifuging the suspension at 200 Gs for five minutes discard the supernatant and resuspend the cells in 175 microliters of cell culture medium.

Add the cell solution to the microcentrifuge tube to get a pre polymer cell solution of 5%GelMA, 0.5%photoinitiator and about six million 3T3 cells per milliliter. After mixing load 100 microliters of cell prepolymer into a 100 microliter microsyringe. Manually align a piece of the photomask onto the bottom slide of the sterilized gradient strain chip.

And simply fix the position using a small drop of deionized water in between. Connect the 100 microliter microsyringe, loaded with prepolymer cell solution to the inlet of the chip. Then place 50 microliters of prepolymer cell solution in the flow channel using the microsyringe and plug the outlet using a PTMS plug.

Inject an extra 40 microliters of solution to create a convex bulge in the circular PTMS membrane. Move the chip with the photomask, microsyringe, and PTMS plug to under a UV lamp. And expose it for 30 to 45 seconds to cross link the concentric circular hydrogel in the fluidic chamber.

After cross linking remove the PTMS plug and the microsyringe to release the liquid pressure. Use a one milliliter syringe loaded with prewarmed DPBS to wash out uncrosslinked resins three times by flushing from the inlet to the outlet. Next fill the flow channel with about 100 microliters of cell culture medium.

Place the chip in a sterilized culture dish and culture in a 5%carbon dioxide atmosphere at 37 degrees Celsius for a week. Refresh the medium every day. Take images of three chips on day zero after four hours of incubation as the control group.

And three chips on day three as the experimental set using a microscope with a 20 times objective. Measure the line width of each hydrogel from line one to line 12 using software to calculate the compress stains. Shown here are representative results of hydrogel patterns with zero microliter and 40 microliters over injection, after UV cross linking and four hour incubation.

The line width of the 3D hydrogel in the non over injection control looks uniform as expected. However the hydrogel patterns with 40 microliters over injection gives a gradient line width of hydrogels decreasing from line one to line 12. After three days of incubation, the cells in the hydrogel extend to different directions.

The cells in line one align along the radial direction due to the stimulation of hydrogel elongation. On the contrary the cells in line 12 align along the hydrogel shape where the geometry cue dominates the cellular alignment. There is no specific cell alignment in line 7 where the two cellular alignment cues of line one and line 12 balance each other out.

Once measuring the micro environment of gradient strains on 3D hydrogel can be done by the gradient strain chip in two hours if it's performed properly. While attempting this procedure, it is important to remember to sterilize the gradient strain chip before loading the cell prepolymer solution. Also thoroughly wipe the surface of the chip with 75%ethanol before placing the chips into the cell culture incubator.

After watching this video you should have a very good understanding of how to generate a gradient strain chip to investigate cell behaviors under different strains imperial.