261
J. Anat. (1979), 129, 2, pp. 261-272 With 7 figures Printed in Great Britain
Isolation and characterization of endothelial cells from rat and cow brain white matter P. PHILLIPS, PAT KUMAR, S. KUMAR AND M. WAGHE
Clinical Research Laboratories, Christie Hospital and Holt Radium Institute, Withington, Manchester M20 9BX
(Accepted 26 July 1978) INTRODUCTION
The endothelial cell barrier between the blood and tissues plays a key role in inflammatory and immunological changes and may also be important in neoplasia. Studies of the nature and mode of action of stimuli which can influence such endothelial cell activities as proliferation and permeability in vivo have been limited by the lack of suitable techniques. During the past few years it has become easier to understand the biology of endothelial cells, because, for example, cells from the human umbilical cord can be cultured for various lengths of time and those containing Weibel-Palade bodies can be identified as endothelial cells with certainty (Weibel & Palade, 1964; Jaffe, Nachman, Becker & Minnick, 1973; Gimbrone, Cotran & Folkman, 1974; Haudenschild, Cotran, Gimbrone & Folkman, 1975). However, such cultures are invariably contaminated by other cells of mesenchymal origin (Gimbrone, 1976; Pollack, 1969). We report here a new source (the brain) which yields much purer cultures of endothelial cells. MATERIALS AND METHODS
Tissue culture In order to initiate a culture, several adult Wistar rats (each weighing approximately 170 g) were killed with anaesthetic ether and their brains aseptically removed and placed in balanced salt solution. The grey matter was discarded, and the white matter was carefully dissected out and finely minced before undergoing trypsinization for 5 minutes with 0-5 % pancreatic bovine trypsin (Difco). Sufficient fetal calf serum was then added to stop the action of trypsin, and the cell suspension was centrifuged at 500 rev/min for 10 minutes. The supematant was discarded and the cells were washed with growth medium (Medium 199, with 20% fetal calf serum,
penicillin and streptomycin) and incubated in Falcon tissue culture flasks after gassing with a 95 % air, 5 % CO2 mixture. They were left undisturbed for a week and thereafter the growth medium was changed every 3 days. After the cells had become confluent they were harvested, using a trypsin-EDTA mixture, and plated into either Falcon flasks, Leighton tubes or Falcon microtest II plates. No attempt was made to grow cells beyond 6 weeks after their initiation. Cow brains were obtained from an abattoir, and cultures of white matter were initiated by the same method as used for the rat brain white matter cultures.
0021-8782/2828-6360 $02.00 © 1979 Anat. Soc. G.B. & 1.
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P. PHILLIPS, PAT KUMAR, S. KUMAR AND M. WAGHE
Microscopical examination For phase contrast and time-lapse studies, coverslip cultures were sealed on to a stainless steel chamber with a rectangular well. The preparations were examined under a phase or interference microscope fitted with a thermostatically controlled stage. Electron microscopy Tissue fragments of 1-2 mm3 of rat brain white matter, and pellets of cells cultured from trypsinized tissue, were fixed in 2 5 %, glutaraldehyde buffered with M/15 Sorensen's phosphate buffer pH 7-4 for 2 hours. Fixed tissues were twice washed with the above buffer, post-fixed in 2 % osmic acid in buffer for 1 hour, dehydrated with graded ethanols and propylene oxide, and embedded in an Araldite-Epon mixture. Ultrathin sections were cut and stained with uranyl acetate and lead citrate before examination.
Autoradiography and scintillation counting In order to determine the labelling index, cultures grown on coverslips were exposed to 055,Ci/ml of tritiated thymidine (3H-TdR) (specific activity 5 Ci/mM, Radiochemical Centre, Amersham, England) for 2 hours and then fixed in aceticalcohol (1: 3). Autoradiographs were prepared by a dipping technique (Baserga & Malamud, 1969) using Gevaert Scintia 7-15 emulsion (Agfa Gevaert). In order to find out the proportion of proliferating cells in the population ('growth fraction'), Leighton tube cultures were continuously labelled for 24 hours with 3H-TdR and autoradiographs were prepared as above. For scintillation counting, varying numbers of cells were plated in the wells of microtest II plates (Falcon Plastics) and incubated overnight. The medium was changed and 2 hours prior to sampling 0 5 Ci/ml 3H-TdR (sp.act. 5 Ci/mM) in 25 ,ul balanced salt solution (BSS) was added to replicate wells. After 2 hours labelling the cultures were washed with BSS, fixed in acetic-ethanol (1: 3) for 10 minutes, rinsed with distilled water, washed successively with cold 10% trichloroacetic acid (TCA) and 5 % TCA for 15 minutes, and air dried. Each microwell with its adherent monolayer was then punched out of the plate into a vial containing 10 ml scintillation fluid and counted in a scintillation counter.
Immunological and histochemical studies Cryostat sections (5 ,um) of rat brain white matter (RBWM), and monolayers of cells grown on glass coverslips, were stained for alkaline phosphatase by the naphthol-ASTR-phosphate method (Burstone, 1958). They were also fixed in cold acetone for 10 minutes for immunofluorescence studies. One of the classical criteria used to identify human and bovine endothelial cells in culture is the presence of factor VIII antigen on the surface of the cells (Bleich, Boro & Jaffe, 1977; Gospodarowicz, Brown, Birdwell & Zetter, 1978). Because of technical difficulties an antiserum to rat factor VIII has not been produced by anyone. However, an antiserum to human factor VIII is commercially available, and it is known that human and bovine factor VIII share common antigenic determinants. If it could be shown by staining with anti-human factor VIII that cow brain white matter cultures were composed of endothelial cells, it would be reasonable to infer that rat brain white matter cultures initiated in the same manner were also of endo-
Endothelial cells from white matter
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Fig. 1. (A) Rat brain white matter after four days in culture. Two cells in this figure have migrated away from the cell clump seen at the bottom right hand corner. Phase contrast. x 880. (B) A confluent culture of rat brain white matter. Phase contrast. x 1000.
thelial origin. In order to demonstrate the presence of factor VIII, acetone-fixed monolayers of cow brain white matter cells were treated for 20 minutes at room temperature with a 1: 9 dilution of rabbit anti-human factor VIII antiserum (Hoechst). After thorough washing the slides were further incubated with fluorescein isothiocyanate (FITC) labelled anti-rabbit 1 g serum (1:30) (Wellcome Laboratories) for 20 minutes at room temperature and mounted in glycerol-saline (1:1) for incident light fluorescence microscopy (Kumar & Taylor, 1975). RESULTS
Cultures from rat and cow brain white matter showed similar growth patterns. Three to four days after the initiation of a culture, isolated flattened cell clumps were noticed (Fig. 1 A). Further growth was rapid, and the cells migrated out as a loose
264
P. PHILLIPS, PAT KUMAR, S. KUMAR AND M. WAGHE
Fig. 2. A typical cow brain endothelial cell (CBEC) in tissue culture, showing interdigitations with other cultured cells. x 9000.
sheet. Migration of cells continued for 2-3 weeks, by which time most of the cultures became confluent (Fig. 1 B). Confluence was accompanied by a cessation of cell movement and multiplication. This growth pattern was remarkably uniform. Cells were polygonal or fusiform (75 ,um x 40 /tm) and had a poorly defined cell boundary. Their cytoplasm contained a few optically dense granules, the majority of which were situated in the perinuclear region. The round or oval nucleus was placed centrally and contained one or two spherical nucleoli. Multinucleate forms were extremely
265
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Time-lapse cinematography showed the cells to be sluggishly motile. Perinuclear phase-dense particles exhibited a little of both saltatory and brownian movements. Mitochondria exhibited moderate to intense activity, especially during the early phase of growth. Nuclear rotation was absent, but a limited amount of pinocytosis was noticed. The growth characteristics and cell morphology of tissuecultured diploid skin fibroblasts differed from those of the endothelial cells. Fibroblasts were spindle-shaped with well-defined cell boundaries, and often grew closely packed in sheets, whorls and overlapping layers. This fact alone is not enough to prove the identity of our cells in culture, but suggests that they are not fibroblasts. An initial innoculum of 1 x 106 cells into T-75 tissue culture flasks (surface area 75 CM2) usually resulted in confluent growth (approximately 3 x 106 cells) in 7-1 0 days. Normally cultures were grown for up to 6 weeks. Although 2-3 subcultures were necessary to avoid overcrowding during this period, the appearance and growth characteristics of the cells remained similar to those of the primary cultures. rare.
Electron microscopy It was observed that our presumptive endothelial cells in culture, whether from rat brain or cow brain, were alike in having areas of cytoplasm with a prominent Golgi apparatus and associated vesicles, and areas showing mainly a sparse network of microfilaments and rough endoplasmic reticulum. Glycogen was absent (Fig. 2). Several different kinds of intracytoplasmic inclusion have been described in the literature as occurring only in endothelial cells. A brief summary of these is included here so that our results can be related to the published literature. Weibel & Palade (1964) described electron-dense cylindrical organelles up to 0 ,tm long and approximately 0-1I tm wide consisting of a tubular or fibrillar matrix bounded by a unit membrane (Weibel-Palade bodies). Such bodies are found only in endothelial cells, and have been described in the arteries and veins of many
~3
266
P. PHILLIPS, PAT KUMAR, S. KUMAR AND M. WAGHE
Fig. 4. Weibel-Palade bodies in tissue-cultured cow brain endothelial cells. (A) x 120000. (B) x 120000.
mammalian and amphibian organs. Some authors considered them to be absent from normal human brain capillaries (Hirano, 1974) whereas others claimed to have found them in very small numbers (Herrlinger, Anzil, Blinzinger & Kronski, 1974). They are reported to be quite common in the blood capillaries of human brain tumours (Hirano, 1974; P. Kumar, unpublished observations) but to vary greatly in their density. Immature Weibel-Palade bodies are small and pale with few visible tubules (Figs. 3, 5). Weibel & Palade (1964) also reported the presence of endothelial cells of "discoid bodies resembling dilated cisternae of endoplasmic reticulum with a relatively pale granular matrix". Kawamura, Kamijyo, Sunaga & Nelson (1974) mentioned three types of organelle in the capillaries of a renal carcinoma metastasis in the human brain. Type I was dense and fibrillar and corresponded to Weibel-Palade bodies. Type II (called tubular bodies) were less dense with prominent and distinct tubules, and often were attached to coated vesicles: they were considered immature forms of
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Fig. 5. Longitudinal profiles of Weibel-Palade bodies (arrowed) in endothelial cells from: (A) Normal human umbilical artery. x 90000. (B) Wilms' tumour capillary. x 90000. (C) Rat brain endothelial cell in tissue culture. x 90000.
267
268
P. PHILLIPS, PAT KUMAR, S. KUMAR AND M. WAGHE
Table 1. The effect of crowding on the uptake of 3H-TdR by rat brain endothelial cells in tissue culture Mean number of counts per 1000 cells ± S.D. 1 day after Nos. of 3 days after 5 days after cells/well medium change medium change medium change 1. 12500 541-8+78-0 383-0±30-9 142-6±9-9 2. 25000 431-2+71-0 88-8± 17-6 2734+±14-7 3. 50000 1713±+ 36-9 95 0±10-8 37 0+± 86 4. 100000 68-8+18-4 41-5±7-5 18-9±3-9 Statistical analysis: The cpm for cell densities 25000, 50000 and 100000 were divided by 2, 4 and 8 respectively. A two factor analysis of variance was carried out on the log transformed data.
Source of variation
Sums of squares
D.F.
Mean squares
F
Between days Between cell densities
24 500
2 3
12-250 16-068
284-12 371-75
48&083
Significance P < 0 001 P < 0.001
Type I. Type III were large, very pale vacuoles with an amorphous matrix containing a few tubules and bounded by a unit membrane, and it was suggested that they were swollen Type I bodies. It is possible that the Type III bodies correspond to the discoid bodies of Weibel & Palade (1964) and Steinsieppe & Weibel (1970). Multivesicular bodies, as described by Florey (1966), are found in many cell types, including endothelial cells, and were observed occasionally in our tissue-cultured cells. They were different from Weibel-Palade bodies in that their tubular contents were of irregular size and mixed with membranous debris. Figure 4 shows the Weibel-Palade bodies that were found in our tissue-cultured rat and cow brains. The majority of these bodies were pale with few tubules (compare Fig. 3). Longitudinal sections of Weibel-Palade bodies from normal human umbilical artery, a Wilms' tumour, and our tissue-cultured rat brain cells, can be seen in Figure 5. Such longitudinal sections were found only rarely in our cultured cells; most of the profiles were round.
Autoradiography and scintillation counting In order to calculate the thymidine labelled index, no. of labelled cells x 100 total no. of cells at least 500 cells were counted per slide. The mean percentage labelling index after a 2 hour pulse label, when more than 4000 cells were counted, was 12-6 ±1 09 standard error (S.E.). In cultures that were labelled continuously for 24 hours the mean percentage of labelled cells was 24-46 ±1-7 S.E. The effect of an increase in cell density on the uptake of 3H-TdR is shown in Table 1. The mean cpm when expressed per 1000 cells dropped significantly as the number of cells exceeded 12500 per mnicrowell, showing that the cell growth was density-dependent.
269
Endothelial cells from white matter
6A
........ ...
....
..6 B
Fig. 6. (A) A cryostat section of rat brain white matter showing the localization of alkaline phosphatase in the capillaries. x 800. (B) A monolayer of rat brain white matter endothelial cells stained for alkaline phosphatase. All the cells in culture were weakly positive for alkaline phosphatase, apart from approximately 1 % which showed moderate activity of the enzyme (one such cell is shown here). x 2000.
Immunological and histochemical studies Alkaline phosphatase was strongly positive in the capillaries of the rat and cow brain white matter (Fig. 6A). All of our cells in culture were positive for alkaline phosphatase, although the staining reaction was weaker than in cryostat sections. Approximately 1 % of the tissue cultured cells contained moderate amounts of the enzyme, and in the remaining cells the reaction was weak (Fig. 6B). Every cell in the
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P. PHILLIPS, PAT KUMAR, S. KUMAR AND M. WAGHE
Fig. 7. Indirect immunofluorescent staining of tissue-cultured cells derived from cow brain white matter with rabbit/anti-human factor VIII antiserum (Hoechst).
cultures of cow brain white matter stained intensely with rabbit anti-human factor VIII antiserum (Fig. 7). DISCUSSION
In the mammalian brain large blood vessels are almost entirely limited to the grey matter, and the blood supply to the white matter is mainly via capillaries. Furthermore, normal nerve and glial cells from adult animals will rarely grow in short term cultures (Kumar et al. 1973). The white matter, therefore, should be a unique source of endothelial cells, and this has been substantiated by our success in obtaining homogenous cultures which appeared to be free of fibroblastoid and glial elements. The latter two can be easily identified by their characteristic morphology alone (Lumsden, 1963; Waghe, Kumar & Steward, 1973). Fibroblasts can also be recognized by being relatively more motile than endothelial cells. Our tissue-cultured cells showed a density-dependent inhibition of growth, a characteristic that has been reported for cultured human and animal endothelial cells (Jaffe et al. 1973; Haudenschild et al. 1975; Gospodarowicz, Moran, Braun & Birdwell, 1976). Gluszoz (1963) found that the only cells in blood vessel walls to show alkaline phosphatase activity were endothelial cells, and Kuwabara & Cogan (1963) reported that pericytes lacked this enzyme. We found it to be strongly positive in the cells lining capillaries in cryostat sections of rat and cow brain white matter. All our cells in culture were positive also, although the staining reaction was less intense than in cryostat sections. The likely reason for the weaker staining of cultured cells is that these cells are very flattened on glass coverslips, whereas cryostat sections are approximately 5 pm thick.
Endothelial cells from white matter
271
That the cells grown from cow brain white matter were endothelial was confirmed by their staining with anti-factor VIII antiserum, a feature which is diagnostic for endothelial cells (Hoyer, de los Santos & Hoyer, 1973; Bleich et al. 1977; Gospodarowicz et al. 1978). Further evidence that the brain cells we grew were endothelial was provided by transmission electron microscopy. The great majority of the cells grown from cow and rat brain contained pale immature Weibel-Palade bodies, which are not found in any other cell type (Gimbrone, 1976; Gospodarowicz et al. 1978). Furthermore, our tissue-cultured cells were similar in morphology to endothelial cells in vivo in brain white matter. The fact that our cultured endothelial cells did not contain particularly large numbers of mature Weibel-Palade bodies is not surprising, since these were not demonstrable in the normal rat brain white matter capillaries from which the cultures were derived. Although normal adult human brain capillaries very rarely contain Weibel-Palade bodies (Hirano, 1974; Herrlinger et al. 1974) these bodies are known in a few pathological conditions and are especially common in brain tumours (Kawamura et al. 1974; Hirano, 1974; Hirano & Matsui, 1975, and our unpublished observations). Their function is unknown, although Burri & Weibel (1968) have suggested a connexion with thromboplastic activities. The alkaline phosphatase activity reported for endothelial cells does not reside in these bodies; nor do they show any 5'nucleotidase activity. They are unlike lysosomes in that they do not show any acid phosphatase activity. They seem to arise in the Golgi apparatus (Sengel & Stoebner, 1970). It is possible that the presence of Weibel-Palade bodies in brain endothelial cells is related to cell proliferation, since our cultured cells contained them but the endothelial cells in rat brain white matter capillaries did not. It is important to determine how soon in culture the bodies appear, since apart from their value for identifying endothelial cells, they may prove to be an early indicator of increased metabolic activity or proliferation of endothelial cells. This would be useful in studies of angiogenesis. We hope that the availability of pure endothelial cultures will facilitate the study of factors which influence the structure and function of these cells. We plan to use the cultured cells to investigate if tumour angiogenesis factor, which stimulates endothelial cell proliferation in vivo, can influence their growth in vitro (Folkman, 1976; Phillips, Steward & Kumar, 1976). SUMMARY
A procedure is described for obtaining pure cultures of endothelial cells from rat and cow brain white matter. The morphology of the tissue-cultured cells was studied by both light and transmission electron microscopy. Weibel-Palade bodies, described in the literature as specific for endothelial cells, appeared in small numbers in our cultured cells. Autoradiographic, scintillation counting, immunological and histochemical studies were performed. The usefulness of pure endothelial cell cultures is discussed. This work was supported by the Cancer Research Campaign and the Children's Research Fund.
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BASERGA, R. & MALAMUD, D. (1969). Autoradiography: Techniques and Application. New York: Harper and Row. BLEICH, H. L., BORO, E. S. & JAFFE, E. A. (1977). Endothelial cells and the biology of factor VIII. New England Journal of Medicine 296, 377-383. BURRI, P. H. & WEIBEL, E. R. (1968). Beeinflussung einer spezifischen cytoplasmatischen Organelle von Endothelzellen durch Adrenalin. ZeitschriftfiurZellforschung undmikroskopische Anatomie 88,426-440. BURSTONE, M. S. (1958). Histochemical comparison of napthol AS-phosphates for the demonstration of phosphatase. Journal of the National Cancer Institute 20, 601-615. FLOREY, L. (1966). The endothelial cell. British Medical Journal ii, 487-490. FOLKMAN, J. (1976). The vascularisation of tumours. Scientific American 234, 59-73. GIMBRONE, M. A. Jr. (1976). Culture of vascular endothelium. Progress in Hemostasis and Thrombosis vol. in, pp. 1-28. New York: Grune & Stratton Inc. GIMBRONE, M. A. Jr., COTRAN, R. S. & FOLKMAN, J. (1974). Human vascular endothelial cells in culture. Journal of Cell Biology 60, 673-684. GLUSZOZ, A. (1963). A histochemical study of some hydrolytic enzymes in tumours of the nervous system. Acta neuropathologie 3, 184-193. GOSPODAROWICZ, D., MORAN, J., BRAUN, D. & BIRDWELL, C. (1976). Clonal growth of bovine vascular endothelial cells: fibroblast growth-factor as a survival agent. Proceedings of the National Academy of Sciences 73, 4120-4124. GOSPODAROWICZ, D., BROWN, K. D., BIRDWELL, C. R. & ZETTER, B. R. (1978). Control of proliferation of human vascular endothelial cells. Journal of Cell Biology 77, 774-788. HAUDENSCHILD, C. C., COTRAN, R. S., GIMBRONE, M. A. Jr. & FOLKMAN, J. (1975). Fine structure of vascular endothelium in culture. Journal of Ultrastructure Research 50, 22-32. HERRLINGER, H., ANZIL, A. P., BLINZINGER, K. & KRONSKI, D. (1974). Endothelial microtubular bodies in human brain capillaries. Journal of Anatomy 118, 205-209. HIRANO, A. (1974). Fine structural alterations of small vessels in the nervous system. Pathology of Microcirculation (ed. J. Cervos-Navarro), p. 203. Berlin: de Gruyter Co. HIRANO, A. & MATSUI, T. (1975). Fine structure of intercellular junctions and blood vessels in medulloblastomas. Human Pathology 6, 611-621. HOYER, L. W., DE LOS SANTOS, R. P. & HOYER, J. P. (1973). Anti-hemophilic factor antigen: Localisation in endothelial cells by immunofluorescent microscopy. Journal of Clinical Investigation 523, 2737-2744. JAFFE, E. A., NACHMAN, R. L., BECKER, C. G. & MINNICK, R. C. (1973). Culture of human endothelial cells derived from umbilical veins. Journal of Clinical Investigation 52, 2745-2756. KAWAMURA, J., KAMIJYO, Y., SUNAGA, T. & NELSON, E. (1974). Tubular bodies in vascular endothelium of a cerebellar neoplasm. Laboratory Investigation 30, 358-365. KUWABARA, T. & COGAN, D. G. (1963). Retinal vascular patterns. VI. Neural cells. Archives of Ophthalmology 69, 492-527. KUMAR, S. & TAYLOR, G. (1975). Non-organ-specific and tumour specific antibodies in children with Wilms' tumour. International Journal of Cancer 16, 448-455. KUMAR, S., TAYLOR, G., STEWARD, J. K., WAGHE, M. A. & MORRIS-JONES, P. (1973). Cell mediated immunity and blocking factors in patients with tumours of the central nervous system. International Journal of Cancer 12, 194-205. LUMSDEN, C. E. (1963). Pathology of Tumours ofthe Nervous System (ed. D. S. Russell & L. J. Rubinstein), p. 281. London: E. Arnold. PHILLIPS, P., STEWARD, J. K. & KUMAR, S. (1976). Tumour angiogenesis factor (TAF) in human and animal tumours. International Journal of Cancer 17, 549-558. POLLACK, 0. J. (1969). Tissue Cultures. Monographs on Atherosclerosis, vol. 1, p. 26. New York: S. Karger. SENGEL, A. & STOEBNER, P. (1970). Golgi origin of tubular inclusions in endothelial cells. Journal of Cell Biology 44, 223-226. STEINSIEPPE, K. F. & WEIBEL, E. R. (1970). Elektronenmikroskopische Untersuchungen am spezifischen Organellen von Endothelzellen des Frosches (Rana temporaria). Zeitschrift fur Zellforschung und mikroskopische Anatomie 108, 105-126. WAGHE, M., KUMAR, S. & STEWARD, J. K. (1973). Tissue culture studies of children's tumours. Journal of Pathology 111, 117-124. WEIBEL, E. R. & PALADE, G. E. (1964). New cytoplasmic components in arterial endothelia. Journal of Cell Biology 23, 101-112.
J. Anat. (1979), 129, 2, pp. 261-272 With 7 figures Printed in Great Britain
Isolation and characterization of endothelial cells from rat and cow brain white matter P. PHILLIPS, PAT KUMAR, S. KUMAR AND M. WAGHE
Clinical Research Laboratories, Christie Hospital and Holt Radium Institute, Withington, Manchester M20 9BX
(Accepted 26 July 1978) INTRODUCTION
The endothelial cell barrier between the blood and tissues plays a key role in inflammatory and immunological changes and may also be important in neoplasia. Studies of the nature and mode of action of stimuli which can influence such endothelial cell activities as proliferation and permeability in vivo have been limited by the lack of suitable techniques. During the past few years it has become easier to understand the biology of endothelial cells, because, for example, cells from the human umbilical cord can be cultured for various lengths of time and those containing Weibel-Palade bodies can be identified as endothelial cells with certainty (Weibel & Palade, 1964; Jaffe, Nachman, Becker & Minnick, 1973; Gimbrone, Cotran & Folkman, 1974; Haudenschild, Cotran, Gimbrone & Folkman, 1975). However, such cultures are invariably contaminated by other cells of mesenchymal origin (Gimbrone, 1976; Pollack, 1969). We report here a new source (the brain) which yields much purer cultures of endothelial cells. MATERIALS AND METHODS
Tissue culture In order to initiate a culture, several adult Wistar rats (each weighing approximately 170 g) were killed with anaesthetic ether and their brains aseptically removed and placed in balanced salt solution. The grey matter was discarded, and the white matter was carefully dissected out and finely minced before undergoing trypsinization for 5 minutes with 0-5 % pancreatic bovine trypsin (Difco). Sufficient fetal calf serum was then added to stop the action of trypsin, and the cell suspension was centrifuged at 500 rev/min for 10 minutes. The supematant was discarded and the cells were washed with growth medium (Medium 199, with 20% fetal calf serum,
penicillin and streptomycin) and incubated in Falcon tissue culture flasks after gassing with a 95 % air, 5 % CO2 mixture. They were left undisturbed for a week and thereafter the growth medium was changed every 3 days. After the cells had become confluent they were harvested, using a trypsin-EDTA mixture, and plated into either Falcon flasks, Leighton tubes or Falcon microtest II plates. No attempt was made to grow cells beyond 6 weeks after their initiation. Cow brains were obtained from an abattoir, and cultures of white matter were initiated by the same method as used for the rat brain white matter cultures.
0021-8782/2828-6360 $02.00 © 1979 Anat. Soc. G.B. & 1.
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P. PHILLIPS, PAT KUMAR, S. KUMAR AND M. WAGHE
Microscopical examination For phase contrast and time-lapse studies, coverslip cultures were sealed on to a stainless steel chamber with a rectangular well. The preparations were examined under a phase or interference microscope fitted with a thermostatically controlled stage. Electron microscopy Tissue fragments of 1-2 mm3 of rat brain white matter, and pellets of cells cultured from trypsinized tissue, were fixed in 2 5 %, glutaraldehyde buffered with M/15 Sorensen's phosphate buffer pH 7-4 for 2 hours. Fixed tissues were twice washed with the above buffer, post-fixed in 2 % osmic acid in buffer for 1 hour, dehydrated with graded ethanols and propylene oxide, and embedded in an Araldite-Epon mixture. Ultrathin sections were cut and stained with uranyl acetate and lead citrate before examination.
Autoradiography and scintillation counting In order to determine the labelling index, cultures grown on coverslips were exposed to 055,Ci/ml of tritiated thymidine (3H-TdR) (specific activity 5 Ci/mM, Radiochemical Centre, Amersham, England) for 2 hours and then fixed in aceticalcohol (1: 3). Autoradiographs were prepared by a dipping technique (Baserga & Malamud, 1969) using Gevaert Scintia 7-15 emulsion (Agfa Gevaert). In order to find out the proportion of proliferating cells in the population ('growth fraction'), Leighton tube cultures were continuously labelled for 24 hours with 3H-TdR and autoradiographs were prepared as above. For scintillation counting, varying numbers of cells were plated in the wells of microtest II plates (Falcon Plastics) and incubated overnight. The medium was changed and 2 hours prior to sampling 0 5 Ci/ml 3H-TdR (sp.act. 5 Ci/mM) in 25 ,ul balanced salt solution (BSS) was added to replicate wells. After 2 hours labelling the cultures were washed with BSS, fixed in acetic-ethanol (1: 3) for 10 minutes, rinsed with distilled water, washed successively with cold 10% trichloroacetic acid (TCA) and 5 % TCA for 15 minutes, and air dried. Each microwell with its adherent monolayer was then punched out of the plate into a vial containing 10 ml scintillation fluid and counted in a scintillation counter.
Immunological and histochemical studies Cryostat sections (5 ,um) of rat brain white matter (RBWM), and monolayers of cells grown on glass coverslips, were stained for alkaline phosphatase by the naphthol-ASTR-phosphate method (Burstone, 1958). They were also fixed in cold acetone for 10 minutes for immunofluorescence studies. One of the classical criteria used to identify human and bovine endothelial cells in culture is the presence of factor VIII antigen on the surface of the cells (Bleich, Boro & Jaffe, 1977; Gospodarowicz, Brown, Birdwell & Zetter, 1978). Because of technical difficulties an antiserum to rat factor VIII has not been produced by anyone. However, an antiserum to human factor VIII is commercially available, and it is known that human and bovine factor VIII share common antigenic determinants. If it could be shown by staining with anti-human factor VIII that cow brain white matter cultures were composed of endothelial cells, it would be reasonable to infer that rat brain white matter cultures initiated in the same manner were also of endo-
Endothelial cells from white matter
W1i
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_
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lB .t
D L-~
263
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Fig. 1. (A) Rat brain white matter after four days in culture. Two cells in this figure have migrated away from the cell clump seen at the bottom right hand corner. Phase contrast. x 880. (B) A confluent culture of rat brain white matter. Phase contrast. x 1000.
thelial origin. In order to demonstrate the presence of factor VIII, acetone-fixed monolayers of cow brain white matter cells were treated for 20 minutes at room temperature with a 1: 9 dilution of rabbit anti-human factor VIII antiserum (Hoechst). After thorough washing the slides were further incubated with fluorescein isothiocyanate (FITC) labelled anti-rabbit 1 g serum (1:30) (Wellcome Laboratories) for 20 minutes at room temperature and mounted in glycerol-saline (1:1) for incident light fluorescence microscopy (Kumar & Taylor, 1975). RESULTS
Cultures from rat and cow brain white matter showed similar growth patterns. Three to four days after the initiation of a culture, isolated flattened cell clumps were noticed (Fig. 1 A). Further growth was rapid, and the cells migrated out as a loose
264
P. PHILLIPS, PAT KUMAR, S. KUMAR AND M. WAGHE
Fig. 2. A typical cow brain endothelial cell (CBEC) in tissue culture, showing interdigitations with other cultured cells. x 9000.
sheet. Migration of cells continued for 2-3 weeks, by which time most of the cultures became confluent (Fig. 1 B). Confluence was accompanied by a cessation of cell movement and multiplication. This growth pattern was remarkably uniform. Cells were polygonal or fusiform (75 ,um x 40 /tm) and had a poorly defined cell boundary. Their cytoplasm contained a few optically dense granules, the majority of which were situated in the perinuclear region. The round or oval nucleus was placed centrally and contained one or two spherical nucleoli. Multinucleate forms were extremely
265
Endothelial cells from white matter
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n
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,
41~~~~~~~~~~~~~~~~~4
Fig. 3. Weibel-Palade bodies of varying density in an endothelial cell of a Wilms' tumour capillary. An immature, pale form is arrowed. x 9000.
Time-lapse cinematography showed the cells to be sluggishly motile. Perinuclear phase-dense particles exhibited a little of both saltatory and brownian movements. Mitochondria exhibited moderate to intense activity, especially during the early phase of growth. Nuclear rotation was absent, but a limited amount of pinocytosis was noticed. The growth characteristics and cell morphology of tissuecultured diploid skin fibroblasts differed from those of the endothelial cells. Fibroblasts were spindle-shaped with well-defined cell boundaries, and often grew closely packed in sheets, whorls and overlapping layers. This fact alone is not enough to prove the identity of our cells in culture, but suggests that they are not fibroblasts. An initial innoculum of 1 x 106 cells into T-75 tissue culture flasks (surface area 75 CM2) usually resulted in confluent growth (approximately 3 x 106 cells) in 7-1 0 days. Normally cultures were grown for up to 6 weeks. Although 2-3 subcultures were necessary to avoid overcrowding during this period, the appearance and growth characteristics of the cells remained similar to those of the primary cultures. rare.
Electron microscopy It was observed that our presumptive endothelial cells in culture, whether from rat brain or cow brain, were alike in having areas of cytoplasm with a prominent Golgi apparatus and associated vesicles, and areas showing mainly a sparse network of microfilaments and rough endoplasmic reticulum. Glycogen was absent (Fig. 2). Several different kinds of intracytoplasmic inclusion have been described in the literature as occurring only in endothelial cells. A brief summary of these is included here so that our results can be related to the published literature. Weibel & Palade (1964) described electron-dense cylindrical organelles up to 0 ,tm long and approximately 0-1I tm wide consisting of a tubular or fibrillar matrix bounded by a unit membrane (Weibel-Palade bodies). Such bodies are found only in endothelial cells, and have been described in the arteries and veins of many
~3
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P. PHILLIPS, PAT KUMAR, S. KUMAR AND M. WAGHE
Fig. 4. Weibel-Palade bodies in tissue-cultured cow brain endothelial cells. (A) x 120000. (B) x 120000.
mammalian and amphibian organs. Some authors considered them to be absent from normal human brain capillaries (Hirano, 1974) whereas others claimed to have found them in very small numbers (Herrlinger, Anzil, Blinzinger & Kronski, 1974). They are reported to be quite common in the blood capillaries of human brain tumours (Hirano, 1974; P. Kumar, unpublished observations) but to vary greatly in their density. Immature Weibel-Palade bodies are small and pale with few visible tubules (Figs. 3, 5). Weibel & Palade (1964) also reported the presence of endothelial cells of "discoid bodies resembling dilated cisternae of endoplasmic reticulum with a relatively pale granular matrix". Kawamura, Kamijyo, Sunaga & Nelson (1974) mentioned three types of organelle in the capillaries of a renal carcinoma metastasis in the human brain. Type I was dense and fibrillar and corresponded to Weibel-Palade bodies. Type II (called tubular bodies) were less dense with prominent and distinct tubules, and often were attached to coated vesicles: they were considered immature forms of
Endothelial cells from white matter
.y. '^
:
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Nl
,. rwckl, |P.3r$fwVW .P
Fig. 5. Longitudinal profiles of Weibel-Palade bodies (arrowed) in endothelial cells from: (A) Normal human umbilical artery. x 90000. (B) Wilms' tumour capillary. x 90000. (C) Rat brain endothelial cell in tissue culture. x 90000.
267
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P. PHILLIPS, PAT KUMAR, S. KUMAR AND M. WAGHE
Table 1. The effect of crowding on the uptake of 3H-TdR by rat brain endothelial cells in tissue culture Mean number of counts per 1000 cells ± S.D. 1 day after Nos. of 3 days after 5 days after cells/well medium change medium change medium change 1. 12500 541-8+78-0 383-0±30-9 142-6±9-9 2. 25000 431-2+71-0 88-8± 17-6 2734+±14-7 3. 50000 1713±+ 36-9 95 0±10-8 37 0+± 86 4. 100000 68-8+18-4 41-5±7-5 18-9±3-9 Statistical analysis: The cpm for cell densities 25000, 50000 and 100000 were divided by 2, 4 and 8 respectively. A two factor analysis of variance was carried out on the log transformed data.
Source of variation
Sums of squares
D.F.
Mean squares
F
Between days Between cell densities
24 500
2 3
12-250 16-068
284-12 371-75
48&083
Significance P < 0 001 P < 0.001
Type I. Type III were large, very pale vacuoles with an amorphous matrix containing a few tubules and bounded by a unit membrane, and it was suggested that they were swollen Type I bodies. It is possible that the Type III bodies correspond to the discoid bodies of Weibel & Palade (1964) and Steinsieppe & Weibel (1970). Multivesicular bodies, as described by Florey (1966), are found in many cell types, including endothelial cells, and were observed occasionally in our tissue-cultured cells. They were different from Weibel-Palade bodies in that their tubular contents were of irregular size and mixed with membranous debris. Figure 4 shows the Weibel-Palade bodies that were found in our tissue-cultured rat and cow brains. The majority of these bodies were pale with few tubules (compare Fig. 3). Longitudinal sections of Weibel-Palade bodies from normal human umbilical artery, a Wilms' tumour, and our tissue-cultured rat brain cells, can be seen in Figure 5. Such longitudinal sections were found only rarely in our cultured cells; most of the profiles were round.
Autoradiography and scintillation counting In order to calculate the thymidine labelled index, no. of labelled cells x 100 total no. of cells at least 500 cells were counted per slide. The mean percentage labelling index after a 2 hour pulse label, when more than 4000 cells were counted, was 12-6 ±1 09 standard error (S.E.). In cultures that were labelled continuously for 24 hours the mean percentage of labelled cells was 24-46 ±1-7 S.E. The effect of an increase in cell density on the uptake of 3H-TdR is shown in Table 1. The mean cpm when expressed per 1000 cells dropped significantly as the number of cells exceeded 12500 per mnicrowell, showing that the cell growth was density-dependent.
269
Endothelial cells from white matter
6A
........ ...
....
..6 B
Fig. 6. (A) A cryostat section of rat brain white matter showing the localization of alkaline phosphatase in the capillaries. x 800. (B) A monolayer of rat brain white matter endothelial cells stained for alkaline phosphatase. All the cells in culture were weakly positive for alkaline phosphatase, apart from approximately 1 % which showed moderate activity of the enzyme (one such cell is shown here). x 2000.
Immunological and histochemical studies Alkaline phosphatase was strongly positive in the capillaries of the rat and cow brain white matter (Fig. 6A). All of our cells in culture were positive for alkaline phosphatase, although the staining reaction was weaker than in cryostat sections. Approximately 1 % of the tissue cultured cells contained moderate amounts of the enzyme, and in the remaining cells the reaction was weak (Fig. 6B). Every cell in the
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P. PHILLIPS, PAT KUMAR, S. KUMAR AND M. WAGHE
Fig. 7. Indirect immunofluorescent staining of tissue-cultured cells derived from cow brain white matter with rabbit/anti-human factor VIII antiserum (Hoechst).
cultures of cow brain white matter stained intensely with rabbit anti-human factor VIII antiserum (Fig. 7). DISCUSSION
In the mammalian brain large blood vessels are almost entirely limited to the grey matter, and the blood supply to the white matter is mainly via capillaries. Furthermore, normal nerve and glial cells from adult animals will rarely grow in short term cultures (Kumar et al. 1973). The white matter, therefore, should be a unique source of endothelial cells, and this has been substantiated by our success in obtaining homogenous cultures which appeared to be free of fibroblastoid and glial elements. The latter two can be easily identified by their characteristic morphology alone (Lumsden, 1963; Waghe, Kumar & Steward, 1973). Fibroblasts can also be recognized by being relatively more motile than endothelial cells. Our tissue-cultured cells showed a density-dependent inhibition of growth, a characteristic that has been reported for cultured human and animal endothelial cells (Jaffe et al. 1973; Haudenschild et al. 1975; Gospodarowicz, Moran, Braun & Birdwell, 1976). Gluszoz (1963) found that the only cells in blood vessel walls to show alkaline phosphatase activity were endothelial cells, and Kuwabara & Cogan (1963) reported that pericytes lacked this enzyme. We found it to be strongly positive in the cells lining capillaries in cryostat sections of rat and cow brain white matter. All our cells in culture were positive also, although the staining reaction was less intense than in cryostat sections. The likely reason for the weaker staining of cultured cells is that these cells are very flattened on glass coverslips, whereas cryostat sections are approximately 5 pm thick.
Endothelial cells from white matter
271
That the cells grown from cow brain white matter were endothelial was confirmed by their staining with anti-factor VIII antiserum, a feature which is diagnostic for endothelial cells (Hoyer, de los Santos & Hoyer, 1973; Bleich et al. 1977; Gospodarowicz et al. 1978). Further evidence that the brain cells we grew were endothelial was provided by transmission electron microscopy. The great majority of the cells grown from cow and rat brain contained pale immature Weibel-Palade bodies, which are not found in any other cell type (Gimbrone, 1976; Gospodarowicz et al. 1978). Furthermore, our tissue-cultured cells were similar in morphology to endothelial cells in vivo in brain white matter. The fact that our cultured endothelial cells did not contain particularly large numbers of mature Weibel-Palade bodies is not surprising, since these were not demonstrable in the normal rat brain white matter capillaries from which the cultures were derived. Although normal adult human brain capillaries very rarely contain Weibel-Palade bodies (Hirano, 1974; Herrlinger et al. 1974) these bodies are known in a few pathological conditions and are especially common in brain tumours (Kawamura et al. 1974; Hirano, 1974; Hirano & Matsui, 1975, and our unpublished observations). Their function is unknown, although Burri & Weibel (1968) have suggested a connexion with thromboplastic activities. The alkaline phosphatase activity reported for endothelial cells does not reside in these bodies; nor do they show any 5'nucleotidase activity. They are unlike lysosomes in that they do not show any acid phosphatase activity. They seem to arise in the Golgi apparatus (Sengel & Stoebner, 1970). It is possible that the presence of Weibel-Palade bodies in brain endothelial cells is related to cell proliferation, since our cultured cells contained them but the endothelial cells in rat brain white matter capillaries did not. It is important to determine how soon in culture the bodies appear, since apart from their value for identifying endothelial cells, they may prove to be an early indicator of increased metabolic activity or proliferation of endothelial cells. This would be useful in studies of angiogenesis. We hope that the availability of pure endothelial cultures will facilitate the study of factors which influence the structure and function of these cells. We plan to use the cultured cells to investigate if tumour angiogenesis factor, which stimulates endothelial cell proliferation in vivo, can influence their growth in vitro (Folkman, 1976; Phillips, Steward & Kumar, 1976). SUMMARY
A procedure is described for obtaining pure cultures of endothelial cells from rat and cow brain white matter. The morphology of the tissue-cultured cells was studied by both light and transmission electron microscopy. Weibel-Palade bodies, described in the literature as specific for endothelial cells, appeared in small numbers in our cultured cells. Autoradiographic, scintillation counting, immunological and histochemical studies were performed. The usefulness of pure endothelial cell cultures is discussed. This work was supported by the Cancer Research Campaign and the Children's Research Fund.
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