Methods In Enzymology Vol 254

Copyright © 1995 by Academic Press, Inc. All rights of reproduction in any form reserved.

and neurogenesis; the adult brain may in fact retain this capacity, albeit at a lower level.1"3 Diversified neuronal phenotypes (e.g., projection neurons, interneurons) arise from neuroblasts (precursor cells for neurons) which in turn arise from neural precursor cells. The precursor cells in the CNS can be stem cells or progenitor cells. In analogy to the hematopoietic system, it has been proposed that CNS stem cells are the original multipotent cell type, with extended self-renewing capacity, and the ability to give rise to progenitor cells.4 The latter cell type, which has a limited life span, also has self-renewing properties and can give rise to either neuroblasts or glioblasts (precursor cells of neurons and glia, respectively).

The mechanism(s) for production of neuroblasts from precursor cells and their differentiation to diversified neuronal cell types can be better understood by studying individual cells in culture, where the environment can be easily manipulated. Since the establishment of long-term neuroblast cell cultures has been achieved only recently, previous studies have used immortalized neuronal cell lines.5 7 The immortalization process arrests cells at specific stages of development and halts their terminal differentiation. Cells at these intermediate stages of development can be propagated indefinitely. Although immortalized cells offer a number of advantages, these cells do not always represent their primary counterparts. Thus, studies have devised methods to grow precursor cells which give rise to neuroblasts and glioblasts from embryonic and adult CNS in vitro}-9,In addition, long-term cultures of neuroblasts have been established from both embryonic and adult rat CNS.1,21213 The parameters important for generation and maintenance of neural cells in culture are discussed in this chapter and the strategies and techniques to culture both immortalized and primary precursor cells and neuroblasts are detailed.

1 B. A. Reynolds and S. Weiss, Science 255, 1707 (1992).

2 L. J. Richards, T. J. Kilpatrick, and P. F. Bartlett, Proc. Natl. Acad. Sci. U.S.A. 89,8591 (1992).

3 H. A. Cameron, C. S. Woolley, B. S. McEwen, and E. Gould, Neuroscience 56, 337 (1993).

7 U. Lendhal and R. D. G. McKay, Trends Neurosci. 13, 132 (1990).

s D. L. Stemple and D. J. Anderson, Cell (Cambridge, Mass.) 71, 973 (1992).

9 T. J. Kilpatrick and P. F. Bartlett, Neuron 10, 255 (1993).

10 B. A. Reynolds, W. Tetzlaff, and S. Weiss, J. Neurosci. 12, 4564 (1992).

11 A. L. Vescovi, B. A. Reynolds, D. D. Fraser, and S. Weiss, Neuron 11, 951 (1993).

12 J. Ray, D. A. Peterson, M. Schinstine, and F. H. Gage, Proc. Natl. Acad. Sci. U.S.A. 90, 3602 (1993).

13 J. Ray and F. H. Gage, J. Neurosci. 14, 3548 (1994).

Neurogenesis during Development: Development of Neurons in Different Brain Regions /Choice of Age

The mammalian CNS is generated from the neural ectoderm which invaginates to form the neural tube. The neural tube consists of undifferentiated neuroepithelial cells which are in different phases of the mitotic cycle. Once a cell has gone through its final division it leaves the lining of the proliferative ventricular zone and migrates to its position within an individual brain region. In general, development of the mammalian brain proceeds in a caudal to rostral fashion, with phylogenetically older parts of the brain appearing earlier during ontogenesis. In most brain regions, neurogenesis takes place during a discrete prenatal period in which all the cells from that region are born. For example, the cells of the ventral mesencephalon are produced between Ell and E15, with the peak of cell division occurring between Ell and E13.14 In contrast, neurogenesis takes place before and after birth in brain regions such as the cortex, hippocampus, and cerebellum. In the hippocampus, pyramidal neurons are produced during the prenatal period, whereas neurogenesis of the granule cells of the dentate gryus starts before birth and continues into adulthood.3'15

The choice of age for culturing cells derived from different brain regions depends on the cell of interest. Early embryonic time points (E10 in the mouse) have been used to culture undifferentiated neuroepithelial cells from the telencephalon and mesencephalon.916 Additionally, striatal pri-mordia from E13.5 to E14.5 rodent (the time point when neurogenesis begins in this region17) has been used to isolate stem or precursor cell populations.1'11'18 In addition to isolating undifferentiated neuroepithelial cells in vitro, conditions for establishing neuroblast cell cultures derived from different regions of embryonic rat brain have been established. Establishment of short-term cultures of neuroblasts from the cerebral hemispheres (E13) or spinal cord (E14) has been described.19'20 We have established long-term cultures of neuroblasts derived from different embryonic brain regions. Cells were isolated during periods of neurogenesis from the hippocampus,12 spinal cord,13 and ventral mesencephalon (VM)21 at E16-18, E14-16, and E14, respectively. Based on differences in the development

14 J. M. Lauder and F. E. Bloom, J. Comp. Neurol. 155, 469 (1974).

16 M. Murphy, J. Drago, and P. F. Bartlett, J. Neurosci. Res. 25, 463 (1990).

17 S. A. Bayer, Int. J. Dev. Neurosci. 2, 163 (1984).

18 E. Cattaneo and R. McKay, Nature (London) 347, 762 (1990).

19 J. C. Deloulme, J. Baudier, and M. Sensenbrenner, J. Neurosci. Res. 29, 499 (1991).

20 C. Gensburger, G. Labourdette, and M. Sensenbrenner, FEBS Lett. 217, 1 (1987).

21 H. K. Raymon, J. Ray, D. A. Peterson, and F. H. Gage, Soc. Neurosci. Abstr. 19,654 (1993).

of cells in discrete brain regions, it is important to determine the time of neurogenesis for the brain region and the cell type of interest.

Factors That Influence Generation and Survival of Precursor Cells and Neuroblasts

Neural tissue is composed of not only neurons and glial cells but also other nonneuronal cells, connective and vascular tissues. When neural tissue is removed from the brain, dissociated, and transferred to culture conditions, the cells no longer retain their three-dimensional structures due to loss of anchorage, physiological connections with other cells, and the humoral environment. In vitro culture conditions have been developed to provide artificial substratum for anchorage and to imitate the humoral environments through the use of culture medium and gas exchange. Exogenous factors like substratum, culture medium, gaseous environment, and other parameters play an important role in the survival, proliferation, and differentiation of precursor cells and neuroblasts. Prior to establishing cell cultures, all the parameters for generating an optimum environment for the cells should be considered.

Effects of Substratum

The composition of the substratum is important for the adhesion, survival, proliferation, and differentiation of neural cells. Neural cells can be cultured with or without precoated substratum in serum-containing medium. Components in serum facilitate the attachment of cells to uncoated plastic. However, culturing in serum-free medium requires the presence of substratum for efficient cell attachment. Polyornithine (PORN), poly-l-lysine (pLL), poly-d-lysine (pDL), and PORN/laminin are the most common substratum used for neural cultures. Attachment and clustering of cells to substratum are variable depending on the composition and pH of the buffer used in the coating procedure. Neuronal cells tend to form clumps when plated on dishes precoated with pLL dissolved in water, whereas cells cultured in dishes precoated with pLL in 0.1 M boric acid-NaOH buffer, pH 8.4, remain mostly isolated.19 In addition to survival, proliferation, and differentiation, composition of the substratum is important for the fate choice of a precursor cell. For example, subclones of neural crest stem cells established on fibronectin (FN) generate neurons when plated on FN/pDL but only generate astrocytes when plated on FN-coated substratum.8


Methods for Coating Culture Substrata

All solutions and buffers used for coating dishes should be sterile, and the procedure is usually done in a laminar flow hood. The most commonly used methods are outlined next.

Coating with Poly-D-lysine

Although the method for coating with pDL22 is described here, a similar method can be used to coat plates with pLL.

1. Dissolve pDL (Sigma, St. Louis, MO) in sterile water or in sterile 0.1 M boric acid-NaOH buffer, pH 8.4, to make a stock solution of 1.0 mg/ml. Filter through a 0.22-/xm filter (Millipore, Bradford, MA). Store in small aliquots at -20°. Dilute the stock solution in appropriate buffer or water to make a solution of the desired concentration. The concentrations of pDL may vary from 10 to 50 ¡xg/ml.

2. Add enough pDL solution into the plates to cover the surface and allow to incubate at 37° for 2-24 hr. Wash plates with sterile water three to four times and allow to dry. Plates can be used for several weeks.

Coating Plates with Polyornithine

1. Make a 1- to 10-mg/ml stock solution of polyornithine hydrobromide (Sigma) in sterile water or in 0.1 M boric acid-NaOH buffer, pH 8.4.23 Filter through a 0.22-^m filter. Store aliquots at -20°. Dilute the stock to make a 10-/xg/ml solution. Add enough PORN to entirely cover the surface of the dish and incubate at room temperature for 24 hr.

2. Wash with sterile water two to three times, cover with enough water and store at -20° sealed in plastic bags for future use. If coating with laminin, wash one to two times with phosphate-buffered saline (PBS).

3. Alternatively, after coating with PORN, the dishes can be air dried at room temperature in a laminar flow hood. Before plating the cells, wash plates two to three times with water followed by a wash with the medium.

Coating with Laminin

1. Make a 5-mg/ml stock solution of laminin (mouse or rat, GIBCO/ BRL, Gaithersberg, MD) in sterile PBS. Store in small aliquots at -80°. Laminin should not be freeze-thawed more than once or twice.

22 B. H. Juurlink, in "Protocols for Neural Cell Cultures" (S. Fedroff and A. Richardson, eds.), p. 49. Humana Press, Totowa, NJ, 1992.

23 B. Rogister and G. Moonen, in "Protocols for Neural Cell Cultures" (S. Fedroff and A. Richardson, eds.), p. 10. Humana Press, Totowa, NJ, 1992.

2. Add a solution of 5 /Ag/ml laminin in PBS to PORN-coated plates. Incubate at 37° for 24 hr. Store the plates sealed in plastic bags at -20°.

3. Alternatively, wash plates two to three times in PBS, cover with PBS, and store in plastic bags at -20°. Do not let the plates dry out. The plates can be stored for 1-2 months.

Tissue Procurement

The following procedure is used in the authors' laboratory for obtaining tissues from fetal rat CNS. Deeply anesthetize timed pregnant Fisher 344 rats with the following anesthesia cocktail: ketamine (44 mg/kg), acepro-mazine (4.0 mg/kg), and Rompun (0.75 mg/kg). Remove uterine horns by cesarean section and place immediately on ice. Remove embryos from their individual sacs and place in sterile Dulbecco's PBS (PBS-D). Measure the crown-rump length to verify the embryonic age.

Dissection of Tissues from Different Regions of Brain

The clean dissection of tissues is extremely important in obtaining pure cell cultures. The presence of connective tissue can increase the nonneuronal cell population which will eventually overtake the cultures. Dissection procedures for a few regions of the rat CNS are described next.


Long-term hippocampal cell cultures are derived from rats between E16 and E18. The brain from each embryo is removed and placed in sterile Dulbecco's PBS-D. Using forceps to stabilize the brain, the cortex on one side is peeled back from the midline and laid out flat. The hippocampus, lying just underneath the cortex, is removed with a pair of erridectomy scissors and a sharp forcep, using the scissors to cut out the hippocampus and the forceps to hold onto the tissue piece. The procedure is repeated on the contralateral side. Once dissected free from the embryo, any meningeal membranes or blood vessels that remain attached to the tissue are removed with sharp forceps.

Spinal Cord

Spinal cord cell cultures are established from rats between E14 and E16. The embryo is placed on its side in sterile PBS-D. An initial cut is made lateral to the spinal canal with a pair of erridectomy scissors. Subsequent cuts are made on the opposite side and at the level of the cervical and lower lumbar regions. Once freed from the embryo, residual connective tissue is removed from the spinal cord. At the earlier time points, the connective tissue is firmly attached to the spinal cord, making it difficult to remove without damage to the cord.

Ventral Mesencephalon

Long-term ventral mesencephalon (VM) cell cultures are established from E14 rats. The embryo is placed on its side in sterile PBS-D. The soft skull covering the mesencephalic flexure is peeled away. The block of tissue lying immediately dorsal to the mesencephalic flexure is removed by making two cuts on either side of the flexure and a third cut above the flexure. Mesenchymal tissue is carefully removed from the tissue piece with sharp forceps. Ventral mesencephali from 25 to 30 embryos are pooled to increase the cell yield following dissociation.

Medium for Neuronal Cell Cultures

The composition of the medium influences the survival, proliferation, and differentiation into neuronal cells in vitro. Neuronal cultures are generally maintained at pH 7.2-7.6 and at the appropriate osmolarity. Synthetic base media containing different amounts of inorganic salts, vitamins, amino acids, and buffering agents have been designed to optimize the survival of cultured neuronal cells.24'25 The most commonly used base medium for culturing neuronal cells is Dulbecco's modified Eagle's medium (DMEM), often combined (1:1, v/v) with Ham's F12 medium. Cells are grown in this medium either in the absence (serum-free) or in the presence of serum [usually 10% fetal bovine serum (FBS), but horse serum has also been used]. One disadvantage of growing cells in serum is that the components of the serum are undefined, making analysis and interpretation of results difficult. When exogenous factors are added to serum-containing media, it is not clear whether the effects are due to the added factors or to an interaction of the factor with substances present in the serum. The alternative to using serum is to supply the cells with known amounts of ingredients that support cell survival. A number of serum-free media supplements containing hormones, transport proteins, and vitamins have been developed; N225 is the most common (Table I). To make N2 medium, DMEM: F12 medium containing l-glutamine is filtered through a 0.22-ju.m filter (Corning, Inc., Corning, NY) and the N2 supplement is added to the medium. This medium can be stored for 1 month at 4°. The N2 supplement

24 R. P. Saneto and J. De Vellis, in "Neurochemistry: A Practical Approach" (A. J. Turner and H. S. Bachelard, eds.), p. 27. IRL Press, Oxford, 1987.

25 J. E. Bottenstein and G. Sato, Proc. Natl. Acad. Sci. U.S.A. 76, 514 (1979).

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