Simple Lithography-Free Single Cell Micropatterning using Laser-Cut Stencils

We'll describe a stencil-based lithography-free single-cell micropattern method that overcomes many obstacles associated with photolithography and soft lithography. The stencils are reusable and the process is inexpensive and customizable. This method opened a new avenue for non-engineer researchers to study cell shape and area-induced mechanotransduction in various physiological and pathological contexts.

Furthermore, this protocol allows for micropatterning of thousands of cells which can be utilized for high-throughput drug screening and disease modeling in a dish. Visual demonstration will facilitate the implantation of two crucial steps:detachment of coverslip from hydrogel, drying hydrogel sufficiently before placing the stencil. Prepare the polyacrylamide hydrogel solution in a new 50 milliliter propylene centrifuge tube as described in the manuscript.

Next, to prepare the 5%photoinitiator solution, dissolve the ingredients in 700 microliters of 100%ethanol. The ingredients are not water soluble so be sure to fully dissolve by vortexing. Add 300 microliters of PBS for a final volume of one milliliter.

Working on ice, dilute basement membrane matrix protein stock solution with ice cold DMEM F12 media or 1X PBS to the optimized dilution ratio. Keep on ice for later use. When working with basement membrane matrix, make sure to always work on ice and use ice cold solutions in chilled pipette tips and tubes.

Place a UV bench lamp in a biological safety hood. Next, place the sterilized paraffin films prepared previously. If the paraffin films are not completely dry, use a vacuum aspiration system to remove any residual liquid.

Using autoclaved forceps, place six autoclaved coverslips on the paraffin film six in each Petri dish. To prepare the UV cross-linkable polyacrylamide final solution, dilute the photoinitiator solution with the polyacrylamide hydrogel precursor solution. Sterilize the solution using a 50 milliliter filter unit with a 0.22 micrometer pore size.

Then dispense 200 microliters of UV cross-linkable polyacrylamide final solution onto each of the six glass coverslips in the Petri dishes. Using autoclaved forceps, carefully cover each coverslip with a second coverslip trapping a layer of the solution between the two coverslips. Place the Petri dishes containing the coverslips under the UV bench lamp with the lid unclosed.

The coverage of white paper on the non-white surface is important to minimize the UV intensity loss. For protection from UV radiation, cover the lamp with aluminum foil. Photopolymerize the polyacrylamide hydrogels for five minutes.

Use a razor blade to carefully separate each pair of coverslips. Fill the reservoir containing six hydrogel coverslip composites with PBS. After rinsing for five minutes, return the hydrogel coverslip composites to the paraffin film in the Petri dish.

Obtain a tube of Sulfo-SANPAH from storage at minus 80 degrees Celsius. Add 1, 200 microliters of pre-chilled PBS to the tube and mix by pipetting up and down. Dispense 200 microliters of the diluted Sulfo-SANPAH onto the paraffin film in the Petri dish.

Place a hydrogel coverslip composite over the Sulfo-SANPAH with the hydrogel contacting the Sulfo-SANPAH. Next, place the hydrogel coverslip composites under the UV lamp and expose them for five minutes to activate the Sulfo-SANPAH. If the Sulfo-SANPAH turns from orange to brown indicating successful activation and incorporation, proceed to the next steps within 10 minutes.

Replenish the disposable polypropylene reservoir with fresh PBS. Transfer the activated hydrogel coverslip composites to the reservoir and rinse them briefly to remove unbound Sulfo-SANPAH. Place the hydrogel coverslip composites on the paraffin film in the Petri dishes and carefully dab them with sterile lint-free wipes.

Place a stencil on top of each hydrogel coverslip composite. To ensure a tight seal between the stencil and the composite, remove excess moisture by dabbing with autoclaved lint-free wipes. Carefully dispense the diluted basement membrane matrix protein solution placing 200 microliters on top of each hydrogel coverslip composite.

Incubate the constructs overnight at 37 degrees Celsius. The next day, transfer the composites to a six-well plate. A tight seal between hydrogel and stencil can be easily validated by observing whether the matrix protein solution has been retained on top of the stencil.

Failure to form a tight seal leads to quick leakage of the protein solution through the holes. Fully immerse the composites in PBS. Using autoclaved tweezers, carefully detach the stencils from the hydrogels without tearing the hydrogels.

Remove the PBS from the wells and refill the wells with DMEM. Incubate the plate overnight at 37 degrees Celsius. After removing the plate from the incubator, examine the DMEM.

If the DMEM is not cloudy, the hydrogel coverslip composites are ready to be used for cell plating. Stencils were fabricated containing arrays of squares and rectangles. Micropatterned hydrogel substrates were created from the stencils and islands of matrix protein were obtained.

Cell islands were also obtained. For example, islands of human-induced pluripotent stem cell-derived cardiomyocytes. Matrix protein concentration played an essential role in generating proper patterns.

An optimal concentration led to a homogenous distribution of proteins. Suboptimal matrix protein solution concentrations led to suboptimal patterning. It is critical to use the front side of the stencil.

If the back side of the stencil is used, the size of the matrix protein islands increases due to the direction of the laser cutting. While using the back side of the stencil did not significantly affect the width of the rectangular patterns, the height significantly increased leading to a reduction in the intended aspect ratio. Stencil-based patterning was applied to silicon elastomer substrates.

The patterned cardiomyocytes on silicon elastomer substrates were visualized by immunocytochemistry of cardiac troponin T at low magnification and by sarcomeric protein alpha-actinin at high magnification. The stencil-based patterning was also applied to pattern two cardiomyocytes side by side on hydrogels with different stiffnesses. The discovery of human-induced pluripotent stem cell and corresponding differentiation protocols has made this an ideal in vitro human model for studying oncogenesis and pathogenesis.

However, a major limitation of using IPS system is the absence of structure to the microenvironment which has particular biochemical composition and stiffness to interact with cells. The patterning can be combined with traction force microscopy and immunocytochemistry assays to characterize the structure and function of the cardiomyocytes. Analysis of IPS-derived cell types, in our case cardiomyocyte, in a more physiological morphology opens opportunities to study challenging cardiomyocytes in culture dish.

The technique can also be applied to many other systems such as and high content screening plates. The shape and substrate can be readily adjusted to the application.