Patterning the Geometry of Human Embryonic Stem Cell Colonies on Compliant Substrates to Control Tissue-Level Mechanics

This protocol allows researchers to culture stem cell colonies on hydrogels with complete control over both the stiffness of the hydrogel and the geometry of the colonies. The main advantage of this technique is that it allows researchers to culture stem cell colonies in conditions that better mimic the physiological environment than tissue culture plastic. When performing this protocol for the first time, be sure to generate a few extra of every material and use a fluorescent ligand so that you can troubleshoot and optimize at each step.

This technique involves multiple steps that involve the precise positioning of various materials. It's much easier to understand the written instructions for this protocol once you've seen it performed. To begin this procedure, mix PDMS base with PDMS curing agent at a 10 to one ratio by weight and mix thoroughly.

Degas this mixture in a desiccator for 30 to 60 minutes or until all of the air bubbles are removed. Slowly and evenly pour the PDMS mixture over the surface of the prepared wafer. Tap the dish on the work surface to release any air bubbles from the surface of the wafer.

Bake the PDMS at 70 degrees Celsius for two hours and allow it to cool to room temperature. Then cut the PDMS into roughly 10 by 10 millimeter squares, each containing the features for a single experimental condition. These will be referred to as stamps for the remainder of the protocol.

After this, mix PDMS base with PDMS curing agent at a ratio of eight to one by weight, and mix thoroughly. Degas the mixture in a desiccator for 30 to 60 minutes or until all of the air bubbles are removed. Next, slowly and evenly pour the PDMS into a clean 100 millimeter dish.

Tap the dish on the work surface to release any air bubbles. Bake the PDMS at 70 degrees Celsius for two hours and allow it to cool to room temperature. For each previously generated stamp, cut a 15 by 15 millimeter square of PDMS.

These will be referred to as flat slabs for the remainder of the protocol. Invert each generated stamp onto a flat slab of PDMS such that the features on the stand for in contact with a flat slab of PDMS. Gently press on the top of the stamp with forceps to ensure even contact.

Next, place a small drop of UV-curable polymer at the top interface of each stamp and slab pair. The polymer will be wicked between the two by surface tension assisted by gravity. Place a small drop of the UV-curable polymer around the edges of the stamp and slab interface.

This creates a border that will hold the ligand solution. Then carefully place the stamp and slap pairs into a UV sterilization box. Use the sterile STR power setting and expose for 10 minutes.

After this, remove the pairs from the UV sterilization box and use two pairs of forceps to gently remove the stamp while holding down the flat slab and stencil that formed from the UV-curable polymer. Using forceps, carefully remove the stencil and invert it such that the surface that is in contact with the flat slab of PDMS is facing up. Place the inverted stencils back into the UV sterilization box.

Use the sterile STR power setting and expose for three minutes. Using forceps, carefully placed each stencil flat side down onto an acid washed coverslip making sure that features are centered on the cover slip. Place a small piece of laboratory film on top of each stencil.

Firmly and evenly press down on the stencil to create strong contact between the stencil and the coverslip. Place the cover slips with stencils in a dish. Place this dish into a plasma cleaner and apply high-power plasma for 30 seconds.

Then, pipette the ligand solution onto the surface of each stencil. For stencils made from 10 by 10 millimeter square stamps, apply 100 microliters per stencil. After this, wrap the dish containing the cover slips in laboratory film and incubate at four degrees Celsius overnight.

First, wash the coverslip with stencil by using forceps to briefly submerge it in a dish containing sterile PBS. Submerge the coverslip with stencil into a second stage of PBS to wash it again. Remove the stencil from the surface of the coverslip being careful to not break the coverslip.

Next, briefly submerge the coverslip in a dish containing ultrapure water to remove salts from the PBS washes. Touch the edge of the coverslip to a delicate task wipe to wick away excess water. Dry the cover slip under an inert gas such as nitrogen.

Then, prepare a polyacrylamide solution to obtain the desired hydrogel elasticity. For each patterned coverslip, prepare a glutaraldehyde activated bottom covers lip with a spacer placed on top. Pipette between 75 and 150 microliters of polyacrylamide solution to the center of each glutaraldehyde activated coverslip.

Using forceps, place a patterned to cover slip onto each glutaraldehyde activated covers slip with polyacrylamide and spacer such that the patterned ligand faces the polyacrylamide solution. Carefully place each polyacrylamide sandwich into a cap holder with threads that are compatible with 15 milliliter conical bottom tubes. Screw a 15 milliliter conical bottom tube into each cap holder to hold the polyacrylamide sandwiches in place.

Centrifuge to polyacrylamide sandwiches in the tubes in swing buckets at 200 times times G for 10 minutes at room temperature. Remove the tubes from the centrifuge and place them in tube racks to maintain orientation for an additional 50 minutes to ensure full polymerization. Then, remove the sandwiches from the tubes and submerge them in PBS.

Wrap the dish in laboratory film and incubate at room temperature for three hours or at four degrees Celsius overnight. After this, use a scalpel and forceps to carefully remove the pattern to cover slips and the spacer from the polyacrylamide sandwich while it remains submerged in PBS. Place each coverslip with patterned polyacrylamide into a cap holder.

Place of gasket on top of the cover slip and around the polyacrylamide where the spacer was located. Screw a sawed-off 15 millimeter conical bottom tube into the cap holder, forming a well with the patterned polyacrylamide at the bottom. These assemblies fit into the wells of a standard 12 well plate for easy handling.

When performing this technique, use a fluorescent ligand to ensure the desired pattern is created on the surface of the glass cover slip, and successfully transferred to the surface of the polyacrylamide hydrogel during polymerization. The ultimate measure of success for this method is the ability to culture hESCs in desired geometries on the patterned hydrogels. When cultured as outlined in the text protocol, the hESCs typically proliferate to complete the patterned geometries by 48 to 72 hours once the Y27632 is completely removed from the media.

Culturing hESC colonies in confined geometries on polyacrylamide hydrogels permits the measurement of cell-generated traction forces using traction force microscopy. These measurements are made by embedding fluorescence microspheres in the hydrogel and imaging the positions of these beads before and after seeding hESCs. The images of the bead positions can be used to generate maps of bead displacements which can subsequently be used to calculate the underlying traction stresses.

In circular colonies of hESCs, the largest traction stresses are found near the peripheral edge of the colonies, while the center of the colonies display uniformly low traction stresses. Despite the observed non-uniform distributions of traction stresses, hESCs cultured as patterned circles in maintenance conditions, display uniform expression of the pluripotency marker Oct 3/4 and cell adhesion molecule E-cadherin. This technique will allow researchers to better model human embryos using human embryonic stem cells ultimately leading into important insights into how humans develop.

This procedure can be adapted for any application in which a researcher aims to culture cells in defined geometries on soft hydrogels. This procedure is compatible with most biochemical acids, allowing researchers to answer questions about how things such as tissue geometry and cell-generated forces affect protein expression and cell signaling. Many researchers are now using human embryonic stem cells to model early human development.

This technique will allow researchers to answer these important questions in conditions that better mimic a physiological environment.