This ex vivo co-culture system can be used to identify novel cellular and molecular mechanisms of human glioma cell migration and could potentially be used for in vitro drug efficacy testing. The main advantage of this technique is the ability to study interactions between human glioblastoma cells and axons in real time. Demonstrating the procedure will be John Zepecki, a graduate student from my lab.
To assemble compartmented culture dishes, dilute collagen stock solution to 500 micrograms per milliliter in sterile distilled water and mix thoroughly. With a sterile transfer pipette, fill a 35 millimeter culture dish with two milliliters of the diluted collagen solution. Then slightly tilt the dish and remove the solution leaving a thin film of collagen behind.
Dispense the solution into the next 35 millimeter dish. Repeat this process, adding more collagen solution as needed until all dishes have been coated. To polymerize the collagen, in a laminar flow hood, wet gauze pads with three milliliters of concentrated ammonium hydroxide and cover the trays for 15 minutes.
Remove gauze pads and allow the 35 millimeter dishes to dry. Next, use a metal file to file off the point of an 18 gauge needle to make a blunt tip. Soak the needle in 70%ethanol to sterilize.
Apply silicone grease to the bottom of the compartmented chamber. Ensure the grease is placed neatly and overlaps at all corners. This technique can be very challenging so we suggest that you take extra time to practice applying the correct amount of grease before setting up the compartmented chambers in order to ensure excellent growth.
It is also very important to work slowly. Set up more chambers than you think you will need so you have extras should something go wrong. Remove the lid from a 35 millimeter dish, invert the dish and place the scratches over the chamber.
Tap down on the bottom of the dish gently with a pair of forceps. When applying the grease chambers to the dish, do not tap too hard. Otherwise, the axons will not be able to grow underneath into the adjacent chamber.
Use the hemostatic forceps to gently flip the dish over. Release the forceps. Place a mound of grease at the base of the center compartment.
Fill each chamber with supplemented neuronal basal medium. Check for leaks and seal leaks with silicone grease as needed. Continue assembling all culture dishes and store overnight at 37 degrees Celsius and 5%carbon dioxide.
The compartmented chamber allows the use of different pharmacological reagents as well as the ability to compare and contrast results between two wells on the same plate. To seed the prepared cultures containing a GBM neurosphere, first replace the medium with supplemented NBF containing 10%FBS in each distal compartmented chamber. Next, with a P20 pipette set to 10 microliters, draw one GBM neurosphere from the culture dish.
Place the tip of the pipette in the distal chamber closest to the center chamber without touching the axon and expel the GBM neurosphere slowly so that it gently falls onto the axons. Leave the culture in the biosafety cabinet for one hour at room temperature to allow the GBM neurosphere to attach. This method could be used to test pharmacological compounds for the treatment of brain tumors.
This method could be also applied to other diseases of the peripheral and central nervous system such as demyelinating neuropathies and multiple sclerosis. In this protocol, purified DRG axons were seeded with HCGs which formed double positive GFAP and KI67 tumor-like structures integrated within the axonal network indicated by the red tumor markers GFAP while individual HGCs expressing the green fluorescent protein migrated either in association or between the axons. Addition of HGCs on the myelinated DRG oligodendrocyte co-cultures showed that HGCs migrate in association with the red stained myelinated axons and away from the tumor mass through the formation of pseudopodia.
Once this technique was perfected, we were able to utilize it to study migration of human GBM stem cells in real time. We were also able to study effects on cell migration after addition of novel therapeutic small molecule inhibitors. The GBM sphere invading a myelinated oligodendrocyte DRG axon culture is shown here.
The protocol described here could serve as a foundation for other biomimetic three-dimensional ex vivo systems designed to assess the effects of novel drug treatments. One should always use caution when handling human-derived cells. Appropriate PPE should be worn and bloodborne pathogen training should be completed so one understands the risks of working with patient-derived tissues.
Ammonium hydroxide should be handled with care and you should wear gloves, a lab coat, and work in a hood to prevent exposure to this chemical.
