Journal of Neuroscience Research 32562-568 (1992)
Neuronal Cells Mature Faster on Polyethyleneimine Coated Plates Than on Polylysine Coated Plates I.H. Lelong, V. Petegnief, and G. Rebel Laboratory of Cell Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland (I.H.L.); Centre de Neurochimie, Strasbourg Cedex, France (V.P.,G.R.) Cell morphology, proteinlDNA ratio, ganglioside analysis, taurine uptake, and the activity of neurone specific enolase showed that neuronal cells mature faster when grown on polyethyleneimine coated plates compared to cells grown on polylysine coated plates. Our results also show higher protein/DNA ratio and total and neuron specific enolase activities in cells grown in serum supplemented medium, when compared to their counterparts grown in synthetic medium. Moreover, our results show that only some specific markers can be used to determine the early and late events of cell maturation, whereas other markers continuously vary with time. 0 1992 Wiley-Liss, Inc.
Key words: neurons, cultures, maturation
cinodehydrogenase activity are also altered by the traces of polylysine remaining in the cell homogenates when cells are scraped from the polylysine coated plates or when an equal amount of the polycation is added as a control in the assays (personal communication, unpublished data). Recently, another synthetic polycation, polyethyleneimine (PEI), has been reported to support growth and maturation of rat neuroblasts (Ruegg and Hefti, 1984). Neuronal cultures grown on polyethyleneimine, polylysine, or polyornithine display similar choline acetyltransferase activity and substance P binding site number (Ruegg and Hefti, 1984). In the present work, using chicken neuronal cultures which can be obtained quite pure (Pettmann et al., 1979), we have studied several neuronal characteristics in cells grown either on polylysine or on PEL
INTRODUCTION Cell adhesion mediates a variety of cellular functions, such as morphological changes, growth, and maturation. A variety of “coating materials” (fibronectin, collagen, extracellular matrix, or isolated cellular matrix components) to increase cell adhesion to tissue culture plates have been developed (Letourneau, 1975; Kleinman et al., 1987). As shown by Maciera-Coehlo and Avrameas (1972) the physical properties of the coating polymer, such as positive or negative charges, alter cell growth in vitro. As shown by these authors, more or less pure cultures of a single cell type could be obtained from heterogeneous cell populations derived from an organ. For instance, purified neuronal cell culture was derived by plating the heterogeneous population of cells from mechanically dissociated telencephalon on synthetic polycationic matrices achieved either with polylysine or polyornithine coated plates (Yavin and Yavin, 1974; Sensenbrenner et al., 1978). However, interferences with biological functions due to polylysine were recently reported, such as the increased phosphorylation of lipocortin by the EGF receptor kinase (Abdel-Ghany et al., 1989). Moreover, some enzymatic activities such as suc0 1992 Wiley-Liss, Inc.
MATERIALS AND METHODS Cell Cultures Polyamines were dissolved in 0.1 M sodium borate buffer, pH 8.4, at a concentration of 2 mg/100 ml for polylysine (poly-L-lysine hydrobromide, mol .wt . 55,000, Sigma) and of 20 mg/100 ml for polyethyleneimine (Polymin P 50% in water, Mr 600,000-1000,000, Fluka). Solutions were sterilized by filtration (0.22 Fm, Millipore filter). Aliquots of 8 ml were added to polystyrene culture dishes of 100 mm diameter (Falcon F3003). The plates were incubated overnight at 37°C to allow proper polymerization of the polycation. Prior to plating the cells, the polycation containing solutions were removed and the coated dishes were washed once with Dulbecco modified Eagle medium (GIBCO, 0741600) buffered with 24 mM sodium bicarbonate Received June 6, 1991; revised March 20, 1992; accepted March 24, 1992. Address reprint requests to Dr. Isabelle Lelong, Centre de Neurochimie, 5, rue Blaise Pascal, 67084 Strasbourg Cedex, France.
Polyethyleneimine vs. Polylysine Coated Plates
(DMEM). Cultures of dissociated neuronal cells from 8 day-old chick embryo telencephalon (chick embryo from the local hen, strain “Vedette blanc”) were established according to Pettman et al. (1979). Cells were plated at an initial density of 5 x lo6 per 100 mm dish in DMEM supplemented with 20% heat inactivated selected fetal calf serum (Intermed or GIBCO), penicillin (50 U/ml), and streptomycin (50 pg/ml). On the 1st day of culture, media were either renewed or replaced by synthetic media consisting of DMEM supplemented with insulin (5 pg/ml) , transfemn ( 10 pg/ml) , progesterone (20 nM) , selenium (30 nM), and putrescine (100 pM) (Bottenstein and Sato, 1979). The cultures were maintained at 37°C in a 5% CO,/air atmosphere. Media were changed every other day. For ganglioside analysis, cultures were washed with isotonic cold saline solution, scraped, and freeze-dried. The cells were homogenized in doubly distilled water and aliquots were withdrawn for DNA (Labarca and Paigen, 1980) and protein (Lowry et al., 1951) determination.
Ganglioside Analysis Lipids were extracted according to Suzuki (1964) with slight modifications to avoid loss of GM3 (Ciesielsky-Treska et al., 1977). Gangliosides in Folch’s upper phase were purified on Sephadex G-25 superfine column chromatography followed by separation by high performance thin layer chromatography (Dreyfus et al., 1980) and quantification by densitometry (Smid and Reisinova, 1973). Total lipid sialic acid (Neu Ac) was measured according to Miettinen and Takki-Luukkainen (1959).
563
taurine was ended by washing the plates 3 times with cold saline and treated as follows: the plates were frozen at -20°C then 1 ml of doubly distilled water was added to each plate and the plates were incubated at 37°C for 1 hour. After incubation the plates were frozen again at -20°C and thawed quickly (Rago et al., 1990). The cell lysate was briefly homogenized (gentle pipetting), 500 p1 was used to determine radioactivity, and 50-100 p1 of homogenate was withdrawn for DNA determination according to a combination of the method described by Rago et al. (1990) and the method of Labarca and Paigen (1980). To 100 p1 of cell homogenate in water, 100 p1 of TNE buffer (Rago et al., 1990) containing 1 pg/ml Hoechst 33258 was added. Fluorescence was measured at 458 nm.
Catecholamines Presence of catecholamines was determined by fluorescence using the formaldehyde-glyoxylic method (Chiba et al., 1981).
RESULTS AND DISCUSSION Cell Morphology Figure 1 shows that after 1 day in culture a greater number of cells bearing short processes were observed on cells plated on PEI than in their counterparts on polylysine. Indeed, the number of processes bearing cells was twice as large on PEI than on polylysine. This difference is less marked on the 2nd day. After 5 days no differences were observed in both types of culture. This last result agrees with previous observations reported by Ruegg and Hefti (1984) showing no Enolase Determination differences between 10 day-old cultures of fetal rat brain Enolase determination was performed as follows. neurons grown either on polylysine, polyornithine, or The culture plates were washed with isotonic cold saline PEI. The capacity of PEI to induce faster neuritogenesis and frozen at -20°C. The frozen samples were homog- than polylysine could be related to a higher adhesion of enized with a Dounce homogeneizer in phosphate buffer the neuroblasts on PEI. As previously shown (Lecontaining 4 mM MgSO, (Scarna et al., 1980). Homoge- tourneau, 1975) adhesion plays an important role in neunates were centrifuged at 100,OOOg and neuron specific ronal morphogenesis . However, we cannot exclude the enolase (NSE) was measured according to Scarna et a1 fact that PEI has a greater affinity than polylysine for (1980). neuritogenic factor(s) present either in the culture media or secreted by the neuroblasts themselves, therefore inTaurine Uptake creasing neuronal sprouting as previously observed with Taurine uptake was measured using a modification early cervical ganglionic neurons (Adler and Varon, of a previously reported method (Lleu and Rebel, 1989). 1981; Lander et al., 1985). Neuronal cells were grown on Costar plates (3 cm diameter) coated with 2ml of either the PEI or the polylysine Protein/DNA Ratio polymer. For the uptake, cells on the culture plates were Previous studies have shown that the protein/DNA washed twice with 3 ml of Krebs-Ringer phosphate ratio of neuronal cells increases with time in culture buffer and preincubated at 37°C with 2 ml of the same (Yavin and Yavin, 1974; Pettmann et al., 1979). This buffer for 5 minutes. One milliliter of the buffer contain- increase parallels an increase in protein synthesis in the ing 0.4 pCi 3H-taurine and 4 pM cold taurine was then nonproliferating neurons and is considered as an index of added and incubation continued for 10 min. Uptake of maturation. Our results (Fig. 2) are in agreement with
564
Lelong et al.
Fig. 1. Chick embryo neurons grown on plates coated either with polyethyleneimine(PEI) or with polylysine. A cell counting of five different fields show that cells grown either on PEI or on polylysine with processes longer than the cell body represent 35 2 5% and 16 f 4%, respectively, of the total cells (Student’s t test: P < . O l ) . DIC = days in culture.
these reported data. Our comparative studies performed on cells grown either on PEI or on polylysine however show that the protein/DNA ratio increase was faster in cells plated on PEL This effect was even more pronounced for cells grown in serum supplemented medium.
Ganglioside Pattern Total lipid sialic acid versus DNA content was slightly higher in 2 day-old neurons grown on polylysine 1.00 and 7.45 2 1.25 pg Neu than on PEI: 12.20 Ac/mg DNA, respectively. This difference became smaller in 5 day-old neurons: 21.90 ? 1.10 and 19.70 1.20 pg Neu Ac/mg DNA, respectively. The Neu Ac/ DNA ratio we found for cultured chick embryo neurons was significantly greater than the one reported by other authors for chicken neurons (Dreyfus et al., 1980) or for
*
*
rat embryo neurons (Yavin and Yavin, 1979). These differences may result from the specificities of the methods used for DNA determination. Indeed, interference by RNA, as observed in some cases, accounts for nucleic acid content increase and thus results also in a decrease in the ganglioside/DNA ratio. In addition the high ratio reported in our study cannot result from contamination of the neuronal ceIl population by glial cells since these latter are almost absent in our cultures (less than 2%). Furthermore, the net increase of ganglioside content that was found between day 2 and 5 in culture was similar to that previously reported. Table I shows the ganglioside pattern of 2 and 5 day-old neuronal cells. In agreement with previous reports (Yavin and Yavin, 1979; Dreyfus et al., 1980; Mandel et al., 1980; Seifert and Fink, 1984) all major brain gangliosides were present (Fig. 3). However, some
Polyethyleneimine vs. Polylysine Coated Plates
2
565
20-
.
0
.-E Q)
r
2
l0-
n
O
L
2
0
4
8
6
10
Day in culture
Fig. 2 . ProteidDNA ratio of neurons cultured either on PEI (squares) or on polylysine (circles) during development. Solid symbols: cells in serum supplemented medium; open symbols: cells in synthetic medium. Two other experiments gave similar curves. Each value is the mean of two determinations which differed by less than 10%. TABLE I. Distribution of Ganglioside Sialic Acid Expressed in Percent (%) of Total Ganglioside Sialic Acid Content of 2 and 5 Day-Old Neurons* Polylysine 2DIC GM, GM, GM, GD, GDI, GDlb GT,, GQ,,
+
Traces ND 5 0.1 40.2 2.3* 22.3 t 0.2 7.2 t 0.6* GTL 30,1 5
0.1
Polyethy leneimine
5DIC
2DIC
5DIC
1.3 t 0.2 ND 2.0 24.9 25.7 t 1.2 20.4 2 1.0 25,4 o,l
0.2 f 0.1 ND 0.4 o.l 27.2 20.7 2 1.0 27.2 f 1.5 24,2
'
0.5 f 0.1 ND 2.0 l.l 25.1 0.7 21.8 f 2.6 24.4 t 2.5 28,0 3,1
0.1
0.2 t 0.1
*
0.3 f 0.1
5
*The difference for GD3 and GDlb of 2 DIC cells grown either on PEI or on polylysine is significant (P < ,001) using Student's t test. DIC = day in culture, ND = not detected.
quantitative differences between our results and published data were noted. GM3 is a major ganglioside of cultured glial cells (Robert et al., 1975; Dreyfus et al., 1980; Dimpfel, 1982; Seifert and Fink, 1984). The trace amount of GM3 detected in our neuronal cultures confirms the absence of glial cells, even in the latter stages of the cultures. We observed no significant differences in the ganglioside pattern of 5 day-old neurons grown either on PEI or on polylysine. In contrast, a notable difference was observed in 2 day-old cultures for GD3 and GDlb. GD3 is considered as a marker of nerve cell immaturity, its amount decreasing with age, both in vivo and in vitro
Fig. 3. Thin-layer chromatogram showing ganglioside patterns from 5 day-old neurons cultured either on polylysine (lane A) or on PEI (lane B). Authentic ganglioside standards were run in lane C: GM3 was obtained from C, glioma cells and all other gangliosides were from pig brain. Spots running ahead of GM3 did not stain purple with resorcinol reagent.
(Engel et al., 1979; Yavin and Yavin, 1979; Dreyfus et al., 1980; Irwin et al., 1980; Goldman et al., 1984). Furthermore, G D l b has been suggested to be a part of the tetanus toxin receptor, a marker of mature neuronal cells (Fishman, 1982). The ganglioside pattern of the 2 day-old neuronal culture on polylysine was characteristic of immature nerve cells. This was not the case for cells grown on PEI which display a pattern quite similar to the pattern found in 5 day-old cultures. This observation is also in agreement with data we reported previously (Lelong et al., 1991). indeed, in this previous paper, we showed a higher level of radiolabeling of G D l b in plasma membranes from 1 day old neurons for cells grown on PEI compared to their counterparts grown on polylysine.
566
Lelong et al. TABLE 111. Taurine Uptake*
TABLE 11. Total Enolase and Neuron Specific Enolase (U/mg DNA)+ Day in culture
Neurone specific enolaset
Total enolase Polylysine
PEI
Polylysine
Day in culture
PEI
2
A
2 4 6
27.1 & 3.4 45.1 f 7.6** 0.10 -+ 0.02 0.18 2 0.04* 40.9 6.9 62.0 f 8.7** 0.42 f 0.05 0.54 0.05 1.40 0.09 1.52 C 0.07 103.0 C 13.1 118.2 8.9
*
*
B
2 4
6
25.0 f 1.9 36.1 f 3.8 50.0 2 8.9
27.3 f 7.0 49.3 f 6.2* 69.9 6.7*
*
0.63 f 0.07 0.81 0.46 f 0.04 0.61 0.51 f 0.06 0.57
f 0.07*
* 0.07 f
0.03
tCells were grown either in serum supplemented medium (A) or in synthetic medium (B) since day 1. One unit of enolase is the amount of enzyme catalysing the transformation of 1 pmole of substrate/min at 30°C. Similar results were obtained with another set of cultures. Values represent the mean of duplicates derived from the same experiment. Statistical comparison: Cultures on PEUpolylysine: *P < .05; **P < .01 (Student’s t test). Total enolase: DIC 4 to DIC 6 in serum or synthetic medium: 5%; DIC 2 to DIC 6 in serum or synthetic medium: 1% . Neuron specific enolase: DIC 2 to DIC 4 on polylysine + serum, DIC 4 to DIC 6 on PEI and polylysine + serum: 5%; DIC 2 to DIC 6 in serum: 1% (ANOVA test).
4 7
Taurine uptake (pmole/mg DNNmin) in cells grown on Polylysine
PEI
22.9 [19.4-31.41 38.3 [31.7-48.11 215.7 [172.3-273.91
47.3 [31.7-62.81 88 .o [82.9-91.11 193.3 [ 130.2-260.51
*Cells were grown in DMEM supplemented with 20% fetal calf serum. Each value (pmoles/mg DNNmin) is the mean value for quadruplicates. Nos. in brackets represent the extreme values. Statistical difference between polylysine and PEI: 2 and 4 days in culture: P < .05; 7 days in culture: not significant.
Taurine Uptake Taurine uptake was low in 2 day-old cultures, increasing thereafter (Table 111). During the maturation period (days 1-5) a higher uptake was obtained with cells grown on PEL Despite a wide variation in the uptake by brain cells (previously discussed: Lleu and Rebel, 1989), a higher taurine uptake was found during the maturation period (days 1-5) in cells grown on PEI compared to cells grown on polylysine. Few data on taurine uptake in cultured neuronal cells can be found. Adler reported the Neurone Specific Enolase (NSE) existence of an uptake in chicken retinal neurones having Similar variations in total enolase activity in neu- the main characteristics of those found in mammalian rons between day 2 and 6 were observed with cells cells (Adler, 1983). By autoradiography, he demongrown either on PEI or on polylysine (Table 11). How- strated that 3 day-old cultures are able to take up taurine. ever, the total specific activity of enolase was always Our results also agree with those of Kishi et al. reporting of taurine uptake by mouse neuhigher in cells grown in serum supplemented media than a doubling of the v,, ronal cells between the 3rd and the 7th day when the in cells grown with synthetic media. More surprising was uptake reaches a plateau (Kishi et al., 1988). the evolution of the neurone specific enolase activity. While this activity increased in cells grown in media Catecholamine Fluorescence supplemented with serum, no change was observed in Whether cells were grown on PEI or on polylysine cells grown in synthetic media. As observed for total in serum supplemented medium, similar fluorescence enolase activity, the type of polycation used to favor the was observed in 2 day-old cultured neurons (data not anchorage of the neurons had no effect on the activity. shown). The number of positive cells was similar. This Indeed, 2 day-old neuronal cell cultures usually have low similarity was also found on the other days where cateNSE activity (Secchi et al., 1980; Weyhenmeyer and cholamine fluorescence was checked. Bright, 1983; Schilling et al., 1988, 1989). Previous Very few cells having tyrosine hydroxylase (TH) studies on the changes of NSE with time have been ob- activity and catecholamines have been found in 3 day-old tained with cultures grown in serum supplemented me- cultures of avian neural crest cells (Christie et al., 1987). dium (Schengrund and Marangos, 1980; Schilling et al., However, 2 day-old cultures of chicken telencephalic 1988, 1989). These authors report, as we have also ob- neurons are able to take up tyrosine and dopamine from served, a notable increase of NSE activity during the first the medium and have a noticeable TH activity (Louis et days in culture. Surprisingly, no increase of NSE was al., 1981, 1983). As for cultured rat neuronal cells (Iaobserved in cultures grown in serum free medium, when covitti et al., 1987), chicken neuronal cells are able to results were expressed by mg of DNA instead of protein. synthesize dopa and dopamine (Louis et al., 1983). Though the NSE activity was always higher in cells grown on PEI, difference with cells grown on polylysine Methodological Considerations Our results also show that the protein amount per was less marked than those found for the other criteria of cell changed every day, thereby confirming previously neuronal maturation studied.
Polyethyleneimine vs. Polylysine Coated Plates
published data (Yavin and Yavin, 1974; Pettmann et al., 1979). Moreover, this variation is also dependent on the growth conditions. These observations emphasize that protein content cannot be used as a reference for expressing results in pharmacological or enzymatical studies performed on neuronal cells. DNA (or cell number) should therefore be used to normalize results. To measure taurine uptake we have replaced the method of Labarca and Paigen (1980) with an easier protocol combining the last procedure with that of Rago et al. (1990). However, using the concentration of Hoechst 33258 recommended by Rag0 et al. (1990) leads to high background in the determination of low amounts of DNA. This can be overcome by decreasing the concentration of Hoechst 33258. A comparison of this modified method with that of Labarca and Paigen has given identical results.
General Conclusions Comparison of chicken brain neuronal cells grown on polylysine or on PEI coated plates showed that PEI coated plates allow faster maturation as determined by morphology, proteidDNA ratio, ganglioside pattern, or taurine uptake taken as index. A slight difference between the two kinds of cultures was observed with neuron specific enolase (NSE). No apparent difference was obtained with catecholamine fluorescence. Our results could discriminate between events linked probably with the end of the differentiation process (catecholamine synthesis), and with early (morphology, ganglioside pattern) and late events (taurine uptake, NSE) linked to maturation. By differentiation, we mean the steps which occur during the development of cells, in which a pluripotent cell (neural crest cell, for example) changes into a cell destined to give only one type of mature cell. Maturation, in contrast, includes the step which allows a differentiated embryologic cell (neuroblast) to gain numerous characteristics found in vivo in the cells of the adult organ (neurons). ProteiniDNA ratio represents another criteria which changes continuously with time. When this criterion or the NSE activity was used to address neuronal maturation, maturation was accelerated in serum supplemented medium compared to that obtained in serum free medium. This undoubtedly related to the presence in the serum of some trophic factors which are missing from the synthetic media. The faster maturation observed on PEI could be related to the cellular adhesion as discussed above, but also to the higher capacity of PEI, as compared to polylysine, to retain some trophic factors contained in the media. Adsorption on plastic (Buchou et al., 1988), on polycations (Lander et al., 1985), or on extracellular matrix (Vigny et al., 1988; Flaumenhaft et al., 1989; Presta et al., 1989) of factors present in culture
567
media is well known. However, this fact raises the problem of selecting sera which allow the growth and survival of neuronal cells, but which do not favor growth of glial cells. Our results extend the observations of Ruegg and Hefti (1984) confirming that neuronal cells can be easily selected and grown on polyethyleneimine coated surfaces. Experiments are in progress to generalize this study with other mammalian cells. Moreover, PEI may represent a useful alternative in cases where polylysine interferes in some enzymatic assays (unpublished data).
ACKNOWLEDGMENTS We wish to thank Dr. M.M. Gottesman (N.I.H.) for helpful comments on the manuscript.
REFERENCES Abdel-Ghany M, Kole HK, Abou El Saad M, Racker E (1989): Stimulation of phosphorylation of lipocortin at theonine residues by epidermal growth factor (EGF) and the EGF receptor: Addition of protein kinase P, with polylysine inhibits this effect. Proc Natl Acad Sci USA 86:6072-6076. Adler R (1983): Taurine uptake by chick embryo retinal neurons and glial cells in purified culture. J Neurosci Res 10:369-379. Adler R, Varon S (1981): Neuritic guidance by polyornithine-attached materials of ganglionic origin. Dev Biol 81:l-11. Bottenstein JE, Sato GH (1979): Growth of a rat neuroblastorna cell line in serum-free supplemented medium. Proc Natl Acad Sci USA 761514-5 17. Buchou T, Charollais PH, Mester J (1988): Involvement of serum factor(s) adsorbed to the dish in the response of cycloheximidepretreated BP-A31 cells to serum pulses. Exp Cell Res 174: 41 1-420. Chiba T, Murata Y, Koike T (1981): Plasticity of pheochromocytoma (PC12) cells demonstrated by nerve growth factor or glucocorticoid treatment: A catecholamine fluorescence and electron microscopic investigation. Biomed Res 2:618-628. Christie DS, Forbes E, Maxwell GD (1987): Phenotypic properties of catecholamine-positive cells that differentiate in avian neural crest cultures. J Neurosci 7:3749-3763. Ciesielsky-Treska J, Robert J, Rebel G, Mandel P (1977): Gangliosides of active and inactive neuroblastoma clones. Differentiation 8:31-37. Dimpfel W (1982): Differential distribution of gangliosides in nervecell cultures. Cell Mol Neurobiol 2:105-113. Dreyfus H, Louis J , Harth S , Mandel P (1980): Gangliosides in cultured neurons. Neuroscience 5: 1647-1655. Engel EL, Wood JG, Byrd FI (1979): Ganglioside patterns and cholera toxin-peroxidase labelling of aggregating cells from the chick optic tectum. J Neurobiol 10:429-440. Fishman PH (1982): Role of membrane gangliosides in the binding and action of bacterial toxins. J Membr Biol 69:85-97. Flaumenhaft R, Moscatelli D, Saksela 0, Rifkin DB (1989): Role of extracellular matrix in the action of basic fibroblast growth factor: Matrix as a source of growth factor for long-term stimulation of plasminogen activator production and DNA synthesis. J Cell Physiol 140:75-81. Goldman JE, Hirano M, Yu RK, Seyfried TN (1984): GD, ganglio-
568
Lelong et al.
side is a glycolipid characteristic of immature neuroectodermal cells. J Neuroimmunol 7:179-192. Iacovitti L, Lee J, Joh TH, Reis DJ (1987): Expression of tyrosine hydroxylase in neurons of cultured cerebral cortex: Evidence for phenotypic plasticity in neurons of the CNS. J Neurosci 7: 1264-1270. Irwin LN, Michael DB, Irwin CC (1980): Ganglioside patterns of fetal rat and mouse brain. J Neurochem 34:1527-1530. Kishi M, Ohkuma S, Kimori M, Kuriyama K (1988): Characteristics of taurine transport system and its developmental pattern in mouse cerebral cortical neurons in primary culture. Biochim Biophys Acta 939:615-623. Kleinman HK, Luckenbill-Edds L, Cannon FW,Sephel G (1987): Use of extracellular matrix components for cell culture. Anal Biochem 166:l-13. Labarca C, Paigen K (1980): A simple and sensitive DNA assay procedure. Anal Biochem 102:344-352. Lander AD, Fujii DK, Reichardt LF (1985): Purification of a factor that promotes neurite outgrowth: Isolation of laminin and associated molecules. J Cell Biol 101398-913. Lelong I, Frechard E, Creme1 G, Langley K, Rebel G, Vincendon G, Mersel M (1991): Expression of plasma membrane and cell surface phospholipids and gangliosides of chick embryo neurons grown in primary cultures: Developmental studies. Dev Neurosci 13:54-60. Letourneau PC (1975): Possible roles for cell-to-substratum adhesion in neuronal morphogenesis. Dev Biol 44:77-91. Lleu PL, Rebel G (1989): Effect of HEPES on the taurine uptake by cultured glial cells. J Neurosci Res 23:78-86. Louis JC, Pettmann B, Courageot J , Rumigny JF, Mandel P, Sensenbrenner M (198 1): Developmental changes in cultured neurones from chick embryo cerebral hemispheres. An ultrastructural and neurochemical study. Exp Brain Res 42:63-72. Louis JC, Langley K, Anglard P, Wolf M, Vincendon G, (1983): Long-term culture of neurones from human cerebral cortex in serum-free medium. Neurosci Lett 41:313-319. Lowry OH, Rosebrough NJ, Farr AI, Randall RJ (1951): Protein measurement with the Folin-phenol reagent. J Biol Chem 193: 265-275. Maciera-Coelho A, Avrameas S (1972): Modulation of cell behavior in vitro by the substratum in fibroblastic and leukemic mouse cell lines. Proc Natl Acad Sci USA 69:2469-2473. Mandel P, Dreyfus H, Yusufi ANK, Sarlieve L, Robert J, Neskovic N, Harth S , Rebel G (1980): Neuronal and glial cell cultures, a tool for investigation of ganglioside function. In Svennerholm L, Mandel P, Dreyfus H, Urban PF (eds): “Structure and Function of Gangliosides.” Plenum Pub Corp. pp 515-531. Miettinen T, Takki-Luukainen IT (1959): Use of butyl acetate in determination of sialic acid. Acta Chem Scand [B] 132356858. Pettmann B, Louis JC, Sensenbrenner M (1979): Morphological and biochemical maturation of neurons cultured in the absence of glial cells. Nature 281:378-380. Presta M, Maier JA, Rusnati M, Ragnoti G (1989): Basic fibroblast growth factor is released from endothelial extracellular matrix in a biologically active form. J Cell Physiol 140:68-74.
Rag0 R, Mitchen J, Wilding G (1990): DNA fluorometric assay in a 96-well tissue culture plates using Hoechst 33258 after cell lysis by freezing in distilled water. Anal Biochem 191:31-34. Robert J , Freysz L, Sensenbrenner M, Mandel P, Rebel G (1975): Gangliosides of glial cells: A comparative study of normal astroblasts in tissue culture and glial cells isolated on sucroseficoll gradients. FEBS Lett 50:144-146. Riiegg UT, Hefti F (1984): Growth of dissociated neurons in culture dishes coated with synthetic polymeric amines. Neurosci Lett 49:319-324. Scarna H, Keller A, Pujol JF (1980): MicromCthode de mesure des trois isoenzymes cCrCbrales de 1’Cnolase. CR Acad Sci Paris 291 :397-400. Schengrund CL, Marangos PJ (1980): Neuron-specific enolase levels in primary cultures of neurons. J Neurosci Res 5:305-311. Schilling K, Scherbaum C, Pilgrim C (1988): Developmental changes in neuron-specific enolase and neurofilament proteins in primary neural culture. Histochemistry 89295-299. Schilling K, Barco EB, Rhinehart D, Pilgrim C (1989): Expression of synaptophysin and neuron-specific enolase during neuronal differentiation in vitro: Effects of dimethyl sulfoxide. J Neurosci Res 24:347-354. Secchi J, Lecaque D, Cousin MA, Lando D, Legault-Demare L, Raynaud JP (1980): Detection and localization of 14-3-2 protein in primary cultures of embryonic rat brain. Brain Res 184: 455 -466. Seifert W, Fink HJ (1984): In vitro and in vivo studies on gangliosides in the developing and regenerating hippocampus of the rat. In Ledeen R, Yu R, Rapport M, Suzuki K, (eds): “Gangliosides: Structure, Function and Biochemical Potential.” Plenum Pub Corp., pp 535-545. Sensenbrenner M, Maderspach K, Latzkovits L, Jaros GG (1978): Neuronal cells from chick embryo cerebral hemispheres cultivated on polylysine-coated surfaces. Dev Neurosci 1:90101. Smid F, Reisinova J (1973): A densitometric method for the determination of gangliosides after their separation by thin-layer chromatography and detection with resorcinol reagent. J Chromatogr 86:200-204. Suzuki K (1964): A simple and accurate micromethod for quantitative determination of ganglioside patterns. Life Sci 3: 1227-1233. Vigny M, Ollier-Hartmann MP, Lavigne M, Fayen N, Jeanny JC, Laurent M, Courtois Y (1988): Specific binding of basic fibroblast growth factor to basement membrane-like structures and to purified heparan sulfate proteoglycan of the EHS tumor. J Cell Physiol 137:321-328. Weyhenmeyer JA, Bright MJ (1983): Exprssion of neuron specific enolase in cultured neurons from the fetal rat. Neurosci Lett 43:303-308. Yavin E, Yavin Z (1974): Attachment and culture of dissociated cells from rat embryo hemispheres on polylysine coated surface. J Cell Biol 62540-546. Yavin E, and Yavin Z (1979): Ganglioside profiles during neural tissue development. Acquisition in the prenatal rat brain and cerebral cell cultures. Dev Neurosci 2:25-37.
Neuronal Cells Mature Faster on Polyethyleneimine Coated Plates Than on Polylysine Coated Plates I.H. Lelong, V. Petegnief, and G. Rebel Laboratory of Cell Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland (I.H.L.); Centre de Neurochimie, Strasbourg Cedex, France (V.P.,G.R.) Cell morphology, proteinlDNA ratio, ganglioside analysis, taurine uptake, and the activity of neurone specific enolase showed that neuronal cells mature faster when grown on polyethyleneimine coated plates compared to cells grown on polylysine coated plates. Our results also show higher protein/DNA ratio and total and neuron specific enolase activities in cells grown in serum supplemented medium, when compared to their counterparts grown in synthetic medium. Moreover, our results show that only some specific markers can be used to determine the early and late events of cell maturation, whereas other markers continuously vary with time. 0 1992 Wiley-Liss, Inc.
Key words: neurons, cultures, maturation
cinodehydrogenase activity are also altered by the traces of polylysine remaining in the cell homogenates when cells are scraped from the polylysine coated plates or when an equal amount of the polycation is added as a control in the assays (personal communication, unpublished data). Recently, another synthetic polycation, polyethyleneimine (PEI), has been reported to support growth and maturation of rat neuroblasts (Ruegg and Hefti, 1984). Neuronal cultures grown on polyethyleneimine, polylysine, or polyornithine display similar choline acetyltransferase activity and substance P binding site number (Ruegg and Hefti, 1984). In the present work, using chicken neuronal cultures which can be obtained quite pure (Pettmann et al., 1979), we have studied several neuronal characteristics in cells grown either on polylysine or on PEL
INTRODUCTION Cell adhesion mediates a variety of cellular functions, such as morphological changes, growth, and maturation. A variety of “coating materials” (fibronectin, collagen, extracellular matrix, or isolated cellular matrix components) to increase cell adhesion to tissue culture plates have been developed (Letourneau, 1975; Kleinman et al., 1987). As shown by Maciera-Coehlo and Avrameas (1972) the physical properties of the coating polymer, such as positive or negative charges, alter cell growth in vitro. As shown by these authors, more or less pure cultures of a single cell type could be obtained from heterogeneous cell populations derived from an organ. For instance, purified neuronal cell culture was derived by plating the heterogeneous population of cells from mechanically dissociated telencephalon on synthetic polycationic matrices achieved either with polylysine or polyornithine coated plates (Yavin and Yavin, 1974; Sensenbrenner et al., 1978). However, interferences with biological functions due to polylysine were recently reported, such as the increased phosphorylation of lipocortin by the EGF receptor kinase (Abdel-Ghany et al., 1989). Moreover, some enzymatic activities such as suc0 1992 Wiley-Liss, Inc.
MATERIALS AND METHODS Cell Cultures Polyamines were dissolved in 0.1 M sodium borate buffer, pH 8.4, at a concentration of 2 mg/100 ml for polylysine (poly-L-lysine hydrobromide, mol .wt . 55,000, Sigma) and of 20 mg/100 ml for polyethyleneimine (Polymin P 50% in water, Mr 600,000-1000,000, Fluka). Solutions were sterilized by filtration (0.22 Fm, Millipore filter). Aliquots of 8 ml were added to polystyrene culture dishes of 100 mm diameter (Falcon F3003). The plates were incubated overnight at 37°C to allow proper polymerization of the polycation. Prior to plating the cells, the polycation containing solutions were removed and the coated dishes were washed once with Dulbecco modified Eagle medium (GIBCO, 0741600) buffered with 24 mM sodium bicarbonate Received June 6, 1991; revised March 20, 1992; accepted March 24, 1992. Address reprint requests to Dr. Isabelle Lelong, Centre de Neurochimie, 5, rue Blaise Pascal, 67084 Strasbourg Cedex, France.
Polyethyleneimine vs. Polylysine Coated Plates
(DMEM). Cultures of dissociated neuronal cells from 8 day-old chick embryo telencephalon (chick embryo from the local hen, strain “Vedette blanc”) were established according to Pettman et al. (1979). Cells were plated at an initial density of 5 x lo6 per 100 mm dish in DMEM supplemented with 20% heat inactivated selected fetal calf serum (Intermed or GIBCO), penicillin (50 U/ml), and streptomycin (50 pg/ml). On the 1st day of culture, media were either renewed or replaced by synthetic media consisting of DMEM supplemented with insulin (5 pg/ml) , transfemn ( 10 pg/ml) , progesterone (20 nM) , selenium (30 nM), and putrescine (100 pM) (Bottenstein and Sato, 1979). The cultures were maintained at 37°C in a 5% CO,/air atmosphere. Media were changed every other day. For ganglioside analysis, cultures were washed with isotonic cold saline solution, scraped, and freeze-dried. The cells were homogenized in doubly distilled water and aliquots were withdrawn for DNA (Labarca and Paigen, 1980) and protein (Lowry et al., 1951) determination.
Ganglioside Analysis Lipids were extracted according to Suzuki (1964) with slight modifications to avoid loss of GM3 (Ciesielsky-Treska et al., 1977). Gangliosides in Folch’s upper phase were purified on Sephadex G-25 superfine column chromatography followed by separation by high performance thin layer chromatography (Dreyfus et al., 1980) and quantification by densitometry (Smid and Reisinova, 1973). Total lipid sialic acid (Neu Ac) was measured according to Miettinen and Takki-Luukkainen (1959).
563
taurine was ended by washing the plates 3 times with cold saline and treated as follows: the plates were frozen at -20°C then 1 ml of doubly distilled water was added to each plate and the plates were incubated at 37°C for 1 hour. After incubation the plates were frozen again at -20°C and thawed quickly (Rago et al., 1990). The cell lysate was briefly homogenized (gentle pipetting), 500 p1 was used to determine radioactivity, and 50-100 p1 of homogenate was withdrawn for DNA determination according to a combination of the method described by Rago et al. (1990) and the method of Labarca and Paigen (1980). To 100 p1 of cell homogenate in water, 100 p1 of TNE buffer (Rago et al., 1990) containing 1 pg/ml Hoechst 33258 was added. Fluorescence was measured at 458 nm.
Catecholamines Presence of catecholamines was determined by fluorescence using the formaldehyde-glyoxylic method (Chiba et al., 1981).
RESULTS AND DISCUSSION Cell Morphology Figure 1 shows that after 1 day in culture a greater number of cells bearing short processes were observed on cells plated on PEI than in their counterparts on polylysine. Indeed, the number of processes bearing cells was twice as large on PEI than on polylysine. This difference is less marked on the 2nd day. After 5 days no differences were observed in both types of culture. This last result agrees with previous observations reported by Ruegg and Hefti (1984) showing no Enolase Determination differences between 10 day-old cultures of fetal rat brain Enolase determination was performed as follows. neurons grown either on polylysine, polyornithine, or The culture plates were washed with isotonic cold saline PEI. The capacity of PEI to induce faster neuritogenesis and frozen at -20°C. The frozen samples were homog- than polylysine could be related to a higher adhesion of enized with a Dounce homogeneizer in phosphate buffer the neuroblasts on PEI. As previously shown (Lecontaining 4 mM MgSO, (Scarna et al., 1980). Homoge- tourneau, 1975) adhesion plays an important role in neunates were centrifuged at 100,OOOg and neuron specific ronal morphogenesis . However, we cannot exclude the enolase (NSE) was measured according to Scarna et a1 fact that PEI has a greater affinity than polylysine for (1980). neuritogenic factor(s) present either in the culture media or secreted by the neuroblasts themselves, therefore inTaurine Uptake creasing neuronal sprouting as previously observed with Taurine uptake was measured using a modification early cervical ganglionic neurons (Adler and Varon, of a previously reported method (Lleu and Rebel, 1989). 1981; Lander et al., 1985). Neuronal cells were grown on Costar plates (3 cm diameter) coated with 2ml of either the PEI or the polylysine Protein/DNA Ratio polymer. For the uptake, cells on the culture plates were Previous studies have shown that the protein/DNA washed twice with 3 ml of Krebs-Ringer phosphate ratio of neuronal cells increases with time in culture buffer and preincubated at 37°C with 2 ml of the same (Yavin and Yavin, 1974; Pettmann et al., 1979). This buffer for 5 minutes. One milliliter of the buffer contain- increase parallels an increase in protein synthesis in the ing 0.4 pCi 3H-taurine and 4 pM cold taurine was then nonproliferating neurons and is considered as an index of added and incubation continued for 10 min. Uptake of maturation. Our results (Fig. 2) are in agreement with
564
Lelong et al.
Fig. 1. Chick embryo neurons grown on plates coated either with polyethyleneimine(PEI) or with polylysine. A cell counting of five different fields show that cells grown either on PEI or on polylysine with processes longer than the cell body represent 35 2 5% and 16 f 4%, respectively, of the total cells (Student’s t test: P < . O l ) . DIC = days in culture.
these reported data. Our comparative studies performed on cells grown either on PEI or on polylysine however show that the protein/DNA ratio increase was faster in cells plated on PEL This effect was even more pronounced for cells grown in serum supplemented medium.
Ganglioside Pattern Total lipid sialic acid versus DNA content was slightly higher in 2 day-old neurons grown on polylysine 1.00 and 7.45 2 1.25 pg Neu than on PEI: 12.20 Ac/mg DNA, respectively. This difference became smaller in 5 day-old neurons: 21.90 ? 1.10 and 19.70 1.20 pg Neu Ac/mg DNA, respectively. The Neu Ac/ DNA ratio we found for cultured chick embryo neurons was significantly greater than the one reported by other authors for chicken neurons (Dreyfus et al., 1980) or for
*
*
rat embryo neurons (Yavin and Yavin, 1979). These differences may result from the specificities of the methods used for DNA determination. Indeed, interference by RNA, as observed in some cases, accounts for nucleic acid content increase and thus results also in a decrease in the ganglioside/DNA ratio. In addition the high ratio reported in our study cannot result from contamination of the neuronal ceIl population by glial cells since these latter are almost absent in our cultures (less than 2%). Furthermore, the net increase of ganglioside content that was found between day 2 and 5 in culture was similar to that previously reported. Table I shows the ganglioside pattern of 2 and 5 day-old neuronal cells. In agreement with previous reports (Yavin and Yavin, 1979; Dreyfus et al., 1980; Mandel et al., 1980; Seifert and Fink, 1984) all major brain gangliosides were present (Fig. 3). However, some
Polyethyleneimine vs. Polylysine Coated Plates
2
565
20-
.
0
.-E Q)
r
2
l0-
n
O
L
2
0
4
8
6
10
Day in culture
Fig. 2 . ProteidDNA ratio of neurons cultured either on PEI (squares) or on polylysine (circles) during development. Solid symbols: cells in serum supplemented medium; open symbols: cells in synthetic medium. Two other experiments gave similar curves. Each value is the mean of two determinations which differed by less than 10%. TABLE I. Distribution of Ganglioside Sialic Acid Expressed in Percent (%) of Total Ganglioside Sialic Acid Content of 2 and 5 Day-Old Neurons* Polylysine 2DIC GM, GM, GM, GD, GDI, GDlb GT,, GQ,,
+
Traces ND 5 0.1 40.2 2.3* 22.3 t 0.2 7.2 t 0.6* GTL 30,1 5
0.1
Polyethy leneimine
5DIC
2DIC
5DIC
1.3 t 0.2 ND 2.0 24.9 25.7 t 1.2 20.4 2 1.0 25,4 o,l
0.2 f 0.1 ND 0.4 o.l 27.2 20.7 2 1.0 27.2 f 1.5 24,2
'
0.5 f 0.1 ND 2.0 l.l 25.1 0.7 21.8 f 2.6 24.4 t 2.5 28,0 3,1
0.1
0.2 t 0.1
*
0.3 f 0.1
5
*The difference for GD3 and GDlb of 2 DIC cells grown either on PEI or on polylysine is significant (P < ,001) using Student's t test. DIC = day in culture, ND = not detected.
quantitative differences between our results and published data were noted. GM3 is a major ganglioside of cultured glial cells (Robert et al., 1975; Dreyfus et al., 1980; Dimpfel, 1982; Seifert and Fink, 1984). The trace amount of GM3 detected in our neuronal cultures confirms the absence of glial cells, even in the latter stages of the cultures. We observed no significant differences in the ganglioside pattern of 5 day-old neurons grown either on PEI or on polylysine. In contrast, a notable difference was observed in 2 day-old cultures for GD3 and GDlb. GD3 is considered as a marker of nerve cell immaturity, its amount decreasing with age, both in vivo and in vitro
Fig. 3. Thin-layer chromatogram showing ganglioside patterns from 5 day-old neurons cultured either on polylysine (lane A) or on PEI (lane B). Authentic ganglioside standards were run in lane C: GM3 was obtained from C, glioma cells and all other gangliosides were from pig brain. Spots running ahead of GM3 did not stain purple with resorcinol reagent.
(Engel et al., 1979; Yavin and Yavin, 1979; Dreyfus et al., 1980; Irwin et al., 1980; Goldman et al., 1984). Furthermore, G D l b has been suggested to be a part of the tetanus toxin receptor, a marker of mature neuronal cells (Fishman, 1982). The ganglioside pattern of the 2 day-old neuronal culture on polylysine was characteristic of immature nerve cells. This was not the case for cells grown on PEI which display a pattern quite similar to the pattern found in 5 day-old cultures. This observation is also in agreement with data we reported previously (Lelong et al., 1991). indeed, in this previous paper, we showed a higher level of radiolabeling of G D l b in plasma membranes from 1 day old neurons for cells grown on PEI compared to their counterparts grown on polylysine.
566
Lelong et al. TABLE 111. Taurine Uptake*
TABLE 11. Total Enolase and Neuron Specific Enolase (U/mg DNA)+ Day in culture
Neurone specific enolaset
Total enolase Polylysine
PEI
Polylysine
Day in culture
PEI
2
A
2 4 6
27.1 & 3.4 45.1 f 7.6** 0.10 -+ 0.02 0.18 2 0.04* 40.9 6.9 62.0 f 8.7** 0.42 f 0.05 0.54 0.05 1.40 0.09 1.52 C 0.07 103.0 C 13.1 118.2 8.9
*
*
B
2 4
6
25.0 f 1.9 36.1 f 3.8 50.0 2 8.9
27.3 f 7.0 49.3 f 6.2* 69.9 6.7*
*
0.63 f 0.07 0.81 0.46 f 0.04 0.61 0.51 f 0.06 0.57
f 0.07*
* 0.07 f
0.03
tCells were grown either in serum supplemented medium (A) or in synthetic medium (B) since day 1. One unit of enolase is the amount of enzyme catalysing the transformation of 1 pmole of substrate/min at 30°C. Similar results were obtained with another set of cultures. Values represent the mean of duplicates derived from the same experiment. Statistical comparison: Cultures on PEUpolylysine: *P < .05; **P < .01 (Student’s t test). Total enolase: DIC 4 to DIC 6 in serum or synthetic medium: 5%; DIC 2 to DIC 6 in serum or synthetic medium: 1% . Neuron specific enolase: DIC 2 to DIC 4 on polylysine + serum, DIC 4 to DIC 6 on PEI and polylysine + serum: 5%; DIC 2 to DIC 6 in serum: 1% (ANOVA test).
4 7
Taurine uptake (pmole/mg DNNmin) in cells grown on Polylysine
PEI
22.9 [19.4-31.41 38.3 [31.7-48.11 215.7 [172.3-273.91
47.3 [31.7-62.81 88 .o [82.9-91.11 193.3 [ 130.2-260.51
*Cells were grown in DMEM supplemented with 20% fetal calf serum. Each value (pmoles/mg DNNmin) is the mean value for quadruplicates. Nos. in brackets represent the extreme values. Statistical difference between polylysine and PEI: 2 and 4 days in culture: P < .05; 7 days in culture: not significant.
Taurine Uptake Taurine uptake was low in 2 day-old cultures, increasing thereafter (Table 111). During the maturation period (days 1-5) a higher uptake was obtained with cells grown on PEL Despite a wide variation in the uptake by brain cells (previously discussed: Lleu and Rebel, 1989), a higher taurine uptake was found during the maturation period (days 1-5) in cells grown on PEI compared to cells grown on polylysine. Few data on taurine uptake in cultured neuronal cells can be found. Adler reported the Neurone Specific Enolase (NSE) existence of an uptake in chicken retinal neurones having Similar variations in total enolase activity in neu- the main characteristics of those found in mammalian rons between day 2 and 6 were observed with cells cells (Adler, 1983). By autoradiography, he demongrown either on PEI or on polylysine (Table 11). How- strated that 3 day-old cultures are able to take up taurine. ever, the total specific activity of enolase was always Our results also agree with those of Kishi et al. reporting of taurine uptake by mouse neuhigher in cells grown in serum supplemented media than a doubling of the v,, ronal cells between the 3rd and the 7th day when the in cells grown with synthetic media. More surprising was uptake reaches a plateau (Kishi et al., 1988). the evolution of the neurone specific enolase activity. While this activity increased in cells grown in media Catecholamine Fluorescence supplemented with serum, no change was observed in Whether cells were grown on PEI or on polylysine cells grown in synthetic media. As observed for total in serum supplemented medium, similar fluorescence enolase activity, the type of polycation used to favor the was observed in 2 day-old cultured neurons (data not anchorage of the neurons had no effect on the activity. shown). The number of positive cells was similar. This Indeed, 2 day-old neuronal cell cultures usually have low similarity was also found on the other days where cateNSE activity (Secchi et al., 1980; Weyhenmeyer and cholamine fluorescence was checked. Bright, 1983; Schilling et al., 1988, 1989). Previous Very few cells having tyrosine hydroxylase (TH) studies on the changes of NSE with time have been ob- activity and catecholamines have been found in 3 day-old tained with cultures grown in serum supplemented me- cultures of avian neural crest cells (Christie et al., 1987). dium (Schengrund and Marangos, 1980; Schilling et al., However, 2 day-old cultures of chicken telencephalic 1988, 1989). These authors report, as we have also ob- neurons are able to take up tyrosine and dopamine from served, a notable increase of NSE activity during the first the medium and have a noticeable TH activity (Louis et days in culture. Surprisingly, no increase of NSE was al., 1981, 1983). As for cultured rat neuronal cells (Iaobserved in cultures grown in serum free medium, when covitti et al., 1987), chicken neuronal cells are able to results were expressed by mg of DNA instead of protein. synthesize dopa and dopamine (Louis et al., 1983). Though the NSE activity was always higher in cells grown on PEI, difference with cells grown on polylysine Methodological Considerations Our results also show that the protein amount per was less marked than those found for the other criteria of cell changed every day, thereby confirming previously neuronal maturation studied.
Polyethyleneimine vs. Polylysine Coated Plates
published data (Yavin and Yavin, 1974; Pettmann et al., 1979). Moreover, this variation is also dependent on the growth conditions. These observations emphasize that protein content cannot be used as a reference for expressing results in pharmacological or enzymatical studies performed on neuronal cells. DNA (or cell number) should therefore be used to normalize results. To measure taurine uptake we have replaced the method of Labarca and Paigen (1980) with an easier protocol combining the last procedure with that of Rago et al. (1990). However, using the concentration of Hoechst 33258 recommended by Rag0 et al. (1990) leads to high background in the determination of low amounts of DNA. This can be overcome by decreasing the concentration of Hoechst 33258. A comparison of this modified method with that of Labarca and Paigen has given identical results.
General Conclusions Comparison of chicken brain neuronal cells grown on polylysine or on PEI coated plates showed that PEI coated plates allow faster maturation as determined by morphology, proteidDNA ratio, ganglioside pattern, or taurine uptake taken as index. A slight difference between the two kinds of cultures was observed with neuron specific enolase (NSE). No apparent difference was obtained with catecholamine fluorescence. Our results could discriminate between events linked probably with the end of the differentiation process (catecholamine synthesis), and with early (morphology, ganglioside pattern) and late events (taurine uptake, NSE) linked to maturation. By differentiation, we mean the steps which occur during the development of cells, in which a pluripotent cell (neural crest cell, for example) changes into a cell destined to give only one type of mature cell. Maturation, in contrast, includes the step which allows a differentiated embryologic cell (neuroblast) to gain numerous characteristics found in vivo in the cells of the adult organ (neurons). ProteiniDNA ratio represents another criteria which changes continuously with time. When this criterion or the NSE activity was used to address neuronal maturation, maturation was accelerated in serum supplemented medium compared to that obtained in serum free medium. This undoubtedly related to the presence in the serum of some trophic factors which are missing from the synthetic media. The faster maturation observed on PEI could be related to the cellular adhesion as discussed above, but also to the higher capacity of PEI, as compared to polylysine, to retain some trophic factors contained in the media. Adsorption on plastic (Buchou et al., 1988), on polycations (Lander et al., 1985), or on extracellular matrix (Vigny et al., 1988; Flaumenhaft et al., 1989; Presta et al., 1989) of factors present in culture
567
media is well known. However, this fact raises the problem of selecting sera which allow the growth and survival of neuronal cells, but which do not favor growth of glial cells. Our results extend the observations of Ruegg and Hefti (1984) confirming that neuronal cells can be easily selected and grown on polyethyleneimine coated surfaces. Experiments are in progress to generalize this study with other mammalian cells. Moreover, PEI may represent a useful alternative in cases where polylysine interferes in some enzymatic assays (unpublished data).
ACKNOWLEDGMENTS We wish to thank Dr. M.M. Gottesman (N.I.H.) for helpful comments on the manuscript.
REFERENCES Abdel-Ghany M, Kole HK, Abou El Saad M, Racker E (1989): Stimulation of phosphorylation of lipocortin at theonine residues by epidermal growth factor (EGF) and the EGF receptor: Addition of protein kinase P, with polylysine inhibits this effect. Proc Natl Acad Sci USA 86:6072-6076. Adler R (1983): Taurine uptake by chick embryo retinal neurons and glial cells in purified culture. J Neurosci Res 10:369-379. Adler R, Varon S (1981): Neuritic guidance by polyornithine-attached materials of ganglionic origin. Dev Biol 81:l-11. Bottenstein JE, Sato GH (1979): Growth of a rat neuroblastorna cell line in serum-free supplemented medium. Proc Natl Acad Sci USA 761514-5 17. Buchou T, Charollais PH, Mester J (1988): Involvement of serum factor(s) adsorbed to the dish in the response of cycloheximidepretreated BP-A31 cells to serum pulses. Exp Cell Res 174: 41 1-420. Chiba T, Murata Y, Koike T (1981): Plasticity of pheochromocytoma (PC12) cells demonstrated by nerve growth factor or glucocorticoid treatment: A catecholamine fluorescence and electron microscopic investigation. Biomed Res 2:618-628. Christie DS, Forbes E, Maxwell GD (1987): Phenotypic properties of catecholamine-positive cells that differentiate in avian neural crest cultures. J Neurosci 7:3749-3763. Ciesielsky-Treska J, Robert J, Rebel G, Mandel P (1977): Gangliosides of active and inactive neuroblastoma clones. Differentiation 8:31-37. Dimpfel W (1982): Differential distribution of gangliosides in nervecell cultures. Cell Mol Neurobiol 2:105-113. Dreyfus H, Louis J , Harth S , Mandel P (1980): Gangliosides in cultured neurons. Neuroscience 5: 1647-1655. Engel EL, Wood JG, Byrd FI (1979): Ganglioside patterns and cholera toxin-peroxidase labelling of aggregating cells from the chick optic tectum. J Neurobiol 10:429-440. Fishman PH (1982): Role of membrane gangliosides in the binding and action of bacterial toxins. J Membr Biol 69:85-97. Flaumenhaft R, Moscatelli D, Saksela 0, Rifkin DB (1989): Role of extracellular matrix in the action of basic fibroblast growth factor: Matrix as a source of growth factor for long-term stimulation of plasminogen activator production and DNA synthesis. J Cell Physiol 140:75-81. Goldman JE, Hirano M, Yu RK, Seyfried TN (1984): GD, ganglio-
568
Lelong et al.
side is a glycolipid characteristic of immature neuroectodermal cells. J Neuroimmunol 7:179-192. Iacovitti L, Lee J, Joh TH, Reis DJ (1987): Expression of tyrosine hydroxylase in neurons of cultured cerebral cortex: Evidence for phenotypic plasticity in neurons of the CNS. J Neurosci 7: 1264-1270. Irwin LN, Michael DB, Irwin CC (1980): Ganglioside patterns of fetal rat and mouse brain. J Neurochem 34:1527-1530. Kishi M, Ohkuma S, Kimori M, Kuriyama K (1988): Characteristics of taurine transport system and its developmental pattern in mouse cerebral cortical neurons in primary culture. Biochim Biophys Acta 939:615-623. Kleinman HK, Luckenbill-Edds L, Cannon FW,Sephel G (1987): Use of extracellular matrix components for cell culture. Anal Biochem 166:l-13. Labarca C, Paigen K (1980): A simple and sensitive DNA assay procedure. Anal Biochem 102:344-352. Lander AD, Fujii DK, Reichardt LF (1985): Purification of a factor that promotes neurite outgrowth: Isolation of laminin and associated molecules. J Cell Biol 101398-913. Lelong I, Frechard E, Creme1 G, Langley K, Rebel G, Vincendon G, Mersel M (1991): Expression of plasma membrane and cell surface phospholipids and gangliosides of chick embryo neurons grown in primary cultures: Developmental studies. Dev Neurosci 13:54-60. Letourneau PC (1975): Possible roles for cell-to-substratum adhesion in neuronal morphogenesis. Dev Biol 44:77-91. Lleu PL, Rebel G (1989): Effect of HEPES on the taurine uptake by cultured glial cells. J Neurosci Res 23:78-86. Louis JC, Pettmann B, Courageot J , Rumigny JF, Mandel P, Sensenbrenner M (198 1): Developmental changes in cultured neurones from chick embryo cerebral hemispheres. An ultrastructural and neurochemical study. Exp Brain Res 42:63-72. Louis JC, Langley K, Anglard P, Wolf M, Vincendon G, (1983): Long-term culture of neurones from human cerebral cortex in serum-free medium. Neurosci Lett 41:313-319. Lowry OH, Rosebrough NJ, Farr AI, Randall RJ (1951): Protein measurement with the Folin-phenol reagent. J Biol Chem 193: 265-275. Maciera-Coelho A, Avrameas S (1972): Modulation of cell behavior in vitro by the substratum in fibroblastic and leukemic mouse cell lines. Proc Natl Acad Sci USA 69:2469-2473. Mandel P, Dreyfus H, Yusufi ANK, Sarlieve L, Robert J, Neskovic N, Harth S , Rebel G (1980): Neuronal and glial cell cultures, a tool for investigation of ganglioside function. In Svennerholm L, Mandel P, Dreyfus H, Urban PF (eds): “Structure and Function of Gangliosides.” Plenum Pub Corp. pp 515-531. Miettinen T, Takki-Luukainen IT (1959): Use of butyl acetate in determination of sialic acid. Acta Chem Scand [B] 132356858. Pettmann B, Louis JC, Sensenbrenner M (1979): Morphological and biochemical maturation of neurons cultured in the absence of glial cells. Nature 281:378-380. Presta M, Maier JA, Rusnati M, Ragnoti G (1989): Basic fibroblast growth factor is released from endothelial extracellular matrix in a biologically active form. J Cell Physiol 140:68-74.
Rag0 R, Mitchen J, Wilding G (1990): DNA fluorometric assay in a 96-well tissue culture plates using Hoechst 33258 after cell lysis by freezing in distilled water. Anal Biochem 191:31-34. Robert J , Freysz L, Sensenbrenner M, Mandel P, Rebel G (1975): Gangliosides of glial cells: A comparative study of normal astroblasts in tissue culture and glial cells isolated on sucroseficoll gradients. FEBS Lett 50:144-146. Riiegg UT, Hefti F (1984): Growth of dissociated neurons in culture dishes coated with synthetic polymeric amines. Neurosci Lett 49:319-324. Scarna H, Keller A, Pujol JF (1980): MicromCthode de mesure des trois isoenzymes cCrCbrales de 1’Cnolase. CR Acad Sci Paris 291 :397-400. Schengrund CL, Marangos PJ (1980): Neuron-specific enolase levels in primary cultures of neurons. J Neurosci Res 5:305-311. Schilling K, Scherbaum C, Pilgrim C (1988): Developmental changes in neuron-specific enolase and neurofilament proteins in primary neural culture. Histochemistry 89295-299. Schilling K, Barco EB, Rhinehart D, Pilgrim C (1989): Expression of synaptophysin and neuron-specific enolase during neuronal differentiation in vitro: Effects of dimethyl sulfoxide. J Neurosci Res 24:347-354. Secchi J, Lecaque D, Cousin MA, Lando D, Legault-Demare L, Raynaud JP (1980): Detection and localization of 14-3-2 protein in primary cultures of embryonic rat brain. Brain Res 184: 455 -466. Seifert W, Fink HJ (1984): In vitro and in vivo studies on gangliosides in the developing and regenerating hippocampus of the rat. In Ledeen R, Yu R, Rapport M, Suzuki K, (eds): “Gangliosides: Structure, Function and Biochemical Potential.” Plenum Pub Corp., pp 535-545. Sensenbrenner M, Maderspach K, Latzkovits L, Jaros GG (1978): Neuronal cells from chick embryo cerebral hemispheres cultivated on polylysine-coated surfaces. Dev Neurosci 1:90101. Smid F, Reisinova J (1973): A densitometric method for the determination of gangliosides after their separation by thin-layer chromatography and detection with resorcinol reagent. J Chromatogr 86:200-204. Suzuki K (1964): A simple and accurate micromethod for quantitative determination of ganglioside patterns. Life Sci 3: 1227-1233. Vigny M, Ollier-Hartmann MP, Lavigne M, Fayen N, Jeanny JC, Laurent M, Courtois Y (1988): Specific binding of basic fibroblast growth factor to basement membrane-like structures and to purified heparan sulfate proteoglycan of the EHS tumor. J Cell Physiol 137:321-328. Weyhenmeyer JA, Bright MJ (1983): Exprssion of neuron specific enolase in cultured neurons from the fetal rat. Neurosci Lett 43:303-308. Yavin E, Yavin Z (1974): Attachment and culture of dissociated cells from rat embryo hemispheres on polylysine coated surface. J Cell Biol 62540-546. Yavin E, and Yavin Z (1979): Ganglioside profiles during neural tissue development. Acquisition in the prenatal rat brain and cerebral cell cultures. Dev Neurosci 2:25-37.
