TITLE: Implanting PDX Tissue into SCID mice 
AUTHORS: Lindsey Abel, Saakshi  Kaushik, Kwangok  P Nickel, Randall Kimple

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Protocol for the establishment of patient derived xenograft from fresh patient tissue to SCID mouse or to passage from one mouse to another</div></div>

[STEPS]
?. [Human to mouse implant- Preparation]
Place fresh tissue (should be passaged within 48 hours of removal from patient ; stored at 4 degrees C) in 60mm cell culture plate filled with 2-4 ml cold 1X PBS. Trim and clean tissue to remove areas of necrosis, fat, muscle.
?. [Human to mouse implant- Preparation]
While cleaning tissue, set out matrigel on ice to thaw. Do not allow to reach room temp, as it will solidify and be useless.
?. [Human to mouse implant- Preparation]
Examine tissue and determine if you have enough to save to prepare fast frozen and/or slow frozen. 1g of total tissue should be enough to passage and save tissue. (See Patient and PDX Tissue Biobanking)
?. [Xenograft tissue establishment]
For the tissue that will be passaged to a new mouse, transfer to a 1.2ml Eppendorf tube filled with 200-400ul of PDX media.
?. [Xenograft tissue establishment]
Use sharp curved scissors to thoroughly dissociate tissue with constant cutting while in tube
?. [Xenograft tissue establishment]
Mixture should be well dissociated so that no large chunks remain. This mixture will need to pass through an 18g needle
?. [Xenograft tissue establishment]
Add additional media based on the amount of patient tissue you began with. Generally samples smaller than 0.5g need 200-350 ul of total media, and samples bigger than 0.5g need 300-400 ul of total media.
?. [Xenograft tissue establishment]
Once the final volume of media has been added, add matrigel equal to the amount of media in tube (1:1 ratio). Mix thoroughly by pipetting up and down 10x or until mixture appears homogenous. Keep on ice to prevent solution from solidifying.
?. [Anesthesia and Injection]
Perform injections on anesthesized mice (using isofluorane).While mouse is being anesthesized, prepare work area
?. [Anesthesia and Injection]
Once mouse is anesthesized, place mouse dorsal side up with nose securely in nose cone. Inject the mouse subcutaneously in 1-4 sites, 100-200ul/site depending on the volume in syringe and the number of sites desired.
?. [Anesthesia and Injection]
Place the mouse backin the cage for recovery before returning cage to colony room (mouse should be awake within 2 minutes of removal from anesthesia.
?. [Anesthesia and Injection]
Draw up tissue-matrigel/media mixture into 1ml syringe with 18g needle already attached.
?. [Anesthesia and Injection]
Prepare ear tag applicator, ear punch or any other tagging method.
?. [Anesthesia and Injection]
Immediately after injecting into each site, pinch skin for 10 seconds – 18g needle leaves a large puncture and the mixture tends to leak.
?. [Anesthesia and Injection]
Apply ear tag while the mouse is still under anesthesia, mark cage with relevant information
?. [Anesthesia and Injection]
Monitor mouse 1-2x a week for tumor growth
?. [Anesthesia and Injection]
Euthanize mouse when tumor size is 600mm^3 or when other human end point is reached.
?. [Human to mouse implant- Preparation]
Ensure that there is enough PDX media (used to mix with tissue sample in later steps) to perform injections :PDX Media Recipe: 13.5ml DMEM (choose preferred brand)1.5ml FBS 75ul of 10,000 U/mL penicillin/streptomycin150ul of 250 μg/ml Amphotericin BMix together in 15ml tube, yields 15ml – recommended to use within 2 weeks
?. [Mouse to mouse xenograft Implant - Preparation]
Determine and confirm the mouse with tumor ready for passage. Place the mouse in CO2 chamber and turn the CO2 tank rate to 1 liter per minute.
?. [Mouse to mouse xenograft Implant - Preparation]
Once the mouse stops breathing, perform secondary method of euthanasia as described in the protocol under use. Setup the BSC for tumor harvest during CO2euthanasia. Clean the working area of BSC with 70% ethanol. Also clean all the materials required for the procedure with 70% ethanol before placing it inside the hood.
?. [Tissue harvest]
Use a pair of blunt scissors to cut open the midline of the euthanized mouse while making sure the tumor is untouched.
?. [Tissue harvest]
Excise the tumor by snipping around the base of the tumor with sharp scissors, by light snips around the base of the tumor. Tumor will be held in place by fascia and blood vessels. Cut all around that to collect as clean tumor as possible.
?. [Tissue harvest]
Place excised tumors into a cell culture dish with PBS. Rinse and clean the tumor further in PBS. Measure the size of the tumor with Vernier Calipers for your records. Place the tumor into a dish with PDX media.
?. [Tissue harvest]
Move the clean tissue into a 1.5ml tube with 200 μl PDX media. Dissociate it with sharp sterile scissors into fine matter. No solid matter should be remaining in the solution. Add more media if the solution seems too thick to be handled in a syringe.
?. [Tissue harvest]
Follow the steps above for xenograft establishment, and anesthesia and injection once a fine solution is prepared.