The overall goal of this procedure is to develop a simple and efficient strategy for immobilizing any protein of interest on poly acrylamide hydrogels in order to independently control various parameters of the cell microenvironment. This is accomplished by first spreading the protein solution across the PDMS stamp surface. The second step is to carefully dry the structured surface of the PDMS stamp with a steady flow of high purity nitrogen gas.
Next, the protein coated stamp is placed with a structured surface in contact with the dried hydrogel surface and brief pressure points are applied on the top of the PDMS stamp to ensure a good contact between the stamp micro features and the hydrogel surface, the final step is to gently remove the PDMS stamp from the hydrogel surface and to clean the stamp After ation of the non-print zones. With BSA cells can be seated on the micro pattern hydrogel surface. Ultimately, an inverted fluorescence microscope is used to observe the protein micro features.
This method can help answer key question in the mechano transduction field, such as the relative contribution of extracellular matrix properties, for instance, stiffness, cell ligon density, or protein nature. In the ano transaction process, It is recommended that this procedure be performed in a chemical fume hood. Start by placing 25 millimeter diameter circular glass cover slips in a Petri dish and smearing 0.1 molar sodium hydroxide solution on them for five minutes.
Remove the sodium hydroxide solution and fully immerse the cover slips in sterile DD H2O gently rock for 20 minutes on a rocking plate, drain the sterile DDH two O add more sterile DDH two O to fully immerse the cover slips and gently rock for another 20 minute. Remove the cover slips with sterile tweezers and placed them in a new Petri dish. With the activated side faced up dry the cover slips with a steady flow of high purity nitrogen gas.
Once the cover slips are dry, take them back to the sterile culture hood and smear a thin layer of three trimethyl propyl acrl on the activated side of each cover. Slip for one hour after one hour. Wash the glass cover slips extensively with sterile DD H2O.
Then immerse the cover slips in sterile DD H2O in a new Petri dish. Seal the Petri dish with para film and place it on a rocking plate on under gentle agitation for 10 minutes. Lastly, use sterile tweezers with fine tips to transfer the cover slips from the DDH two O to a new Petri dish.
With the activated face up. Cover the dish with aluminum foil to avoid dust from sticking to the cover slips and store at room temperature in a dry place. To begin this procedure, prepare five milliliters of a sterile hydroxy poly acrylamide hydrogel solution and 100 microliters of a fresh 10%ammonium per sulfate solution.
As described in the protocol text, add 2.5 microliters of tetra methylene diamine, and 25 microliters of the ammonium per sulfate solution to the hydroxy poly acrylamide hydrogel solution. To initiate the polymerization, mix the solution by three successive pipetting without introduction of bubbles under sterile conditions under a sterile hood, place a 25 microliter drop of the hydroxy poly acrylamide hydrogel solution on a 25 millimeter circular cover slip and immediately place a 22 millimeter circular glass cover slip on top of the droplet to squeeze the hydroxy poly acrylamide hydrogel solution. The 22 millimeter glass cover slip was previously activated by a seven minute exposure in a UV ozone cleaner.
Center the glass cover slip with sterile tweezers and smooth out any bubbles. Allow the hydroxy poly acrylamide hydrogel to polymerize in room temperature for 15 minutes. Invert manually the remaining hydroxy poly acrylamide hydrogel solution in the tube.
To follow the completion of the polymerization process, fully immerse the cover slips with sterile D DH two o. Carefully separate the 22 millimeter glass cover slip by inserting the edge of a razor blade between the 22 millimeter glass cover slip and the hydroxy poly acrylamide hydrogel layer. Wash the hydroxy poly acrylamide hydrogel three times with sterile PBS and leave the gel fully immerse in sterile PBS to maintain hydration.
The poly dimethyl soane or PDMS micro stamps used in this procedure were fabricated as described in the text protocol. Place the PDMS micro stamps in a 50 to 50 ethanol water solution and sonicate for 15 minutes. Dry the stamps with a steam of nitrogen flow and place them pattern up in a UV ozone cleaner for seven minutes.
Under a sterile hood place a 150 microliter drop of a desired protein solution onto the micro structured surface of A-P-D-M-S. Stamp FI nin is used in this demonstration. Spread the protein solution across the stamp surface by moving it with a sterile pipette tip toward each corner of the stamp.
Leave the protein solution to absorb on the PDMS stamp for 60 minutes. Under this sterile hood, turn off the lamps to avoid protein damage. Next, transfer the hydroxy poly acrylamide hydrogel coated cover slips into a new Petri dish.
Remove excess PBS from the surface of the hydroxy poly acrylamide hydrogel substrates with the low nitrogen stream under sterile conditions. Stop the procedure. As soon as no evidence of standing water on the gel surface is observed.
The gel should not be dried thoroughly at this stage. Carefully dry the structured surface of the PDMS stamp with a steady flow of high purity nitrogen gas. Grasp the protein coated stamp with dressing tissue forceps and place a structured surface in contact with the dried hydrogel surface.
Apply brief pressure points with the tip of the tweezers on the top of the PDMS stamp to ensure good contact between the stamp micro features and the hydrogel surface, leave the PDMS stamp on the hydrogel surface for one hour at room temperature after one hour. Gently remove the PDMS stamp from the hydroxy poly acrylamide hydrogel with dressing tissue forceps and clean the stamp by fornicating in an ethanol water solution for 15 minutes. Wash the stamped hydroxy poly acrylamide hydrogel extensively by three exchanges of PBS in sterile conditions for 10 minutes per exchange.
After the last PBS wash, add a sterile solution of BSA and incubate overnight at four degrees Celsius under gentle agitation on a rocking plate. This will ate the non-print zones on the following day. Wash the stamped hydroxy poly acrylamide hydrogel extensively by three exchanges of PBS in sterile conditions for 10 minutes per exchange.
Stamped hydroxy poly acrylamide hydrogels can be stored at four degrees Celsius for up to one week. Results show a linear correlation between the evolution of the stiffness of hydroxy poly acrylamide hydrogels and the amount of bis acrylamide crosslinker epi fluorescent images of laminate rectangular micro patterns deposited on hydroxy poly acrylamide Hydrogels of three different stiffnesses demonstrate the independent tuning of micropattern geometry and matrix stiffness. The cell ligand density can be modulated by varying the concentration of the protein solution used to incubate the PDMS stamps.
Shown here are two fluorescent images from sequential micro contact printings. Stripes of fibronectin in red and laminin in green, crossed at 90 degrees and fibrin laminin and collagen stripes micro printed sequentially on a 25 kilo pascal hydroxy poly acrylamide hydrogel substrate. When primary cells were plated on homogenously coated and micro pattern hydroxy poly acrylamide hydrogel surfaces, cell viability was at least 80%up to three days post plating.
It was also observed that the cells proliferate more on stiff substrates compared to soft substrates. When plated on rectangular fibronectin coated micro patterns deposited on hydroxy poly acrylamide hydrogels of three different stiffnesses. Primary endothelial cells exhibit low actin fiber density and a rounded nucleus on the soft.
The substrates on stiffer substrates, actin fibers are straighter and thicker and the nucleus is deformed After its development. This technique paved the way for researcher in the field of mechanical transduction to explore the individual role of the parameters of the cell microenvironments in a wide range of cellular systems such as primary or stem cells, tissue level, model organism, fashion, demographic, or organ systems.
