The overall goal of this procedure is to illustrate a technically simple, yet highly reproducible protocol for successful primary culture of human vestibular schwannoma tumors. This is accomplished by first timely processing the tumor sample with attention to sterile technique. The second step is to mince the tumor sample into sub-millimeter cubed fragments in ice cooled conditions.
Next, the tumor is further dissociated by enzymatic digestion and serial tation. The final step is to plate the tumor suspension on pretreated culture plates. Ultimately, immunofluorescence microscopy is used to evaluate culture purity by evaluating cell phenotype and to evaluate changes in functional outcomes such as proliferation and apoptosis.
So the main advantages of this technique of existing methods are first, the simplicity of the technique. Second, the decreased time commitment that's involved in preparing the cultures. Third, the reliability of being able to produce highly purified cultures.
And fourth, the fact that the cell phenotype and behavior in vitro in these cultures mimics the behavior of the tumor in the patient. Begin the setup by coating the cell culture plates working in a sterile culture hood. Add enough 0.01%poly L ornithine solution to cover the bottom of a polystyrene culture dish.
After replacing the lids, let the dishes sit at room temperature for at least two hours. After two hours, suction off the poly L ornithine solution and allow the plates to dry completely in a sterile culture hood. Then add enough laminate solution to completely cover the culture plates and replace the lids.
After wrapping the plates in plastic wrap to prevent evaporation, place them on a rocker in a four degrees Celsius refrigerator overnight the day of the tumor harvest. Remove the cell culture plates from the refrigerator and allow them to warm up at room temperature. Then prepare the culture media as described in the text protocol and store it at four degrees Celsius using one sterile 50 milliliter conical tube per tumor to be harvested.
Fill each tube with 25 milliliters of HBS plus plus. Place the tubes in an ice filled cooler and take the cooler to the operating room. Estimating the volume of the tumor based on the preoperative MRI image.
Prepare one sterile two milliliter round bottom tube per 0.5 centimeters cubed of tumor, and add in a few more tubes as spares. Fill the tubes with one milliliter of HBSS plus plus and place them on ice. These tubes will be used for sharp dissection of the tumor samples.
Next, sterilize the tissue scissors, small forceps scalpel handle Petri dish P 1000 pipet men and pipet men tips under ultraviolet light for approximately 15 minutes. In the meantime, the surgical team harvests the tumor specimen in the operating room immediately following surgical resection of each tumor specimen. Place the specimens into a tube with ice cold HBSS plus plus as quickly as possible.
Once all the tissue samples are collected, transport the samples on ice from the operating theater to the lab in a laminar flow hood. Wash the tumor specimen by inverting the conical tube 50 times. Allow the fragments to fall to the bottom of the tube and then remove the HBSS plus plus.
Refill the tube with 30 milliliters of HBSS plus plus and invert another 50 times. Repeat the wash and inversion twice more for a total of four times after resus, suspending the tumor fragments in 30 milliliters of HBSS plus plus. For the last time, pour the mixture into a sterile 100 millimeter Petri dish and separate the tumor into 0.5 centimeter cubed groupings.
If any larger tumor fragments are encountered, use the tissue, scissors, or a scalpel to cut them into smaller pieces. Remove and discard any heavily cauterized or charred tissues as well as the fibrotic tissues of the tumor capsule. Then use forceps to place the 0.5 centimeter cubed tissue groups into the individual HBSS plus plus filled two milliliter round bottom conical tubes all on ice.
Next, use tissue scissors to mince the tumor fragments into smaller fragments of less than one millimeter cubed. Mince the tumor only until the majority of the fragments are less than one millimeter cubed. Do not over mince the tumor samples.
When finished, the suspension should have a snow globe appearance. Place the tube with the completely minced tissue back on ice and proceed to mince the tumors in the next specimen tube. The next step is to use a P 1000 pipe eman with a cut sterile tip to transfer all of the tumor fragments to a single 15 milliliter conical tube.
Use HBSS plus plus to flush the two milliliter tubes to make sure all tumor fragments have been collected in the 15 milliliter tube.Centrifuge. The mixture at 23 degrees Celsius at 73 times G for five minutes. After aspirating the HBSS plus plus supernatant resuspend the cell pellet in a one-to-one mixture of 0.25%trypsin 0.2%collagenase adding just enough to keep the tumor fragments in a tight suspension.
After replacing the screw top, incubate the tube at 37 degrees Celsius for 30 minutes. Also incubate the schwannoma culture media and the cell culture plates at this time. Agitate the tumor cell mixture at least once during the incubation to keep the fragments in suspension.
After incubation, add 100 microliters of FBS to each tube to inactivate the trypsin, and then centrifuge the sample at 23 degrees Celsius, 73 times G for five minutes. Using a pipet carefully suction off as much supernatant as possible without disturbing the cell pellet. Next, add two milliliters of warmed culture media.
For every one milliliter of tumor fragments. Prepare for tumor tation by cutting several P 1000 pipette tips to diameters between one to two millimeters. Then slowly tate the cell suspension using progressively smaller and smaller diameter pipette tips.
Being careful to not over tate the sample. If a single larger piece of tissue blocks aspiration during tation, tap the pipette tip on the bottom of the conical tube to dislodge it. Stop the tation when the solution can be easily pipetted through an unaltered P 1000 pipette tip.
After retrieving the culture plates from the incubator, remove the laminate solution from the culture plates, but do not let the plates dry. Next, make up a master mix by dissolving the fragmented tumor in warmed culture Media at a ratio of 0.5 centimeter cube tumor fragment in 10 liters of warmed culture media. If plating in 100 millimeter dishes, add 10 milliliters of the master mix per dish.
If plating in four, well slides add 750 microliters of master mix per well. If plating eight, well slides add 400 microliters of master mix per well. Then incubate the cultures in 37 degrees Celsius, 100%humidity and 6%carbon dioxide.
Leave the cultures alone for at least 36 hours and then change the media if the solution is yellowed or acidic. Otherwise, change the media at 72 hours then every two to three days or when the media becomes acidic. If the media becomes acidic daily for several days in a row, this is generally an indication of potential bacterial or yeast contamination on culture.
Day 10, spindled schwannoma cell outgrowth radiates from the tumor fragment, which is outlined in red in the lower right corner. Immuno staining of primary vestibular schwannoma cultures with anti S 100 antibodies demonstrates the typical culture and cell morphologies in this image taken on culture. Day 14, a bridging outgrowth is observed between two different tumor fragments.
The spindled morphology of schwannoma cells is observed in this day 14 culture stained with anti S 100 antibodies. Culture purity and cell identity of the primary vestibular schwannoma cultures are verified by labeling the nuclei with DPI and immuno staining with anti S 100 antibodies or anti P 75 NTR antibodies. When attempting this procedure, it's important to file a strict tail technique it to wash the tumor fragments vigorously prior to any dissociation steps.
This will decrease the risk of contamination from both the culture environment as well as from the skin floor of the patient undergoing the tumor resection.
