Culture of purified rat astrocytes in serum-free medium supplemented with mitogen

Brain Research, 274 (1983) 79--86 Elsevier

79

Culture of Purified Rat Astrocytes in Serum-Free Medium Supplemented with Mitogen SEUNG U. KIM*, JANET STERN, MYONG W. KIM and DAVID E. PLEASURE

Department of Neurology, Universityof Pennsylvania and Division of Experimental Child Neurology, Children's Hospital of Philadelphia, Philadelphia, Pennsylvania19104 (U.S.A.) (Accepted January 18th, 1983)

Key words: astrocyte - - mitogen supplement - - cell culture

We have obtained a highly purified astrocyte population in cultures originating from neonatal (2-5 days) rat cerebrum by use of the selection process provided by a serum-free chemicallydefined medium (DM). The addition of a glial growth factor isolated from bovine pituitary glands to DM induced in these astrocyte cultures both a stimulation of astrocytic proliferation and a morphological transformation of the astroeytes from fiat fibroblastic form to multipolar stellate form. INTRODUCTION

MATERIALS AND METHODS

The isolation and culture of specific cell types from the nervous system have been the focus of several previous studies3,6A3,16-18,20-22,36,37,4°. There have also been attempts to develop serum-free synthetic media specifically designed for neurons and glia cells in which the cells could be grown in a completely defined microenvironment2,5,s,10A9,23,25,26,30,34,35,41. Such defined media have two potential advantages: (1) the effects on cellular function of hormones, substrates, cofactors, and mitogens can be studied without the variables introduced by unknown components of serum or embryo extract; and (2) serum-free media can be formulated to select one specific cell type out of the heterogeneous cell population of the nervous system. In the present communication, we describe longterm culture of purified rat astrocytes in a serumfree, defined medium (DM) and demonstrate that addition of a glial growth factor isolated from bovine pituitary glands (GGF-BP) 4 induces both astrocytic proliferation and a morphological transition of the astrocyte from flat cells with fine glial filaments to typical multipolar, stellate forms.

Cell culture Primary dissociated cell cultures of neonatal rat (2-5 days) cerebrum that also included corpus callosum were prepared by trypsinization as previously describedn. Single cells were plated onto 13 mm round glass coverslips (105 cells) that had been immersed in 10/,g/ml polylysine for 20 min, washed once in water, and then placed in 35 m m plastic dishes (4 coverslips/dish). The nutrient medium was Eagle's minimum essential medium (MEM) plus 10% fetal bovine serum, 5 mg/ml D-glucose, 50 units/ml penicillin and 50/~g/ml streptomycin. After 24 h, coverslips were washed twice with H a m ' s F12 medium and fed with serum-free defined medium consisting of Ham's F12 synthetic medium containing 2 ml/100 ml MEM essential amino acid solution (50x), 1 ml/100 ml MEM vitamin solution (100x), 10/~g/ml bovine insulin (Iletin, Eli Lily), 10 /~g/ml human transferrin (Sigma), 100 nM hydrocortisone (Sigma), 0.01 nM triidothyronine (Sigma), 30 nM sodium selenite (Collaborative Research), 50 units/ml penicillin, and 50/~g/ml streptomycin. Other cultures were fed by DM supplemented with 100/~g/ml crude GGF-BP prepared by ammonium sulfate fractionation of a bo-

* To whom correspondence should be addressed at: Division of Neurology, Acute Care Hospital, University of British Columbia, Vancouver, B.C. V6TlW5, Canada.

0006-8993/83/$03.00© 1983 Elsevier Science Publishers B.V.

80 vine pituitary extract, 5 ~g/ml of GGF-BP partially purified by carboxymethyl-cellulose column chromatography, or 1 btg/ml of GGF-BP further purified by phosphocellulose column chromatography4. Preliminary studies had demonstrated that these concentrations of GGF-BP produced maximal stimulation of astroglial [3H]thymidine incorporation. The cultures were incubated at 37 °C in a humidified atmosphere of 95% air and 5% CO2. The medium was renewed completely every 3--4 days.

lmmunofluorescence Cells growing on 13 mm glass coverslips were identified by indirect immunofluorescence using cell-type specific antibodies as described by previous authors7, 28. Rabbit antiserum to glial fibrillary acidic protein (GFAP), a specific marker for astrocytes 1 was supplied by Dr. L. Eng. Rabbit antiserum to neurofilaments, a specific marker for neurons 32 was provided by Dr. W. Schlaepfer. Rabbit antiserum to galactocerebroside, a specific surface marker for oligodendrocytes16.29, was supplied by Dr. D. Silberberg. Rabbit antiserum to fibronectin, a marker for fibroblasts 38, and rhodamine coupled goat anti-rabbit immunoglobulin were purchased from Cappel Laboratories. The cell surface antigens, galactocerebroside and fibronectin, were demonstrated by first incubating living cells in antiserum for 20 min at room temperature followed by rhodamine-coupled goat anti-rabbit immunoglobulin (used 1:40) for 20 min at room temperature. After 3 changes of washing in F12 medium buffered with 10 mM Hepes (pH 7.4), the cells were fixed in 5% acetic acid in 95% ethanol at --20 °C for 10 min, washed and mounted in glycerolPBS (phosphate buffered saline) (140 mM NaCI, 5 mM phosphate, pH 7.4). For demonstrating intracytoplasmic localization of GFAP and neurofilaments, cells were fixed in cold acetone (--20 °C) for 2 min before being incubated in antiserum and rhodamine coupled goat anti-rabbit immunoglobulin (as above). The cells were examined on a Zeiss Universal fluorescence microscope equipped with phase contrast, fluorescein and rhodamine optics and epi-illumination.

[3H] Thymidine incorporation and autoradiography Coverslips carrying cells were treated for 24 h with [3H]thymidine (1/zCi/ml, 16 Ci/mmol, New England

Nuclear). The coverslips were washed twice in PBS and the cells stained by indirect immunofluorescence prior to mounting on glass slides with permount. The slides were dipped in Kodak NTB-2 emulsion, dried, and stored in the dark at 4 °C for 5 days. The slides were then developed in Kodak D19 developer for 5 min at 20 °C and fixed in Kodak rapid fixer for 5 min. After the slides were washed and dried, a second coverslip (22 mm diameter) was placed over the emulsion-covered cells using a drop of glycerol-PBS, and sealed with nail polish. Cells were examined by phase contrast and fluorescence microscopy. Cells having more than 15 silver grains over the nucleus were scored as dividing cells. Primary dissociated cell cultures of 2-5-day-old rat cerebrum were grown in 24 well-uncoated Linbro plates. After 7-10 days in DM or DM plus pituitary extract, [3H]thymidine (1/zCi/ml, 16 Ci/mmol) was included in the medium for 24 h. Then the medium was removed and the cells were incubated with cold 10% (w/v) TCA for 15 min on ice. The 10% TCA was removed, and the wells were washed once with icecold 5% TCA, washed twice with cold phosphatebuffered saline (PBS) and air-dried. The contents of the wells were then solubilized in 1% (w/v) SDS in 0.3 N NaOH 9, and this solution was added to Aquasol (New England Nuclear) for liquid scintillation spectrometry. RESULTS Two distinct cell types were recognized among the cells grown for 2-3 days in serum-free chemically defined medium (DM) under the phase optics: one was small, phase-bright, process-bearing with short, thin processes, and the other was flat and fibroblast-like. At 6 days in vitro and onward, the majority of cells in the DM were flat and fibroblast-like. Almost all of these cells were positively identified as astrocytes by immunolabeling with anti-glial fibrous acidic protein (GFAP) serum, a cell-type specific marker for the astrocytesl,28 (Fig. t). A small number of process-bearing cells which were also GFAP ÷ were found among the 'flat' astrocytes (Fig. 2). These cells amounted to less than 10% of the total astrocyte population. Their small size and scanty GFAP ÷ cytoplasm suggest that these cells are immature astrocytes emerging from the mitotic cycle. The selection of astrocytes by the

81 TABLE I Indirect immunofluorescence labeling of cultured astrocytes with various antisera Antiserum

Cell type

Dilution

% Cells labeled

Rabbit anti-GFAP Rabbit anti-galactocerebroside Rabbit anti-fibronectin Rabbit anti-neurofilament

astrocytes oligodendrocytes fibroblasts

1:40

>95

1:40 1:40

<2 <1

neurons

1:40

0

were intensely fluorescent with a distinctive intracellular, fibrillar pattern typical of cultured astrocytes (Fig. 1). At the higher magnification, fine individual G F A P + fibrils could be visualized to extend into the periphery of the cell contour (Fig. 1). To establish whether astrocytes grown in the D M

Fig. 1. Staining of purified astrocytes grown in serum-free defined medium for 10 days by rabbit anti-glial fibrons acidic protein (GFAP). All cells in the field were GFAP + astrocytes, and were flat and fibroblastic in morphology. A: phase contrast microscopy, x400. B: GFAP-rhodamine immunofluorescence. x400.

D M was achieved during the first few days in vitro and neurons and fibroblasts died and disappeared during this period. Oligodendrocytes, on the other hand, did survive in the DM, though in far smaller number than the astrocytes. At 6 days, 95% of cells in culture were astrocytes as they were G F A P ÷ by indirect immunofluorescence (Table I). NF (neurofilament) ÷ neurons were absent and FN (fibronectin)+ fibroblasts and GC (galactocerebroside) ÷ oligodendrocytes amounted to less than 5% of all cell populations grown in the DM. In better preparations in which there were fewer G F A P - - cells which were either FN ÷ fibroblasts or G C ÷ oligodendrocytes, 98--99% of cells were identified as astrocytes. At 14 days in vitro and beyond, the cultures were generally 95-99% G F A P ÷ astrocytes. These cells were mostly flat and fibroblastic in morphology, yet

Fig. 2. [3H]thymidine incorporation by astrocytes grown in serum-free defined medium for 7 days. After 6 days in defined medium, cells were treated with [3H]thymidine for 24 h, washed in the medium, stained with rabbit anti-glial fibrous acidic protein (GFAP) followed by rhodamine conjugated goat antirabbit immunoglobulins, and then processed for autoradiography. Both fiat, GFAP + cells and process-bearing, small GFAP + cells were labeled by [3H]thymidine autoradiography. A: phase contrast microscopy, x400. B: GFAP-rhodamine immunofluorescence, x400.

82 were proliferating, the rate of [3H]thymidine incorporation into astrocytes was analyzed by autoradiography. It was found that 10-12% of GFAP ÷ astrocytes incorporated the isotope as shown by silver grains localized on nuclei (Fig. 2). It has been demonstrated that astrocytes could be stimulated to divide by a factor present in extract of bovine pituitary glands4, 27, by fibroblast growth factor (FGF) 27, by epidermal growth factor (EGF)14, 39, by platelet-derived growth factor (PDGR)9, or by myelin basic protein 33. Recently, Pruss et al. have examined various mitogens for cultured astrocytes and shown that the 'glial growth factor' extracted from bovine pituitary (GGF-BP) is the most effective mitogen for the cultured astrocytes 27. In order to generate a large yield of astrocytes using the purified population of astrocytes selected in the DM, we decided

Fig. 4. [3H]thymidine incorporation by astrocytes grown in serum-free defined medium plus glial growth factor isolated from bovine pituitary (GGF-BP) for 7 days. After 6 days in defined medium plus GGF-BP, cells were treated with [3H]thymidine for 24 h, washed in the medium, stained with rabbit anti-glial fibrous acidic protein (GFAP) followed by rhodamine conjugated goat anti-rabbit immunoglobulins, and then processed for autoradiography. Most of GFAP ÷ cells were labeled by [3H]thymidine autoradiography. A: phase contrast microscopy. x400. B: GFAP-rhodamine immunofluorescence, x400.

Fig. 3. Purified astrocytes grown in serum-free defined medium supplemented with glial growth factor isolated from bovine pituitary for 10 days were stained with rabbit anti-glial fibrous acidic protein (GFAP). All of the GFAP + cells had the multipolar steUate form typical of mature, well-differentiated astrocytes. A: phase contrast microscopy, x400. B: GFAP-rhodamine immunofluorescence, x400.

to supplement the DM with GGF-BP. When crude GGF-BP (100~g/ml) was added to the DM from the first day (second day in vitro), there was a remarkable increase in the number of cells, so that by 6 days in vitro, GFAP ÷ astrocytes virtually covered all the surface of the coverslips (Fig. 3). The pituitary extract also imposed on astrocytes certain morphological changes, namely transformation of 'flat' astrocytes into a typical multipolar astrocytic morphology (Fig. 3). In contrast to rapid (less than 2 h) conversion of flat to process-bearing cells by simultaneous serum withdrawal and addition of dibutyryl cyclic AMP noted by Manthorpe et a1.18 serial phase contrast observations of astrocytes grown in our DM indicated the morphological conversion by

83 GGF-BP is considerably slower, not reaching completion for at least 24 h. In order to establish a stimulating effect of G G F - B P on astrocytic proliferation, we combined immunofluorescence with autoradiography as described previously. The results showed that 42-55% of the G F A P + astrocytes labeled with [3H]thymidine (Fig. 4), while in astrocytes grown in the DM only, 10-12% of the G F A P ÷ cells were labeled during a single 24 h pulse. Identical astrocytic proliferation and morphological transformation were observed with 5/zg/ml of GGF-BP purified by carboxymethyl cellulose column chromatography or with 1/~g/ml of GGF-BP further purified by phosphocellulose column chromatography 4. In parallel experiments using liquid scintillation spectrometry to measure incorporation of [3H]thymidine into cellular DNA, these concentrations of GGF-BP produced a five-to-eight-fold enhancement (n = 10) in radiolabeling over control wells cultured without GGF-BP. In control cultures grown in serum-supplemented medium (10% fetal bovine serum), fibroblasts proliferated extensively and formed a dense layer covering all the available coverslip surface by 7 days in vitro, and neurons, astrocytes and oligodendrocytes

were found anchoring on the fibroblast monolayer. When these cultures were processed for G F A P immunofluorescence, the astrocytes appeared as intensely fluorescent multipolar cells with G F A P + fibrils extending into the processes (Fig. 5). DISCUSSION By the use of the selection process provided by the serum-free chemically defined medium (DM), we were able to obtain a highly purified astrocyte population in cultures from newborn rat cerebrum. Similar experiments using newborn mouse cerebrum also produced astrocyte-enriched cultures indicating that the selection regimen described could be used for species other than the rat as well. By addition of GGF-BP to the DM, we were able to obtain large numbers of astrocytes suitable for biochemical studies. The identification and the purity of the astrocyte population in the DM was ascertained by G F A P immunohistochemistry. It was demonstrated that 95-99% of the cells grown in DM or in DM plus pituitary extract were G F A P + astrocytes. The rest of the cell population were either FN + fibroblasts or GC ÷ oligodendrocytes. In the present study, it was demonstrated that 10-12% of GFAP ÷ astrocytes in the DM incorporated [3H]thymidine with a single 24 h pulse, while nearly 50% of the GFAP ÷ astrocytes were labeled when the DM was supplemented with GGF-BP (Table II). This confirms a recent report by Brockes et al. 4 that GGF-BP stimulated astrocytes to incorporate [t25I]iododeoxyuridine 5-fold over the control TABLE II [3H]Thymidine uptake by GFAP ÷ astrocytes studied by autoradiography Medium

No. o f No. o f GFAP ÷ cells GFAP ÷ cells with labeled nucleus

(%)

DM

409 417 373

10 11 12

Fig. 5. Staining of astrocytes grown in medium containing 10%

fetal bovine serum for 10 days by rabbit anti-glial fibrous acidic protein. Astrocytes were found on the top of fibroblast monolayer. The GFAP + cells had multipolar stellate form typical of mature astrocytes. A: phase contrast microscopy. ×400. B: GFAP-rhodamine immunofluorescence, x400.

DM + pituitary extract

481 416 448

41 46 46 203 215 246

42 52 55

84 value. Moreover, we found that the presence of serum is not required for the stimulatory effect of GGFBP on astroglial proliferation. There have been several reports of serum-free media designed for neural tissue culture; most of these support the growth of mixed cell populations derived from nervous tissue 5,10,23,26,30,34,41. Morrison and De Vellis z5 have reported that astrocyte-enriched cultures can be obtained from newborn rat brain by use of a serum-free medium (Table III). Purity, as judged by GFAP immunofluorescence, was similar to that we obtained. Their procedure requires longer total time in vitro, a shaking step to remove oligodendrocytes, and subculture of astrocytes employing a trypsinization-harvesting step, and hence is somewhat less straightforward than our method. They demonstrated that FGF and prostaglandine F2a were effective astrocytic mitogen in serum-free medium. It is noteworthy that they described a morphological transformation of G F A P ÷ cells from fiat or polygonal to branched forms, using serum-free medium containing FGF and prostaglandine F2a, similar to that which we observed with GGF-BP. We did not investigate the effects of these mitogens in this study. In addition to the mitogens listed above, myelin basic protein (BP) has been reported to be an astrocytic mitogen33. Although we did not investigate the stimulative effect of BP in our astrocyte cultures, a recent TABLE

study by Pruss et a l p indicated that BP was not effective as FGF or pituitary extract as a mitogen for the astrocytes. A recent study by Fischer et al. also reported the formulation of a defined medium (Table III) for astrocytes derived from newborn mouse cerebellum s. These authors observed that 'immature astrocytes' generated in their cultures were almost completely GFAP-negative, although these cells became GFAPpositive when transferred to serum-supplemented medium. Results from our study indicate that the astrocytes grown in our defined medium express GFAP in their cytoplasm as early as 2 days in culture, the earliest time examined for G F A P immunoreaction. It is possible that a factor or factors present in the medium of Fischer et al. adversely prevent the expression of GFAP in their 'immature astrocytes'. It is interesting to note that the 'fiat' G F A P + astrocytes grown in the DM change their morphology into a typical multipolar, stellate form, when exposed to GGF-BP. A similar morphological change of astrocytes has been reported by previous authors when 'flat' astrocytes grown in serum-supplemented medium are subjected to either dibutyryl cyclic AMP alonO 2 or to a combination of serum withdrawal and the addition of dibutyryl cyclic AMP 15,18,24,31 or are cultured in serum with a crude brain extract 15. The significance of these astrocytic morphological

III

Formulation of serum-free defined media for astrocytes Medium

Hormones and growth factors Glial growth factor bovine pituitary Insulin Epidermal growth factor Fibroblast growth factor P r o s t a g l a n d i n F2. Triiodothyronine Hydrocortisone Other factors Transferrin Selenic a c i d Putrescine Hyaluronic acid Aprotinin

Kim et al. (rat and mouse)

Morrison and DeVellis 25 (rat)

Fischer et al. ~ (mouse)

Ham's F-12

Ham's F-12 + DulbeccoVogt Eagle's modification

BME-Earle's + 2 m M glutamine

See text 10 ~ug/ml

-50 ~ug/ml --

-10 ~ g / m l 3 nM

--0.01 n M 100 n M

100 n g / m l 500 n g / m l -50 n M

-----

10/~g/ml 30 n M ----

--100 n M ---

lOOpg/ml 30 n M lOpg/ml 1ug/ml

85 changes is presently u n k n o w n .

ACKNOWLEDGEMENTS

In the future, we plan to investigate the metabolic Supported by grants from the National Multiple

alterations induced in these DM-cultured astrocytes by G G F - B P and other mitogens, and will use the cells, as well, for biochemical studies of astrocytic

Sclerosis Society, Muscular Dystrophy Association, and U S P H S (NS-10648, NS-15205, NS-17752, H D -

function.

08536, NS-11037, and NS-08075).

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