MEM medium could be stored at 4°C or -20°C for at least a month before being ... ~Ligand: [N%(3-(3'-[EI2Sl]-4'-hydroxyphenyl)propionyl)Lys 3] substance P (ref.
Neuroscience Letters, 49 (1984) 319-324
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Elsevier Scientific Publishers Ireland Ltd.
NSL 02903
G R O W T H OF D I S S O C I A T E D N E U R O N S IN C U L T U R E D I S H E S C O A T E D WITH SYNTHETIC POLYMERIC AMINES
U.T. RI~IEGG and F. HEFTI
Preclinical Research, Sandoz Ltd., 4002 Basel (Switzerland) (Received March 23rd, 1984; Revised version received June 15th, 1984; Accepted June 21st, 1984)
Key words: cell culture - neurons - coating
Polyornithine and polylysine, the most c o m m o n l y used coating materials in the preparation of neuronal cultures, can be replaced by polyethyleneimine (PEI) and other synthetic polymeric amines. PEI supports attachment and growth of neurons from fetal rat brain equally well as polyornithine. Furthermore, differentiation of the cultured neurons, as judged by the expression of choline acetyltransferase and of binding sites for substance P, was very similar in cultures grown in dishes coated with PEI or polyornithine.
Primary cultures of dissociated neurons are used frequently in biochemical and electrophysiological studies. Dissociated neurons tend to form aggregates and do not attach to the usual glass and plastic surfaces of c o m m o n culture dishes. Monolayer cultures o f neurons are obtained, when culture dishes are coated with polylysine [9] or polyornithine [4]. These basic polyamino acids are thought to form a molecular layer on the culture dish and to interact with negatively charged lipids or polysaccharides on the cell surface. The interaction between cells and coating material competes with the forces leading to cell aggregation. Expenses for polyamino acids can be substantial when large quantities of cultures are prepared. We therefore investigated whether other polymers can serve as coating materials for neuronal cultures. The search was focused on polyamines which, like polyornithine and polylysine, carry a positive charge at neutral pH. Polyamines used for coating were dissolved in 0.15 M sodium borate buffer, p H 8.5, at a concentration of 1 m g / m l . The sources and commercial names of the polymers are given in Table III. The solutions were sterilized by filtration (0.22 #m, Millipore filter). Aliquots of 250/~1 were added to polystyrene culture dishes of 16 m m diameter (multiwell dishes, Costar); aliquots of 1 ml were added to 35 m m dishes (Falcon). Glass cover slips were cleaned by washing in acetone and water and were then coated in culture dishes. After 2-12 h of exposure at room temperature, the polyamine solutions were removed and culture dishes were washed twice with minimal essential medium (MEM, Gibco). Coated and washed dishes containing 0304-3940/84/$ 03.00 © 1984 Elsevier Scientific Publishers Ireland Ltd.
c: low density culture in PEI-coated dish; d: same visual field after staining for tetanus toxin.
Fig. 1. Growth of neurons in dishes coated with polyethyleneimine (PEI). Neurons were dissociated from fetal rat brains and grown for 10 days in a modified L-15 medium. Growth of non-neuronal cells was suppressed by adding cytosine arabinoside, a: dish coated with polyornithine: b: dish coated with PEI;
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MEM medium could be stored at 4°C or - 2 0 ° C for at least a month before being used. Dissociated neurons were obtained from fetal rat brains at embryonal day 17. Pieces o f brain tissue were incubated in 0.5°7o trypsin (Gibco) and 0.05°7o DNAase (type I, Sigma) in Dulbecco's phosphate-buffered saline for 10 min at 37°C. The pieces were washed twice with MEM. Cells were dissociated by mild trituration in the same medium using a fire-polished Pasteur pipet. They were cultured for 7-10 days in a modified L-15 medium [3], containing 10% horse serum and 1°70 fetal calf serum (Gibco), in a 5°70 CO2 incubator. Growth of non-neuronal cells was suppressed by adding 10-5 M cytosine arabinoside to the medium. Cultures were examined using phase contrast optics. The total number of neurons per dish was established by counting them in randomly distributed visual fields corresponding to 2°7o of the total area. (For the determination of protein content, the cells were lysed with 0.1 °T0 sodium dodecylsulfate, pH 7.0.) Proteins were measured by the method of Lowry et al. [5] using bovine serum albumin as standard. Binding of substance P was determined as described earlier [8]. The activity of choline acetyltransferase (CHAT) was determined according to Fonnum [2]. Binding of tetanus toxin was demonstrated using the method of Mirsky et al. [6]. Dishes coated with polyethyleneimine (PEI), a simple polymer containing secondary amines, supported attachment and growth of neurons equally well as dishes coated with polyornithine or polylysine (Table I). Cultures grown on either PEI or polyornithine had the same morphological appearance and contained an equal number of neuron-like cells (Fig. 1). In uncoated dishes and in the presence of cytosine arabinoside no cells survived. All cells grown in PEI-coated dishes were labelled with tetanus toxin, suggesting that they indeed were neurons [6]. (In addition to neurons, tetanus toxin has also been shown to label a subpopulation of astrocytes (type 2 astrocytes) [7]. However, as judged by immunocytochemical visualization of glial fibrillary acid protein, which specifically is expressed by astrocytes, our cultures contained less than 1°10 of astrocytic cells. Furthermore, the
TABLE I G R O W T H OF NE URONS D I S S O C I A T E D FROM FET A L RAT BRAINS IN DISHES (16 mm D I A M E T E R ) C O A T E D W I T H P O L Y O R N I T H I N E , P O L Y L Y S I N E OR P O L Y E T H Y L E N E I M I N E (PE1) Cultures were analyzed 10 days after plating. Growth of non-neuronal cells was suppressed by adding cytosine arabinoside (10 -5 M). Values are mean _+ S.E.M., n = 6.
Number of neurons per dish ( × 1 0 -3 ) Protein content, #g per dish
Polyornithine
Polylysine
PEI
174 _+ 14
180 + 14
189 _+ 6
120 _+ 3
122 _
116 _+ 7
3
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TABLE 11 EXPRESSION OF SUBSTANCE P BINDING SITES AND C H O L I N E ACETYLTRANSFERASE (CHAT) ACTIVITY BY NEURONS CULTURED IN DISHES WITH P O L Y O R N I T H I N E OR PEI Neurons dissociated from whole fetal rat brains or from the septal area were grown for 10 days before assay. Values are mean + S.D., n 4-12. Polyornithine
F'EI
108 + 7 4.9 + 0,3
105 5.0
121 97
103 ~ 5 83 :~ 6
Whole brain neurons protein content (gg/dish) substance P binding ~ (fmol/mg prot.) Septal neurons protein content (~.g/dish) (7hAT activity ( p m o l / m i n / m g prof.)
_+ 7 ± 11
± 8 + 0.4
~Ligand: [N%(3-(3'-[EI2Sl]-4'-hydroxyphenyl)propionyl)Lys3] substance P (ref. 8).
proliferation of astrocytes and of other types of non-neuronal cells was suppressed by cytosine arabinoside.) To investigate whether neurons in PEI- and polyornithine-coated dishes differentiate equally well, we measured the expression of a peptide receptor and of a TABLE I l l G R O W T H OF NEURONS ON POLYMERS
DISHES (16 mm DIAMETER) COATED
WITH
VARIOUS
Dissociated neurons from fetal rat brains were plated, grown for 7 days and then taken for protein determination. Growth of non-neuronal cells was suppressed by the addition of cytosine arabinoside (10-5 M) to the medium. Mean ~- S.D.; n = 6; *significantly lower than polyornithine group, P < 0 . 0 5 it-test). Coating material
f'rotein content (/Lg/dish)
No coaling Poly-L-ornithine, MW 100,000 (Sigma) Polyethyleneimine (Sigma) Polyethyleneimine, MW 15,000 (Corcat P 150)~ Polyethyleneimine, MW 60,000 (Corcat P 600) ~' Polyethyleneimine, MW 40,000 (Polymin P; Fluka) Polyctheramine, MW 3000 (PAM; Sandoz) Polyamideamine, MW 3000 (FAMO; Sandoz) Poly-(N,N-dimethyl-3,5-dimethylen-piperidinium chloride) (EGA; Aldrich Poly-(2-vinylpyridine) (EGA; Aldrich) 1,5-Dimethyl(1,5-diazaundecamethylene)polymethabromide (Polybrene, EGA; Aldrich) Polyoxyethyltallowamine (Noramox Q15) ~'
4 112 96 106
"Cordowa Chem. Corp. Muskegon, MI, U.S.A. bPierrefitte-Auby, Paris, France.
~ I* ! I0 ' 5 * 7
104
t
3
113
~
16
~ 14 100 ~ 5 108 +_ 7 85 + 8* 110
68 +_ 14" 29 ~ 3*
323 transmitter-specific enzyme in such cultures. A specific binding site for substance P is present in cultures of neurons dissociated f r o m murine [1] and from rat brains [8]. For rat neurons, the average number of receptors per cell was found to be 4000 and the Ka value for the ligand used was approximately 30 pM [8]. In the present study, we measured equal amounts of specifically bound substance P to both cultures grown on P E I - or polyornithine-coated dishes (Table II). As an example for a transmitter-specific enzyme, we measured the activity of choline acetyltransferase (CHAT) in cultures of neurons obtained from the septal area of fetal rat brains. As shown earlier, C h A T activity in these cultures reflects the presence of cholinergic neurons forming the septo-hippocampal pathway in vivo [3]. Cultures grown in P E I - and polyornithine-coated dishes expressed the same C h A T activity (Table II). Besides P E I , several other commercially available polymers were tested with respect to their ability to support attachment and growth of neurons in culture. Neuronal growth was quantified by measuring protein content of cultures 7 days after plating. Polyethyleneimines of various molecular weights and various commercial sources were equally effective as coating agents (Table III). Besides PEI, three other polyamines were found to be useful (Table III): PAM, a branched polyether with side chains containing the polyethyleneimine structure; F A M O , a straightchain polyamideamine with secondary amino groups, and poly-(N,Ndimethyl-3,5-dimethylen-piperidium chloride), a polyquarternary amine. The fact that poly-(2-vinylpyridine), a polyaromatic amine carrying no charge at neutral pH, was less effective, confirms the view that interactions between positively charged amines and negatively charged groups on the cell surface are responsible for the attachment of neurons to polyamine coating materials. However, not all polyamines are effective coating materials. For example, polybrene, a polyquarternary amm o n i u m salt, or n o r a m o x Q 15, a polytertiary amine, were ineffective. These two latter compounds possibly failed to attach to the surface of the culture dishes in sufficient quantity. Our results indicate that P E I and other polyamines can serve as effective coating materials for cultures of dissociated CNS neurons. The replacement of the frequently used coating materials polyornithine and polylysine by PEI, a very inexpensive chemical, should help to reduce the cost of culture techniques. We thank Dr. J. Runyon for samples of some of the polymers and Mrs. B. Burckhardt, Ms. C. Messer and Mr. J.F. Zuber for skillful technical assistance. 1 Beaujouan, J.C., Torrens, Y., Herbet, A., Daguet, M.C., Glowinsky, J. and Prochiantz, A., Specific binding of an immunoreactive and biologically active [~251]labelledsubstance P derivative to mouse mesencephalic cells in primary culture, Molec. Pharmacol., 22 (1982) 48-55. 2 Fonnum, F., A rapid radiochemical method for the determination of choline acetyltransferase, J. Neurochem., 24 (1975) 407-409. 3 Hefti, F., Hartikka, J., Eckenstein, F., Gnahn, H., Heumann, R. and Schwab, M.E., Nerve growth
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4 5 6 7
8 9
factor (NGF) increases choline acetyltransferase but not survival or fiber outgrowth of cultured septal cholinergic neurons, Neuroscience, in press. Letourneau, P.C., Possible roles for cell to substratum adhesion in neuronal morphogenesis, Develop. Biol., 44 (1975) 77-86. Lowry, O . H . , Rosebrough, N.J., Farr, A.L. and Randall, R.J., Protein measurement with the folin-phenol reagent, J. biol. Chem., 193 (1951) 265-275. Mirsky, R., Wendon, L.M.B., Black, P., Stolkin, C. and Bray, D., Tetanus toxin: a cell surface marker for neurons in culture, Brain Res., 148 (1978) 251-259. Raft, M.C., Abney, E.R., Cohen, J., Lindsay, R. and Noble, M., Two types of astrocytes in cultures of developing rat white matter: differences in morphology, surface gangliosides, and growth characteristics, J. Neurosci., 3 (1983) 1289-1300. R(iegg, U.T., A binding site for substance P on dissociated cells from cultures of rat CNS. In P. Skrabanek and D. Powell (Eds.), Substance P, Dublin 1983, P. Boole Press, Dublin, 1983, pp. 65-66. Yavin, E. and Yavin, Z., Attachment and culture of dissociated cells from rat embryo hemispheres on poly-lysine coated surface, J. (?ell Biol., 62 (1974) 540-546.
