- Email: [email protected]
Purification of astroglial-cell cultures from rat spinal cord: the use of d -valine to inhibit fibroblast growth
Neurochem. Int. Vol. 15, No. 3, pp. 365 369, 1989 Printed in Great Britain. All rights reserved
019%0186/89 $3.00 + 0.00 Copyright ~ 1989 Pergamon Press plc
P U R I F I C A T I O N OF A S T R O G L I A L - C E L L C U L T U R E S F R O M RAT SPINAL CORD: THE USE OF D-VALINE TO INHIBIT FIBROBLAST G R O W T H A. J. CHOLEWINSKI,J. C. REID,A. M. MCDERMOTT and G. P. WILKIN Biochemistry Department, Imperial College, London SW7 2AZ, England (Received 21 February 1989; accepted 19 May 1989) Abstract--Primary astrocyte cultures from spinal cord were purified from contaminating fibroblasts by growth in Dulbecco's modified Eagle's medium (DMEM) in which L-valine was substituted by D-valine. This medium was not supportive for growth of fibroblasts and inhibited their proliferation. The culture purity was assessed using immunofluorescence labelling with specific antibodies to various cell markers. Cultures contained predominantly astrocytes with greater than 92% of this cell type in D-valine medium as opposed to approx. 70% in D,L-valine DMEM medium. This procedure enables primary cultures to be obtained with a larger percentage of astrocytes by a simple modification to the growth medium.
The predominant class of cells which make up the C N S are the astroglia, but, due to the cellular heterogeneity, difficulties exist in obtaining a homogeneous astroglial (or other cell type) population from the CNS. Using primary cell culture techniques various investigators have been able to obtain highly enriched populations of neurones, oligodendrocytes or astrocytes (Booher and Sensenbrenner, 1972; Chapman and Rumsby, 1982; Juurlink and Devon, 1987; Mains and Patterson, 1973; McCarthy and de Vellis, 1980; Suzumura et al., 1984). In primary cultures, obtained from rat or mouse brain, relatively pure astrocyte cultures can be obtained from the various regions such as cortex or cerebellum, with greater than 90% G F A P positive cells (McCarthy and de Vellis, 1980; Pearce et al., 1985; Raizada et aL, 1987). This in part is aided by the relative ease of dissection in obtaining the various regions free of meninges which are a major source of contaminating fibroblasts. In some of our studies we have examined astrocyte regional heterogeneity in the C N S and included cultures from spinal cord (Cholewinski et al., 1988; Cholewinski and Wilkin, 1988). Here we report on a relatively simple method whereby spinal cord culture purity (with respect to astrocytes) can be increased from approx. 70% to over 92% by following a procedure modified from Address correspondence and reprint requests to: Dr A. J. Cholewinski, Department of Biochemistry, Imperial College, London SW7 2AZ, England. 365
that first described by Estin and Vernadakis (1986) and substituting D-valine for L-valine in normal D M E M . Part of this modification is the use of a medium which inhibits fibroblast growth as these cells lack the enzyme o-amino acid oxidase and are thus unable to convert and utilize o-valine for growth (Gilbert and Migeon, 1975). EXPERIMENTAL PROCEDURES
Materials Dulbecco's modified Eagle's medium (DMEM) and foetal calf serum (FCS) were purchased from Imperial Laboratories, England, while gentamycin and 24 well multiwell plates were obtained from Flow Laboratories, England. The following were purchased from Sigma Chemical Co., England; bovine pancreatic DNase-I; bovine pancreatic trypsin type III; bovine serum albumin (BSA); poly-L-lysine (MW > 300,000); and L-valine. All other chemicals were of analytical grade from BDH, England. Anti-GFAP antibody was obtained from DAKO and anti-Thy 1.1 from Serotec. Anti-neurofilament (NF) antibody was a gift from Dr J. Wood, Sandoz, England and anti-galactocerebroside (GC) was a gift from Dr B. Ranscht, La Jolla, Calif., U.S.A. All remaining antibodies and conjugates were purchased from either Cappel, Nordic or Serotec. The rats were of the Sprague-Dawley strain and obtained from the animal unit at Imperial College. Culture procedure Glial cell cultures were prepared from 2-day-old rat pup spinal cords. This age of animal was found to give the least amount of cell debris upon dissociation with very few or no viable neurones present. The spinal cords were dissected out and the meninges removed, where possible. The tissue was then chopped into 4 mm 2 blocks and incubated in Earle's
366
A.J. CHOLEWINSKIet al.
balanced salts containing; 0.3% (BSA), 0.025% bovine pancreatic trypsin and 40/~ g/ml bovine pancreatic DNase-I for 15 minutes at 3T'C in a shaking waterbath. Trypsinization was terminated by the addition of at least an equal volume of DMEM containing 10% FCS and 40#g/ml gentamycin. After spinning down the suspension, the pellet was resuspended in Earle's balanced salts solution without trypsin and triturated 10-15 times using a Pasteur pipette. The suspended cells were collected and the resultant clumps further triturated 5 l0 times. The suspended cells were then pooled together and once again spun down and resuspended in a known volume of DMEM culture medium. The cell number was adjusted to give a plating density of l05 cells/ cm 2. The cells were then plated onto 13 mm diameter glass coverslips pre-coated with 5 #g/ml poly-L-lysine. Cultures were maintained at 37'C in a water-saturated atmosphere of 5% CO 2 in air. Culture media
Two types of culture media were used; DMEM with or without L-valine. Essentially the DMEM was identical to a commercial preparation except for the essential amino acid valine. Instead of L-valine, the isomer D-valine was included at twice the normal L-valine concentration. This D-valinecontaining DMEM medium was made to order by Imperial Laboratories, England. To obtain "normal" DMEM, Lvaline was added to the D-valine medium at the concentration usually present in commercial DMEM (93.6 mg/1). The resultant O,L-valine DMEM behaved as commercial DMEM with respect to both astrocyte and fibroblast growth (Estin and Vernadakis, 1986). Before being added to o-valine DMEM the foetal calf serum was first dialysed and then heat inactivated. This procedure was carried out in order to remove any L-valine from the serum (Estin and Vernadakis, 1986). The dialysis tubing was first activated by boiling in sodium carbonate (2%), EDTA (0.5%) and distilled water (four changes). The serum was then dialysed for 48 h against three changes of normal saline (1:50 serum:saline) and then filter sterilized and heat-inactivated at 56'C for 1 h. Thus the D-valine medium contained dialysed serum while the D,L-valine medium contained untreated serum. In experiments comparing glial cell growth in D,L-valine medium containing either untreated serum or dialysed serum, no differences in cell numbers were observed (data not shown).
few cells survived, although none were Thy 1.1 positive. The growth rate and development o f the astrocytes appeared stunted when c o m p a r e d to cells grown in O,L-valine m e d i u m (Estin and Vernadakis, 1986). Subsequently the cells were plated in D,L-valine medium and grown for up to 5 days before switching to D-valine medium. This enabled the cells to divide and better survive the change to D-valine medium and still reduce the numbers o f fibroblasts in confluent 12 day old cultures In all subsequent experiments cells were grown in D,L-valine D M E M for 5 days after which time the medium in half o f the wells was changed to o-valine D M E M (with dialysed FCS at 10%) with further medium changes at 8 and 10 days in culture. Cells were taken for immunoftuorescence staining at 6, 8, 10 and 12 days in culture and culture purity assessed. Table 1 shows the percentage o f labelled cells under the two culture conditions with respect to astrocytes and fibroblasts. Initially at 6 days in culture there was little difference in fibroblast numbers in both cultures although fewer fibroblasts appeared to be present in D-valine D M E M . With time in culture fibroblast n u m b e r s decreased in D-valine D M E M and by 12 days in culture they n u m b e r e d below 3% o f total cells. In O,L-valine D M E M fibroblast numbers remained high at approx. 20% o f the total cell number. We also found that oligodendrocytes accounted for less than 5% o f the total cell n u m b e r at 12 days in culture in both o-valine D M E M and O,L-valine D M E M , as stained with antibodies against galactocerebroside (data not shown). N o neurones could be detected at 12 days in culture based on their characteristic m o r p h o l o g y or when staining was carried out with anti-neurofilament antibodies. Staining with normal rabbit serum and ingestion o f 1.0 # m latex beads labelled < 2.3 and < 5%, respectively, o f cells
lrnmunofluorescence
The different cell types present in spinal cord cultures were identified with light microscopic immunofluorescence (Reichart, Polyvar) using the following antibodies; anti-glial fibrillary acidic protein (GFAP)-astrocytes (Bignami and Dahl, 1974), anti-Thy l.l-fibroblasts (Stern, 1973; Raft et al., 1979), anti-galactocerebroside (GC)-oligodendrocytes (Ranscht et al., 1982), and anti-neurofilament (NF)neurones (Anderton et al., 1980). Neurons could also be identified by their morphology. Normal rabbit serum followed by rhodamine-conjugated anti-rabbit antibody was used to label macrophages together with ingestion of 1.0/~ m latex beads (Raft et al., 1979). RESULTS Initial studies showed that when cells were grown in o-valine m e d i u m immediately after plating, very
Table 1. Percentageof cells labelled with antibodies against GFAP and Thy 1.1 under two different media conditions D,L-Valine DMEM Days in culture 6 8 10 12
GFAP 69.0+3.7 71.0 + 2.7 71.3_+3.1 73.4_+3.6
Thy 1.1 22.8+3.3 22.8 + 3.4 25.3+2.6 20.7_+2.2
D-Valine DMEM GFAP
Thy 1.1
77.9+ 1.2 14.1 + 1.9 86.7 + 0.9 5.7 + 1.6 91.2_+0.6 3.4_+0.3 92.1 + 1.0 2.7_+0.6
Cells obtained from 2-day-oldrat spinal cords and grown in culture for 5 days. The medium was then changed in half of the wells to D-valinecontaining DMEM and again at 8 and 10 days. Cells stained with antibodies to GFAP and Thy 1.1 at 6, 8, 10 and 12 days in culture. Results are expressedas the mean + SEM of 4 separate experimentsfor days 6, 8 and 10 and as mean + SEM of 3 separate experiments for day 12. In each experiment l0 random fields were counted on duplicate coverslips.
B
A
C
D
Fig. 1, Immunofluorescent staining of 12 day old spinal cord cultures grown in either O,L-valine DMEM (A) and (B) or D-valine DMEM (C) and (D). Cells were stained with antibodies against GFAP (A) and (C) and antibodies against Thy 1.1 (B) and (D). Bar = 35/~m. 367
368
A.J. CHOLEWINSKIet al.
believed to be macrophages (Raft et al., 1979) (data not shown).
DISCUSSION The purification step described in this paper utilizes a modification of the D M E M growth medium with a substitution of D-valine (at twice the L-valine concentration) for L-valine. Using this procedure spinal cord cultures can be enriched in astrocytes with greater than 92% purity and contaminating fibroblasts reduced to below 3%. Retardation offibroblast growth appears to be based on their inability to convert D-valine to the L-isomer as they lack the enzyme D-amino acid oxidase (Gilbert and Migeon, 1975). This procedure is modified from that first described by Estin and Vernadakis (1986) in that the cells are changed to D-valine containing D M E M medium following 5 days in culture in D,L-valine D M E M . It is important that freshly dissociated cells are grown in a normal medium for the first few days in culture in order to both rapidly increase their numbers, and to prevent the marked degree of stunted growth observed when astrocytes were plated directly after dissociation in D-valine D M E M (Estin and Vernadakis, 1986). The very small percentage of other contaminating cells (apart from fibroblasts) following this procedure were either oligodendrocytes ( < 5%) and their precursors (Levi et al., 1987; Curtis et al., 1988), or macrophages ( < 5%) and or microglia. Should absolutely pure astrocyte cultures be required then oligodendrocytes can be removed by orbital shaking (McCarthy and de Vellis, 1980). Macrophages and or microglia can be removed by passage as they do not proliferate (DuBois et al., 1986) or selective chemical destruction using L-leucine methyl ester (Giulian et al., 1988). We have now adopted the procedure described here for obtaining fibroblast-free cultures from other C N S regions.
REFERENCES
Anderton B. H., Thorpe R., Cohen J., Selvendron S. and Woodhams S. P. (1980) Specific neuronal localisation by immunofluorescene of l0 nm filament polypeptide. J. Neurocytol. 9, 835-844. Bignami A. and Dahl D. (1974) Astrocyte-specific protein and neuroglial differentiation. An immunofluorescence study with antibodies to the glial fibrillary acidic protein. J. comp. Neurol. 153, 27-37. Booher J. and Sensenbrenner M. (1972) Growth and cultivation of dissociated neurones and glial cells from embry-
onic chick, rat and human brain in flask cultures. Neurobiology 2, 97 105.
Chapman J. and Rumsby M. (1982) A syringe modification for the simple and rapid dissociation of post-natal rat cerebral tissue for preparing primary cultures of mixed glial cells. Neurosci. Lett. 34, 307 313. Cholewinski A. J. and Wilkin G. P. (1988) Astrocytes from forebrain, cerebellum and spinal cord differ in their responses to vasoactive intestinal peptide. J. Neurochem. 51, 16261633. Cholewinski A. J., Hanley M. R. and Wilkin G. P. (1988) A phosphoinositide-linked peptide response in astrocytes: evidence for regional heterogeneity. Neurochem. Res. 13, 389-394. Curtis R., Cohen J., Fok-Seang J., Hanley M. R., Gregson N. A., Reynolds R. and Wilkin G. P. (1988) Development of macroglial cells in rat cerebellum. I. Use of antibodies to follow early in vivo development and migration of oligodendrocytes. J. Neuroeytol. 17, 43-54. DuBois J. H., Bolton C. and Cuzner M. L. (1986) The production of prostaglandin and the regulation of cell division in neonate rat primary mixed glial cultures. J. Neuroimmun. 11, 277 285. Estin C. and Vernadakis A. (1986) Primary glial cells and brain fibroblasts Interactions in culture. Brain Res. Bull. 16, 723 731. Gilbert S. F. and Migeon B. R. (1975) D-Valine as a selective agent for normal human and rodent epithelial cells in culture. Cell5, 11 17. Giulian D., Young D. G., Woodward J., Brown D. C. and Lachman L. B. (1988) Interleukin-I is an astroglial growth factor in the developing brain. J. Neurosci. 8, 709-714. Juurlink B. H. J. and Devon R. M. (1987) Procedure for establishing oligodendroglial cells in primary cultures based on developmental parameters. Int. J. dev. Neurosci. 5, 327 336. Levi G., Aloisi F. and Wilkin G. P. (1987) Differentiation of cerebellar bipotential glial precursors into oligodendrocytes in primary culture. Development profile of surface antigens and mitotic activity. J. Neurosci. Res. 18, 407~J, 17. Mains R. E. and Patterson P. H. (1973) Primary cultures of dissociated sympathetic neurones. I. Establishment of long-term growth in culture and studies of differentiated properties. J. Cell Biol. 59, 329-345. McCarthy K. D. and de Vellis J. (1980) Preparation of astroglial and oligodendroglial cell cultures from rat cerebral tissue. J. Cell Biol. 85, 890 902. Pearce B., Cambray-Deakin M. A., Morrow C., Grimble J. and Murphy S. (1985) Activation of muscarinic and of ~-adrenergic receptors on astrocytes results in the accumulation of inositol phosphates. J. Neurochem. 45, 1534-1540. Raft M. C., Fields K. L., Hakomori S. I., Mirsky R., Pruss R. M. and Winter J. (1979) Cell-type-specific markers for distinguishing and studying neurones and the major classes of glial cells in culture. Brain Res. 174, 283 308. Raizada M. K., Phillips M. I., Crews F. T. and Sumners C. (1987) Distinct angiotensin II receptor in primary cultures ofglial cells from rat brain. Proc. natn. Acad. Sci. U.S.A. 84, 4655~4659. Ranscht B., Clapshaw P. A., Price J., Noble M. and Seifert
Purification of astroglial-cell cultures from rat spinal cord W. (1982) Development of oligodendrocytes and Schwann cells studied with a monoclonal antibody against galactocerebroside. Proc. natn. Acad. Sci. U.S.A. 79, 2709 2713. Stern P. L. (1973) 0 Alloantigen on mouse and rat fibroblasts. Nature New Biol. 246, 76-78.
369
Suzumura A., Bhat S., Eccleston A., Lisak R. P. and Silberberg D. H. (1984) The isolation and long-term culture of oligodendrocytes from newborn mouse brain. Brain Res. 324, 379 383.
019%0186/89 $3.00 + 0.00 Copyright ~ 1989 Pergamon Press plc
P U R I F I C A T I O N OF A S T R O G L I A L - C E L L C U L T U R E S F R O M RAT SPINAL CORD: THE USE OF D-VALINE TO INHIBIT FIBROBLAST G R O W T H A. J. CHOLEWINSKI,J. C. REID,A. M. MCDERMOTT and G. P. WILKIN Biochemistry Department, Imperial College, London SW7 2AZ, England (Received 21 February 1989; accepted 19 May 1989) Abstract--Primary astrocyte cultures from spinal cord were purified from contaminating fibroblasts by growth in Dulbecco's modified Eagle's medium (DMEM) in which L-valine was substituted by D-valine. This medium was not supportive for growth of fibroblasts and inhibited their proliferation. The culture purity was assessed using immunofluorescence labelling with specific antibodies to various cell markers. Cultures contained predominantly astrocytes with greater than 92% of this cell type in D-valine medium as opposed to approx. 70% in D,L-valine DMEM medium. This procedure enables primary cultures to be obtained with a larger percentage of astrocytes by a simple modification to the growth medium.
The predominant class of cells which make up the C N S are the astroglia, but, due to the cellular heterogeneity, difficulties exist in obtaining a homogeneous astroglial (or other cell type) population from the CNS. Using primary cell culture techniques various investigators have been able to obtain highly enriched populations of neurones, oligodendrocytes or astrocytes (Booher and Sensenbrenner, 1972; Chapman and Rumsby, 1982; Juurlink and Devon, 1987; Mains and Patterson, 1973; McCarthy and de Vellis, 1980; Suzumura et al., 1984). In primary cultures, obtained from rat or mouse brain, relatively pure astrocyte cultures can be obtained from the various regions such as cortex or cerebellum, with greater than 90% G F A P positive cells (McCarthy and de Vellis, 1980; Pearce et al., 1985; Raizada et aL, 1987). This in part is aided by the relative ease of dissection in obtaining the various regions free of meninges which are a major source of contaminating fibroblasts. In some of our studies we have examined astrocyte regional heterogeneity in the C N S and included cultures from spinal cord (Cholewinski et al., 1988; Cholewinski and Wilkin, 1988). Here we report on a relatively simple method whereby spinal cord culture purity (with respect to astrocytes) can be increased from approx. 70% to over 92% by following a procedure modified from Address correspondence and reprint requests to: Dr A. J. Cholewinski, Department of Biochemistry, Imperial College, London SW7 2AZ, England. 365
that first described by Estin and Vernadakis (1986) and substituting D-valine for L-valine in normal D M E M . Part of this modification is the use of a medium which inhibits fibroblast growth as these cells lack the enzyme o-amino acid oxidase and are thus unable to convert and utilize o-valine for growth (Gilbert and Migeon, 1975). EXPERIMENTAL PROCEDURES
Materials Dulbecco's modified Eagle's medium (DMEM) and foetal calf serum (FCS) were purchased from Imperial Laboratories, England, while gentamycin and 24 well multiwell plates were obtained from Flow Laboratories, England. The following were purchased from Sigma Chemical Co., England; bovine pancreatic DNase-I; bovine pancreatic trypsin type III; bovine serum albumin (BSA); poly-L-lysine (MW > 300,000); and L-valine. All other chemicals were of analytical grade from BDH, England. Anti-GFAP antibody was obtained from DAKO and anti-Thy 1.1 from Serotec. Anti-neurofilament (NF) antibody was a gift from Dr J. Wood, Sandoz, England and anti-galactocerebroside (GC) was a gift from Dr B. Ranscht, La Jolla, Calif., U.S.A. All remaining antibodies and conjugates were purchased from either Cappel, Nordic or Serotec. The rats were of the Sprague-Dawley strain and obtained from the animal unit at Imperial College. Culture procedure Glial cell cultures were prepared from 2-day-old rat pup spinal cords. This age of animal was found to give the least amount of cell debris upon dissociation with very few or no viable neurones present. The spinal cords were dissected out and the meninges removed, where possible. The tissue was then chopped into 4 mm 2 blocks and incubated in Earle's
366
A.J. CHOLEWINSKIet al.
balanced salts containing; 0.3% (BSA), 0.025% bovine pancreatic trypsin and 40/~ g/ml bovine pancreatic DNase-I for 15 minutes at 3T'C in a shaking waterbath. Trypsinization was terminated by the addition of at least an equal volume of DMEM containing 10% FCS and 40#g/ml gentamycin. After spinning down the suspension, the pellet was resuspended in Earle's balanced salts solution without trypsin and triturated 10-15 times using a Pasteur pipette. The suspended cells were collected and the resultant clumps further triturated 5 l0 times. The suspended cells were then pooled together and once again spun down and resuspended in a known volume of DMEM culture medium. The cell number was adjusted to give a plating density of l05 cells/ cm 2. The cells were then plated onto 13 mm diameter glass coverslips pre-coated with 5 #g/ml poly-L-lysine. Cultures were maintained at 37'C in a water-saturated atmosphere of 5% CO 2 in air. Culture media
Two types of culture media were used; DMEM with or without L-valine. Essentially the DMEM was identical to a commercial preparation except for the essential amino acid valine. Instead of L-valine, the isomer D-valine was included at twice the normal L-valine concentration. This D-valinecontaining DMEM medium was made to order by Imperial Laboratories, England. To obtain "normal" DMEM, Lvaline was added to the D-valine medium at the concentration usually present in commercial DMEM (93.6 mg/1). The resultant O,L-valine DMEM behaved as commercial DMEM with respect to both astrocyte and fibroblast growth (Estin and Vernadakis, 1986). Before being added to o-valine DMEM the foetal calf serum was first dialysed and then heat inactivated. This procedure was carried out in order to remove any L-valine from the serum (Estin and Vernadakis, 1986). The dialysis tubing was first activated by boiling in sodium carbonate (2%), EDTA (0.5%) and distilled water (four changes). The serum was then dialysed for 48 h against three changes of normal saline (1:50 serum:saline) and then filter sterilized and heat-inactivated at 56'C for 1 h. Thus the D-valine medium contained dialysed serum while the D,L-valine medium contained untreated serum. In experiments comparing glial cell growth in D,L-valine medium containing either untreated serum or dialysed serum, no differences in cell numbers were observed (data not shown).
few cells survived, although none were Thy 1.1 positive. The growth rate and development o f the astrocytes appeared stunted when c o m p a r e d to cells grown in O,L-valine m e d i u m (Estin and Vernadakis, 1986). Subsequently the cells were plated in D,L-valine medium and grown for up to 5 days before switching to D-valine medium. This enabled the cells to divide and better survive the change to D-valine medium and still reduce the numbers o f fibroblasts in confluent 12 day old cultures In all subsequent experiments cells were grown in D,L-valine D M E M for 5 days after which time the medium in half o f the wells was changed to o-valine D M E M (with dialysed FCS at 10%) with further medium changes at 8 and 10 days in culture. Cells were taken for immunoftuorescence staining at 6, 8, 10 and 12 days in culture and culture purity assessed. Table 1 shows the percentage o f labelled cells under the two culture conditions with respect to astrocytes and fibroblasts. Initially at 6 days in culture there was little difference in fibroblast numbers in both cultures although fewer fibroblasts appeared to be present in D-valine D M E M . With time in culture fibroblast n u m b e r s decreased in D-valine D M E M and by 12 days in culture they n u m b e r e d below 3% o f total cells. In O,L-valine D M E M fibroblast numbers remained high at approx. 20% o f the total cell number. We also found that oligodendrocytes accounted for less than 5% o f the total cell n u m b e r at 12 days in culture in both o-valine D M E M and O,L-valine D M E M , as stained with antibodies against galactocerebroside (data not shown). N o neurones could be detected at 12 days in culture based on their characteristic m o r p h o l o g y or when staining was carried out with anti-neurofilament antibodies. Staining with normal rabbit serum and ingestion o f 1.0 # m latex beads labelled < 2.3 and < 5%, respectively, o f cells
lrnmunofluorescence
The different cell types present in spinal cord cultures were identified with light microscopic immunofluorescence (Reichart, Polyvar) using the following antibodies; anti-glial fibrillary acidic protein (GFAP)-astrocytes (Bignami and Dahl, 1974), anti-Thy l.l-fibroblasts (Stern, 1973; Raft et al., 1979), anti-galactocerebroside (GC)-oligodendrocytes (Ranscht et al., 1982), and anti-neurofilament (NF)neurones (Anderton et al., 1980). Neurons could also be identified by their morphology. Normal rabbit serum followed by rhodamine-conjugated anti-rabbit antibody was used to label macrophages together with ingestion of 1.0/~ m latex beads (Raft et al., 1979). RESULTS Initial studies showed that when cells were grown in o-valine m e d i u m immediately after plating, very
Table 1. Percentageof cells labelled with antibodies against GFAP and Thy 1.1 under two different media conditions D,L-Valine DMEM Days in culture 6 8 10 12
GFAP 69.0+3.7 71.0 + 2.7 71.3_+3.1 73.4_+3.6
Thy 1.1 22.8+3.3 22.8 + 3.4 25.3+2.6 20.7_+2.2
D-Valine DMEM GFAP
Thy 1.1
77.9+ 1.2 14.1 + 1.9 86.7 + 0.9 5.7 + 1.6 91.2_+0.6 3.4_+0.3 92.1 + 1.0 2.7_+0.6
Cells obtained from 2-day-oldrat spinal cords and grown in culture for 5 days. The medium was then changed in half of the wells to D-valinecontaining DMEM and again at 8 and 10 days. Cells stained with antibodies to GFAP and Thy 1.1 at 6, 8, 10 and 12 days in culture. Results are expressedas the mean + SEM of 4 separate experimentsfor days 6, 8 and 10 and as mean + SEM of 3 separate experiments for day 12. In each experiment l0 random fields were counted on duplicate coverslips.
B
A
C
D
Fig. 1, Immunofluorescent staining of 12 day old spinal cord cultures grown in either O,L-valine DMEM (A) and (B) or D-valine DMEM (C) and (D). Cells were stained with antibodies against GFAP (A) and (C) and antibodies against Thy 1.1 (B) and (D). Bar = 35/~m. 367
368
A.J. CHOLEWINSKIet al.
believed to be macrophages (Raft et al., 1979) (data not shown).
DISCUSSION The purification step described in this paper utilizes a modification of the D M E M growth medium with a substitution of D-valine (at twice the L-valine concentration) for L-valine. Using this procedure spinal cord cultures can be enriched in astrocytes with greater than 92% purity and contaminating fibroblasts reduced to below 3%. Retardation offibroblast growth appears to be based on their inability to convert D-valine to the L-isomer as they lack the enzyme D-amino acid oxidase (Gilbert and Migeon, 1975). This procedure is modified from that first described by Estin and Vernadakis (1986) in that the cells are changed to D-valine containing D M E M medium following 5 days in culture in D,L-valine D M E M . It is important that freshly dissociated cells are grown in a normal medium for the first few days in culture in order to both rapidly increase their numbers, and to prevent the marked degree of stunted growth observed when astrocytes were plated directly after dissociation in D-valine D M E M (Estin and Vernadakis, 1986). The very small percentage of other contaminating cells (apart from fibroblasts) following this procedure were either oligodendrocytes ( < 5%) and their precursors (Levi et al., 1987; Curtis et al., 1988), or macrophages ( < 5%) and or microglia. Should absolutely pure astrocyte cultures be required then oligodendrocytes can be removed by orbital shaking (McCarthy and de Vellis, 1980). Macrophages and or microglia can be removed by passage as they do not proliferate (DuBois et al., 1986) or selective chemical destruction using L-leucine methyl ester (Giulian et al., 1988). We have now adopted the procedure described here for obtaining fibroblast-free cultures from other C N S regions.
REFERENCES
Anderton B. H., Thorpe R., Cohen J., Selvendron S. and Woodhams S. P. (1980) Specific neuronal localisation by immunofluorescene of l0 nm filament polypeptide. J. Neurocytol. 9, 835-844. Bignami A. and Dahl D. (1974) Astrocyte-specific protein and neuroglial differentiation. An immunofluorescence study with antibodies to the glial fibrillary acidic protein. J. comp. Neurol. 153, 27-37. Booher J. and Sensenbrenner M. (1972) Growth and cultivation of dissociated neurones and glial cells from embry-
onic chick, rat and human brain in flask cultures. Neurobiology 2, 97 105.
Chapman J. and Rumsby M. (1982) A syringe modification for the simple and rapid dissociation of post-natal rat cerebral tissue for preparing primary cultures of mixed glial cells. Neurosci. Lett. 34, 307 313. Cholewinski A. J. and Wilkin G. P. (1988) Astrocytes from forebrain, cerebellum and spinal cord differ in their responses to vasoactive intestinal peptide. J. Neurochem. 51, 16261633. Cholewinski A. J., Hanley M. R. and Wilkin G. P. (1988) A phosphoinositide-linked peptide response in astrocytes: evidence for regional heterogeneity. Neurochem. Res. 13, 389-394. Curtis R., Cohen J., Fok-Seang J., Hanley M. R., Gregson N. A., Reynolds R. and Wilkin G. P. (1988) Development of macroglial cells in rat cerebellum. I. Use of antibodies to follow early in vivo development and migration of oligodendrocytes. J. Neuroeytol. 17, 43-54. DuBois J. H., Bolton C. and Cuzner M. L. (1986) The production of prostaglandin and the regulation of cell division in neonate rat primary mixed glial cultures. J. Neuroimmun. 11, 277 285. Estin C. and Vernadakis A. (1986) Primary glial cells and brain fibroblasts Interactions in culture. Brain Res. Bull. 16, 723 731. Gilbert S. F. and Migeon B. R. (1975) D-Valine as a selective agent for normal human and rodent epithelial cells in culture. Cell5, 11 17. Giulian D., Young D. G., Woodward J., Brown D. C. and Lachman L. B. (1988) Interleukin-I is an astroglial growth factor in the developing brain. J. Neurosci. 8, 709-714. Juurlink B. H. J. and Devon R. M. (1987) Procedure for establishing oligodendroglial cells in primary cultures based on developmental parameters. Int. J. dev. Neurosci. 5, 327 336. Levi G., Aloisi F. and Wilkin G. P. (1987) Differentiation of cerebellar bipotential glial precursors into oligodendrocytes in primary culture. Development profile of surface antigens and mitotic activity. J. Neurosci. Res. 18, 407~J, 17. Mains R. E. and Patterson P. H. (1973) Primary cultures of dissociated sympathetic neurones. I. Establishment of long-term growth in culture and studies of differentiated properties. J. Cell Biol. 59, 329-345. McCarthy K. D. and de Vellis J. (1980) Preparation of astroglial and oligodendroglial cell cultures from rat cerebral tissue. J. Cell Biol. 85, 890 902. Pearce B., Cambray-Deakin M. A., Morrow C., Grimble J. and Murphy S. (1985) Activation of muscarinic and of ~-adrenergic receptors on astrocytes results in the accumulation of inositol phosphates. J. Neurochem. 45, 1534-1540. Raft M. C., Fields K. L., Hakomori S. I., Mirsky R., Pruss R. M. and Winter J. (1979) Cell-type-specific markers for distinguishing and studying neurones and the major classes of glial cells in culture. Brain Res. 174, 283 308. Raizada M. K., Phillips M. I., Crews F. T. and Sumners C. (1987) Distinct angiotensin II receptor in primary cultures ofglial cells from rat brain. Proc. natn. Acad. Sci. U.S.A. 84, 4655~4659. Ranscht B., Clapshaw P. A., Price J., Noble M. and Seifert
Purification of astroglial-cell cultures from rat spinal cord W. (1982) Development of oligodendrocytes and Schwann cells studied with a monoclonal antibody against galactocerebroside. Proc. natn. Acad. Sci. U.S.A. 79, 2709 2713. Stern P. L. (1973) 0 Alloantigen on mouse and rat fibroblasts. Nature New Biol. 246, 76-78.
369
Suzumura A., Bhat S., Eccleston A., Lisak R. P. and Silberberg D. H. (1984) The isolation and long-term culture of oligodendrocytes from newborn mouse brain. Brain Res. 324, 379 383.