Fabrication of Custom Agarose Wells for Cell Seeding and Tissue Ring Self-assembly Using 3D-Printed Molds

The overall goal of this procedure is to create custom cell seeding molds that are used to make self-assembled tissue rings of various dimensions. This method allows us to create three dimensional tissue rings from human cells that can be used to evaluate tissue structure, strength, and function. The primary advantage of this technique is that it is a simple method for fabricating molds for self assembled tissues with custom dimensions.

To begin the procedure, use computer-aided design software to create a drawing of a mold in the desired dimensions. Send the CAD file to a high resolution 3D printer. Print the mold in a plastic with a glossy finish that is stable at 50 degrees celsius.

Thoroughly wash the printed mold with a brush, detergent, and water. Rinse the mold with distilled water, and let it air dry in a rack. It is important to choose a 3D printed plastic that is stable at the PDMS curing temperature.

Residues that transfer onto PDMS during heating may affect ring formation. Next, measure 25 grams of PDMS base in a disposable weigh boat. Add curing agent in a 1:10 ratio by weight and stir vigorously until the base and curing agent are thoroughly mixed.

Then, fix laboratory tape around the sides of the mold to form a wall about one centimeter high around the face with the printed wells. Ensure that there are no gaps in the tape wall. Pour the PDMS mixture into the 3D printed mold.

De-gas the PDMS in a vacuum chamber for 30 to 60 minutes or until no bubbles remain. Cure the PDMS at 50 degrees celsius for two to four hours or until the PDMS is sufficiently solid to be removed from the mold. Then, remove the tape, and carefully separate the PDMS negative from the mold.

Incubate the PDMS negative in an oven at 60 degrees celsius for one hour to ensure that it is fully cured. Then, thoroughly wash the negative with detergent and water. Rinse the mold with distilled water and let it air dry.

Prior to fabricating the agarose mold, autoclave the PDMS negative, a pair of blunt forceps, and any other desired tools. To begin fabrication, prepare and autoclave at least 50 milliliters of two percent agarose in DMEM. Prepare agarose wells one day prior to ring seeding.

Pipette four milliliters of molten agarose into the autoclaved PDMS negative, being careful not to overfill the mold. Then, pipette molten agarose into the cavities for the agarose well posts. Use the pipette tip to remove all air bubbles.

The final agarose surface must be flat. Allow the agarose to cool for about 10 minutes. Then, use blunt forceps to carefully separate the cooled agarose mold from the PDMS negative.

Place the mold face up in one well of a six well plate. Add the appropriate complete culture medium to the plate well so that the agarose wells are completely submerged. Equilibrate the agarose wells at 37 degrees celsius over night before use.

First, in a 15 centimeter culture dish, culture Rat Aortic Smooth Muscle Cells at 37 degrees celsius in a 5 percent carbon dioxide atmosphere until 70 percent confluence is achieved. Once the desired agarose mold has been prepared and equilibrated over night, rinse the culture dish twice with five milliliters of phosphate-buffered saline. After removing PBS, add three milliliters of 0.25 percent tripzin to the dish.

Incubate at 37 degrees celsius for two to three minutes or until the cells have lifted from the dish. Neutralize the tripzin with three milliliters of the complete culture medium. Transfer the cells to a conical tube, and gently pipette the cells until thoroughly resuspended.

Dilute an aliquat of the cell suspension with an equal volume of trypan blue dye, and count the cells with a hemocytometer. Then, centrifuge the suspension at 200 times G for five minutes. Remove the supernatant and resuspend the cells in complete culture medium to achieve a concentration of 10 million cells per milliliter.

The most important steps to ensure consistent ring formation are optimizing the cell number and culture medium for the cell line that you are using. Next, obtain the equilibrated agarose wells. Carefully aspirate all medium from both around the outside of the agarose, and within the agarose wells, being careful not to puncture the bottoms of the wells.

Pipette 50 microliters of the cell suspension into each well. Add two milliliters of fresh medium around the outside of the agarose wells without letting the medium spill into the wells. Incubate the plate overnight at 37 degrees celsius in a five percent CO2 atmosphere to allow cells to aggregate.

Then, aspirate the medium from outside the agarose. Add 4.5 milliliters of fresh medium to the plate well so that the agarose wells are filled and the entire agarose mold is completely submerged. Continue incubating the plate until the tissue rings are sufficiently developed, replacing the medium each day.

Then, gently slide each tissue ring from its post using forceps. Immediately, either fix the rings for histology, or use the rings for functional or mechanical assessments. 3D printing allowed more flexible mold design.

The post diameters were easily varied, as shown my rat smooth muscle cell rings fabricated around posts with diameters of two, four, and twelve millimeters. Tissue rings have also been prepared from primary human SMCs human mesenchymal stem cells, and SMCs derived from induced pluripotent stem cells. The rings were used as in vitro tissue models for assessments of tissue function and mechanical strength.

Although this method was originally designed to fabricate and model vascular tissue, it can also be used to fabricate segments of other tissue types as well, such as cartilage, skeletal muscle, and cardiac tissue. This method provides a simple way for researchers to create custom molds to fabricate 3D tissue rings with different dimensions. These rings can be used to assess structure, mechanical properties and function of engineered tissues made from different cell types.