Isolation and Long‐Term Expansion of Functional, Myelinating Oligodendrocyte Progenitor Cells from Neonatal Rat Brain

Bangfu Zhu1, Chao Zhao2, Fraser I. Young1, Robin J.M. Franklin2, Bing Song1

1 Cardiff Institute of Tissue Engineering and Repair, School of Dentistry, College of Biomedical and Life Sciences, Cardiff University, Cardiff, 2 Wellcome Trust—Medical Research Council Cambridge Stem Cell Institute, University of Cambridge, Cambridge
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 2D.17
DOI:  10.1002/9780470151808.sc02d17s31
Online Posting Date:  November, 2014
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Oligodendrocytes are the myelinating cells of the central nervous system (CNS). The isolation of purified oligodendrocyte progenitor cells (OPCs) in large numbers has been sought after as a source of cells for repair following CNS‐demyelinating diseases and injuries, such as multiple sclerosis (MS) and spinal cord injury (SCI). Methods for isolation of OPCs from rodent neonatal brains are well established and have formed the basis for research in myelin repair within the CNS for many years. However, long‐term maintenance of OPCs has been a challenge owing to small cellular yields per animal and spontaneous differentiation within a short period of time. Much effort has been devoted to achieving long‐term culture and maintenance of OPCs, but little progress has been made. Here, protocols are presented for preparation of highly enriched rat OPC populations and for their long‐term maintenance as oligospheres using mixed‐glial‐conditioned medium. Functional myelinating oligodendrocytes can be achieved from such protocols, when co‐cultured with primary neurons. This approach is an extension of our normal shaking method for isolating OPCs, and incorporates some adaptations from previous OPC culture methods. © 2014 by John Wiley & Sons, Inc.

Keywords: oligodendrocytes; oligodendrocyte progenitor cells; oligospheres; differentiation; myelination

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Table of Contents

  • Introduction
  • Basic Protocol 1: Generation of OPC Cultures from Neonatal Rat Brain
  • Basic Protocol 2: Culture of Oligospheres
  • Support Protocol 1: In Vitro Differentiation and Funtional Identification of Cultured OPCs
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Generation of OPC Cultures from Neonatal Rat Brain

  • 5 μg/ml poly‐D‐lysine hydrobromide (Sigma, cat. no. P6407)
  • Postnatal day 0 to 2 (P0‐2) rat pups
  • 70% (v/v) ethanol in a spray bottle
  • Dissection medium (see recipe), 4°C
  • Papain solution (see recipe), 37°C
  • Ovomucoid solution (see recipe), 37°C
  • Mixed‐glial culture medium (see recipe), 37°C
  • OPC culture medium (see recipe), 37°C
  • Human platelet‐derived growth factor (PDGF‐AA; Peprotech, cat. no. 100‐13A)
  • Basic fibroblast growth factor (bFGF; Peprotech, cat. no. 100‐18B)
  • T75 tissue culture flasks with plug seals (Sarstedt, cat. no. 83.1813.3002)
  • Laminar‐flow hood (advanced biological safety cabinet, e.g., Microflow, Bioquell Service)
  • Humidified tissue culture incubator (e.g., Binder, VWR) at 37°C, 95% air/5% CO 2
  • Sharp, sterile surgical and microdissection tools:
    • Small dissecting scissors (Dumont)
    • Curved microdissecting scissors
    • Forceps, straight and angled
    • Spatula
    • Fine‐tipped forceps
  • 100 × 20−mm polystyrene tissue culture dish (Sarstedt, cat. no. 83.1802)
  • Dissecting microscope (KL1500, Nikon)
  • 7‐ml Bijou tube (Greiner Bio‐One, cat. no. 189171)
  • Sterile 0.22‐μm syringe filters (Elkay)
  • Sterile 2‐ to 5‐ml syringe
  • 15‐ml polystyrene conical tubes (Sarstedt, cat. no. 62.554.502)
  • Sterile 50‐ml tubes
  • Orbital shaker incubator (Stuart, cat. no. S1500)
  • 100‐mm non‐TC‐treated culture dishes (Corning, cat. no. 430591)
  • Hemacytometer (e.g., Labor Optik)
  • Additional reagents and equipment for euthanasia (e.g., by CO 2 asphyxiation)

Basic Protocol 2: Culture of Oligospheres

  • Poly 2‐hydroxyethyl methacrylate (Poly‐HEMA, Sigma, cat. no. P3932)
  • OPCs (see protocol 1, step 34)
  • OPC culture medium (see recipe), 37°C
  • Human platelet‐derived growth factor (PDGF‐AA; Peprotech, cat. no. 100‐13A)
  • Basic fibroblast growth factor (bFGF; Peprotech, cat. no. 100‐18B)
  • Oligosphere culture medium: 75% OPC culture medium plus 25% mixed‐glial‐conditioned medium (see protocol 1; combined medium can be stored up to 1 week at 4°C)
  • StemPro Accutase (Invitogen, cat. no. A11105‐01)
  • 6‐well tissue culture plates or T25 tissue culture flasks (Sarstedt, cat. no. 83.1839.300 or 83.1810.3002)
  • 70‐μm nylon mesh cell strainer (BD Falcon, cat. no. 352350)

Support Protocol 1: In Vitro Differentiation and Funtional Identification of Cultured OPCs

  • 5 μg/ml poly‐D‐lysine hydrobromide (Sigma, cat. no. P6407)
  • 10 μg/ml laminin (Invitrogen, cat. no. 23017‐015)
  • Isolated OPCs or oligospheres (see protocol 1 or 2)
  • StemPro Accutase (Invitogen, cat. no. A11105‐01)
  • Phosphate‐buffered saline (e.g., PBS tablets, Fisher Scientific; autoclave before use)
  • Oligodendrocyte differentiation medium (see recipe)
  • Primary antibodies, such as:
    • Anti‐MBP, clone SKB3 (Millipore, cat. no. 05‐675)
    • Anti‐Olig2 (Millipore, cat. no. AB9610)
    • Anti‐A2B5, clone A2B5‐105 (Millipore, cat. no. MAB 312R)
    • Anti‐NG2 (Millipore, cat. no. AB5320)
    • Anti‐Nkx2.2 (Developmental Studies Hybridoma Bank, 74.5A5)
    • Anti‐MAP2 (Cell Signaling, cat. no. 4542S)
  • Cortical neuron culture medium (see recipe), 37°C
  • 24‐well tissue culture plates (Sarstedt, cat. no. 83.1836.300)
  • Glass chamber slides or glass coverslips (optional)
  • Hemacytometer (e.g., Labor Optik)
  • Additional reagents and equipment for fixing cells and immunocytochemistry
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Literature Cited

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