Establishing a Three-dimensional Co-culture Model of Murine Dorsal Root Ganglia and Cancer Cells: An In Vitro Model To Study Perineural Cancer Cell Invasion
Transcrição
To prepare the semi-solid matrix droplets, transfer one glass well-bottom plate from ice onto an ice block under the operating microscope. Place the tip of a no more than 10-microliter pipette directly onto the glass at a 45-degree angle and carefully dispense a 1.5-microliter droplet of matrix onto the glass. Slowly move the pipette tip away from the glass as the matrix becomes engaged with the plate.
Deposit one droplet of matrix onto each of the four corners of the plate and transfer the plate to a room temperature surface for about 1 minute. When the matrix has slightly stiffened, return the plate to the ice block and use closed microscopic forceps to scoop the DRG gently with the left hand.
Using the right hand, carefully transfer the nerve tissue to the tip of a 21G needle and use the needle to gently insert the DRG into the middle of the matrix droplet. The DRG should release easily into the matrix for central positioning with the needle.
After inserting a DRG piece into each of the four droplets, place the plate into a 37 degrees Celsius incubator for 3 minutes. When all of the DRG have been embedded, hold the plates one at a time at a slight angle and slowly add 4 milliliters of DMEM supplemented with 10% FBS to the plates such that the medium only gradually comes into contact with the matrix-DRG units.
Return the plates to the incubator for another 48 to 72 hours. When the neurites reach at least 75% of the way to the edge of the matrix, aspirate the medium from the wells without removing the matrix-DRG units. Then, using a 200-microliter pipette with a smooth trigger, place two drops of 3 times 10 to the fifth cells per milliliter of medium over each matrix DRG assay and return the plates to the incubator. After one hour, gently add 4 milliliters of DMEM with 10% FBS along the sidewall of the glass-bottom plate and return the plates to the incubator until their next microscopic evaluation.
