Comparative Human and Rat “Neurosphere Assay” for Developmental Neurotoxicity Testing

Jenny Baumann1, Marta Barenys1, Kathrin Gassmann2, Ellen Fritsche2

1 Both authors contributed equally to this unit, 2 IUF–Leibniz Research Institute for Environmental Medicine, Düsseldorf
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 12.21
DOI:  10.1002/0471140856.tx1221s59
Online Posting Date:  February, 2014
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The developing nervous system is highly vulnerable to the adverse effects of chemical agents. Currently, there is an increasing need for testing and regulating chemical compounds in general use and, due to the lack of available data, to identify those which are developmental neurotoxicants. In this context, alternative testing strategies are needed in order to allow fast and cost‐efficient screening and to reduce the number of animal experiments usually required. In this unit we present an in vitro three‐dimensional model for developmental neurotoxicity screening based on human and rat neural progenitor cells. This model enables the detection of disturbances in basic processes of brain development, such as proliferation, migration, differentiation and apoptosis, and allows the distinction of these specific disturbances from general cytotoxicity. Furthermore, the comparison of human and rat data provides useful insights into species differences for toxicodynamics of compounds contributing to human risk assessment of developmental neurotoxicants. Curr. Protoc. Toxicol. 59:12.21.1‐12.21.24. © 2014 by John Wiley & Sons, Inc.

Keywords: developmental neurotoxicity; neural progenitor cell; in vitro; brain development; species differences

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Table of Contents

  • Introduction
  • Basic Protocol 1: Cultivation of Human and Rat NPCs as Neurospheres
  • Basic Protocol 2: Plating and Chemical Exposure of Human and Rat Neurospheres
  • Basic Protocol 3: Investigation of Developmental Neurotoxicity Endpoints Under Proliferating and Differentiating Conditions
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Cultivation of Human and Rat NPCs as Neurospheres

  • Phosphate‐buffered saline with Ca2+ and Mg2+ (PBS; Gibco, Life Technologies GmbH), sterile
  • Minimal essential medium (MEM; Biochrom AG)
  • PND 5 rat pups (in this protocol, the delivery day of the pups is considered as PND 0)
  • Tissue digestion solution (see recipe)
  • Dulbecco's modified Eagle medium (DMEM; Gibco GlutaMAX High Glucose, Life Technologies GmbH)
  • Ovomucoid solution (see recipe)
  • Proliferation medium (see recipe)
  • Cryovial of normal human neural progenitor (NHNP) cells (Lonza)
  • 70% (v/v) ethanol
  • 100% acetone
  • 6‐ and 10‐cm petri dishes
  • 15‐ and 50‐ml tubes
  • Scissors
  • Forceps
  • 37°C incubator
  • 1000‐µl pipet tips
  • Centrifuge
  • McIlwain tissue chopper
  • Double‐edged razor blade
NOTE: All procedures should be performed in a laminar‐flow hood.

Basic Protocol 2: Plating and Chemical Exposure of Human and Rat Neurospheres

  • Poly‐D‐lysine (see recipe)
  • Sterile water
  • 1 mg/ml laminin (Sigma‐Aldrich, cat. no. L2020)
  • Phosphate‐buffered saline with Ca2+ and Mg2+ (PBS; Gibco, Life Technologies GmbH), sterile
  • Proliferation medium (see recipe)
  • Differentiation medium (see recipe)
  • Test compound stock solution with respective solvent
  • Solutions for the endpoint‐specific controls (Table 12.21.1)
  • Chopped human and rat neurosphere (from protocol 1)
  • Ethanol
  • Cleaning solution (for COPAS Large Particle Sorter; Union Biometrica)
  • 8‐chamber slides
  • 37°C incubator
  • Round‐bottom 96‐well plates
  • 6‐ and 10‐cm petri dishes
  • 100‐µl tips
  • Binocular microscope
  • Micrometer
  • COPAS Large Particle Sorter (Union Biometrica)
NOTE: All procedures should be performed in a laminar‐flow hood.
Table 2.1.1   MaterialsMaterial Needed to Prepare the Endpoint‐Specific Control Solutions

Assay conditions Endpoint Material
Proliferating Proliferation by diameter increase Proliferation medium without growth factors
Proliferation by BrdU incorporation Proliferation medium without growth factors
Viability (Alamar Blue Assay) Proliferation medium (at the end of the assay Triton X solution will be added)
Cytotoxicity (Lactate Dehydrogenase assay; LDH assay) Proliferation medium (at the end of the assay Triton X solution will be added)
Differentiating Differentiation to neurons Differentiation medium with 20 ng/ml EGF
Differentiation to astrocytes Differentiation medium with 20 ng/ml EGF
Differentiation to oligodendrocytes Differentiation medium with 50 ng/ml FGF‐2
Migration Differentiation medium with 10 µM PP2
Viability (Alamar Blue Assay) Differentiation medium (at the end of the assay Triton X solution will be added)
Cytotoxicity (LDH assay) Differentiation medium (at the end of the assay Triton X solution will be added)
Apoptosis (Live Dead assay) Differentiation medium (16 hr prior to the end of the experiment, Staurosporine will be added)

Basic Protocol 3: Investigation of Developmental Neurotoxicity Endpoints Under Proliferating and Differentiating Conditions

  • Pictures of rat or human proliferating neurospheres taken at day 0 of culture (from protocol 2)
  • 96‐well plate with rat or human neurospheres cultured under proliferating conditions (from protocol 2)
  • CytoTox‐One Reagent (see recipe)
  • Triton X solution (see recipe)
  • CytoTox‐One kit (Promega GmbH) containing Assay Buffer and Substrate Mix
  • CellTiter‐Blue Reagent (CTB; Promega GmbH)
  • Proliferation medium without growth factors (see recipe)
  • BrdU cell proliferation ELISA kit (Roche Applied Sciences; chemiluminescent) containing:
    • BrdU labeling reagent
    • Anti‐BrdU stock solution
    • Antibody dilution solution
    • FixDenat solution
    • 10× washing solution
    • Substrate component A solution
    • Substrate component B solution.
  • BrdU labeling solution (see recipe)
  • Accutase
  • Anti‐BrdU‐POD working solution (see recipe)
  • Washing solution (see recipe)
  • Substrate solution (see recipe)
  • 8‐chamber slide with rat or human neurospheres cultured under differentiating conditions (from protocol 2)
  • Differentiation medium (see recipe)
  • 12% (w/v) paraformaldehyde (PFA; see recipe)
  • 1× PBS (see recipe)
  • PBS‐T (see recipe)
  • Goat serum
  • Primary and secondary antibodies (see Table 12.21.2)
  • Nuclear stain (see Table 12.21.2)
  • Distilled water
  • Mounting medium: Aqua Poly/Mount (Polysciences Europe GmbH)
  • Staurosporine (Sigma‐Aldrich)
  • Propidium iodide (1 mg/ml solution in water; Sigma‐Aldrich)
  • ImageJ software
  • Incubator
  • Flat‐bottom transparent 96‐well plate
  • Fluorescence reader with excitation 530 to 570 nm and emission 580 to 620 nm filter pair
  • Water bath
  • Black 96‐well plate
  • Multichannel pipet
  • Hairdryer
  • Paper towels
  • Luminescence reader
  • Phase‐contrast microscope with camera
  • Parafilm
  • Coplin jar
  • Humidified box
  • Cotton sticks
  • Slide cover slips
  • Graph Pad Prism
Table 2.1.2   MaterialsAntibody Dilutions Needed for Immunocytochemical Staining

Dilution factor Supplier
Primary antibodies
Rabbit IgG anti‐GFAP 1:100 Sigma‐Aldrich
Mouse IgG anti‐β(III)tubulin 1:100 Sigma‐Aldrich
Mouse IgM anti‐O4 1:200 R&D Systems
Secondary antibodies
Alexa Fluor 488 anti‐mouse IgG 1:250 Life Technologies GmbH
Alexa Fluor 546 anti‐rabbit IgG 1:100 Life Technologies GmbH
Alexa Fluor 488 anti‐mouse IgM 1:250 Life Technologies GmbH
Nuclear stain
Hoechst 33258 (see recipe) 1:100 Sigma‐Aldrich

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