Establishment, Maintenance, and In Vitro and In Vivo Applications of Primary Human Glioblastoma Multiforme (GBM) Xenograft Models for Translational Biology Studies and Drug Discovery

Brett L. Carlson1, Jenny L. Pokorny1, Mark A. Schroeder1, Jann N. Sarkaria1

1 Mayo Clinic, Department of Radiation Oncology, Rochester, Minnesota
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 14.16
DOI:  10.1002/0471141755.ph1416s52
Online Posting Date:  March, 2011
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Abstract

Development of clinically relevant tumor model systems for glioblastoma multiforme (GBM) is important for advancement of basic and translational biology. One model that has gained wide acceptance in the neuro‐oncology community is the primary xenograft model. This model entails the engraftment of patient tumor specimens into the flank of nude mice and subsequent serial passage of these tumors in the flank of mice. These tumors are then used to establish short‐term explant cultures or intracranial xenografts. This unit describes detailed procedures for establishment, maintenance, and utilization of a primary GBM xenograft panel for the purpose of using them as tumor models for basic or translational studies. Curr. Protoc. Pharmacol. 52:14.16.1‐14.16.23. © 2011 by John Wiley & Sons, Inc.

Keywords: glioblastoma; xenograft; mouse models

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Implantation of Patient Tumor Samples Into Nude Mice
  • Basic Protocol 2: Serial Passage of Flank Tumor Xenografts
  • Basic Protocol 3: Cryopreservation of Xenograft Tissue
  • Basic Protocol 4: Establishing Short‐Term Explant Cultures From Xenograft Lines
  • Basic Protocol 5: Intracranial and Flank Tumor Implantation Using Short‐Term Explant Cultures
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Implantation of Patient Tumor Samples Into Nude Mice

  Materials
  • Tumor sample
  • Hanks' balanced salt solution (HBSS; Irvine Scientific)
  • Wet ice
  • Growth Factor Reduced BD Matrigel Matrix (BD Bioscience; subsequently referred to as Matrigel through the remainder of this unit)
  • Anesthetic (e.g., Isoflurane, Novaplus)
  • 4‐ to 5‐week‐old female athymic nude mice (Harlan Sprague Dawley Athymic Nude‐Foxn1nu mice—this mouse colony originated from NCI Frederick)
  • 10% povidone‐iodine (Betadine) or rubbing alcohol
  • Centrifuge
  • 100‐mm petri dishes, sterile
  • 1‐ml syringes
  • 16‐G, 11/ 2‐in. needles
  • Scalpels, optional
  • Bell jar desiccator for anesthesia (Fisher Scientific)
  • Fume hood
  • Paper towels
  • Animal ear‐tag punch (Fisher Scientific)
  • Sterile gown, gloves, and mask
  • Animal cages
NOTE: Ideally, 1‐cm3 of tumor tissue is used for implantation, although tumor samples as small as 0.125‐cm3 have been employed to implant a single mouse. Tissue obtained in the operating room using a Cavitron ultrasonic surgical aspirator (CUSA) works well for tumor implantation.

Basic Protocol 2: Serial Passage of Flank Tumor Xenografts

  Materials
  • Mouse bearing tumor ( protocol 1)
  • CO 2 source
  • Betadine
  • OCT medium (Sakura Tissue‐Tek)
  • Dry ice
  • 10% buffered formalin (Fisher Scientific)
  • BD Bioscience Growth Factor Reduced BD Matrigel Matrix
  • Wet ice
  • Liquid nitrogen, optional
  • Scalpels
  • 100‐mm culture plates, sterile
  • Tissue Path disposable base molds (Fisher Scientific)
  • Forceps
  • −80°C freezer
  • 2‐oz specimen containers (Kendall)
  • 1.8‐ml cryotube (Nunc or Corning)
  • 1‐ml syringes
  • 1.5‐ml microcentrifuge tubes, optional
  • Additional reagents and equipment for euthanizing the mouse (Donovan and Brown, )

Basic Protocol 3: Cryopreservation of Xenograft Tissue

  Materials
  • Mouse bearing tumor measuring 1 to 1.5 cm in greatest dimension (for cryopreservation; see Basic Protocols protocol 11 or protocol 22)
  • Betadine
  • Freezing medium (see recipe)
  • BD Bioscience Growth Factor Reduced BD Matrigel Matrix (BD Biosciences)
  • Phosphate‐buffered saline (PBS; Cellgro), sterile
  • Recipient mouse (for restoration of cryopreserved tumor tissue)
  • 1.8‐ml cryotube (Corning or Nunc)
  • Scalpels
  • 1‐ml syringe
  • Cryo 1°C freezing container (Nalgene)
  • Liquid nitrogen storage tank
  • Wet ice
  • 16‐G hypodermic needle
  • Additional reagents and equipment for euthanizing the mouse (Donovan and Brown, )

Basic Protocol 4: Establishing Short‐Term Explant Cultures From Xenograft Lines

  Materials
  • BD Bioscience Growth Factor Reduced BD Matrigel Matrix
  • Wet ice
  • Sterile‐filtered complete DMEM containing 2.5% fetal bovine serum (FBS) and 1% penicillin/streptomycin
  • Mouse bearing tumor measuring 1 to 1.5 cm in greatest dimension (see protocol 2)
  • Betadine
  • Sterile‐filtered complete DMEM containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin
  • 150‐cm2 tissue culture flasks (Corning, cat. no. 430825)
  • Scalpels
  • 100‐mm petri dish (Sarstedt)
  • 1‐ml syringes
  • 37°C, 5% CO 2 incubator
  • Pasteur pipet, optional
  • Additional reagents and equipment for euthanizing the mouse (Donovan and Brown, )

Basic Protocol 5: Intracranial and Flank Tumor Implantation Using Short‐Term Explant Cultures

  Materials
  • Short‐term explant cultured tumor cells (from protocol 4)
  • Trypsin/EDTA (Cellgro; 0.05% trypsin/0.53 mM EDTA in HBSS)
  • Complete DMEM (10% FBS and 1% penicillin/streptomycin)
  • Sterile phosphate‐buffered saline (PBS; Cellgro)
  • Wet ice
  • BD Bioscience Growth Factor Reduced BD Matrigel Matrix
  • Trypan blue
  • Children's liquid Tylenol (32 mg/ml)
  • 100 mg/ml ketamine
  • 20 mg/ml xylazine
  • Saline
  • Spore‐klenz or similar disinfectant
  • Betadine
  • Artificial tears (Petrolatum opthalmic ointment, Puralub Vet Ointment, Dechra)
  • 100% ethanol
  • Triple antibiotic (Bacitracin, Neomycin, Polymyxin B sulfate, G&W Laboratories)
  • 15‐ml and 50‐ml conical tubes (Falcon)
  • Hemacytometer
  • Centrifuge
  • 18‐G × 11/ 2‐in. hypodermic needles
  • 1‐ml syringes
  • 0.5‐ml tuberculin syringes equipped with 28‐G needles
  • Scalpels
  • Dremel drill with a #7 or #8 bit
  • 10‐µl Hamilton syringe with a 26‐G needle
  • Towels, sterile
  • Stereotactic frame (ASI Instruments) with a neonatal rat adaptor (Stoelting)
  • 4‐0 vicryl with rb‐1 needle (Ethicon J30 4H)
  • Radiofrequency identification (RFID) chips and reader (optional; Datamars Companion Animal ID; www.datamars.com)
  • Paper towels
  • Animal cages with bedding chips
  • Heating pad, optional
  • Additional reagents and equipment for anesthetizing the mice and injecting tumor cells ( protocol 1)
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Figures

Videos

Literature Cited

Literature Cited
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