CFU‐GM Assay for Evaluation of Drug Myelotoxic Activity

Augusto Pessina1, Arianna Bonomi1

1 University of Milan, Milan, Italy
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 20.2
DOI:  10.1002/0471140856.tx2002s34
Online Posting Date:  November, 2007
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Abstract

To study hematotoxicity of compounds on the myeloid cell compartment, the authors describe a standard procedure developed as a workable good laboratory practices–compliant protocol to determine the in vitro myelotoxic effect of drugs and chemicals. Specific protocols are presented to prepare human and murine myeloid progenitors (CFU‐GM) for testing in a validated CFU‐GM assay. Details are given for performing a screening test when toxicity data are not available and for passing on to an accurate inhibitory concentration–determination phase. To quantify the potential hematotoxicity of xenobiotics from their direct adverse effects on CFU‐GM, the unit describes how to manage the results by means of an algorithm able to predict the acute xenobiotic exposure levels that cause maximum tolerated decreases (MTD) in absolute neutrophil count (ANC). A protocol describes a miniaturized application of the procedure in 96‐well plates for high‐throughput screening of compounds or for testing compounds that are available in very small quantities. Curr. Protoc. Toxicol. 34:20.2.1‐20.2.18. © 2007 by John Wiley & Sons, Inc.

Keywords: CFU‐GM assay; acute neutropenia; maximum tolerated dose; phase I trial; myelotoxicity

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Determining the In Vitro Myelotoxic Effects of Drugs and Chemicals
  • Support Protocol 1: Preparation of Human Hematopoietic Progenitor Cells
  • Support Protocol 2: Preparation of Murine Hematopoietic Progenitors (m‐BM) Cells
  • Support Protocol 3: Preparation of Test Toxicant Dilutions
  • Support Protocol 4: Interpretation of Results from CFU‐GM Assay
  • Alternate Protocol 1: Miniaturized CFU‐GM Assay
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Tables
     
 
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Materials

Basic Protocol 1: Determining the In Vitro Myelotoxic Effects of Drugs and Chemicals

  Materials
  • Methylcellulose culture medium (MCM; see recipe)
  • Test compound, vehicle, and diluent
  • Iscove's Modified Dulbecco's medium (IMDM), with 2 mM L‐glutamine, containing 30% (v/v) fetal bovine serum (FBS)
  • Isolated cells (see Support Protocols protocol 21 and protocol 32)
  • 10‐ml syringes and 18‐G needles
  • 14‐ml round‐bottom polystyrene tubes (BD Falcon, cat. no. 352057)
  • 35‐mm petri dishes
  • 1‐ml insulin syringes with 19‐G needles
  • 37°C, 5% CO 2 incubator
  • 60‐mm petri dishes
  • 60‐mm gridded tissue culture dishes
  • Inverted microscope
NOTE: For tissue culture, high‐performance‐quality plastic must be used and testing it is advisable in order to guarantee high quality level.

Support Protocol 1: Preparation of Human Hematopoietic Progenitor Cells

  Materials
  • Citrate‐phosphate‐dextrose (CPD) solution
  • Fresh human cord blood (hu‐CB) or frozen isolated cord blood cells (hu‐CBC)
  • Calcium‐ and magnesium‐free Dulbecco's phosphate‐buffered saline (CMF‐DPBS; appendix 2A)
  • Ficoll‐Paque (Research Grade; GE Healthcare; store at 4°C protected from direct light)
  • 0.2% (w/v) trypan blue in Hanks' balanced salt solution (HBSS; appendix 2A)
  • Turk solution (see recipe)
  • Iscove's modified Dulbecco's medium (IMDM), with 2 mM L‐glutamine, containing 40% (v/v) fetal bovine serum (FBS)
  • Freezing medium (see recipe)
  • 70% (v/v) ethanol
  • Iscove's modified Dulbecco's medium (IMDM), with 2 mM L‐glutamine, containing 30% (v/v) fetal bovine serum (FBS)
  • 15‐ml and 50‐ml centrifuge tubes
  • Centrifuge
  • 1.5‐ or 2‐ml cryotubes
  • Additional reagents and equipment for determining cell viability by trypan blue exclusion ( appendix 3B)

Support Protocol 2: Preparation of Murine Hematopoietic Progenitors (m‐BM) Cells

  Materials
  • Male BDF/1 (C57Bl/6 × DBA‐2) mice 8 to 12 weeks old
  • 70% (v/v) ethanol
  • Iscove's modified Dulbecco's medium (IMDM), with 2 mM L‐glutamine, supplemented with 100 U/ml penicillin and 100 µg/ml streptomycin
  • IMDM, with 2 mM L‐glutamine, without antibiotics
  • IMDM, with 2 mM L‐glutamine, containing 30% (v/v) FBS (without antibiotics)
  • 0.2% (w/v) trypan blue in Hanks' balanced salt solution (HBSS; appendix 2A)
  • Turk solution (see recipe)
  • Gauze
  • Surgical instruments including small scissors and surgical tweezers
  • 150‐mm petri dishes
  • 5‐ml syringe and 23‐ to 25‐G needle
  • 15‐ and 50‐ml centrifuge tubes
  • 100‐µm disposable cell strainer
  • Additional reagents and equipment for euthanasia of mice (Donovan and Brown, )

Support Protocol 3: Preparation of Test Toxicant Dilutions

  Materials
  • Test toxicant
  • 0.5% (v/v) dimethylsulfoxide (DMSO)

Support Protocol 4: Interpretation of Results from CFU‐GM Assay

  • 4‐ml round‐bottom tubes
  • Flat‐bottom 96‐well plates
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Literature Cited

Literature Cited
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