May 16, 2020
Implanting PDX Tissue into SCID mice
- Lindsey Abel1,
- Saakshi Kaushik1,
- Kwang Nickel1,
- Randall J Kimple2
- 1University of Wisconsin - Madison;
- 2University of Wisconsin - Madison, UW Carbone Cancer Center
Protocol Citation: Lindsey Abel, Saakshi Kaushik, Kwang Nickel, Randall J Kimple 2020. Implanting PDX Tissue into SCID mice . protocols.io https://dx.doi.org/10.17504/protocols.io.qmpdu5n
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: June 01, 2018
Last Modified: May 16, 2020
Protocol Integer ID: 12687
Keywords: Patient derived xenograft, PDX
Abstract
Protocol for the establishment of patient derived xenograft from fresh patient tissue to SCID mouse or to passage from one mouse to another
Guidelines
Notes:
This protocol has been used for the passaging of head and neck tumors as well as lung and lung/brain tumors
Tissue should be transferred from OR to lab in either a base media or PBS (not formalin or ethanol)
Fast frozen – place tissue in 1.2ml cryovial – label. Place cryovial in LN2, move to -80 freezer for storage
Slow Frozen – If slow freezing, chop entire sample (minus fast frozen tissue), dissociate halfway. Take amount intended for freezing and place in cryovial. Bring volume to 600ul, add 60ul of DMSO. Immediately place in Mr. Frosty Nalgene freezing container (or use controled rate freezer) and place in -80 for 24 hours. Move to LN2 after 24 hours.
Matrigel- Matrigel has to be kept on ice before and while using as it solidifies at room temperature.
Drawing tissue into syringe – Be sure to draw up mixture through needle and syringe. If you draw mixture through syringe and then add needle you may draw up large chunks that will not pass through needle.
Scissors “any” These scissors are for cutting through the mouse skin to allow for excision of tumors. Use scissors that will not be used for excising tumors or chopping tissue. Cutting through the skin dulls the blades quickly.
Materials
MATERIALS
Phosphate Buffered SalineThermo Fisher ScientificCatalog #28374
Matrigel, Phenol Red Free, 10mlCatalog #CB 40234C
PDX Media
1.2ml Eppendorf TubesFisher Scientific
Straight, sharp/sharpRobozCatalog #5906
Scissors - Curved, sharp/sharpCatalog #5908
18G needleBecton Dickinson (BD)Catalog #305196
Amphotericin BThermo Fisher ScientificCatalog #15290026
Penicillin-Streptomycin (10,000 U/mL)Thermo FisherCatalog #15140148
Corning™ DMEM with L-Glutamine 4.5g/L Glucose and Sodium PyruvateThermo Fisher ScientificCatalog #10013CV
1 ml syringeBecton Dickinson (BD)Catalog #309628
Ear tags and applicatorFisher ScientificCatalog #10-000-958
STEP MATERIALS
Corning™ DMEM with L-Glutamine 4.5g/L Glucose and Sodium PyruvateThermo Fisher ScientificCatalog #10013CV
Protocol materials
Ear tags and applicatorFisher ScientificCatalog #10-000-958
1.2ml Eppendorf TubesFisher Scientific
18G needleBecton Dickinson (BD)Catalog #305196
Corning™ DMEM with L-Glutamine 4.5g/L Glucose and Sodium PyruvateThermo Fisher ScientificCatalog #10013CV
Straight, sharp/sharpRobozCatalog #5906
Scissors - Curved, sharp/sharpCatalog #5908
Amphotericin BThermo Fisher ScientificCatalog #15290026
Phosphate Buffered SalineThermo Fisher ScientificCatalog #28374
Matrigel, Phenol Red Free, 10mlCatalog #CB 40234C
1 ml syringeBecton Dickinson (BD)Catalog #309628
Corning™ DMEM with L-Glutamine 4.5g/L Glucose and Sodium PyruvateThermo Fisher ScientificCatalog #10013CV
PDX Media
Penicillin-Streptomycin (10,000 U/mL)Thermo FisherCatalog #15140148
Corning™ DMEM with L-Glutamine 4.5g/L Glucose and Sodium PyruvateThermo Fisher ScientificCatalog #10013CV
Safety warnings
all experiments should be approved by institutional IRB and IACUC
Human to mouse implant- Preparation
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.
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)
Corning™ DMEM with L-Glutamine 4.5g/L Glucose and Sodium PyruvateThermo Fisher ScientificCatalog #10013CV
1.5ml FBS
75ul of 10,000 U/mL penicillin/streptomycin
150ul of 250 μg/ml Amphotericin B
Mix together in 15ml tube, yields 15ml – recommended to use within 2 weeks
While cleaning tissue, set out matrigel on ice to thaw. Do not allow to reach room temp, as it will solidify and be useless.
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
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.
Use sharp curved scissors to thoroughly dissociate tissue with constant cutting while in tube
Equipment
Micro Dissecting Scissors 3.5" Curved Sharp/Sharp, Tungsten Carbide, SureCut
NAME
Tool/PDX equipment
TYPE
Roboz
BRAND
RS-5908SC
SKU
LINK
Mixture should be well dissociated so that no large chunks remain. This mixture will need to pass through an 18g needle
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.
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
Anesthesia and Injection
Perform injections on anesthesized mice (using isofluorane).
While mouse is being anesthesized, prepare work area
Draw up tissue-matrigel/media mixture into 1ml syringe with 18g needle already attached.
Prepare ear tag applicator, ear punch or any other tagging method.
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.
Immediately after injecting into each site, pinch skin for 10 seconds – 18g needle leaves a large puncture and the mixture tends to leak.
Apply ear tag while the mouse is still under anesthesia, mark cage with relevant information
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.
Monitor mouse 1-2x a week for tumor growth
Euthanize mouse when tumor size is 600mm^3 or when other human end point is reached.
Mouse to mouse xenograft Implant - Preparation
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.
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
Tissue harvest
Use a pair of blunt scissors to cut open the midline of the euthanized mouse while making sure the tumor is untouched.
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.
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.
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.
Follow the steps above for xenograft establishment, and anesthesia and injection once a fine solution is prepared.