The overall goal of this procedure is to process a human vestibular schwannoma sample received from the operating room for primary cell culture and collection of tumor secretions. This method can help answer key questions in the field of vestibular schwannoma research, such as how best to receive, store and process a primary human tumor sample intended for use in multiple biomolecular essays. The main advantage of this technique is that it makes use of a minimally manipulative culture method to propagate primary human vestibular schwannoma cells.
The implications of this technique extend toward therapy for vestibular schwannoma because promising therapeutic candidates can be tested directly on cultured cells. Begin by transferring the specimen container holding the freshly harvested vestibular schwannoma or great auricular nerve sample from the ice used for transportation from the operating room to a laminar flow hood in the laboratory. Use sterile forceps to carefully transfer the specimen to the first of three 1.5 milliliter tubes filled with 1.4 milliliters of one exterior PBS.
Close each tube once it contains the specimen and shake gently to wash. Place the sample in a dry 60 millimeter petridish and use forceps to remove all dead tissue in areas with prominent blood vessels. Use forceps or a scalpel blade to divide the sample into separate pieces for RNA preservation, protein extraction, secretion collection, fixation and cell culture.
Transfer the piece of the specimen designated for cell culture to a new 60 millimeter culture dish containing five to eight milliliters of cold supplemented DMEM F12 medium. Place the piece of vestibular schwannoma or great auricular nerve designated for the collection of secretions into an empty 1.5 milliliter tube. Begin by pipetting 500 microliters of DMEM not supplemented with FBS, into two 1.5 milliliter tubes.
The first tube will be used to collect the tumor secretions and the second tube will serve as a matched control. Place one of the DMEM containing tubes onto a calibrated digital scale. Tare the scale, place the piece of specimen designated for the collection of secretions into the tub and weigh it.
Note the result, which will represent the weight of the tissue alone. Under a laminar flow hood, adjust the volume of DMEM in the tube to 100 microliters per 10 milligrams of the specimen. Place the tube containing the tissue sample and its matched control into the incubator.
Incubate for at least 72 hours, to collect biologically useful quantities of tumor or nerve secretions. After incubation for 72 hours, use a one milliliter pipette to remove the secretion containing medium from the specimen. To prevent repeated freeze-thaw cycles, aliquot the medium into new tubes according to planned downstream analysis.
Store the original tissue piece, secretions and control DMEM in a minus 80 degree celsius freezer until downstream applications are initiated. Begin the culture procedure by using a pair of forceps in each hand to separate the vestibular schwannoma tissue into approximately one milliliter cubed pieces. Then aspirate the medium and tumor pieces with a 10 millimeter serological pipette and transfer all contents to a 15 milliliter conical tube.
Centrifuge for three minutes at 1000G at 25 degrees celsius. During the centrifugation, prepare disintegration medium by combining 4.7 milliliters of supplemented DMEM F12 medium with 250 microliters of collagenase and 50 microliters of hyaluronidase in a 60 millimeter culture dish. After centrifugation, the tumor pieces will form a palate at the bottom of the conical tube.
Carefully pipette off and discard the supernatant. Then add two milliliters of freshly made enzyme containing disintegration medium to the tumor palate. Pipette up and down several times to mobilize the tissue and then transfer it to the 60 millimeter culture dish.
Place the culture dish into the incubator for 18 hours, the next day, warm DMEM F12 medium supplemented with 10%FBS and 1%penicillin streptomycin in 37 degree celsius water bath. Remove the dish containing the vestibular schwannoma culture from the incubator and transfer to the flow hood. Using a 22 gauge needle attached to a six milliliter syringe, aspirate the culture containing medium and transfer it to a new 15 millimeter conical tube.
Centrifuge for five minutes at 1000G at 37 degree celsius. In the mean time, use sterile forceps to place the required number of polydelycine and laminin coated glass cover slips into a 24 well culture plate. 24 to 32 wells can be cultured from a one centimeter cube tumor, so for most smaller tumors, planning for eight to 16 wells of culture cells is appropriate.
After centrifugation, discard the supernatant and re-suspend the residual cell palate in the pre-warmed medium, to allow one milliliter of cell suspension per planned well. Use a five milliliter serological pipette to aspirate two milliliters of cell suspension and deposit one milliliter into a well of the 24 well plate. Continue aspirating and depositing tumor cell containing medium, aspirating in increments of two milliliters from which one milliliter is deposited into each planned well, until the tumor cell suspension is equally divided between all prepared wells.
Place the 24 well plate, into the 37 degree celsius incubator overnight. Check the plate the next day, if significant debris is noted on the bottom of the wells, carefully change the medium in each well to new supplemented DMEM F12 culture medium, taking care not to dislodge adherent cells. If there is no debris, return the plate to the incubator and change the medium for the first time on day five and every three days thereafter.
This bright field image illustrates the typical organizational pattern of vestibular schwannoma cells in culture. These immunofluorescent images of a primary human vestibular schwannoma culture were taking after 48 hours of exposure to GFP expressing ink 80, shown in green. The red fluorescence is from phalloidin F actin immuno staining, which stains the cytoskeleton and the blue fluorescence is dapi, which stains the nucleus.
The inset, is a higher power image of a region of the low power image. While attempting this procedure, it's important to remember to write down the times you put the secretion sample and the culture sample into the incubator, to ensure later steps are followed after the correct incubation periods. Following this procedure, other methods like viral transduction, fixation, immunofluorescence, drug treatment and RNA extraction can be performed to answer additional questions, like how different viruses are expressed in cells or how gene expression changes after drug treatment.
After its development, this technique paved the way for researchers in the field of vestibular schwannoma research to explore the way primary human cells respond to different drugs in living patients who have undergone surgery.
