Culturing Salispheres from Salivary Gland Tissue: A Method to Develop Salispheres from Human Salivary Gland Derived Epithelial Cells
Transcrição
Within two to four hours after removal from the patient, on a culture plate, use autoclave sterilized dissecting scissors and surgical forceps to mince the salivary gland tissue into fine pieces of the size of 1 to 2 millimeters. Add 6 milliliters of the dispase/collagenase solution to the minced tissue. Then, incubate at 37 degrees Celsius for approximately 30 minutes to one hour.
Disrupt the tissue by pipetting with the 5-milliliter serological pipette, 15 to 20 times. Incubate at 37 degrees Celsius for another 30 minutes to one hour. Repeat the pipetting and incubation step two to three more times or until the tissue resembles a slurry and can easily pass through the pipette opening.
To coat wells with BMM, slowly pipette 500 microliters of overnight thawed BMM into each well of a 6-well plate. Slowly swirl the plate to evenly distribute the BMM over the wells. Then, incubate the coated plates at 37 degrees Celsius for at least 15 minutes prior to use to allow the BMM to solidify.
Transfer the dispase/collagenase solution containing homogenized tissue to a 15-millimeter conical tube. Wash the wall of the plate once with 6 milliliters of DPBS to transfer the remaining cells into the conical tube. Then, through a 70-micrometer nylon mesh cell strainer, filter the homogenate into a new 50-millimeter conical tube to remove undigested tissue.
Centrifuge strained cells for 5 minutes at 500 x g. Aspirate the supernatant. Then, resuspend the cell pellet in 10 milliliters of 1x RBC lysis buffer. Incubate for 5 minutes at 37 degrees Celsius. Then, add 20 to 25 millimeters of DPBS to neutralize the RBC lysis buffer and minimize lysis of salivary cells.
Centrifuge for 5 minutes at 500 x g. White cell pellet indicates complete lysis of all RBC. If the pellet has red color, repeat treatment with RBC lysis buffer. Aspirate the supernatant and resuspend the pellet in 1 to 2 milliliters of BEGM and then transfer the resuspended cells onto BMM-coated wells.
Incubate at 37 degrees Celsius. Once plated on BMM, cells will form into spherical structures or salispheres over a two to three-day period. Cells can be maintained as salispheres for about five to seven days before the BMM begins to degrade, allowing the cells to access and adhere to the plastic and grow as a monolayer.
