Generating and Maintaining Slice Cultures from Human Glioblastoma Tissue

All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines.

1. Pre-slicing Preparation

1. Prepare "tissue processing" media and "slice culture maintenance" media the day before planned tumor resection and tissue collection (or utilize previously generated media within 2 weeks). Add 5 mL of Penicillin-Streptomycin solution (10,000 U/mL) and 5 mL of 1 M 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) to 500 mL of a High-Glucose Dulbecco's modified Eagle medium (DMEM) to generate the tissue processing medium.
2. Prepare 250 mL of slice culture maintenance media using a base of neuronal medium (e.g., Neurobasal) without phenol red. Supplement this medium with 10 mM HEPES, 1x B-27 supplement, 400 µM L-glutamine, 600 µM L-alanyl-L-glutamine dipeptide, 60 U/mL penicillin, 60 µg/mL streptomycin, and 6 U/mL nystatin.
3. Store all media at 4 °C for no longer than 2 weeks.

2. Day of Surgery: Tissue Acquisition

1. On the day of tissue acquisition, prepare 50 – 100 mL of 1% and 2% (wt/vol %) solutions of low melting temperature agarose in tissue processing media using autoclaved glassware and sterile technique in a laminar flow hood. Both concentrations of agarose are needed to accommodate unpredictable variation in tumor tissue consistency.
2. Heat the agarose suspension, using a microwave, until gentle boiling is observed. Place the agarose solution in a 37 °C water bath to maintain in a liquid state until use.
3. Place 1 mL of slice culture maintenance media in each well of a 6-well plate.
4. Use a sterile forceps to place empty polytetrafluoroethylene (PTFE) culture inserts in each well. Place the plate in a humidified, water-jacketed, tissue culture incubator with a 5% CO₂ atmosphere maintained at 37 °C.
5. Using a sterile Pasteur pipette in a laminar flow hood, bubble a mixture of 95% O₂/5% CO₂ gas into a flask containing ice cold tissue processing media for approximately 15 to 30 min, prior to tumor tissue acquisition. The use of supra-oxygenated media minimizes hypoxia in the bulk tissue during processing.
6. Prepare labeled 50 mL conical tubes (enough for desired number of individual tumor regions to be isolated) containing 20 mL aliquots of supra-oxygenated ice-cold processing media to transport tissue between the operating room and slicing facility.
7. In conjunction with a neurosurgeon, pre-operatively plan the tumor region for sample acquisition. Select a tumor region with contrast enhancement as demonstrated on the patient's clinical imaging (T1 post-gadolinium magnetic resonance imaging (MRI) sequences). Previous experience suggests this area yields viable tumor tissue as opposed to necrotic tissue.
   NOTE: Generation of slices from the surrounding (peritumoral) white matter was attempted; however, background autofluorescence and decreased tumor cell density limited the experimental utility of these slices.
8. Obtain tumor tissue towards the beginning of tumor resection. Avoid collection of tumor tissue exposed to extensive bipolar cautery, which may compromise tissue viability secondary to thermal injury. Tissue samples acquired during piecemeal tumor resection demonstrate enhanced viability when compared to tissue acquired from lengthy en bloc resections. This observation may relate to differential tumor tissue deprivation of blood flow as a result of surgical resection and extended time to acquisition.
9. If desired, label and store tumor tissue in separate conical tubes to isolate samples from distinct tumor regions. (i.e. superficial vs. deep tumor tissue). Precise tissue locations can be recorded if/when intraoperative surgical navigation is available.

3. Slice Culture Preparation

1. Place tumor tissue pieces in a Petri dish with ice-cold processing media. To wash the tumor tissue and minimize adherent red blood cells, use a pipette to gently exchange and discard the media in the Petri dish three times.
2. Using a scalpel, cut tumor pieces into rectangular box shapes. Trim tissue pieces to approximate 3 mm x 3 mm x 10 mm. Carefully remove any attached vessels through excision or gentle traction with fine forceps.
3. Pipette 5 – 7 mL of 37 °C agarose into a small cube-shaped (~ 2 cm³) plastic embedding mold. Confirm temperature of agarose solution is no greater than 37 °C with a sterile thermometer before use.
4. Allow agarose to sit for approximately 1 min on a bed of ice.
5. Place 2 – 4 tumor tissue "strips" into agarose with long axis oriented vertically. NOTE: Tissue strips will tend to "sink" in the agarose before solidification. To avoid this complication, temporarily hold the tissue strip in vertical orientation until agarose is further solidified.
6. Keep the tissue containing agarose mold on a bed of ice for 2 – 5 min to facilitate solidification.
7. Remove mold from ice and gently remove the agarose block by cutting the sides of the form with a scalpel. Avoid placing excessive force on the block, which may fracture the agarose.
8. Use a generous drop of cyanoacrylate glue to affix the agarose block to the vibratome specimen plate. Let glue set for approximately 1 – 2 min.
9. Fill the vibratome reservoir with ice-cold processing media to submerge the agarose block affixed to the specimen plate.
10. During slicing, bubble 95% O₂ / 5% CO₂ gas mixture into the vibratome reservoir through a trimmed sterile plastic pipette.
11. Set the vibratome slice thickness to 300 – 350 µm.
12. Adjust blade advance speed and blade amplitude according to tissue consistency. "Stiffer" glioblastoma tissue requires slower blade advance speeds and higher amplitude. Exact blade speed and amplitude settings will vary according to vibratome specifications.
    NOTE: Several issues commonly arise during slicing. If the tissue strip dislodges from the agarose block, attempt embedding the tissue in a higher concentration agarose. If tumor slices are thicker than desired (machine set), or are asymmetric, increase the amplitude and decrease  speed of vibratome blade. To ensure tumor tissue is not stuck to or "dragged" by the blade holder, as slicing occurs, carefully position the microspatula between the blade holder and slice, as the blade moves.
13. Transfer tissue slices to Petri dish with ice-cold processing media using a stainless steel microspatula.
14. Obtain 6-well plates containing PTFE inserts and equilibrated slice culture media from incubator (as prepared in section 2, step 4).
15. Plate tissue slices using a stainless steel microspatula. Minimize direct contact and manipulation of tissue by using a small fine bristle paintbrush to generate a "fluid wave" to gently push the slice off the spatula and onto the culture insert.
    NOTE: Minimize the amount of processing media introduced on top of the tissue culture insert. If excessive amounts of media are transferred causing the slices to float, use a sterile Pasteur pipette to remove media.
16. Return the 6-well plates containing tumor slices to an incubator maintained at 37 °C with a 5% CO₂ atmosphere.
    NOTE: The entire embedding and slicing protocol should be completed within 90 min of tumor tissue acquisition from the operating room.

4. Slice Culture Maintenance

1. After 12 – 24 h, transfer the slice cultures by grasping each insert rim with a sterile forceps, and transfer to plates containing fresh slice culture maintenance media. Ensure the slice culture maintenance media aliquoted into each well equilibrates in the incubator for at least 15 min before transferring inserts.
2. Move inserts to new 6-well plates with fresh equilibrated slice culture media every 48 h.
3. If desired, aliquot old slice culture media with a pipette for immediate use in biochemical assays or freeze at -80 °C for future use.