Use of a Linear Accelerator for Conducting In Vitro Radiobiology Experiments

Summary

Clinical linear accelerators can be used to determine biologic effects of a wide range of dose rates on cancer cells. We discuss how to set up a linear accelerator for cell-based assays and assays for cancer stem-like cells grown as tumorspheres in suspension and cell lines grown as adherent cultures.

Abstract

Radiation therapy remains one of the cornerstones of cancer management. For most cancers, it is the most effective, nonsurgical therapy to debulk tumors. Here, we describe a method to irradiate cancer cells with a linear accelerator. The advancement of linear accelerator technology has improved the precision and efficiency of radiation therapy. The biological effects of a wide range of radiation doses and dose rates continue to be an intense area of investigation. Use of linear accelerators can facilitate these studies using clinically relevant doses and dose rates.

Introduction

Radiotherapy is an effective treatment for many types of cancer^1,2,3,4. Extra high dose rate irradiation is relatively new in radiation therapy and is made possible by recent technological advances in linear accelerators⁵. Clinical advantages of extra high dose rate over standard dose rate irradiation include shortened treatment time and improved patient experience. Linear accelerators also provide a clinical setting for cell culture based radiation biology studies. The biological and therapeutic implications of radia....

Protocol

1. Cell preparation for suspension cell culture

1. Culture glioma stem-like cells in stem cell culture media at approximately 5 x 10⁶ cell/10 cm plates in a cell culture incubator with 5% CO₂, 95% relative humidity at 37 °C.
   NOTE: The cell culture condition is the same throughout all the procedures. The media used in the protocol are complete media.
2. Two days before scheduled irradiation, collect glioma stem-like cells from the culture plate with a sterile 5 mL pipette .......

Discussion

Radiotherapy is an integral part of cancer management. Ongoing efforts seek to improve the efficacy and efficiency of radiation treatment. Advancements in linear accelerator technology have provided the opportunity to treat patients with unprecedented accuracy and safety. Because most patients are treated with high energy X-rays from linear accelerators, studies examining the biologic effects of a large range of dose rates performed on linear accelerators may be readily applied to patients. There have been several report.......

Materials

Name	Company	Catalog Number	Comments
Material
glioma stem-like cell 4121			gift from Dr. Jeremy Rich
293 cells	ATCC	CRL-1573
neuron stem cell culture media	Thermo Fisher Scientific	21103049	NeurobasalTM media
DMEM	Thermo Fisher Scientific	10569044
Fetal Bovine Serum	Thermo Fisher Scientific	16000044
Penicillin/Streptomycin	Thermo Fisher Scientific	15140-122
Recombinant Human EGF Protein	R&D Systems	236-EG-01M
Recombinant Human FGF basic	R&D Systems	4114-TC-01M
B-27™ Supplement	Thermo Fisher Scientific	17504044
Sodium Pyruvate	Thermo Fisher Scientific	11360070
L-Glutamine	Thermo Fisher Scientific	25030164
Tripsin-EDTA	Thermo Fisher	25200056
extracellular proten matrix	Corning	354277	MatrigelTM
Ethanol	Fisher chemical	A4094
Equipment
10 cm cell culture dish	Denville	T1110
3.5 cm cell culture dish	USA Scientific Inc.	CC7682-3340
22x22mm glass cover slip	electron microscopy sciences	72210-10
15 ml centrifuge tube	Thomas Scientific	1159M36
50 ml centrifuge tube	Thomas Scientific	1158R10
5 ml Pipette	Fisher Scientific	14-955-233
pipet aid	Fisher Scientific	13-681-06
Vortex mixer	Fisher Scientific	02-215-414
Centrifuge	Eppendorf	5810R
Linear Accelerator	Varian	n/a
water equivalent material	Sun Nuclear corporation	557	Solid waterTM
Reagent preparation
DMEM media	10% fetal bovine serum (FBS), 2 mM L-glutamine, 100 units/mL penicillin G, 100 µg/mL streptomycin in 500 ml DMEM media
stem cell culture media	10 ml B27 supplement, 20 µg hFGF, 20 µg hEGF, 2 mM L-glutamine, 100 units/mL penicillin G, 100 µg/mL streptomycin in 500 ml Neurobasal media