Generation of Multicue Cellular Microenvironments by UV-Photopatterning of Three-Dimensional Cell Culture Substrates

The influence of microenvironmental factors on cell behavior is often studied using oversimplified in vitro platforms, that isolate single cues. Our approach creates cell culture substrates that combine multiple of these cues. This approach is highly versatile and enables a systematic study using a wide variety of cell culture materials, contact guidance patterns, cellular readouts, and proteins.

The procedure will be demonstrated by two PSE candidates from our lab. CAS VAN DER PUTTEN, and MIRKO D'URSO First, create a negative glass mold using a femtosecond laser direct right technique containing all features of interest. Then, carefully place the negative glass mold on the bottom of a Petri dish and pour the Polydimethylsiloxane pre polymer on top of the negative glass mold to cover it completely.

Place this Petri dish in the vacuum desiccator and start the vacuum pump. Once a vacuum is achieved, wait for 5 minutes to remove all bubbles present at the interface between the mold surface and the Polydimethylsiloxane pre polymer. Cure the PDMS pre polymer overnight in the oven at 65 degrees Celsius.

After curing, use a spatula to lift the edges of the PDMS. Remove the newly cured positive PDMS chip from the negative glass mold, and if the positive PDMS chip tends to stick to the negative glass mold, add ethanol or water to the edges of the imprint while lifting. Then, place the positive Polydimethylsiloxane chip in a desiccator next to a small vial with a droplet of silanization agent, and leave the chip under vacuum overnight.

Pour PDMS pre polymer into the negative PDMS mold to produce multiple cell culture chips of 5 millimeter thickness. Next, remove the bubbles using the desiccator and cure for three hours at 65 degrees Celsius. Use a razor blade to cut the chip to the final size and store the chips at room temperature For substrate passivation using tweezers, place the chip in the basket of the plasma Asher.

Then, use oxygen plasma to activate the hydroxyl groups on the surface of the chip and vent the ashing chamber using nitrogen. Take the chip out of the basket and place it in a small Polydimethylsiloxane container. Now, use a pasteur pipette to add 500 microliters of Poly-L-lysine on the top of the chip, to completely immerse surface in the solution and incubate for 30 minutes at room temperature.

Then, remove 450 microliters of the Poly-L-lysine from the cell culture chip with a pipette. Next, rinse the chip surface trice with 500 microliters of 0.1 molar HEPES buffer. Maintain the final pattern quality by leaving a small volume of liquid on the Polydimethylsiloxane chip, to prevent sample drying.

Just before use, prepare 500 microliters of 50 milligrams per milliliter methoxy polyethylene glycosic cyanamidal valerate solution in 0.1 molar heaps buffer per cell culture chip and incubate the sample in it for 60 minutes. Using a micro pipette, remove 450 microliters of the methoxy polyethylene glycosic cyanamidal valerate solution. Wash the chip surface five times with PBS by pipetting in and out.

To remove all unbound mPEG-SVA. Place the glass slide with a fluorescent highlighter in the microscope's optical path. Switch to fluorescent mode on the microscope and carefully focus the primo image, ensuring that both the logo and text are in focus.

Click on next to see the calibration data and pattern to finish the calibration procedure. Write down the Z location of the stage when calibrating and use this location as a reference later in the protocol. After removing the calibration slide from the stage place a glass slide containing a droplet of the photo initiator on the stage and place the PDMS cell culture substrate upside down, in the photo initiator droplet.

Ensure that the 3D features on the surface of the cell culture substrate face the glass slide and are fully submerged in the photo initiator, to ensure proper patterning. Focus on the top or bottom of convex or concave structures respectively and focus on the area of the chip in the right location as described in the manuscript. Adjust the patterning settings according to the feature and select a dose of 1000 millijoules per milliliter.

Click on the play button on the right bottom of the screen to start the patterning. Once finished, observe the pattern displayed in green. Using a micro pipette, add two milliliters of PBS to a vial of 20 micrograms of rhodamine labeled fibronectin, to obtain a concentration of 10 micrograms per milliliter.

Pipette gently to avoid the protein clumps and protect from light. Using a micro pipette, add 200 to 500 microliters of the protein solution to the cell culture chip. Adjust the incubation time and temperature depending on the protein of choice, and make sure to cover the sample.

Remove the protein solution and wash the chip five times with 500 microliters of sterile PBS. Make sure to pipette the PBS up and down multiple times above all relevant features of the cell culture chip, to remove any unbound protein. Once the cell suspension is prepared, remove PBS and add 1 milliliter of the cell suspension to the chip.

Then, incubate it for 60 minutes at 37 degrees Celsius. Check the adhesion of the cells on the pattern cell culture chip under a bright field microscope. Look for elongated cell morphologies in the case of line patterns.

And if cells have started adhering outside the patterned area remove these by pipetting the medium up and down directly above the cells on the substrate. After staining, place the stained sample upside down in a droplet of PBS on a glass slide. Then, depending on the required level of detail make Z stacks with a suitable objective and Z spacing to ensure proper image acquisition.

Create a 3D render of Z stack with 3D rendering software. A concave pit was patterned using light induced molecular absorption of proteins and the maximum intensity projection and orthogonal view were visualized. The intensity profile showed high pattern resolution sharp transitions between patterned and non patterned areas and consistent protein intensity.

The patterning using the single focal plane as well as two and three focal planes method showed perfect alignment of the patterns on top of the features. The concave semi cylinders, convex semi cylinders, saddle surface and PID were patterned using lines or concentric circles of various widths. The affect inside of skeleton of the human keratocytes is stained using foliden and visualized.

The dermal fibroblasts were cultured on a patterned concave semi cylinder and visualized. The cells sensed and adhered to the multi cue cell culture substrate and remained viable over time as visualized by F-actin, Vinculin and Nuclei. Also, the cells formed focal adhesions mainly on the fibronectin lines.

The human dermal fibroblasts cultured on a patterned concave cylinder in the presence of rhodamine fibronectin adhered to the multi cue substrate and showed an alignment response, according to the contact guidance pattern. The alignment response and cell viability are maintained throughout the entire culture duration. The most important thing when performing this protocol is to prevent the surface of the cell culture chip from drying out.

The use of different substrate materials, protein coatings and cell types might require additional optimization. Providing multiple environmental cues in 3D can open new possibilities to steer cellular organization in complex engineer tissues.