Brain primary culture — a characterization

Brain Research, 188 (1980) 233-246

233

© Elsevier/North-Holland Biomedical Press

BRAIN PRIMARY CULTURE -- A CHARACTERIZATION

ELISABETH HANSSON, AKE SELLSTROM, LENNAR'I I. PERSSON and LARS RONNB,~CK bzstitute of Neurobiology and (L.I.P andL.R.) Department of Neurology, Ur,iversity of Gb'tebi~rg, G~teborg (Sweden)

(Accepted September 13th, 1979) Key words: brain - - primary cultures -- astrocytes

SUMMARY Primary cultures from rat or mouse brain hemispheres contain predominantly glial cells. These cells accumulated [3H]GABA and showed a specific fluorescence with FITC-labeled anti-S-100 antiserum. Round or elongated cells which reacted positively with antimacrophage antiserum and exhibited phagocytotic activity, were considered mesodermal macrophages. Big flat cells, unreactive to S-100 antiserum or to any other antiserum tested, showed a morphology similar to mesenchymal cells of a low differentiation grade. These cells formed a monolayer upon which other cell types grew. Positive reaction for alkaline phosphatases was used as a criterion to identify endothelial cells, the number of which increased with increasing age of the cultures. Anti-143-2 antiserum gave no specific reaction, indicating the presence of very few or no differentiated neurons in the cultures.

INTRODUCTION Primary cultures of dissociated nervous tissues have been taken as good model systems in experimental cell research of the central nervous system. These cell cultures are potentially important tools for investigating morphological and biochemical aspects of nerve and glial cell differentiation, intracellular regulatory mechanisms, cellular interactions, and drug effects1,5,22, 87. By choosing the appropriate parameters for culturing, a preferential survival of one cell type or other can be obtained. It has been suggested that cell cultures made from brain hemisphere of the newborn rat or mouse will result in a glia cell culture 2. It has also been shown that primary cultures exhibit some of the biochemical and physiological properties characteristic of the central nervous system. However, the charac-

234 terization as to specific cell type is uncertain. Thus, many studies on glial primary cells have been directed towards analyzing the capabilities of these cells, without being particularly concerned with what such studies will tell us about nervous system function. Nevertheless, the testing has been important, since we know that, at least in some respects, the cultures behave similarly to normal nervous tissue. The aim of the present investigation was to define some cell types in a primary astroglial cell culture. Besides neuroectodermal glial cells which constituted the main part of the cells present, macrophages, endothelial and mesenchyme-like cells were seen. MATERIALS AND METHODS

Culture medium The medium is based on Eagle's minimum essential medium, supplied with double concentrations of amino acids (except glutamine), quadruple concentrations of vitamins, 250,000 IU/liter penicillin, 2 mM glutamine, 7 mM glucose, 20 ~o (v/v) fetal calf serum, and having a pH set at 7.3. Materials Dibutyryl cyclic-Y,5'-adenosine-monophosphate (dB-cAMP), amino acids, vitamins, deoxyribonucleic acid (DNA), aminooxyacetic acid (AOAA), 3,5-diaminobenzoic acid dihydrochloride (DABA) and fluorescein diacetate were obtained from Sigma Fine Chemicals, St. Louis, U.S.A. ; [2,3-3H]~-aminobutyric acid (GABA) from N E N Chemicals G m b H ; and emulsion K-2 from |lford. Eagle's minimum essential medium was obtained from Flow Laboratories, U.K. ; fetal calf serum from GIBCO Bio-Cult. Lab Ltd., U.K.; penicillin from Kabi, Sweden; and petri dishes from N U N C A/S, Denmark. Fluorochrome-labeled sheep or goat anti-rabbit immunoglobulin was bought from SBL, Stockholm, Sweden. Munktelt Filter M1 was obtained from Kebo Grave, Sweden. All other chemicals were of reagent grade. Tissue culturing The cultures were made from newborn rat or mouse brains ~,zS. The animal was immersed in 70 ~o ethanol for a short period and decapitated. The skull was opened and the cerebral hemispheres collected. Meningeal material was removed, and the brains were rinsed in culture medium. Two rat or 5 mouse brains were passed through an 80/~m nylon mesh into 10 ml of the medium, by gently pressing with a teflon pestle. One ml of this suspension was pipetted into a plastic petri dish, 50 mm in diameter, prefilled with 3 ml culture medium. The dishes were kept at 37 °C in a humidified atmosphere of 95 ~ oxygen and 5 ~ carbon dioxide for 3 days, whereafter the culture medium was changed 3 times a week. Following a 2-week growing period, the fetal calf serum was removed from the medium, and 1.0 mM dB-cAMP was added to some cultures as a stimulator of morphological differentiation z°,3°,31. Determination of growth rate The growth rate was expressed as the daily increment of increase in protein and

235 D N A content. The petri dishes were scraped with a teflon spatula and homogenized in distilled water (pH 7.2). Measurements of protein were performed according to Lowry et al. 21 and of D N A as described by Kissane and Robins 17. Briefly, trichloroacetic acid (TCA) was added to the cell homogenate to a final concentration of 0.3 M TCA. The homogenate was centrifuged and lipids were extracted from the precipitate by alcoholic potassium acetate (0.1 M), followed by successive washings in absolute ethanol. Standard curves were made from a solution of 0.88 /~g/#l D N A in 1 M N H 4 O H and were linear in the range of 25 to 200 ng DNA//~I. Freshly prepared 2 M DABA was added to the evaporated cell samples, standards and blanks under continuous mixing, and allowed to react for 30 min at 60 °C. Perchloric acid (PCA) was added, mixed and centrifuged. A small volume was transferred to a special fluorometer tube containing PCA in a final concentration of 0.6 M. Fluorescence was assayed in an Aminco-Bowman Spectrophotofluorometer with an activating wavelength of 425 nm and a fluorescent wavelength of 510 nm.

Viability The viability of the cells was evaluated by incubating them in trypan blue and in fluorescein diacetate. Trypan blue was added to the culture medium and the time taken for the cells to become stained was recorded. The staining was taken as an indicator of cell damage. The cells were also incubated in a solution of 10-8 M fluorescein diacetate and 0.32 M sucrose. Fluorescein diacetate does not fluoresce when free in solution, but upon entering a cell it will be metabolized giving the ceil a yellow-green fluorescence when irradiated by UV-light 10.

Autoradiography The uptake of [aH]GABA by the cells of the 14-day-old primary cultures was studied by autoradiography. The cells were incubated in 37 °C with medium containing 35 mM Tris.HC1 (pH 7.4); 140 mM NaC1 ; 3 mM KC1 ; 2.5 mM MgC12; 20 mM glucose; 1.5 mM CaCIz; and 5 × 10-5 M AOAA. [aH]GABA was added to a final concentration of 10-7 M. After incubation for 10 rain, the cells were washed twice in incubation medium containing no radioactivity, and fixed in a medium containing 3 ~o glutaraldehyde for 5 min. The fixed cells were washed twice in incubation medium and dehydrated in 50 ~ ethanol. Emulsion Ilford K-2 diluted 1 : 1 with distilled water, was layered at a temperature of 43 °C. The emulsion was filtered and deaerated prior to use. The film was exposed for 5, 10, 14, 21 or 28 days, and developed in Kodak D19, rinsed and fixed in a 30 ~ solution of Na2S207.

Production of the antisera Antisera against the proteins S-100 and 14-3-2 were produced as previously describedT, 8. The purity of the antigens was controlled, in three different electrophoretic systems, prior to their use for immunisation. The purification of the antibodies was performed as described by Hyd6n and R6nnb~ick la. The specificity of the antigen-antibody reactions was tested in the following ways.

236 (1) Single precipitation bands were obtained when the antisera were tested by double diffusion in ! ~ agar against purified S-100 and 14-3-2, respectively. (2) Immunoelectrophoresis in agarose according to Stavrou et al. a9 against different organ extracts, such as liver, lung and kidney, revealed a 10,000-fold increase of S100 and a 200-fold increase of 14-3-2 antigen in rabbit brain over other tissues studied. The single precipitation lines detected for anti-S-100 and anti-14-3-2 in brain homogenate Were not present in other organs. (3) After immunoabsorption, the pure antibodies showed a specific reaction with the antigen according to Hyd6n and R6nnb/ick 13. The anti-macrophage antiserum was prepared in rabbits against rat peritoneal macrophages, according to Persson and R6nnb/ick and R6nnb/~ck and Persson2~, 33. In short: an immunological cross-reaction was found against blood monocytes and macrophages in the thymus and spleen, as well as against perivascular macrophages in liver, kidney, and lung. Antimacrophage activity was seen neither in spleen lining cells, nor in liver Kupffer cells. In normal brain, there were anti-macrophage antiserum reactive cells among the leptomeningeal macrophages and in a perivascular position around larger blood vessels.

Conjugation of antiserafor immunofluorescence microscopy Anti-S- 100, anti-I 4-3-2 or anti-macrophage antisera were coupled to fluorescein isothiocyanate (FITC) according to the method of Hijmans et al. 11. The conjugates used had an optical density (O.D.) ratio of 6.5-7.3 (ref. 41). Part of the anti-macrophage antisera (ammonium sulfate precipitated IgG-enriched anti-macrophage serum) was conjugated with tetramethylrhodamine isothiocyanate (TRITC) according to the method described by BrandtzaegL The conjugate used had an optical density ratio of 2.0-4.0 (ref. 3). The conjugates of anti-S-100 and anti-14-3-2 antisera were absorbed with liver powder H. The antisera were absorbed with pure S-100 protein, pure 14-3-2 protein or peritoneal macrophages, respectively, by incubation for 1 h at 37 °C in a shaking water bath, and for 12 h at + 4 °C. The absorbed conjugates were then centrifuged for 30 min at 10,000 × g. The procedure was repeated several times, until the antisera were free from antibodies against S-100, 14-3-2 or macrophage antigens, respectively, as controlled by immunoelectrophoresis ~9,39. The absorbed conjugates were always used as controls during immunofluorescence procedures, as were preimmune serum or serum from a non-immunized rabbit. Conjugated sera were kept at + 4 °C in the presence of methiolate (1 : 10,000) or sodium azide (0.02 ~o) and never used later than two weeks after preparation. Fluorochrome-labeled sheep or goat anti-rabbit immunoglobulin was used for indirect staining (see below).

Preparation of culturesfor immunofluorescencemicroscopy The cultures were fixed in 4°/o freshly prepared formaldehyde in 0.15 M cacodylate buffer (pH 7.2) for 60 min. In most experiments, 0.1 ~o picric acid was added to the fixative. After extensive rinsing, the cultures were incubated in the antibodycontaining solutions or control solutions.

237 In experiments with direct labeling, the cultures were incubated in anti-S-100, anti-14-3-2 or anti-macrophage antisera conjugated with F I T C or T R I T C for 20-45 min, dilutions 1 : 10-1 : 250. The cells were repeatedly washed in incubation medium (same as used in autoradiography) for 60 min, and thereafter observed in the fluorescence microscope. During some incubations, bovine serum albumin was added at concentrations of 2 - 1 2 ~ . The following controls were performed: (1) the cells were incubated with antisera repeatedly absorbed with the respective antigen(s) (see above). Antisera and absorbed antisera were diluted serially and were used in the dilution where the difference between test and control fluorescence was optimal; (2) labeled serum of a non-immunized rabbit or pre-immune serum were used; (3) cultures were incubated in unlabeled antisera, and thereafter washed and exposed to labeled antisera; (4) cultures not treated with any antiserum were used to assess the autofluorescence. This was negligible in our experimental set-up. Indirect labeling was used in some experiments. Dilutions were tried from 1 : 10 down to 1 : 250 in buffered saline. After extensive washing the cultures were incubated in the sheep or goat anti-rabbit-IgG conjugated with F I T C or T R I T C for 30-45 min in buffered saline, p H 7 2. The cells were studied after extensive washing. Controls were treated as above, except for the incubation in the specific antisera in one case, or incubation with an absorbed, a pre-immune serum or an anti-albumin serum prior to incubation in the sheep or goat anti-rabbit-IgG antiserum as further control.

Microscopy The fluorescence microscope used was a Zeiss photomicroscope III RS, equipped with a mercury lamp. The sample was irradiated for excitation via the objective and the excitation filters for F I T C or rhodamine were exchangable in a sliding arrangement which permitted rapid photography. Photographs were recorded using phase contrast on K o d a k Tri-X-PAN 27 Din film.

The use of Indian ink to study phagocytotie activity A suspension of Indian ink was prepared by mixing 50 ml of Indian ink with 50 ml of 0.15 M NaC1, p H 7.2. The mixture was heated to 37 °C and filtered 6 times through Munktell filter F 1. The filtrate was centrifuged twice at 2500 × g for 50 rain (ref. 32). To cells at different ages was added 50 #1 of this Indian ink suspension. Cells were allowed to incubate for time periods from 10 min up to 48 h.

Histochemieal method for alkaline phosphatase The cells were incubated for 30 min in r o o m temperature in a solution containing naphthol AS-TR-phosphate, dissolved in dimethylformamide (DMF), 0.2 M Tris-HC1, p H 8.3, and 30 mg Fast red violet LB-salt. The cultures were then rinsed in 0.2 M Tris.HCl, p H 8.3, before microscopy 4.

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Fig. 1. Total protein (mg) (a) and DNA (~g) (b) content in mouse brain cell cultures at different times after culturing. The amounts are the means of 6-7 experiments and values are expressed per whole brain hemisphere. RESULTS

Cell growth The cell suspension formed as a rat brain was passed through the nylon sieves, was added to petri dishes, and the protein and D N A content during subsequent development of the cells in culture is seen in Fig. la, b. These show that the first 6 days in the culture could be considered a recovery period, whereafter the new cells outnumber the dying ones. If the D N A content of these cultures is taken as a measure of the number of cells appearing, the cultures were at confluency at approximately 10 days. The protein content of the cultures also increased, even after the 10th day. At 15 days the calf-serum was withdrawn and dB-cAMP was added to most cultures, resulting in a decrease in the amounts of D N A and protein in parallel with the morphological differentiation of the formation of cell processes.

Viability of cells Since the withdrawal of calf-serum and addition of dB-cAMP profoundly altered the cell morphology, the viability of the cells was evaluated by incubating them in trypan-blue and fluorescein diacetate. All types of cells remained unstained when exposed to trypan blue for 3-4 h at room temperature in air. During this period, the cells also accumulated fluorescein diacetate as judged by their fluorescence. Both these observations indicate that they were viable, Cells incubated in the presence or absence of fetal calf serum and with the addition of 1.0 mM dB-cAMP for 2 days were also tested, but no differences in viability were observed.

Glial cells Cells reacting positively with the anti-S-100 antiserum were the predominating, cell type in the confluent culture (Fig. 2). These cells had an average diameter of 16/tm, were mostly round or spindle-shaped, and had few processes. Their nuclei were round or oval with a diameter of 6/~m and usually contained two nucleoli. When fetal calf serum was removed and 1.0 mM dB-cAMP was added to the culture medium, the cell body decreased in size and long slender processes were formed. The reaction with

239

Fig. 2. a: brain cell culture 14 days old, as described in the text. Most cells are round or spindle-shaped with small slender processes, b: the same culture as in a, incubated with FITC-labeled anti-S-100 antiserum as described in the text. A cytoplasmic specific fluorescence is demonstrated in most of the cells, indicating their neuroectodermal origin. These cells are characterised as glial cells, since they did not show any reaction with anti-14-3-2 antiserum, x 220. the fluorescent labeled anti-S-100 a n t i s e r u m seemed to be localized to the c y t o p l a s m as viewed in the fluorescence m i c r o s c o p e (Fig. 3). C o n t r o l s are s h o w n in Fig. 4. I n some e x p e r i m e n t s cultures were i n c u b a t e d with [ a H ] G A B A a n d the u p t a k e o f this c o m p o u n d was studied b y a u t o r a d i o g r a p h y . I n the confluent culture, the cells which were shown t o be positive for anti-S-100 a n t i s e r u m also a c c u m u l a t e d [aH]G A B A (Figs. 5a a n d b). Negatively testing cells were p r i m a r i l y o f two types: one e l o n g a t e d o r r o u n d with an a p p e a r a n c e similar to those identified as m a c r o p h a g e s (see below, Fig. 5c); a n d a n o t h e r flat a n d very large (see below, Fig. 5d). A l t h o u g h the m o r p h o l o g y o f the cells is t h a t o f astroglial cells, this study does n o t allow a d i s t i n c t i o n to be m a d e b e t w e e n astro- a n d oligodendrocytes.

Macrophages It was early o b s e r v e d t h a t some r o u n d o r e l o n g a t e d cells h a d p h a g o c y t o t i c activity. This was confirmed b y the o b s e r v a t i o n o f p h a g o c y t o s i s o f I n d i a n i n k w h e n a d d e d

Fig. 3. Similar culture as in Fig. 2 at two days after the removal of fetal calf serum and addition of 1.0 mM dB-cAMP. The cells have long slender processes, extending from cell bodies which have diminished in size in comparison to those in Fig. 2. a: light microscopic picture. Phase contrast, x 220. b: fluorescence microscopy demonstrating the presence of S-100 protein using fluorescein-labeledanti-S100 antibodies. The fluorescence is stronger than in Fig. 2, probably indicating that these cells contain more S-100 protein (ref. 6). x 220.

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O Fig. 4. Cells from a 14-day-old brain cell culture, a : phase contrast microscopy. × 320. b: incubation in fluorescein-labeled anti-S-100 antiserum repeatedly absorbed with pure S-100 as a control. A faint, non-specific fluorescence is seen. × 320.

@ Fig. 5. Autoradiography of brain cell cultures after incubation in [3H]GABA as described in the text. a: 14-day-old culture. Many grains indicating the uptake of [SH]GABA is seen in most cells, b: 16day-old culture, 2 days after removal of fetal calf serum and addition of 1.0 mM dB-cAMP. A morphological differentiation with long slender processes is seen as in /Hg. 3. Grains indicating the uptake of [3H]GABA are visualized in most cells suggesting they are glial cells, c: same culture as in a. Two oval cells with two or three long processes are seen, lacking grains, i.e. no [~H]GABA uptake. (This cell type is similar to that in Fig. 6 and was thus considered macrophages.) d, large, fiat cells in culture of a. No [3H]GABA uptake is seen. These cells were considered mesenchymal cells (see text). × 500.

241

Fig. 6.14-day-old culture, incubated with Indian ink. Some round or oval cells have been phagocyting the particles. Phase contrast microscopy, x 500. to the culture medium, as illustrated in Fig. 6. Similar cells showed a positive reaction after incubation with the antiserum against macrophages (Fig. 7). Enzyme histochemistry o f n a p h t h o l AS-esterase, i.e. unspecific esterases or aliesterases, using Fast Red T R as visualizing diazonium salt, showed a close correlation between intense activity and intense anti-macrophage activity as visualized by the fluorescence of F I T C or T R I T C in direct or indirect staining of mononuclear macrophages. Neurons Antiserum against the nervous tissue-specific protein 14-3-2 was added to some cultures. N o cells showed any affinity for the antibodies. The 14-3-2 protein has been reported to be localized exclusively in neurons27, 28. The present findings indicate that very few or no differentiated neurons were present in the cultures.

@ Fig. 7. Brain cell culture, 14 days old, after incubation with fluorescein-labeled anti-macrophage antiserum (see text), a: Phase contrast microscopy, x 400. b: a specific cytoplasmic fluorescence is seen in the fluorescence microscope, x 400.

242

Endothelial cells Since endothelial cells are likely to appear in this type of culture, attempts were made to quantify these cells by staining for alkaline phosphatase 24 (Figs. 8 and 9). The more dense the culture, the more intense was the staining reaction for alkaline phosphatase. Positive-staining cells appeared with a frequency of approximately 15 percent in 14-day cultures, often grouped in patches, while very few or no endothelial cells were found in 1-week-old cultures. The appearance of the cell was sometimes rather similar to that of the astrocytes. The alkaline phosphatase positive cells had, however, shorter processes, giving the cell a polygonal appearance.

Figs. 8 and 9. 14-day-oldbrain cell culture prepared for demonstrating alkaline phosphatase activity as described in the text. A positive reaction (red reaction product) is seen in some polygonal flat cells. x 500.

243 Mesenchymal cells Big, flat cells which did not react with anti-S-100 or any other antiserum tested, were characterized as undifferentiated mesenchymal-like cells. They constituted around 5 ~o of the total cells in a 14-day-old culture, and formed a monolayer upon which other cell types grew. Comments on the immunological studies When the cells were incubated with the two-step method, i.e. with the indirect labeling technique, the intensity of the specific FITC or TRITC fluorescence observed was higher than in cultures incubated with the direct method. There was no difference with respect to the distribution of antigens in the different cells between the two-step and the one-step incubation techniques. No specific fluorescence of FITC or TRITC was observed after incubation with pre-immune serum, with serum of a non-immunized rabbit, with rabbit antiserum to bovine albumin, after omitting incubation in the antisera or after blocking tests, either with the one-step or with the two-step method. There was a significant reduction of the specific fluorescence of FITC or T R I T C after incubation with antisera, repeatedly absorbed with the respective antigen(s). DISCUSSION Despite the large volume of literature that has been published on brain primary cultures, very little is actually known about their cellular composition and their degree of differentiation9,29,3s,4°. Our aim has been to identify by different criteria some of the cellular elements which are present. We began by assuming that all of the cells present in a brain homogenate could exist in the culture. Cells possibly appearing are: neurons; astrocytes and oligodendrocytes; endothelial cells; ependymal cells; macrophages, among these also the microglial cells; mesenchymal cells; and blood cells. The dominating cells in this culture were identified as neuro-glia. These cells reacted positively with anti-S-100 antiserum. They took up [aH]GABA, which has been shown as a glial cell parameter14,15, z4, did not react with anti-macrophage antiserum, and had low phagocytic activity with Indian ink. In our hands these cells constituted about 60 ~o of the cell population. The survival of neurons in this type of culture has been reported to be low2, 23. Therefore, our finding that none of the cells reacted positively with anti-14-3-2 antiserum is not surprising. However, this is not sufficient evidence on which to conclude that the cell culture is absolutely neuron-free. Cells from the capillary endothelium of the cerebral cortex have been successfully grown in tissue culture medium after their partial isolation z4. This indicates a relatively high survival rate of these cells in tissue culture. Thus, their existence in primary cell cultures from the cerebral cortex is not unexpected and has already been reported 1~. Our study confirms their existence and further stresses the necessity of a well controlled culture, if experimental results with these cultures are to be correctly interpreted.

244 It is known that brain injury will cause a massive invasion of reactive macrophages into the brain12,1s, 26. These cells may be derived from blood vessel walls, from the submeningeal or subendothelial layers of the cortex, or the blood itself. The macrophages in the cultures could originate from any of these sources, since no dissection technique would allow for removing all of the meninges or larger blood vessels. Also, since the brain was not perfused before sieving, monocytes from the blood might accidently contaminate the cultures. The phagocytotic activity of the cells becomes evident when the culture is observed in the microscope. The cell debris, evenly distributed throughout the petri dishes, was 'cleaned away' in the vicinity of these macrophages. These cells multiplied rapidly during the first week, probably at the time when the cultures are contaminated by cellular debris. It is likely that the tissue damage caused by the sieving of the brain might induce reactiveness of the macrophages. Large cells, often forming a monolayer upon which the other cells grew, did not accumulate [3H]GABA and did not react with either anti-S-100 or with anti-macrophage antiserum. These cells were called mesenchymal cells, since they had a low degree of morphological differentiation, as has also been proposed by others 9. The cells might, of course, also be some sort of precursor cells, as has also been suggested29,40. The present investigation was undertaken in order to better identify the cellular elements that exist in primary brain cultures. By using different techniques we have been able to assess that 60 % of the cells are astrocytes. This figure is somewhat lower than what has been advocated by others, although definite figures on cell frequencies are seldom expressed. The precise assessment of oligodendrocytes in the culture is pending until antiserum directed against basic-myelin-protein has been tried as a marker. ACKNOWLEDGEMENTS The technical assistance of Miss Ylva Larson is warmly appreciated. The antiserum against the S-100 protein was a gift from Dr. K. G. Haglid, GSteborg, Sweden, and the antiserum against the 14-3-2 protein was a gift from Dr. A. Grasso, Roma, Italy, to whom the authors are grateful. The study was supported by grants from the Medical Faculty of GSteborg, Statens Naturvetenskapliga Forskningsrfid, The Medical Research Council and from Tore Nilsson's Foundation for Medical Research.

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