To begin, place collagen, sodium hydroxide, sterile ultrapure water, 10 times PBS, one micro centrifuge tube, methacrylated hyaluronic acid, and the photo initiator on ice. Then place the tissue culture inserts in the plate and label it. To prepare three milligrams per milliliter collagen solution, combine high concentration collagen, one normal sodium hydroxide, ultrapure water, and 10 times PBS.
Add components to the micro centrifuge tube and keep the pipette tip submerged when mixing to prevent bubble formation. After that, centrifuge cells at 200 G for five minutes at room temperature. Then gently remove excess media from the top of each cell condition solution, leaving the volume of media required for the condition.
Place these solutions on ice. Add the calculated volume of collagen solution to each cell condition solution. Next, add the calculated volume of 1%methacrylated hyaluronic acid, followed by 17 milligrams per milliliter LAP in each cell condition solution.
Mix each condition tube one at a time while keeping the pipette tip submerged to avoid bubble formation and add 100 to 150 microliters to each tissue culture insert. Now turn on the 385 nanometer ultraviolet lamp at a constant current. To photo cross link each gel, expose each well to ultraviolet light for 45 seconds, one at a time.
Then place the plate at 37 degrees Celsius for 30 to 45 minutes to cross link the collagen. Using an astral basal medium with 0.5%and two volume by volume and 1%volume by volume B27 without vitamin A, apply the fluid pressure head to gels. Add media to the well plate and tissue culture inserts for net zero flow.
Place the plate at 37 degrees Celsius, 5%carbon dioxide for at least 18 hours or up to five days.
