Custom Engineered Tissue Culture Molds from Laser-etched Masters

This method can facilitate research in the tissue-engineering field related to tissue development, cell alignment, and mechanical analysis of polymers and engineered tissues. The main advantage of this technique is that it is low-cost, easy to use, and fast. We developed this technique in my lab because geometric control of engineered tissues on the millimeter-to-centimeter scale was not being done in the field, and we saw a critical need to think translationally about scaling up the size of engineered tissues.

This technique allows us to leverage the benefits of geometric queues on cellular physiology and engineered tissues through fabrication techniques using posts and a variety of shapes. This method can provide insight into the production of engineered tissues with controlled size, shape, and aligned. It can also be applied to various disease models, personalized regenerative medicine, and for the translation of engineered tissues to preclinical studies.

Begin by opening a vector graphics program on a computer. To prepare the desired mold geometry and vector format, open a new canvas of appropriate dimensions with the RGB color format. Then, select the shape tool from the left-hand panel and create the desired geometry by entering the dimensions at the top of the window to precisely define shape sizes.

Design the mold geometries to allow for at least a six-millimeter border from the edge of the outermost features and the cutting line to permit trimming of the PDMS meniscus following mold casting. This will allow the finished mold to lay flush with the bottom of the culture dish. Next, open the color picker in the top left-hand corner of the window and define new color swatches that are compatible with the laser cutter software.

Select each path and assign it an appropriate color from the cut swatch. When finished, assign None for the fill color. Similarly, assign swatch colors to etching paths by selecting the object and then selecting the etching path for the fill color and None for the path color from the color picker.

Save designs as either ai or. pdf file formats, depending on which is appropriate for the vector graphics program and laser cutter. Prepare the laser cutter for cutting according to the manufacturer's specification and ensure that sufficient ventilation is used.

Then place a 1/4th-inch sheet of acrylic onto the laser bed. Adjust the bed height so that it is properly calibrated to the acrylic. Next, open the design file on the computer connected to the laser cutter and open the Print menu.

Ensure that the laser cutter is set as the printer and that Do Not Scale is selected in the Scaling dropdown menu to prevent distortion of the design. Then, click the Print button at the bottom of the Printing dialog. In the laser cutter printing utility, click the setting button to set cut etch parameters for each of the design features.

Here, set the power, speed, and pulses per inch parameters for each of the previously chosen colors. Once the settings have been entered, click the large green button in the laser cutter software. This will cause the laser cutter to begin to etch and cut the negative master molds.

Finally, use a small brush or compressed air to remove any residual debris from the negative master molds. This will prepare the surface for PDMS casting. Prepare a PDMS casting mix according to the manufacturer's specifications.

Degas the prepared PDMS in a vacuum chamber connected to a standard line vacuum lab for one hour or until all bubbles have been eliminated. While the PDMS is being degassed, take the outer edge of the mold with vinyl or masking tape, so the tape extends at least three millimeters above the etched face of the negative master mold. Be sure to press the tape firmly against the side of the mold so prevent leaks later on.

Next, pour approximately 0.35 milliliters per centimeter squared of PDMS into the master mold so the PDMS reaches a minimum thickness of 1.5 millimeters. Place the PDMS-coated negative master mold into a vacuum chamber and degas again for one hour or until all bubbles have been eliminated. Then, place the degassed PDMS-coated negative master mold onto a level shelf in a 60 degrees Celsius oven for at least six hours to cure the PDMS.

Once cured, use a razor blade to cut the individual molds apart while they are still on the negative master mold. Then, slowly and carefully peel each PDMS mold off of the negative master mold. Trim off any regions with a meniscus from casting, which would prevent the mold from lying flat, as well as any excess material or debris.

Finally, autoclave the molds using a standard autoclave cycle to sterilize them for tissue culture. To begin, adhere molds to untreated plastic plates. Prepare the fibrinogen, thrombin, and neutralized collagen solutions and harvest the desired cells as described in the accompanying text protocol.

Combine the fibrinogen, neutralized collagen, and cell suspension to create a casting mix. Keep the casting mix on ice. Next, use a handheld high frequency generator to treat the surfaces of the mold that will be exposed to the casting mix to mitigate PDMS hydrophobicity.

Expose the surfaces to the plasma treatment to three to five seconds about five minutes before casting the material. Immediately prior to casting the protein cell mixture, add the thrombin to the casting mix and pipette up and down gently without introducing any bubbles. Working quickly, pipette the casting mix into the molds using care to deposit the mix into all corners and crevices of the mold.

Avoid ejecting the mix beyond the first stop of the pipette to prevent bubble formation inside the constructs. Then, place the samples into a humidified 37 degrees Celsius incubator for 45 minutes. After 45 minutes, return the constructs to the hood and cover them with cell media before returning to the incubator.

Change the cell media every 48 to 72 hours as needed for the cell and construct type. Finally, follow along in the accompanying text protocol to quantity the tissue compaction and tensile properties of the constructs, as well as to prepare various staining techniques and to measure cell alignment. Over time and culture, cellularized constructs compact due to matrix remodeling.

This occurs through both reorganization and degradation of the surrounding matrix and is typically associated with an increase in mechanical stiffness. Directed compaction can also induce stress fields within the tissue, which can be manipulated in cellularized constructs to encourage cell alignment. These two images were taken from cardiac tissue constructs that were grown in three millimeter by 17 millimeter rectangular molds with posts at either end.

And these two images are cardiac tissue constructs that were grown unconstrained. The two constructs that were constrained were only able to compact in the remaining two directions and led to stress-induced cellular alignment. Once mastered, this technique can be used to design, fabricate, and implement custom tissue molds in less than one day.

Following tissue formation, other methods like mechanical testing and histology can be performed to further understand the effects of alignment on cell morphology and tissue function. After watching this video, you should have a good understanding of how to fabricate custom PDMS tissue molds from laser-cut acrylic masters.