A Photopolymerizable Hyaluronic Acid-Collagen Model of the Invasive Glioma Microenvironment with Interstitial Flow

Our work investigates how the tumor microenvironment drives invasion of tumor cells in glioblastoma, the deadliest form of brain cancer. Specifically, we're interested in how interstitial fluid flow driven by increased intratumoral pressures causes tumor cells to invade into the surrounding brain parenchyma. We currently use the 3D hyaluronic acid collagen system mass models, combined with MRI imaging and a computational analysis to study glioma invasion driven by interstitial fluid flow.

For the in vitro approaches, fluid flow is generated by applying pressure on the top of the TME model, mimicking interstitial fluid flow in the brain. Providing the right physical cues to replicate the tissue microenvironment is a continual challenge. Our three dimensional model uses hyaluronic acid and collagen, both found in the brain matrix, and sustained interstitial fluid flow.

Together, these factors provide key physical signals to healthy and cancerous cells. Our recent work indicates that resident brain cells influence how glioma cells respond to interstitial flow. However, this response depends on patient-specific factors.

To study the influence of these factors on glioma invasion, we developed a patient-informed model of the glioma invasive front that mimics native brain tissue. The patient tumor model allows for a highly controlled in vitro experiment that still represents tissue level factors that influence glioma invasion. Further, this model is accessible compared to similar models of glioma invasion and interstitial flow because it does not require tubing or pumps.

And it uses commercially available materials. 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 astro 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.