Neuronal Cell Culture

Special Note: Isolation of primary neurons for cell culture is an invasive procedure which must be conducted in a humane manner. The U.S. Public Health Service Guide for the Care and Use of Laboratory Animals describes procedures for the humane use of live vertebrate animals in biomedical research.


Poly-L-Lysine Treatment of Glass Coverslips

Treatment of culture dishes or coverslips with poly-L-lysine (PLL) improves cell attachment. For cultures of mammalian neurons use PLL of MW = 70 - 150kDa (Sigma P6282). Use PLL of MW >300kDa for cultures of Aplysia neurons (Sigma P5899). Dissolve PLL in sterile distilled water. A concentration of 25 µg/mL is sufficient to treat coverslips for mammalian neuronal culture. For culture of Aplysia neurons use a PLL concentration of 500 µg/mL (Schacher, personal communication). Place 3 coverslips in each of several 35mm plastic culture dishes (Falcon 3001). The coverslips can be sterilized by letting them sit in the hood under UV light for one hour. The type of coverslip glass can apparently affect neuronal culture viability, with German class giving the most satisfactory results (Banker, 1992). I use Assistant coverslips (12 mm diameter, 0.1 mm thickness) manufactured by Deutsche Spiegelglas and available from Carolina Biological Supply. Place several drops of PLL solution on top of each coverslip. Allow ten minutes for the PLL to interact with the glass, which is adequate for mammalian neuronal culture . For Aplysia neuron cultures treat the cover slips (or the plastic dish) with PLL for two hours (Schacher, personal communication). Aspirate off the solution and rinse twice with sterile distilled water. Air dry for one hour. Don't try to UV sterilize coated coverslips since this will cause the PLL to depolymerize. Once the coverslips are dry they can be stored indefinitely at -20° C.

 

 

Culture of Cerebellar Purkinje and Granule Cell Neurons

Note: the culture methods described in this section are based on the work of Slesinger & Lansman (1991), Mount et al (1993), Maconochie et al (1994), Steinbach (1995), and Mount (1996).

Animals - Use E20 - D0 rat pups to select for the most Purkinje cells. D0 pups work well for Purkinje cells. D7 pups are best for granule cell culture. The rationale is to isolate the neurons at the time in development when they first differentiate, which is when they are most adaptable to survive under culture conditions. For isolation of neurons from embryonic rats, timed-pregnant rats can be effectively and humanely anesthetized with 2% halothane and killed by cervical dislocation.
 
Purkinje Cell Isolation - Remove fetuses by Cesarean section and transfer to a sterile dish with phosphate-buffered saline, Ca/Mg-free (PBS) at 4°C. Isolate cerebella and aseptically wash in PBS. Transfer to isolation media (see Plating section). Don't use enzyme dissociation. Take a single folium at a time and mince it with a no. 11 scalpel blade into 500 µm or smaller sections. These sections should stick to poly-L-lysine coated coverslips and flatten out. If they don't, try gentle trituration after mincing the tissue to break it into smaller pieces. Alternatively, you can use the trituration method described by Slesinger and Lansman (1991a) for granule cells. However, don't triturate to a single cell suspension. Stop trituration when you have formed a suspension of small cell clumps.
 
Granule Cell Isolation - Decapitate anesthetized D7 rat pups. Isolate cerebella and aseptically wash with PBS at 4°C. Mince cerebella into 500 µm or smaller sections with a no. 11 scalpel blade. Incubate minced tissue at room temperature for 10 min in trypsin-EDTA (0.05% trypsin, 0.53 mM EDTA; GIBCO 25300-013). Warm the trypsin-EDTA to 37û C before starting the incubation. After trypsination, resuspend the minced tissue in 2 mL isolation media (see Plating section) containing DNase I (0.05% or 1,000 Kunitz/mL; Sigma D-4263). Note: DNase I has an absolute requirement for divalent metal cations. Keep the suspension on ice and triturate though a fire-polished glass pipette with the minimal number of strokes needed to obtain a suspension of single cells. I use three pipettes of decreasing tip diameters, moving the entire suspension through each pipette tip 2-4 times. Triturate slowly and avoid making bubbles. Single-cell suspensions appear milky and free of particulates.
 
Plating - Both Purkinje cells and granule cells are isolated and initially plated in the presence of media that contains 10% horse serum. Plate at a density of about 1 x 105 cells cm2.
 
Isolation Media
- MEM Eagle's with Earle's BSS, 2 mM glutamine (0.292g/L), 10% heat-inactivated horse serum (Hyclone), 6 mg/mL glucose (final concentration), 10 U/mL penicillin, 10 µg/mL streptomycin, 10 µM MK-801 (RBI, cat. no. M-107; use only for Purkinje cell cultures), 25 mM KCl. The addition of potassium will slightly depolarize the cells, which may increase Purkinje cell survival when combined with neurotrophic factors such as NGF (Cohen-Cory et al, 1991). MK-801 is a potent noncompetitive NMDA receptor antagonist (Wong et al, 1986) which has been shown to promote Purkinje cell survival (Mount et al, 1993). Purkinje cells are exquisitely sensitive to glutamate ionotropic receptor-mediated toxicity. In addition, the type of horse serum you use can make a profound difference in neuronal survival. Make sure your horse serum is obtained from a mixed gender herd (Mount, personal communication, 1996). Some commercial serums are obtained from geldings and this serum does not effectively support neuronal survival.
 
Isolation Media, 250 mL
225 mL MEM
25 mL horse serum
1.25 g glucose
0.25 ml pen/strep (100x)
0.466 g KCl
 
Neurobasal Serum Free Media - After a 5 hour period for cell attachment, change to serum-free culture media . The serum-free media is: Neurobasal-SFM Media (GIBCO, cat. no. 21103-015) supplemented with B-27 (GIBCO, cat. no. 17504-010), 10 U/mL penicillin , 10 µg/mL streptomycin , 10 µM MK-801 (for Purkinje cell cultures only), 25 mM KCl, 2 mM glutamine. For Purkinje cell cultures, supplement the media with glial-derived neurotrophic factor (GDNF, 1-10 pg/ml) (Calbiochem, cat. no. 345872; RBI, cat. no. G-128) GDNF promotes survival of Purkinje cells (Mount et al, 1995). Cultures of Purkinje cells or granule cells can be maintained for up to four days without a media change at 37°C in a 95% air, 5% CO2 (v/v) humidified atmosphere. An additional advantage of Neurobasal/B27 is that it is relatively selective for neuronal survival so you don't have to add antimitotics such as arabinosylcytosine to supress proliferation of glial cells. Purkinje cells need glial cells to survive, but unchecked glial cell division will kill neurons.
 
Neurobasal SFM, 250 mL
245 mL Neurobasal-SFM
5 mL B-27
0.25 ml pen/strep (100x)
0.466 g KCl
2.5 mL glutamine (100x)
 
Growth in Culture - The first group of cells to migrate out from the plated cerebellar explants are glial cells, followed by granule cells. The cerebellum explant should flatten out until it is only one or two cell layers thick. The Purkinje cells can be identified within the flattened explant. Purkinje cells stain preferentially with antibodies to calcineurin (Usuda, 1996). But they can be identified morphologically by several criteria: 1) By D10 they produce a single thick dendrite and a small axon. The dendrite may grow arborizations that look like Purkinje cells in vivo; 2) The cell body of a Purkinje cell is > 20 µM in diameter, while granule cells have cell bodies < 10 µM; 3). Using these morphology criteria it is possible by D10 to identify Purkinje cells with about 95% confidence, (Steinbach, personal communication).

 

Isolation and Culture of Cerebellar Granule Neurons
(Slesinger and Lansman, 1991)



Granule Neuron Culture Media
Primary cultures of granule cells require depolarizing media and additional glucose for survival and differentiation. Slesinger and Lansman (1991a) have used 25 mM KCl media with 3 - 11 mM glucose. Other investigators use higher concentrations of glucose. Below are several media formulations for primary cultures of cerebellar granule cells, given in mM. Add 20 µM cytosine arabinoside 48 h after plating to stop glial growth. Also note that glutamine deteriorates by ~10% in 11 days when stored at 4°C.

 

Van Vliet et al. (1989) -1:1 Dulbecco's MEM/F-12 (Gibco); 5% FBS; 5% HS; 25 KCl; 30 glucose; 5 HEPES; 3 NaHCO3; 2 glutamine
 
Holopainen et al. (1991) -10% FBS; 25 KCl; 30 glucose; 7µM PABA; 100mU/L insulin
 
Slesinger and Lansman (1991a) - 180 mL MEM Eagle's @ Earle's BSS; 20 ml HS; 25 KCl (MW=74.551); 0.06% (~3.33mM) glucose (MW=180.16); 2 glutamine
 
Granule Cell Media, 200 mL (BW) - 180 mL MEM @ Earle's BBS, 1.0gL glucose, 2 mM glutamine, 20 mL horse serum, gentamicin 50 - 100 µg/ml, 25 mM KCl, 4.5 mM glucose. Optional: 50 nM insulin.



Isolation and Culture of Rat Hippocampal Neurons
(Huettner and Baughman, 1986; Jahr and Stevens, 1987)
 

Hippocampal Neuron Culture Media

 

Jahr and Stevens (1987) - MEM with Earle's Salts, 20 mM glucose, 0.5 mM glutamine, 50 units/ml penicillin/streptomycin, 5% heat-inactivated rat serum.
 
Toselli and Lux (1989) - BME basal medium, 2 mM glutamine, 10% glucose, 10% horse serum.
 
Slesinger (1991) - For culture of cerebellar granule cells: MEM Eagle's with Earle's basal salts, 2 mM glutamine, 3.3 - 11.1 mM glucose, 10% horse serum, 25 mM KCl.


PC-12 Culture Media

 

PC-12 Growth Media - 170 Dulbecco's Modified Eagle's Media (DME H-21), 20 Horse Serum, 10 Fetal Bovine Serum
 
PC-12 Growth Media (Masiakowski and Shooter, 1988) - 180 Dulbecco's Modified Eagle's Media (DME H-21), 10 Horse Serum, 10 Fetal Bovine Serum, 2 Penicillin (10K units/ml) /Streptomycin (10K µg/ml)
 
5S NGF Stock Solution - Reconstitute NGF with PC-12 Growth Media to yield a 100 µg/ml stock solution. Gently swirl to dissolve but do not shake. Aliquot to sterile tubes and store at -20°C. Avoid repeated freeze/thaw cycles.
 
PC-12 Neurite Extension Media - Add 10ul of 2.5S NGF Stock Solution to 100ml of PC-12 Growth Media. This concentration of NGF (10ng/ml) promotes maximal neurite extension in PC-1 cells.



References

Banker, G. 1992. Culturing neurons on glass coverslips - disaster strikes. Neuroscience Newsletter. July/August.

Cohen-Cory S, Dreyfus CF, Black IB. 1991. NGF and excitatory neurotransmitters regulate survival and morphogenesis of cultured cerebellar Purkinje cells. J. Neurosci, 11, 462-471.

Holopainen I, Louve M, Akerman KE. 1991. Interactions of glutamate receptor agonists coupled to changes in intracellular Ca2+ in rat cerebellar granule cells in primary culture. J. Neurochem., 57, 1729-1734.

Huettner JE; Baughman RW. 1986. Primary culture of identified neurons from the visual cortex of postnatal rats. J. Neurosci., 6, 3044-3060.

Izu YC, Sachs F. 1991. Inhibiting synthesis of extracellular matrix improves patch clamp seal formation. Pflügers Archiv. Eur. J. Physiol., 419, 218-220.

Jahr CE; Stevens CF. 1987. Glutamate activates multiple single channel conductances in hippocampal neurons. Nature, 325, 522-525.

Kay AR, Wong RK. 1986. Isolation of neurons suitable for patch-clamping from adult mammalian central nervous systems. J. Neurosci. Met., 16, 227-238.

Linden D. 1995. Personal communication.

Maconochie DJ; Zempel JM; Steinbach JH. 1994. How quickly can GABAA receptors open? Neuron, 12, 61-71. [describes culture of cerebellar Purkinje cells]

Masiakowski P, Shooter EM. 1988. Nerve growth factor induces the genes for two proteins related to a family of calcium-binding proteins in PC12 cells. Proc Natl Acad Sci, USA, 85,1277-1281.

Mogul DJ, Fox AP. 1991. Evidence for multiple types of Ca2+ channels in acutely isolated hippocampal CA3 neurones of the guinea-pig. J. Physiol, 433, 259-281.

Mount HT, Dreyfus CF, Black IB. 1993. Purkinje cell survival is differentially regulated by metabotropic and ionotropic excitatory amino acid receptors. J Neurosci, 13, 3173-3179.

Mount HT, Dean DO, Alberch J, Dreyfus CF, Black IB. 1995. Glial cell line-derived neurotrophic factor promotes the survival and morphologic differentiation of Purkinje cells Proc Natl Acad Sci, USA, 92, 9092-9026.[published erratum appears in Proc Natl Acad Sci US A 1995 Dec 5;92(215):11945].

Mount HTJ. 1996. Personal communication.

O'Dell TJ, Alger BE. 1991. Single calcium channels in rat and guinea-pig hippocampal neurons. J Physiol, 436, 739-767.

Schacher, S. 1996. Personal communication by email.

Slesinger PA, Lansman JB. 1991. Inactivation of calcium currents in granule cells cultured from mouse cerebellum. J. Physiol., 435, 101-121.

Steinbach JH. 1995. Personal communication.

Toselli M, Lux HD. 1989. GTP-binding proteins mediate acetylcholine inhibition of voltage dependent calcium channels in hippocampal neurons. Pflügers Archiv. Eur J Physiol, 413, 319-321.

Usuda N, Arai H, Sasaki H, Hanai T, Nagata T, Muramatsu T, Kincaid RL, Higuchi S. 1996. Differential subcellular localization of neural isoforms of the catalytic subunit of calmodulin-dependent protein phosphatase (calcineurin) in central nervous system neurons: immunohistochemistry on formalin-fixed paraffin sections employing antigen retrieval by microwave irradiation. J Histochem Cytochem, 44, 13-18.

Van Vliet BJ, Sebben M, Dumuis A, Gabrion J, Bockaert J, Pin JP. 1989. Endogenous amino acid release from cultured cerebellar neuronal cells: effect of tetanus toxin on glutamate release. J Neurochem, 52, 1229-1239.

Wong EH, Kemp JA, Priestley T, Knight AR, Woodruff GN, Iversen LL. 1986. The anticonvulsant MK-801 is a potent N-methyl-D-aspartate antagonist. Proc Natl Acad Sci, USA, 83, 7104-7108.