TITLE: UABMC - PDX Dissection Protocol
AUTHORS: Christopher Willey

[STEPS]
?. [TUMOR HARVEST]
Select 1 to 2 mice that have tumors that measure no more than 8-10mm in greatest diameter, are not ulcerated and appear to have grown in a consistent fashion.
This must be done aseptically using sterile surgical techniques
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Euthanize one mouse at a time with CO2 asphyxiation in designated inhalation chamber attached via a two-stage regulator to a CO2 tank.  Perform cervical dislocation or bilateral thoracotomy to ensure the mouse is dead.
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Spray the mouse with 3% chlorhexidine sanitizing solution (over the sink) and transfer mouse to a sterile convertor cloth. Sanitize the skin around the tumor with betadine scrub solution.  Wait at least  before proceeding.
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Use sterile forceps and one scalpel with a #11 blade to produce an incision in the skin forming a half-circle about 5mm away from the tumor on the ventral (lower) side of the tumor.
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Use the forceps to lift the edge of the skin and use a new sterile scalpel with #11 blade to carefully dissect under the tumor freeing it from the underlying muscle tissues. Blunt dissection works best, where possible.  Use your ring finger of the hand holding the forceps to push the tumor forward during this dissection.
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After you have dissected the tumor free from the body of the mouse, continue to dissect the tumor from the overlying skin.
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Open one of the glass Petri dishes and set the cover aside.  Continue the dissection of the tumor such that it “drops” into the open dish. Cover the Dish and transfer to a laminar flow biological safety cabinet.
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Dice tumor into large pieces (chunks) and dissect out any necrotic (dead) tissue. Necrosis can be white, yellowish or dark red and bloody.  Transfer healthy-looking tumor tissue chunks to a new glass Petri dish, if necessary. Place Petri dish on an ice bath until all tumor chunks have been harvested and processed to this point.
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Place the carcass in the black plastic sack and proceed to euthanize the next mouse and repeat the process.  Ensure that the carcass bag is well tied and disposed in the Morgue in Animal Resource Program space.
?. [PREPARATION OF ENZYME SOLUTION]
Add 35ml of serum-free RPMI 1640 to a 50ml centrifuge tube. Add one aliquot of Enzyme D, Enzyme R and Enzyme A to the tube.
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Sterile filter the enzyme solution using a 5ml syringe equipped with a 0.22um syringe filter into a “C” tube.
?. [DISSOCIATION OF PDX TUMOR TISSUE]
This should be performed aseptically in a laminar flow biological safety cabinet – Be sure to use gloves, sanitize your gloves with 3% chlorhexidine spray and observe strict aseptic, no-touch techniques
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Mince PDX tissue finely using two #11 scalpel blades in a scissor-like action.
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Using sterile curved forceps, transfer the minced tumor pieces to the “C” tube containing filtered dissociation solution
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Place the “C” tube upside down on the GentleMACS Tissue Dissociation machine set to “m_imp_tumor 02.02. Press “START” and let the machine run.
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After , remove the tissue rotator form the incubator and take “C” tube off.
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Place “C” tube on GentleMACS machine set to “m_imp_tumor 03.02”. Press “START” and let the machine run.
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Remove “C” tube from GentlMACS machine and place in centrifuge. Centrifuge
Centrifuge: 300 34, 3 min
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Remove “C” tube from centrifuge and place under laminar flow hood. Resuspend the pellet and enzyme solution by pipetting 8–10 times using a 5ml pipet.
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Place a 40um cell strainer on the top of a 50ml centrifuge tube.
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Add of serum-free RPMI 1640 to the tissues solution in the “C” tube and pipet gently to mix.
10 mL
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Slowly add  of the cell suspension to the strainer, allowing it to drip through.
6 mL
NOTE: This process will take 2 – 3 strainers, as the chunks of non-dissociated tissue will clog the strainer. Monitor the strainer and replace as needed.
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Centrifuge the strained cell suspension  and resuspend the pellet in  of serum-free RPMI 1640 at room temperature.
Centrifuge: 300 34, 7 min
10 mL
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Count the cells to determine total number of viable cells using your preferred method.
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Before any medium is added, disperse the pellet to facilitate resuspension of the cells by flicking the bottom of the tube several times with you forefinger while holding the top of the tube.
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Resuspend the cells inof NeuroBasal Complete medium and place in a T75 ( 10 million viable cells), containing the balance of the appropriate amount of medium for the flask (for T75 and for T225).
10 mL
5 mL
20 mL