Isolation and Culture of Ventral Mesencephalic Precursor Cells and Dopaminergic Neurons from Rodent Brains

Jan Pruszak1, Lothar Just2, Ole Isacson1, Guido Nikkhah3

1 Harvard Medical School, McLean Hospital, Belmont, Massachusetts, 2 Institute of Anatomy, Center for Regenerative Biology and Medicine, Eberhardt‐Karls‐University Tübingen, Tübingen, Germany, 3 Freiburg University Hospital, Freiburg, Germany
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 2D.5
DOI:  10.1002/9780470151808.sc02d05s11
Online Posting Date:  December, 2009
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The ability to isolate ventral midbrain (VM) precursor cells and neurons provides a powerful means to characterize their differentiation properties and to study their potential for restoring dopamine (DA) neurons degenerated in Parkinson's disease (PD). Preparation and maintenance of DA VM in primary culture involves a number of critical steps to yield healthy cells and appropriate data. Here, we offer a detailed description of protocols to consistently prepare VM DA cultures from rat and mouse embryonic fetal‐stage midbrain. We also present methods for organotypic culture of midbrain tissue, for differentiation as aggregate cultures, and for adherent culture systems of DA differentiation and maturation, followed by a synopsis of relevant analytical read‐out options. Isolation and culture of rodent VM precursor cells and DA neurons can be exploited for studies of DA lineage development, of neuroprotection, and of cell therapeutic approaches in animal models of PD. Curr. Protoc. Stem Cell Biol. 11:2D.5.1‐2D.5.21. © 2009 by John Wiley & Sons, Inc.

Keywords: stem cells; cell and tissue culture; neuroscience; isolation; purification; separation; cell and developmental biology; cell therapy

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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Dissection of Ventral Mesencephalon
  • Basic Protocol 2: Preparation of Cell Suspension
  • Basic Protocol 3: Midbrain Neural Culture: Organotypic Culture
  • Alternate Protocol 1: Midbrain Neural Culture: Three‐Dimensional Aggregate Culture
  • Alternate Protocol 2: Midbrain Neural Culture: Adhesion Culture
  • Support Protocol 1: Analysis of VM Neural Precursors and DA Neurons
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Dissection of Ventral Mesencephalon

  • C57B6/J mice (The Jackson Laboratory), embryonic day (E) 11 to E13 or Sprague–Dawley rats (Charles River), E11.5 to E14
  • Hanks balanced salt solution Mg2+, Ca2+‐free (CMF‐HBSS; Invitrogen, cat. no. 14170), ice cold
  • Dissection buffer (see recipe)
  • Microdissecting instruments (sterilized; Fine Science Tools):
    • Small dissecting scissors
    • Medium dissecting scissors
    • Dumont forceps—straight and angled or curved
    • Curved microdissecting scissors
    • Spatula
    • Moria perforated spoon with holes
  • Laminar flow hood, sterilized by cleaning with 70% ethanol or UV‐exposure for 15 min
  • 60‐mm and 100‐mm round dishes (petri dishes), filled with dissection buffer
  • Dissecting microscope (e.g., Leica MZ6 or Zeiss Stemi 2000)
  • Curved scalpel blade (e.g., BD Bard‐Parker no. 23 or 24)
  • 15‐ and 50‐ml conical tubes

Basic Protocol 2: Preparation of Cell Suspension

  • Ventral midbrain tissue ( protocol 1)
  • Dissection medium (see recipe)
  • Dissociation medium (see recipe) or trypsin 0.05% (w/v)/ EDTA (Invitrogen, cat. no. 25300) containing 0.2% (w/v) DNase I (see recipe) or Accutase (Innovative Cell Technologies, cat. no. AT104) or TrypLE Express (Invitrogen, cat. no. 12605)
  • Heat‐inactivated fetal bovine serum (FBS; Hyclone, cat. no. SH30070)
  • Expansion medium (see recipe)
  • Differentiation medium (see recipe)
  • Trypan blue (Invitrogen, cat. no. 15250) or acridine orange/ethidium bromide solution (see recipe)
  • 15‐ml conical tubes
  • Laminar flow hood
  • 37°C water bath
  • Sterile fire‐polished 9‐in. Pasteur pipets (see recipe)
  • 200‐ and 1000‐µl plastic tips and pipettors
  • 70‐µm cell strainer (BD, cat. no. 352350) or round bottom tube with 35‐µm cell strainer caps (BD, cat. no. 352235)
  • 1.5‐ml microcentrifuge tubes
  • Benchtop centrifuge
  • Hemacytometer
  • Microscope for viability dye detection (trypan blue: light microscope with bright field or phase contrast; acridine orange/ethidium bromide: fluorescence microscope with UV excitation and filters appropriate for simultaneous red‐green channel detection; emission max for DNA is 526 nm, for RNA 650 nm)
  • Additional reagents and equipment for determining the cell concentration and viability using trypan blue (unit 1.3)

Basic Protocol 3: Midbrain Neural Culture: Organotypic Culture

  • Ventral midbrain tissue pieces ( protocol 1)
  • Differentiation medium (see recipe)
  • 4% (w/v) paraformaldehyde (PFA) solution
  • Laminar flow hood
  • Pasteur pipet with a fire‐polished widened orifice (see recipe) or curved forceps
  • Forceps or tungsten needles
  • Millicell cell culture inserts (for six‐wells; e.g., Millipore, cat. no. PICM0RG50)
  • 6‐ and 24‐well tissue culture plates (e.g., Fisher, Falcon or Nunc)
  • 37°C water bath
  • Scalpel

Alternate Protocol 1: Midbrain Neural Culture: Three‐Dimensional Aggregate Culture

  • VM cell suspension ( protocol 2)
  • Differentiation or expansion medium (see reciperecipes)
  • 4% (w/v) paraformaldehyde solution
  • 15% (w/v) agar gel
  • 15‐ml conical tubes
  • Shaker/roller tube system (e.g., Miltenyi Biotec, cat. no. 130‐090‐753, MACSmix Tube Rotator)
  • Humidified tissue culture incubator (37°C, 5% CO 2), preferably including low O 2 option
  • Vibratome

Alternate Protocol 2: Midbrain Neural Culture: Adhesion Culture

  • VM cell suspension ( protocol 2)
  • Expansion medium (see recipe)
  • Differentiation medium (see recipe)
  • 24‐well tissue culture plates
  • Laminin/poly‐L‐ornithine coated 12‐mm coverslips (see recipe)
  • 100‐ or 200‐µl pipets
  • Humidified tissue culture incubator (37°C, 5% CO 2), preferably including low O 2 option

Support Protocol 1: Analysis of VM Neural Precursors and DA Neurons

  • Dulbecco's phosphate‐buffered saline (DPBS) Mg+, Ca+‐free (CMF‐DPBS; Invitrogen, cat. no. 14190)
  • Antibodies typically used in a basic VM DA differentiation:
    • Sheep anti‐TH (1:1,000; Pel‐Freez)
    • Mouse anti‐nestin (1:100; Millipore/Chemicon)
    • Rabbit anti‐TuJ1 (Covance 1:1000)
    • Mouse anti‐MAP2 (Millipore/Chemicon 1:500)
    • Mouse anti‐Pitx3 (Zymed 1:1000)
    • Rabbit anti‐Pitx3 (1:250; Invitrogen)
    • Rabbit anti‐glial fibrillary acidic protein (1:500; Dako)
    • Rabbit anti‐Nurr1 (E‐20; 1:300; Santa Cruz Biotechnology)
    • Mouse anti‐engrailed 1 (clone 4G11; 1:40)
    • Rabbit anti‐ki67 (1:2,000; Novocastra/Vector Laboratories)
    • Rabbit anti‐DAT (1:1000; Millipore/Chemicon)
  • Corresponding secondary antibodies
  • Pipets
  • Microscope for cell analysis
  • Vibratome for sectioning of three‐dimensional‐aggregate cultures (Leica VT1000 S; protocol 3)
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