Aggregating Neural Cell Cultures

Paul Honegger1

1 University of Lausanne, Lausanne, Switzerland
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 12.9
DOI:  10.1002/0471140856.tx1209s15
Online Posting Date:  May, 2003
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Abstract

Aggregating Neural Cell Cultures (Paul Honegger, University of Lausanne, Lausanne, Switzerland). When freshly dissociated embryonic tissues are kept under gyratory agitation, the cells aggregate to form three‐dimensional spheroids in which the cells can migrate and organize themselves, attaining maximal cellular differentiation after weeks of culture. The three‐dimensional architecture of the aggregates permits direct cell‐to‐cell interactions and the formation of a natural cell matrix, which is fundamental to the acquisition of the histotypic properties of the aggregates. This unit describes protocols for preparing forebrain cells from embryonic rodents for aggregating cultures and maintaining these cultures to the differentiated state.

     
 
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Table of Contents

  • Basic Protocol 1: Preparation of Aggregating Cell Cultures from Rat Forebrain
  • Alternate Protocol 1: Mouse‐Derived Brain Cell Cultures
  • Alternate Protocol 2: Preparation and Maintenance of Neuron‐Enriched Aggregate Cultures
  • Support Protocol 1: Preparation and Use of Chemically Defined Media
  • Support Protocol 2: Washing and Sterilizing Procedures
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Tables
     
 
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Materials

Basic Protocol 1: Preparation of Aggregating Cell Cultures from Rat Forebrain

  Materials
  • Rats, 16‐day pregnant (e.g., Sprague‐Dawley)
  • Puck's D‐GS (see recipe), 4°C
  • 96% ethanol denatured with 2% (v/v) 2‐butanone, in a squirt bottle
  • 0.4% trypan blue solution
  • Chemically defined medium (complete DMEM; see protocol 4), 4°C
  • Sterile laminar flow hood with horizontal air flow
  • Dissecting block (optional) perfused with cooling liquid and equipped with wells to hold 60‐mm petri dishes
  • Dissecting microscope(s), if required
  • Cold light source to illuminate embryos and tissues during dissection
  • 250‐ml beaker
  • 60‐mm plastic petri dishes, sterile
  • Guillotine for rats
  • Scissors (large; small and pointed; curved, Vannas)
  • Forceps (large; small and curved; fine, curved; extrafine, Dumont no. 5)
  • Disposable absorbent pads
  • Plastic waste bags
  • 50‐ml conical plastic tubes, sterile (e.g., Falcon)
  • Surgical gloves and mask
  • Scalpel holder and no. 11 blade
  • Nylon mesh bag, 200‐µm and 100‐µm pore size, nylon mesh purchased in large sheets from Sefar (see recipe for construction of nylon mesh bags)
  • Glass rods (0.6‐cm diameter, 20 cm‐long) with blunt, fire‐polished ends
  • 1‐ml and 5‐ml sterile serological plastic pipets (e.g., Falcon)
  • Hemacytometer and hand‐held counter
  • 100‐ml or 500‐ml plastic culture flask, graduated and sterile (e.g., Falcon)
  • 25‐ml and 50‐ml modified Erlenmeyer flasks (see step )
  • Gas‐permeable, autoclavable plastic caps for Erlenmeyer flasks (e.g., Bellco)
  • 37°C, 10% to 11% CO 2 humidified incubator (with inside dimensions sufficient to place a gyratory shaker with large platform)
  • Gyratory shaker with shaking speeds up to 80 rpm, 25‐mm shaking diameter with a large platform to hold up to 100 culture flasks that fits inside a CO 2 incubator, functions at 100% humidity, driven by magnetic induction rather than by a driving belt, and does not destabilize the internal temperature of the incubator
  • 37°C water bath
  • Supports with a removable slanted base for media replenishment and transport of flasks—manufactured in alumina to hold 5 flasks, requiring inside dimensions of 27‐cm width, 6‐cm depth, 3‐cm height, with removable insert (27 × 6–cm)
NOTE: All solutions and equipment coming into contact with living cells must be sterile, and aseptic technique should be used accordingly. For additional washing and sterilizing procedures, see protocol 5.

Alternate Protocol 1: Mouse‐Derived Brain Cell Cultures

  • Mice, 14‐day pregnant (e.g., C57/BL)

Alternate Protocol 2: Preparation and Maintenance of Neuron‐Enriched Aggregate Cultures

  • 2000× Ara‐C (see recipe)

Support Protocol 1: Preparation and Use of Chemically Defined Media

  Materials
  • Dulbecco's modified Eagle's medium (DMEM containing high glucose (4.5 g/liter) and high L‐glutamine (580 mg/liter), but no bicarbonate and no pyruvate; powder from GIBCO‐Invitrogen)
  • Ultrapure water
  • Choline chloride (Sigma)
  • L‐Carnitine (Fluka)
  • Lipoic acid (Sigma)
  • Vitamin B12 (Fluka)
  • 50 µM CdSO 4⋅8H 2O (Merck)
  • 100 µM CuSO 4⋅5H 2O (Merck)
  • 50 µM MnCl 2⋅4H 2O (Merck)
  • 150 µM Na 2SeO 3 (Serva)
  • 2.5 mM NaSiO 3⋅5H 2O (Fluka)
  • 5 µM (NH 4) 6Mo 7O 24⋅4H 2O (Sigma)
  • 2.5 µM NiSO 4⋅6H 2O (Merck)
  • 2.5 µM SnCl 2⋅2H 2O (Merck)
  • 50 µM ZnSO 4⋅7H 2O (Merck)
  • Sodium bicarbonate
  • CO 2 gas
  • Dulbecco's modified Eagle's medium (DMEM devoid of glucose, glutamine, phenol red, bicarbonate, and pyruvate powder mix, e.g., Sigma)
  • D‐Glucose
  • Phenol red
  • 100× BME vitamins (GIBCO‐Invitrogen)
  • 1 mg/ml transferrin (Sigma)
  • 30 µM triiodothyronine (Na+ salt; Sigma)
  • 5 mg/ml insulin (Sigma)
  • 20 µM hydrocortisone‐21‐hemisuccinate (Na+ salt; Sigma)
  • 3 mg/ml linoleic acid (Na+ salt; Sigma)
  • Vitamin A and E solution (see recipe)
  • 500× gentamicin sulfate stock solution (see recipe)
  • 170 mM L‐glutamine (see recipe)
  • 10‐liter glass jar (Pyrex)
  • Magnetic plate and stir bar
  • 5‐ml serological pipet
  • Hollow fiber filters (0.2‐µm MediaKap‐10, Spectrum Laboratories)
  • Peristaltic pump
  • 500‐ml bottles with gas‐tight closures (Pyrex)
  • 0.2‐µm syringe filters (e.g., Acrodisc, Gelman), sterile
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Figures

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Literature Cited

Literature Cited
   Braissant, O., Henry, H., Villard, A.M., Zurich, M.‐G., Loup, M., Eilers, B., Parlascino, G., Matter, E., Boulat, O., Honegger, P., and Bachmann, C. 2002. Ammonium‐induced impairment of axonal growth is prevented through glial creatine. J. Neurosci. 22:9810‐9820.
   Choi, H.K., Won, L., and Heller, A. 1993. Dopaminergic neurons grown in three‐dimensional reaggregate culture for periods of up to one year. J. Neurosci. Meth. 46:233‐244.
   Corthésy‐Theulaz, I., Mérillat, A.‐M., Honegger, P., and Rossier, B.C. 1990. Na+‐K+‐ATPase gene expression during in vitro development of rat fetal forebrain. Am. J. Physiol. 258:C1062‐C1069.
   DeLong, G.R. and Sidman, R.L. 1970. Alignment defect of reaggregating cells in cultures of developing brains of reeler mutant mice. Dev. Biol. 22:584‐599.
   Doyle, A., Griffiths, J.B., and Newell, D.G. (eds.) 1994. Cell and Tissue Culture: Laboratory Procedures. John Wiley & Sons, Chichester.
   Eskes, C., Honegger, P., Juillerat‐Jeanneret, L., and Monnet‐Tschudi, F. 2002. Microglial reaction induced by noncytotoxic methylmercury treatment leads to neuroprotection via interactions with astrocytes and IL‐6 release. Glia 37:43‐52.
   Honegger, P. and Richelson, E. 1976. Biochemical differentiation of mechanically dissociated mammalian brain in aggregating cell culture. Brain Res. 109:335‐354.
   Honegger, P. and Matthieu, J.‐M. 1980. Myelination of aggregating fetal rat brain cell cultures grown in a chemically defined medium. In Neurological Mutations Affecting Myelination (N. Baumann, ed.) pp. 481‐488. Elsevier, Amsterdam.
   Honegger, P. and Werffeli, P. 1988. Use of aggregating cell cultures for toxicological studies. Experientia 44:817‐823.
   Honegger, P. and Schilter, B. 1992. Serum‐free aggregate cultures of fetal rat brain and liver cells: Methodology and some practical applications in neurotoxicology. In The Brain in Bits and Pieces. In vitro Techniques in Neurobiology, Neuropharmacology and Neurotoxicology (G. Zbinden, ed.) pp. 51‐79. MTC Verlag, Zollikon, Switzerland.
   Honegger, P. and Pardo, B. 1999. Separate neuronal and glial Na+,K+‐ATPase isoforms regulate glucose utilization in response to membrane depolarization and elevated extracellular potassium. J. Cereb. Blood Flow Metab. 19:1051‐1059.
   Honegger, P. and Monnet‐Tschudi, F. 2001. Aggregating neural cell cultures. In Protocols for Neural Cell Culture, 3rd edition. (S. Fedoroff and A. Richardson, eds.) pp. 199‐228. Humana Press, Totowa, N.J.
   Honegger, P., Lenoir, D., and Favrod, P. 1979. Growth and differentiation of aggregating fetal brain cells in a serum‐free defined medium. Nature 282:305‐308.
   Honegger, P., Braissant, O., Henry, H., Boulat, O., Bachmann, C., Zurich, M.‐G., and Pardo, B. 2002. Alteration of amino acid metabolism in neuronal aggregate cultures exposed to hypoglycemic conditions. J. Neurochem. 81:1141‐1151.
   Kucera, P., Cano, E., Honegger, P., Schilter, B., Zijlstra, J.A., and Schmid, B. 1993. Validation of whole chick embryo cultures, whole rat embryo cultures and aggregating embryonic brain cell cultures using six pairs of coded compounds. Toxic. In Vitro 7:785‐798.
   Monnet‐Tschudi, F., Zurich, M.‐G., Pithon, E., van Melle, G., and Honegger, P. 1995a. Microglial responsiveness as a sensitive marker for trimethyltin (TMT) neurotoxicity. Brain Res. 690:8‐14.
   Monnet‐Tschudi, F., Zurich, M.‐G., Riederer, B.M., and Honegger, P. 1995b. Effects of trimethyltin (TMT) on glial and neuronal cells in aggregate cultures: Dependence on the developmental stage. Neurotoxicol. 16:97‐104.
   Monnet‐Tschudi, F., Zurich, M.‐G., and Honegger, P. 1996. Comparison of the developmental effects of two mercury compounds on glial cells and neurons in aggregate cultures of rat telencephalon. Brain Res. 741:52‐59.
   Monnet‐Tschudi, F., Zurich, M.‐G., and Honegger, P. 1997. Aggregate cell cultures for neurotoxicity testing: The importance of cell‐cell interactions. In Animal Alternatives, Welfare and Ethics (L.F.M. van Zutphen and M. Balls, eds.) pp. 641‐649. Elsevier, Amsterdam.
   Monnet‐Tschudi, F., Zurich, M.‐G., Schilter, B., Costa, L.G., and Honegger, P. 2000. Maturation‐dependent effects of chlorpyrifos and parathion and their oxygen analogs on acetylcholinesterase and neuronal and glial markers in aggregating brain cell cultures. Toxicol. Apl. Pharmacol. 165:175‐183.
   Moscona, A.A. 1961. Rotation‐mediated histogenetic aggregation of dissociated cells: A quantifiable approach to cell interactions in vitro. Exp. Cell Res. 22:455‐475.
   Pardo, B. and Honegger, P. 1999. Selective neurodegeneration induced in rotation‐mediated aggregate cell cultures by a transient switch to stationary culture conditions: A potential model to study ischemia‐related pathogenic mechanisms. Brain Res. 818:84‐95.
   Rose, S.P.R. 1965. Preparation of enriched fractions from cerebral cortex containing isolated, metabolically active neuronal cells. Nature (London) 206:621‐622.
   Seeds, N.W. 1971. Biochemical differentiation in reaggregating brain cell culture. Proc. Natl. Acad. Sci. U.S.A. 68:1858‐1861.
   Varon, S. and Raiborn, C.W., Jr., 1969. Dissociation, fractionation, and culture of embryonic brain cells. Brain Res. 12:180‐199.
   Wilson, S.H., Schrier, B.K., Farber, J.I., Thompson, E.J., Rosenberg, R.N., Blume, A.J., and Nirenberg, M.W. 1972. Markers for gene expression in cultured cells from the nervous system. J. Biol. Chem. 247:3159‐3169.
   Zurich, M.‐G., Honegger, P., Schilter, B., Costa, L.G., and Monnet‐Tschudi, F. 2000. Use of aggregating brain cell cultures to study developmental effects of organophosphorus insecticides. Neurotoxicol. 21:599‐606.
   Zurich, M.‐G., Eskes, C., Honegger, P., Bérode, M., and Monnet‐Tschudi, F. 2002. Maturation‐dependent neurotoxicity of lead acetate in vitro: Implication of glial reactions. J. Neurosci. Res. 70:108‐116.
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