Micro-patterning is a very powerful tool because it allows studying cellular responses to define biochemical and biophysical signals that cells encounter in living organisms. In contrast to other methods, the described is more flexible for pattern generation. For example, it allows production of gradients and micro-patterns of multiple proteins with high reproducibility.
Our group uses LIMAP not only to study neuronal pathfinding, but also how other cells respond to signals that have influence on cell proliferation, differentiation, and migration. Micro-patterning is used to answer fundamental questions in cell biology. The gained knowledge can be used to modify existing devices in regenerative medicine, for example, nerve repair after peripheral nerve injury.
Begin by designing templates for patterning on digital drawing software. Select an image size of 1, 824 pixels in length and 1, 140 pixels in width. Design the template, and save the template as an 8-bit TIFF file.
Prior to patterning, clean and activate the surface with a plasma cleaner using a process pressure of 1, 000 to 1, 300 millitorr and a power of 29.6 watts for one to five minutes. Use a glass-bottom dish with a 20-millimeter inner well size and glass thickness of 0.16 to 0.19 millimeters. Select either a six-well or a single-well dish, depending on the number of conditions being tested.
Cut PDMS stencils under sterile conditions to ensure that they fit in the inner glass-bottom well. Remove the inner microwell fillings with sterile forceps, and stick the stencils on the glass well. Prepare a 0.1-milligrams-per-milliliter PLL-PEG solution in PBS, and add 20 microliters to each microwell.
Then, incubate the dish at room temperature for one hour. After incubation, remove 15 microliters of the PLL-PEG from each well, and wash it five times with 20 microliters of PBS without letting it dry. Prepare one microwell to create a reference pattern by removing 18 microliters of PBS from a single well and adding five microliters of photoinitiator, leaving PBS in the remaining microwells.
Then, use a fluorescent highlighter to mark an empty inner glass well. Position the well above the objective, and adjust the objective focus until the PRIMO logo and the tagline take care of your cells"are in focus. Then, record the Z position of the focal plane.
To create a reference pattern, visualize the edge of the microwell with the photoinitiator using the transmitted light through the camera, and choose the ROI symbol from the right-hand menu. Select PRIMO to upload the desired pattern template, which will be projected on the ROI as a design unit. Navigate to a peripheral region of the microwell, select Lock, and adjust the focus to the Z position of calibration.
Then, select Play to start photopatterning the reference pattern. Wash the reference pattern microwell three times with 20 microliters of PBS to remove the photoinitiator. Then, add 20 microliters of fluorescently labeled extracellular matrix, or ECM, protein solution.
Incubate the dish at room temperature for 10 to 20 minutes, making sure to protect the reference well from light. After incubation, remove 18 microliters of protein solution, and wash the well three times with 20 microliters of PBS. Use an epifluorescence microscope to visualize the reference pattern.
Adjust focus on the pattern edges through the camera path, and record the Z position according to the best focus. This position will be the optimal laser focus used for subsequent patterning. Under sterile conditions, remove 18 microliters of PBS from all the microwells.
Add five microliters of the photoinitiator to each well, and homogenize by pipetting up and down. If patterning multiple proteins in the same microwell, upload all sets of desired pattern templates simultaneously. Then, select the number of rows, columns, and dose.
Save the template configuration as file in the software. Navigate to the area where the reference pattern was produced, and adjust the focus to the optimal Z position. Then, select Play to initiate photopatterning.
The design units turn from red to blue as the photopatterning is completed. Remove the photoinitiator from the microwells by washing them three times with 20 microliters of PBS. After the last wash, remove 18 microliters of PBS from each microwell, and add 20 microliters of ECM protein solution.
Incubate the dish at room temperature for 20 to 30 minutes, making sure to protect it from light. Next, remove 15 microliters of the ECM protein solution, and wash the microwells three times using 20 microliters of PBS. To prevent cross-binding, add 20 microliters of PLL-PEG, or BSA, to the microwells, and incubate the dish at room temperature for one hour, in the dark.
Then, remove 15 microliters of the blocking agent, and wash all microwells three times with 20 microliters of PBS. Remove 18 microliters of PBS, and add 5 microliters of the photoinitiator to each microwell, ensuring that the photoinitiator is homogenous on the whole surface of the microwells. Navigate to the first microwell, and load the template configuration saved previously.
Then, select the actions to be patterned for the second round of photopatterning, making sure to deselect actions from the first round. After photopatterning, remove PLPP by washing with PBS. Then, incubate for 20 minutes with the second ECM protein solution, and wash the microwells three times with 20 microliters of PBS.
Use an epifluorescence microscope to visualize and image the printed patterns. When ready to plate the cells, add one milliliter of medium with cells to the inner glass well, making sure to cover all microwells. And place the culture dish in a 37 degree celsius, and 5%carbon dioxide incubator.
In order to ensure that the generated patterns are a precise representation of the template, fluorescence intensity measurements are obtained along the stripes, and from a corresponding background region above each stripe. Reproducible defined micro-patterns are achieved ranging from 20 to 2 micrometers in width, but an edge effect can be observed when printing features 20 micrometers or wider. Defined protein concentrations are obtained by either incubating with varying concentrations of protein, or varying the laser dose that is used to cleave the antiadhesive film.
Laser intensity is proportional to the grayscale level of the template, generating gradients of UV illumination. The measurement of the fluorescence intensity profile along the gradient stripe, is linear in the pattern template, and in the generated gradient pattern. This protocol can be used to create micro-patterns with multiple proteins in the same microwell, such as cross patterns with horizontal stripes of fibronectin, and vertical stripes of laminin.
But a blocking step is necessary to prevent cross-binding of proteins. The generated cross patterns can be subsequently used for cellular assays with neuronal cell lines, or CAD cells, or primary neurons, rat DRGs. When performing this protocol, the most important aspects to remember are doing a proper system calibration, not letting the microwells drying out during the multiple washing steps, and using the right parameters for protein incubation and blocking.
Analysis of cell behavior typically involve microscopy, such as fluorescence, time-lapse, AFM. Further characterization can comprise other biochemical methods, such as gene expression profiling.
