分化運命の空間組織を探るためのヒト多能性幹細胞のステンシルマイクロパターニング

The overall goal of this stencil-based micropatterning method, for human pluripotent stem cells, is to provide a simple and robust means to generate spatial environmental gradients that control stem cell differentiation patterns. This method can help you answer key questions in tissue patterning using a human stem cell model, such as how spatial gradients in cell-cell and cell matrix adhesion forces can help to organize stem cell differentiation fates. The advantage of this method is that it does not require additional surface modification to generate cell adhesive ECM patterns for cells to attach to.

This means the method is compatible with various ECM and substrate configurations, specific for each cell line. Visual demonstration of this method is critical, as the micropatterning steps are difficult to learn. Because it requires precise and careful handling of the stencil and the cells.

For this procedure, the PTMS stencils must already be designed and fabricated. Likewise, have human EFC cells growing on basement membrane matrix, such that the colonies will be ready for seeding later in the procedure. To begin, load a dish with 700 microliters of 70%analytical-grade ethanol, in ultra-pure water.

Then, place the PDMS stencil on the solution, and set the dish into a bio-safety cabinet to dry out overnight. The next day, check that no bubbles have formed under the stencil. Then, field the dish until the cells can be seeded onto the stencil.

Prepare an aliquot of human ESC-quality basement membrane matrix by adding 16.7 milliliters of DMF12, to make a 1.5x solution and keep it on ice. Treat the substrate with 100 watts of oxygen plasma for 90 seconds. Don't open the dish until it is inside the plasma chamber, and quickly cover it up after the treatment.

This treatment will prevent bubbles from forming when the substrate is immersed in solution. Now, cover the stencil with 450 microliters of prepared 1.5x basement membrane matrix solution. Then, seal the dish with parafilm and incubate it for five hours.

Before proceeding, check over the six well plates of prepared HESC colonies. Identify and aspirate off colonies of differentiated cells that have lost their typical HESC morphology. They should all appear rounded, have a tightly-packed epithelial morphology, and have a high nucleus-to-cytoplasm ratio with prominent nucleoli.

Then, wash each well of plated cells with two milliliters of DMF12 two times. After the second wash, add one milliliter of digestive enzymes to each well, such as accutase and incubate the plate for eight minutes at 37 degrees Celsius. After the incubation, tap the plate gently to detach all the colonies from the substrate.

Next, rinse each well with at least four milliliters of media for every milliliter of digestive enzyme applied to the well, and collect the cell suspensions into a single 15 milliliter conical tube. Centrifuge the cells at 200 g for three minutes at room temperature to form a cell pellet. Then aspirate the supernatant, and re-suspend the cells in 400 microliters of HESC maintenance media, supplemented with 10 micromolar of rock inhibitor.

Triturate the cells gently three times to break up the clumps. Next, correct the suspensions'density. Mix it well, and make a 200 microliter 1:20 dilution in media.

Count the cells in the dilution using a hemocytometer. Then, dilute the main suspension as needed, using media with rock inhibitor. To proceed, add the required number of cells onto each prepared stencil.

Only a mono-layer of cells should be deposited, so spread them out evenly. Let the stencil sit undisturbed for five minutes, so the cells can settle. Then, carefully transfer the loaded stencils to a incubator, keeping the dishes level throughout the transfer.

Let the cells attach to the stencils over an hour-long incubation. After the cells have been allowed to attach, examine them under a microscope to ensure that they did so properly. Transfer the stencil back to the hood, and aspirate the unattached cells off the stencil.

Next, add two milliliters of 0.5%compatible non-ionic surfactant in DMF12 to the area surrounding the stencil. Swirl the solution around to cover the stencil. Then, use a pair of autoclaved forceps to gently peel the stencil off the dish.

After removing the stencil, inspect the micropatterend cells under a microscope. The non-ionic surfactant prevents these cells from drying out. Let the micropatterend cells incubate in the surfactant-supplemented media solution for 10 minutes at 37 degrees Celsius.

After 10 minutes, aspirate away the solution, and wash the plate with two milliliters of normal DMF12 medium, three times. After the washes, add two milliliters of HESC maintenance medium with rock inhibitor. Once again, inspect the cells to check that the micropatterns have formed properly.

Then, incubate the cells at 37 degrees Celsius overnight. The next day, aspirate the medium, wash the cells once with DMF12, and then, add two milliliters of supplemented differentiation medium, to induce the cells to undergo mesoendoderm differentiation. Later, analyze the results.

The fabrication of the cell stencil using a laser cutter, provided 1000 micron features. The stencil was composed of a thin stenciling sheet with micropatterned through holes, and a PDMS gasket used to contain the cells. Using this tool, micropatterened HESCs were investigated during mesoendoderm differentiation.

H9-HESC colony peripheries have higher integrin adhesions than cells at the colony interior. Thus, the cells differentiate into brachiary T-positive mesoendoderm cells, when induced with activan, BMP4, and FGF2. When such cells were patterned into monolayers of different geometries, geometric and isotropiate right angles and at convex curvatures, led to a local concentration of integrin mediated traction forces, and resulted in increased mesoendoderm differentiation.

T expression intensity at different localities within a single colony showed that the extent of meso endoderm induction was indeed higher in sharp corners of square and rectangular colonies, and at the convex curvatures of a semi-circular arc. This demonstrates that cell adhesion mediated mechanical forces can be modulated using the described method. Before attempting the procedure, it is important to optimize the matrix coating time, the cell digestion and attachment time, for the specific human pluripotent stem cell line that you are working with.