Preparation and Maintenance of Organotypic Slice Cultures of CNS Tissue

Beat H. Gähwiler1, Scott M. Thompson2, Dominique Muller3

1 University of Zurich, Zurich, Switzerland, 2 University of Maryland School of Medicine, Baltimore, Maryland, 3 University of Geneva, Geneva, Switzerland
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 6.11
DOI:  10.1002/0471142301.ns0611s09
Online Posting Date:  May, 2001
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Abstract

Organotypic slice cultures are the in vitro method of choice for applications requiring long‐term survival of the preparation and a high degree of cellular differentiation and organization resembling that of the original tissue. Long‐term survival is achieved by culturing slices at the air/liquid interface, either by continuously rotating the preparation (roller‐tube cultures) or by culturing them on semiporous membranes (stationary interface cultures). Both culture techniques yield nerve cells which are highly differentiated in terms of their morphological and physiological characteristics. Because most of these cultures are prepared from 1‐week‐old postnatal animals, in which the cellular and tissue organization is already relatively advanced, the original cytoarchitecture is often remarkably well maintained. Moreover, the presence of a full complement of glial and nerve cells is thought to provide a microenvironment facilitating differentiation of neurons. Slice culture also offers unique advantages for recording from pairs of cells, as a consequence of the high degree of connectivity between nerve cells. Recently, new applications have emerged such as the cultivation of slices from knock‐out animals with limited postnatal survival time or alteration of gene expression by viral vectors.

     
 
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Table of Contents

  • Basic Protocol 1: Roller‐Tube Cultures
  • Alternate Protocol 1: Interface Cultures
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Roller‐Tube Cultures

  Materials
  • 70% and 95% ethanol
  • 25 µg/ml poly‐D‐lysine (30,000 to 70,000 mol. wt.; Sigma), sterile
  • Chicken plasma (Cocalico)
  • 50 U/mg thrombin (Merck)
  • Gey's balanced salt solution (BSS) containing 33.3 mM glucose
  • Experimental rats (fetal to 7‐day‐old)
  • Serum‐free medium (see recipe)
  • Roller tube culture medium (see recipe)
  • Antimitotic drugs: 0.1 to 1.0 µM each of cytosine arabinoside, uridine, and 5‐fluoro‐2‐deoxyuridine (Sigma) in roller tube culture medium
  • Surgical instruments including:
  •  Large scissors
  •  Fine scissors
  •  Razor blades (Martin or Fine Sciences Tools)
  •  2 curved fine forceps (Dumont‐type, #7)
  •  2 straight fine forceps (Dumont‐type, #5)
  •  2 holders for razor blade knives, curved (#BA 290, Aesculap)
  •  4 metal spatulas with polished edges (Merck)
  • Teflon holder (60 × 40 × 20 mm, with grooves for holding 20 coverslips), custom made, for sterilizing coverslips
  • 12 × 24–mm glass coverslips, 1‐mm thickness (Kindler)
  • Bottle‐top filters: e.g., Nalgene Steritop‐GP
  • 40‐, 58‐, and 92‐mm petri dishes
  • Stereo dissecting microscope
  • Plastic foil (Plastoscreen 250 from Mühlebach or Aclar from Ted Pella)
  • McIlwain‐type tissue chopper (Brinkmann)
  • Screw‐top, flat‐sided 16 × 110–mm tissue culture tubes (Nunc, no. 156758)
  • 36°C incubator without CO 2 or humidification
  • Roller‐drum (New Brunswick Scientific, Lab‐Line Instruments, or Schütt Labortechnik)
NOTE: Maintain strictly sterile conditions during all phases of handling since no antibiotics are added to the culture medium. All tissue manipulations are carried out inside a laminar flow hood. Use ethanol followed by flaming to sterilize all open surfaces and bottles.

Alternate Protocol 1: Interface Cultures

  Materials
  • 70% and 100% ethanol
  • Experimental rats (see note below)
  • Dissection medium (see recipe)
  • Interface culture medium (see recipe)
  • Surgical instruments:
  •  Large scissors
  •  Fine scissors
  •  Razor blades
  •  Curved fine forceps (Dumont‐type, #7)
  •  Straight fine forceps(Dumont‐type, #5)
  •  Plastic spatula with polished edges (Merck)
  • Biopore‐CM membrane (10 × 10–cm sheets: Millipore)
  • 8‐mm hole punch
  • 35‐ and 100‐mm petri dishes (Falcon)
  • McIlwain‐type tissue chopper (Brinkmann)
  • Cut, fire‐polished Pasteur pipets
  • 6‐well, 35‐mm multiwell culture plates (Falcon)
  • Millicell‐CM inserts (Millipore)
  • Stereo dissecting microscope
  • 33° and 36°C humidified 5% CO 2 incubators
NOTE: Brain tissue from both rats and mice is routinely used for the preparation of interface slice cultures. The best results in terms of reproducibility, survival, and morphological organization of slice cultures have been obtained with neonatal animals between 0 and 14 days old (Stoppini et al., ). Organotypic cultures can also be prepared from older animals (15 to 22 days), however, and there are even reports of interface slice cultures prepared from adult rats (Del Cerro et al., ).NOTE: Maintain sterile conditions despite the presence of antibiotics in the culture medium. Perform all tissue manipulations inside a laminar flow hood. Use ethanol flame to sterilize all open surfaces and bottles.
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Figures

Videos

Literature Cited

Literature Cited
   Annis, C.M., Edmond, J., and Robertson, R.T. 1990. A chemically‐defined medium for organotypic slice cultures. Neurosci. Methods 32:63‐70.
   Bolz, J., Novak, N., Gotz, M., and Bonhoeffer, T. 1990. Formation of target‐specific neuronal projections in organotypic slice cultures from rat visual cortex. Nature 346:359‐362.
   Buchs, P.‐A., Stoppini, L., and Muller, D. 1993. Structural modifications associated with synaptic development in area CA1 of rat hippocampal organotypic cultures. Dev. Brain Res. 71:81‐91.
   Del Cerro, S., Garcia‐Estrada, J., and Garcia‐Segura, L.M. 1995. Neuroactive steroids regulate astroglia morphology in hippocampal cultures from adult rats. Glia 14:65‐71.
   Gähwiler, B.H. 1981. Organotypic monolayer cultures of nervous tissue. J. Neurosci. Methods 4:329‐342.
   Gähwiler, B.H. 1984. Development of the hippocampus in vitro: Cell types, synapses and receptors. Neuroscience 11:751‐760.
   Gähwiler, B.H. 1988. Organotypic cultures of neural tissue. Trends Neurosci. 11:484‐489.
   Gähwiler, B.H., Capogna, M., Debanne, D., McKinney, R.A., and Thompson, S.M. 1997. Organotypic slice cultures: A technique has come of age. Trends Neurosci. 20:471‐477.
   Gähwiler, B.H., Thompson, S.M., McKinney, R.A., Debanne, D., and Robertson, R.T. 1998. Organotypic slice cultures of neural tissue. In Culturing Nerve Cells (G. Banker and K. Goslin, eds.), Chapter 17. MIT Press, Cambridge, Mass.
   Muller, D., Buchs, P.‐A., and Stoppini, L. 1993. Time course of synaptic development in hippocampal organotypic cultures. Dev. Brain Res. 71:93‐100.
   Stoppini, L., Buchs, P.‐A., and Muller, D. 1991. A simple method for organotypic cultures of nervous tissue. J. Neurosci. Methods 37:173‐182.
   Stoppini, L., Buchs, P.‐A., and Muller, D. 1993. Lesion‐induced neurite sprouting and synapse formation in hippocampal organotypic cultures. Neuroscience 57:985‐994.
   Wray, S., Kusano, K., and Gainer, H. 1991. Maintenance of LHRH and oxytocin neurons in slice explants cultured in serum‐free media: Effects of tetrodotoxin on gene expression. Neuroendocrinology 54:327‐39.
   Zimmer, J. and Gähwiler, B.H. 1984. Cellular and connective organization of slice cultures of the rat hippocampus and fascia dentata. J. Comp. Neurol. 228:432‐446.
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