The overall goal of this methodology is to create immobilized bioactive spatial protein patterns which be used to study cellular responses. This method allows the recapitulating in-vivo signals using biomaterial strategies. This technique allows for accurate mimicry of certain signaling motives that cannot be achieved using standard culturing methods such as, sequential growth factors, cells cells signaling and spatial specific biochemical cues.
This protocol is significant to existing methods as it provides a simplistic method for adjusting substrate stiffness and protein patterning. While this method can further tissue engineering and regenerative medicine technologies, it can also be applied to study other systems such as, tissue development and stem cell fate. To begin, prepare stock solutions for the major hydrogel component, polyethylene glycol diacrylate, the photo initiator, lithium phenyl-2, 4, 6-trimethylbenzoylphosphinate and the ECM protein, fibronectin, under sterile conditions as described in the accompanying text protocol.
Filter sterilize each solution using a 0.22 micron syringe filter prior to using or storing the individual aliquots. Then, wrap the PEG-DA solution with foil to protect it from light. Wrap the photo initiator stock solution in foil to protect it from light and store the prepared solution at four degree celsius for up to several months.
If fibronectin stock solution is frozen, allow sterile aliquot to thaw on ice for several hours or in four degree celsius overnight. Start by autoclaving one polyester sheet for each gel mold. Then, soak two glass slides in three plastic spacers for each gel mold in 70%ethanol for at least two hours.
Additionally, soak five binder clips for each gel mold in 70%ethanol for 10 minutes. Place the glass slides, spacers and binder clips onto a small autoclave sheet in the cell culture hood to allow them to air dry for several hours. Next, prepare the hydrogel molds by placing the 0.5 millimeter thick plastic spacers around the edge of a glass slide.
Place the second glass slide on top and then secure the spacers tightly next to each other with binder clips. Finally, surface sterilize the mold by placing it in the hood and turning on the UV light for 30 minutes prior to use. Halfway through the sterilization process, flip the mold to be suer to expose both surfaces.
Create the gel precursor solutions as described in table one of the accompanying text protocol. Then, mix together the PEG-DA, fibronectin and photo initiator volumes to create the hydrogel. Pipette the solution vigorously to ensure a homogenous solution, but avoid creating bubbles.
Pipette the gel precursor solution carefully between the two glass slides of the gel mold. Then, place the mold under a Uv light and expose it for one to two minutes to form the hydrogel. Once gelled, take off the binder clips and gently remove the top glass slide by applying opposite pressure to the two side spacers.
Then, using appropriately sized biopsy punch to cut out the hydrogel samples. Punch out multiple hydrogels from the gel rectangle to serve as replicates and control samples. Sterilize the photo mask by soaking it in 70%ethanol for 10 minutes.
Then, allow the photo mask to air dry in a cell culture hood. Next, pipette one to two microliters per centimeter square of a thiolated protein solution to the surface of each cut out hydrogel for surface patterning. It is important to spread the protein solution across the surface of the hydrogel evenly to ensure uniform protein immobilization.
Carefully place the photo mask on the surface of the hydrogel. Gently press down on the mask to remove any bubbles that form between the mask and the hydrogel surface. Next, place the hydrogel under the UV light and expose it to a second round of UV light for 30 to 60 seconds.
Wash the hydrogels with PBS to remove any unreacted species and place each hydrogel within the well plate so that the pattern surface is faced up. Then, wash the gels overnight in PBS at four degrees celsius. Remove PBS from wells, then, add 250 microliters of basal EGM2 into each well and incubate the gels for five to 10 minutes at 37 degree celsius.
During incubation, trypsin digest a plate of near-confluent HUVECs into a single cell suspension using standard techniques. Then, spin down the cell suspension and carefully remove the supernatant. Re-suspend the cell pellet and basal EGM2 with no added growth factors and add some of the cell suspension to a hemocytometer.
Count the cells to determine the concentration. Next, spin down the plate containing he hydrogels at 300 x G for three minutes, to ensure that the hydrogels are located at the bottom of the well and are not floating. It is critical that hydrogels are not floating within the wells.
Floating hydrogels will limit success in cell seeding and cause problems with hydrogel imaging. Slowly pipette 75, 000 cells per centimeters squared onto the center of each hydrogel surface so as to not disturb the gels. Then, place the plate into a cell culture incubator at 37 degrees celsius.
For several days, periodically remove the dish to observe the cell migration in response to the protein pattern. Exchange 50%of the media every two to three days. When forming the hydrogels, various precursor PEG-diacrylate concentrations can be used to yield the desired substrate stiffness.
Shown here, 20%PEG-DA solution correlates with the stiffness of over 100 kilopascals. It is also important to consider both photo initiator concentration and UV exposure time as an increase in either variable will decrease the bioactivity of attached molecules, represented here by the bioactivity of lysosome. Minimizing the UV exposure during hydrogel formation is critical to maintaining free acrylate functional groups for subsequent protein immobilization reactions.
Hydrogels exposed to UV light for longer than two minutes are unable to create immobilized protein patterns shown in red. Additionally, as the UV exposure to the protein pattern increases, more proteins react to the surface. When prepared correctly, cells can be cultured onto these patterned hydrogel substrates to manipulate their behavior.
Here, endothelial cells were uniformly seeded onto the surface of VEGF pattern PEG hydrogels. Two days after seeding, the endothelial cells were observed to migrate towards the spatial regions of the hydrogel that contained the immobilized VEGF. Following the development of this protocol, researchers can use it to immobilize any protein or peptide in order to explore new bioactive platforms for in-vitro cell systems.
While attempting this procedure, it is important to remember to minimize photo initiator concentration and UV exposure time to maintain bio activity of immobilized molecules. After watching this video, you should have a good understanding of how to pattern bioactive proteins and peptides onto PEG hydrogels, seed cells uniformly onto hydrogels and observe the consequent cell response.
