Fabricating Complex Culture Substrates Using Robotic Microcontact Printing (R-µCP) and Sequential Nucleophilic Substitution

The overall goal of the following experiment is to generate tissue culture substrates with multiple micro pattern PEG brushes, presenting orthogonal chemistries that enable spatially precise and site-specific immobilization of biological ligands. This is achieved by deposition arrays of acan thiol, A TRP initiator by a robotic micro contact printing on gold coated substrates. As the second step SI, A TRP is performed on the micro pattern substrates, which generates PEG brushes with terminal bromine groups.

Next, the terminal bromine groups are functionalized with one or two chemical reactive groups in order to allow for IM mobilization of appropriately prepared biological ligands. Then the entire process is repeated to functionalize with a second chemical group. The results show microarrays of two orthogonally functionalized, PEG brushes superimposed with 10 micron accuracy.

This is verified using custom image analysis software following conjugation of the reactive groups with fluorescent molecules. The main advantage of this technique over methods like manual micro contact printing is that robotic micro contact printing is capable of superimposing multiple printing steps with micron accuracy, while also enabling maximum flexibility and substrate design. The accuracy afforded by this method, coupled with sequential nucleophilic substitution reactions, allows us to generate tissue culture substrates that are functionalized with multiple biological ligands that are then capable of exerting temporal and spatial control of sulfate on the micron scale.

Prior to S-I-A-T-R-P, A gold coated cover slide should have been patterned by A-P-D-M-S stamp. This relatively common procedure is explained in a text protocol. Begin by removing the patent cover slide from the robot by releasing the vacuum.

Then transfer it to a 50 milliliter sch length flask, seal the flask, and apply a negative 30 PSI vacuum. Next, add 5.5 milliliters of A TRP reaction mixture into the flask using a syringe followed by adding 0.5 milliliters of L sodium ascorbate in water also via syringe. Observe the reaction change color from light green to dark brown.

Allow the reaction to continue for 16 hours under inert gas. When the reaction is complete, remove the cover slide and rinse it with ethanol, then water and then ethanol again. Then dry it gently with nitrogen.

The microarray of grafted P-E-G-M-E-M-A brushes should be visible to the naked eye. Use microscopy to reveal these changes in detail to Azad functionalize, the P-E-G-M-E-M-A chains. Transfer the pattern cover slide to a 20 milliliter glass reaction vial and add six milliliters of DMF containing 100 millimolar sodium azide.

Let the reaction go for 24 hours at 37 degrees Celsius. The next day, rinse the patent cover slide with ethanol and dry it under a gentle nitrogen stream to pacify the remaining bromine functionalized P-E-G-M-E-M-A chains. Transfer the micro pattern cover slide to a 20 milliliter glass reaction vial and add six milliliters of DMSO containing 100 millimolar ethanol amine, and 300 millimolar triethyl amine.

Allow this reaction to proceed for 24 hours at 40 degrees Celsius the following day. Rinse the pattern cover slide with ethanol and dry it gently under a nitrogen stream. The next step is to repeat the robot micro contact printing using a second PDMS stamp and following stamping.

Use SI A TRP again to graph the second micro array PEG brush. Then it is time to acetylene functionalize the second micro array P-E-G-M-E-A chains Francois. The micro pattern cover.

Slide into a 20 milliliter glass reaction vial with six milliliters of 100 milli molar propag amine in DMF. Let the reaction proceed for 24 hours. At room temperature the following day, rinse the patent cover slide with ethanol and dry under a gentle nitrogen stream.

The patent cover slide is then ready for biotin elation. Begin by placing the micro patent cover slide in a 20 milliliter glass reaction vial. First, biotin ate the acetylene terminated chains.

Add six milliliters of copper sulfate with the azide PEG four biotin conjugate to the reaction vial, followed by 1.2 milliliters of 0.15. Millimolar rbic acid in water to initialize the reaction, bubble the reaction with nitrogen for 10 seconds. Then seal the vial with paraform and let the reaction go for 24 hours.

At room temperature the next day, rinse the cover slide with water and transfer it to a 12 well polystyrene dish. Then at room temperature at DPBS with donkey serum for an hour an hour later, stain in the biotin elated groups with strep DEIN 5 46 conjugate at two micrograms per milliliter in DPBS stain for two hours at room temperature two hours later. Rinse the cover slide in DPBS five times with gentle vegetation.

Tobi ITIN aate the azi terminated chains. Put the cover slip in a well of a six well polystyrene plate and applied two milliliters of DPBS containing 20 micromolar D-B-C-O-P-G four biotin. Then incubate the cover slide for 24 hours at room temperature 24 hours later.

Rinse the cover slide as before four and stain it with streptavidin 4 88. Conjugate at two micrograms per milliliter in DPBS. Two hours later.

Rinse the cover slide again, then image it with microscopy. Using this robot micro contact printing protocol. An accurately patterned array of PEG brush ANN UI with terminal alkin groups were patterned within a separate array of larger PEG brush ANN UI with terminal azi groups.

The spatial distribution of azide and alkin groups were viewed using the click chemistry and streptavidin binding of fluorescent probes. An overlay of both fluorescent channels shows how the stamps did not overlap. The accuracy of the alignment was calculated to be sub 10 microns, which is at the limits of the SCAR.

A system used to do the printing. After watching this video, you should have a good understanding of how to generate custom tissue culture substrates using robotic micro contact printing to overlay multiple peg brush arrays. Functionalized with orthogonal chemistries.