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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  •   FigureFigure 2.D0.1 Overview: Isolation and culture of VM precursors and DA neurons. Dissection of rodent midbrain (see ) enables analysis of intact VM neural tissue for organotypic culture (see ), as well as gentle dissociation into single‐cell suspensions (see ). Cell culture options include expansion and/or differentiation as three‐dimensional‐aggregate cultures (see ), or as adherent monolayer cultures (see ). Subsequent detailed analysis of DA neuronal phenotype is customized for the specific experimental paradigm at hand (see ).
  •   FigureFigure 2.D0.2 Dissection of the VM region from embryonic rodent brain. (AF) Isolate the embryo from the uterine sac. (GH) Free the embryo from any remaining placental and amniotic membranes. (IL) Decapitate the embryo, and identify anatomical landmarks of the cranial central nervous system. Arrowheads indicate rostral and caudal borders of the midbrain region. Dotted lines outline the contour of forebrain CNS tissue. Arrow in (K) indicates VM region (lateral view). (LM) Remove the overlying scalp tissue, to isolate the brain (superior view). Cut away the rostral forebrain and the caudal hindbrain regions (dashed lines; lateral view). (N) Open the resulting tube‐like structure along the posterior midline (dashed line; coronal view). Arrow indicates anterior midline and VM region. (O) Trim the resulting butterfly‐shaped structure, removing ∼2/3 of the posterior/lateral tissue on each side (dashed line; view from ventral midline, tissue flattened). Arrow indicates anterior midline of VM region. Also, see the supplemental Video 1 at Abbreviations: fb, forebrain; hb, hindbrain; VM, ventral midbrain.
  •   FigureFigure 2.D0.3 VM region organotypic culture. (A) Transfer of intact VM tissue onto tissue culture insert. (B) Precursor stage (rat E12) VM region organ culture stained for the DA marker TH. (C) Rat E12 VM region organotypic culture after 2 days in vitro, stained for TH. (D) Higher magnification of VM DA neurons in intact VM region tissue culture. Abbreviations: TH, tyrosine hydroxylase; DA, dopaminergic; VM, ventral midbrain; E12, embryonic day 12; div, days in vitro. Scale bars = 100 µm.
  •   FigureFigure 2.D0.4 Three‐dimensional‐aggregate culture. (A) Viability of VM cell suspensions for three‐dimensional‐aggregate cultures, adherent culture systems, and transplantation studies alike is determined by viability dyes. Here, acridine orange/ethidium bromide (fluorescent image showing live cells in green; upper panel). Lower panel: phase contrast image of the identical field. (B) Aggregate formed after 7 days in vitro in the roller tube system. (C) Aggregate cultures stained for nuclear marker DAPI (tightly packed), TuJ1 neuronal marker, dense fiber network surrounding. (D) Aggregate cultures stained for tyrosine hydroxylase. Inset: higher magnification. Sections of aggregates cut on a vibratome after embedding in agarose are shown; C and D display identical areas. Abbreviations: AO, acridine orange; EthBr, ethidium bromide; TuJ1, neuronal marker beta‐III tubulin; DAPI, nuclear marker; TH, tyrosine hydroxylase.
  •   FigureFigure 2.D0.5 Adherent culture system. (A) VM DA precursors (E11‐12) at 1 day in vitro after plating, and (B) at 7 days in vitro of expansion with bFGF. (C) VM DA precursors during the expansion phase stain positive for Nestin (red), a minor fraction of cells stains positive for beta‐III‐tubulin (green). Blue = nuclear Hoechst stain. (D) Expansion and proliferative capacity is monitored by BrdU incorporation assays: here ranging from 44.2% BrdU+ cells at 1 day in vitro, to over 46.4% at 5 days in vitro to 30.8% at 7 days in vitro. Error bars indicate SEM; three independent experiments. (E) Differentiation of DA neurons is induced subsequent to in vitro expansion or alternatively immediately after VM dissection from older embryos (E14), forming a dense network of neuronal processes, staining positive for neuronal markers such as beta‐III‐tubulin (TuJ1, green; F, G) and dopaminergic markers such as tyrosine hydroxylase (TH, red; H).
  •   FigureFigure 2.D0.6 Options for analytical readout of VM DA neurons. Immunocytochemical assays include measures of neurite outgrowth and targeting studies (A), and/ or co‐culture assays of VM DA neurons, here using astroglial feeder cells (B). Detailed analysis of specific DA neuronal subsets is achieved by isolating fixed DA neurons using laser capture microdissection (LCM) (C). Fluorescence‐activated cell sorting (FACS) methods optimized for fragile neural cell types enables isolation of viable VM DA neurons for further in vitro and in vivo analysis in pharmacological, toxicological, and cell transplantation assays (D,E).


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