Mouse N-myc

31, 35

LTR-"oncogene"-pBRori-SVpro-neo-LTR " Adapted from Ryder et al.31

h LTR, long terminal repeat; 3'ss, 3' splice site; SVori, SV40 origin of replication; pBRori, pBR origin of replication; and SVpro. SV40 early promoter and origin of replication.

LTR-"oncogene"-pBRori-SVpro-neo-LTR " Adapted from Ryder et al.31

h LTR, long terminal repeat; 3'ss, 3' splice site; SVori, SV40 origin of replication; pBRori, pBR origin of replication; and SVpro. SV40 early promoter and origin of replication.

4. Remove the PPD solution by aspiration, add 5 ml DMEM/F-12-DNase solution [0.01% (w/v) DNase in DMEM/F-12], Incubate for 15-30 min at 37°, triturate, and centrifuge as described in step 3.

5. Aspirate the medium and resuspend cells in the culture medium (2 ml). Count cells using the trypan blue exclusion method. Adjust the cell concentrations and plate onto prepared plates.

Generation of Immortalized Neural Cell Cultures

Before the development of methods for long-term culturing of neuroblast cells, which give rise to neurons in culture, establishment of such cultures posed a problem due to lack of or slow proliferation of neuronal cells and rapid growth of nonneuronal cells in cultures which eventually outgrew the neurons. As an alternative, immortalized neuronal cell lines have been generated either by transduction of oncogenes (Table II)31-35 into precursor cells or neuroblasts5-7 or by fusion of tumor lines with primary cells.36 A description of procedures for immortalization of cells by both methods is presented next.

31 E. F. Ryder, E. Y. Synder, and C. L. Cepko, J. Neurobiol. 21, 356 (1990).

32 C. Evrard, E. Galiana, and P. Rouget, EMBO J. 5, 3157 (1986).

33 P. S. Jat and P. A. Sharp, Mol. Cell. Biol. 9, 1672 (1989).

34 K. Frederiksen, P. S. Jat, N. Valtz, D. Levy, and R. McKay, Neuron 1, 439 (1988).

35 P. F. Bartlett, H. H. Reid, K. A. Bailey, and O. Bernard, Proc. Natl. Acad. Sci. U.S.A. 85, 3255 (1988).

36 H. J. Lee, D. N. Hammond, T. H. Large, and B. H. Wainer, Dev. Brain Res. 52,219 (1990).

1. Make a single cell suspension from dissected tissues. Plate 5 X 104 to 2 X 105 cells/cm2 onto PORN/laminin-coated plates in DMEM + 10% FBS and incubate at 33°.

2. The oncongenes most commonly used for immortalization are myc oncogenes,31,32 the SV40 large T antigen,31 or a temperature-sensitive hybrid of the SV40 T antigen (SV40 T tsA58) (Table II).33 34 Cells infected with SV40 T tsA58 grow at 33° (permissive temperature) but not at 39° (nonper-missive temperature). The retroviral vector construction and creation of packaging lines have been described in detail elsewhere.37-38 The retroviral vectors contain the neomycin-resistant gene for easy selection of stably infected cells. Incubate the packaging cell line overnight in DMEM + 10-20% FBS. Collect the conditioned medium and filter the medium through a 0.45-/i.m filter.

3. The day after plating, incubate the cells for 2 hr with conditioned medium collected from the packaging cell lines (step 2) in the presence of Polybrene (8 /Ag/ml; Sigma). Replace the virus containing medium with fresh medium and incubate at appropriate temperatures. After 1-2 days, start the selection by growing cells in the presence of 200 /i.g/ml G418 (neomycin analog Genticine, GIBCO). Change medium every 3-5 days.

4. Pick colonies (usually appear in 3-4 weeks) separately using cloning rings and expand. For long-term storage, ~106 cells/cryovial (Nunc, Naper-ville, IL) in DMEM + 10-30% FBS + 10% dimethyl sulfoxide (DMSO) or 10% glycerol can be frozen in liquid nitrogen.

Generation of Immortalized Neuronal Cell Lines by Fusion with Tumorogenic Lines

Somatic cell fusion methods have been used to immortalize neuronal cells and generate cell lines.36 The advantage of this method is that postmitotic cells can be immortalized.

1. Make a single cell suspension from fetal or adult tissues (septal or hippocampal) as described earlier. For postnatal tissue, centrifuge cells at the buoyant density to enrich for viable, single cells. Resuspend cells in phytohemagglutinin (PHAP, Difco Laboratories, Detroit, MI). This treatment enhances the adhesion of cells to the neuroblastoma cells, N18TG2. This is a HPRT-deficient mutant cell line and the neuroblastoma cells that did not fuse with primary cells can be eliminated from cultures by

37 C. L. Cepko, E. F. Ryder, C. P. Austin, C. Walsh, and D. M. Fekete, this series. Vol. 225, p. 933.

3* M. D. Kawaja, L. J. Fisher, M. Schinstine, H. A. Jinnah, J. Ray, L. S. Chen, and F. H. Gage, in "Neural Transplantation" (S. B. Dunnett and A. Bjorklund, eds.), p. 20. Oxford University Press, New York, 1992.

supplementing the medium with HAT (100 ¡jlM hypoxanthine, 0.4 ¡xM aminopterin, and 16 ¡xM thymidine). The neuroblastoma line is usually grown in DMEM + 10% FBS.

2. Layer hippocampi-PHAP cells on logarithmically growing neuroblastoma cells. After 15 min, remove the unattached cells by aspiration. Add 50% polyethylene glycol (PEG 1000, KochLight) in DMEM (v/v) to initiate cell fusion. Remove the solution after 40 sec and, after 60 sec, wash cells and incubate the cultures in DMEM + 10% FBS overnight. Replate cells in HAT-containing medium to select for growth. Remove aminopterin after 4-8 weeks and hypoxanthine and thymidine 4 weeks later.

3. Cells can be cloned, subcloned, and maintained as described earlier.

Immortalized hippocampal and septal cell lines have been generated by this method from embryonic and young mice.36 Phenotypically stable hybrid cell lines express neuronal markers and exhibit electrophysiological properties.

Culturing of Precursor Cells from Embryonic or Adult Brains Using Growth Factors

Neural precursor cells give rise to neuroblasts or glioblasts under appropriate culture conditions. Culturing of precursor cells from fetal or adult brain with mitogenic growth factors (EGF or FGF-2) has been described1'2'9""12 Although EGF has been used in serum-free culture conditions, FGF-2 has been used in 10% FBS-containing medium. EGF-generated clonal cultures gave rise to both neurons and astrocytes, but FGF-2-gener-ated cultures produce either pure neuronal clones or clones containing both neurons and astrocytes.9,1116

Embryonic Tissues10

Suspend striatum (E14 mouse) in DMEM/F-12 medium and mechanically dissociate the tissue as described earlier. Plate cells on PORN-coated glass coverslips in a 24-well plate at a density of 2500/cm2 in DMEM/F-12 medium containing the N2 supplement (the concentrations of insulin and putrescine are 25 ¿ig/ml and 60 fxM instead of 5 ¿ig/ml and 100 /xM, respectively). Add EGF (Collaborative Research) to the N2 medium at 20 ng/ ml prior to use. However, the addition of EGF at the time of plating is not required and can be added up to 7 days in vitro (DIV). Change medium after 10-14 DIV and then every 2-4 days thereafter. Using this method, cells have been maintained up to 25 days in culture.

Adult Brain1

1. Dissect the striatum from mouse brain (3-18 months old), cut 1mm coronal sections, and transfer into normal artificial cerebrospinal fluid (aCSF) containing 124 mM NaCl, 5 mM KC1,1.3 mM MgCl2, 2 mM CaCl2, 26 mM NaHC03, and 10 mM glucose (pH 7.35, ~280 mosmol) aerated with 95% 02-5% C02 at room temperature.

2. After 15 min, transfer the tissues to a spinner flask with a magnetic stirrer (Bellco glass) containing low Ca2+ aCSF (composition same as aCSF except it contains 3.2 mM MgCl2 and 0.1 mM CaCl2, and is aerated at 32-35° with 95% 02-5% C02) and 1.33 mg/ml trypsin (9000 benzoyl-l-arginine ethyl ester units/mg), 0.67 mg/ml hyaluronidase, and 0.2 mg/ml kynurenic acid. After 90 min, transfer tissue sections to normal aCSF, incubate for 5 min, and then triturate.

3. Transfer tissues to DMEM/F-12 medium containing 0.7 mg/ml ovomucoid (Sigma) and triturate with a fire-polished Pasteur pipette. Plate cells onto uncoated 35-mm plates at 1000 cells/plate and culture in serumfree medium containing 20 ng/ml EGF as described for embryonic cells. Cells initially attach on the substratum, but by 6-8 DIV the proliferating cell clusters detach and grow in floating spheres. The plating of cells on PORN-coated substratrum inhibits cell proliferation.

4. If it is necessary for cells to be attached to the substratum, proliferating cell clusters can be transferred with a large-bore Pasteur pipette and plated onto PORN-coated plates for 1 hr for adhesion and growth.

Short-Term Cultures of Neuronal Cells

A variety of media (both serum-containing and serum-free) and culture conditions have been used for culturing neuronal cells for a short period of time. Under these culture conditions cells survive for 1-4 weeks. Briefly, fetal tissues are washed in HBSS and then dissociated as described earlier. A single cell suspension is adjusted to the desired concentration and plated. Approximately, 2 X 105-1 X 106 cells are plated/35-mm dishes.39-40 Cells can be grown in DMEM/F-12 medium containing 10% FBS for 2 weeks. For serum-free conditions, cells are initially plated in the presence of DMEM/F-12 medium containing 10% FBS. After 18 hr of incubation, the medium is changed to serum-free medium and the cells are grown for 2-3 weeks.40 Cells can be directly plated on PORN/laminin-coated plates in serum-free

39 K. Unsiker, H. Reichert-Preibsch, R. Schmidt, B. Pettmann, G. Labourdette, and M. Sensenbrenner, Proc. Natl. Acad. Sci. U.S.A. 84, 5459 (1987).

4,1 R. S. Morrison, A. Sharma, J. De Vellis, and R. A. Bradshaw, Proc. Natl. Acad. Sci. U.S.A. 83, 7537 (1986).

medium with the N2 supplement.41 Neuronal cells survive for 1-2 weeks under these culture conditions. In addition, neuronal cells have been cocul-tured on a feeder layer of astrocytes.42 Under these culture conditions, neurons survive for about 4 weeks.

Long-Term Cultures of Neuroblasts

Utilizing the proliferative property of FGF-2, long-term neuroblast cultures have been generated from both embryonic and adult CNS of rats.

Embryonic Tissues12

Long-term neuroblast cultures have been generated from embryonic hippocampus, VM, locus coeruleus, septum, and spinal cord. Neuroblast cultures generated from hippocampus have survived for 8 months in culture and have been passaged 9 times. The cultures have been generated 35 times from hippocampus with equal consistency.

1. Place the dissected tissues in a 15-ml polypropylene centrifuge tube and wash three to four times with 5 ml PBS-D each time. Resuspend tissues in 1 ml N2 medium containing 20 ng/ml FGF-2 (human recombinant, Syntex-Synergen Consortium or purified bovine, R&D Systems) (N2 + FGF-2). Make a single cell suspension by mechanical dissociation (hippocampus) or by using the PPD solution (VM or spinal cord) as described earlier.

2. Count viable cells in a hemocytometer and plate ~1 X 106 cells/T75 flasks (coated with PORN/laminin) in 8 ml N2 + FGF medium. The medium is changed every 3-4 days.

3. Cells can be passaged when the cultures are 70-80% confluent. During culture and cell growth, cells detach from the substratum and float in the medium. More floating cells are noted when the cell density is >80%. The floating cells can be collected by centrifugation of the culture medium at lOOOg for 3 min and replated on PORN/laminin-coated plates.

To passage cells by trypsinization, add 1 ml ATV trypsin (Irvine Scientific, Santa Ana, CA) to a culture flask. Roll the flask to spread trypsin and immediately hit the flask gently on the side to dislodge the cells. The trypsinization process usually takes <1 min. Add 5 ml N2 medium, transfer

41 P. Walicke, W. M. Cowan, N. Ueno, A. Baird, and R. Guillemin, Proc. Natl. Acad. Sci.

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