The overall goal of this procedure is to produce biomimetic cell culture substrata to engineer the structure and function of macroscopic cardiac tissues. This is accomplished by first fabricating a silicone master with the desired topographic architecture by deep reactive ion etching. The second step is to transfer the desired topographic pattern from the silicon master to a PUA mold using UV assisted capillary force lithography.
Next, the PUA mold is used as a template to fabricate cell culture substrata out of various polymeric materials depending on the desired application via UV assisted or solvent mediated capillary force lithography. The final step is to plate either primary or stem cell derived cardiomyocytes onto the nano pattern cell culture substrata for subsequent analysis. Ultimately, bright field and immunofluorescence microscopy are used to show changes in cell morphology and contractual protein arrangement within the cardiomyocytes.
The main advantage of this technique over existing methods, such as micro contact printing, is that capillary force lithography produces nano pattern cell culture substrata that can induce structural alignment of cardiac tissue monolayers without relying on cell proliferation or migration. Although this method can be used to provide insight into the structure function relationship of cardiac tissues, it can also be applied to other systems such as cancer mechanobiology. To study the impact of matrix topography on cell migration Be, begin this procedure by cleaning the silicon master surface with 100%ethanol or xylene and drying under oxygen nitrogen gas.
Next, place the silicon master pattern side up in a Petri dish for a silicon master with a two centimeter by two centimeters surface pattern pipette 40 microliters of PUA to it. Then place a sheet of four centimeters by four centimeters transparent polyester film over the dispensed PUA press down on the PET sheet and spread the PUA underneath the sheet across the pattern face using a roller or flat edge surface so that the entire pattern is covered by the PUA pre polymer. Then place the silicone master pre polymer and PET approximately 10 centimeters below a 20 watt UV light for 50 seconds.
After curing, remove the PET film slowly with forceps. PUA should attach to the PET film with a negative of the Silicon Master nano pattern. Subsequently cure the P-U-A-P-E-T nano patterns under UV for at least 12 hours prior to use.
To clean the Silicon Masters place another film of PET on top of the master without the addition of PUA. Expose it to the UV light for 50 seconds and remove the PET film. Then rinse the silicon master with 100%ethanol or xylene and dry under oxygen nitrogen gas.
In this procedure, treat 18 millimeter diameter circular glass slides by placing them in an ozone chamber for 10 minutes. Next, place the slides on blocks of PDMS to temporarily secure them for easy handling. Next, apply a layer of glass primer to the surface of the glass slide.
Allow the primer to air dry for 30 minutes. Now place the glass slides on a piece of printer paper. Drop 10 microliters of polyurethane pre polymer to the center of each glass slide and make sure no bubbles are generated.
Then place the PUA mold pattern face down onto the glass slide disperse the pu pre polymer uniformly across the surface of the glass slide by rolling a rubber cylinder roller along the PUA mold. The printer paper will absorb the polymer overflow afterward UV cure them for 60 seconds under a 20 watt UV lamp. Subsequently, remove the sample from the UV light source and carefully peel the PUA mold from the PU coated glass slide polymerization of the PU is considered complete.
When the PUA mold peels cleanly away from the sample and the PU glass slide has an iridescent appearance, then place the samples in a desiccate for storage for as long as a month. Now attach A NFS cover slips to a 35 millimeter tissue culture polystyrene dish pipette 20 microliters of NOA to the bottom of the dish. Gently place the NPPU cover slip on top of the NOA and allow the glue to spread out and cover the entire cover slip bottom after that cure NOA by exposing the dish to UV for 10 minutes.
Next, sterilize the NP SUBSTRATA by rinsing it twice with two milliliters of 70%aqueous ethanol solution each for five minutes. Remove the ethanol by aspiration. Allow the A NFS to completely air dry for about one hour under the UV sterilization lamp in the biological safety cabinet.
To enhance the cellular adhesion, coat the A NFS in fibronectin overnight by first diluting the fibronectin in di water to five micrograms per milliliter. Next, pipette two milliliters of fibronectin solution into a dish. Then place the A NFS in the incubator at 37 degrees Celsius and 5%CO2 overnight.
Obtain NR VMs, HESC CMS or other cardiac cells of interest, centrifuge the cell sample at 1000 RPM for three minutes to pellet the cells. Carefully remove the supernatant by aspiration and make sure not to disturb the pellet. After that, resuspend the cells in culture media to a concentration of 4.6 times 10 to the six cells per milliliter.
Carefully pipette 200 microliters of cell suspension onto the sterilized A NFS and make sure the cell suspension remains on the cover slip. Next, place the cells in the incubator at 37 degrees Celsius and 5%CO2 for four hours to allow the cells to attach to the A NFS. Then add two milliliters of additional warm culture media to the dish and place the cells in the incubator again under the same conditions.
After 24 hours, remove the media and wash with two milliliters of DPBS twice to remove excess cells. After that, add two milliliters of warm culture media to the dish. Place the cells in an incubator under the same conditions and culture.
The cells to confluence replace media every other day. This figure shows the representative brightfield images of NR VMs after seven days of culture and HESC cms. After 48 hours of culture, NR VMs and HESC CMS on the NP surfaces show obvious structural anisotropy and alignment, whereas cardiomyocytes on UN pattern surfaces are randomly oriented.
Immunohistochemical analysis highlights the impact of the nano topography on cell cytoskeletal alignment, actin micro filaments and alpha sarcomeric actin in cells cultured on the A NFS become aligned along the nano ridges, but remain randomly distributed in cells on un patterned substrata. While attempting this procedure, it's important to remember to sterilize your substrata prior to cell seating as contamination can be a big problem after its development. This technique paved the way for researchers in cardiovascular biology to explore cardiac maturation and stem cell derived cardiomyocytes.
After watching this video, you should have a good understanding of how to produce a biomimetic cell culture substrata via capillary force lithography for cardiac tissue engineering applications.
