Br. J. ex). Path. (1975) 56, 8
EN FACE ORGAN CULTURE OF VASCULAR ENDOTHELIUM D. DE BONO From the D)epartment of Surgery, University of Cambridge, Douglas House, Trumpington Road, Cambridge, England Received for publication 5 August 1974
Summary.-A simple technique is described for maintaining in organ culture segments of blood vessel in such a way that the endothelium can be studied en face. The behaviour of such cultures throws further light on the interaction between endothelium and intravascular clotting.
\'ASCULAR endothelium is involved in thrombosis, atherogenesis, allograft rejection and other interesting pathological processes. A simple technique is described for maintaining intact sheets of endothelium in vitro for up to 10 days as part of an " organ culture " of vessel wall. The technique is applicable to a wide variety of vessels and species, and produces virtually an " instant " monolayer of endothelium. It is intended to be complementary to technique3 already described for making cell cultures of endothelium (Tsutsumi and Gore, 1969; Jaffe et al., 1973). MATERIALS AND METHODS
Vessels which have been successfully cultured include human umbilical artery and v-ein, saphenous and mesenteric vein: pig carotid and iliac arteries, jtugular and iliac veins, and inferior vena cava; rabbit vena cava and aorta and rat aorta. Care is needed in dissecting out the vessel. Thiis is best done by sharp dissection and a no-touch technique after tying ligatures at the ends of the segment to be used and injecting a s;nall quantity of heparin (1000 u/ml) into the lumen. Attempts at stripping the adventitia almost always damage the endothelium. The culture technique is based on that of Trow ell (1959). The vessel is slit open with scissors and placed endothelial side upward on a sterile Millipore filter (0-8 pore size). The adventitia adheres to the filter, which helps to support and flatten the vessel wall. Vessel and filter are cut into pieces 5 mm square w;ith a scalpel blade and these are laid endothelial side downwards over 4 mm diameter holes punched
in stainless steel " expanded metal " grids. The grids are 12 mm square with the edges turned down so as to support the culture 3 mm above the floor of the culture dishes. These are 30 mm diameter glass petri dishes filled with culture medium to the level of the grid (2-25 ml). Two or 4 of these are placed on moist filter paper in a 90 mm diameter petri dish, and the whole is incubated in an atmosphere of 100% 02 or 95% 02/5% CO2 depending on the medium used. This was either BGJ 5 medium, kindly supplied by Strangeways Research Laboratory, Cambridge, or Waymouth's medium (Burroughs Wellcome). The medium was buffered to pH 7-3 with either bicarbonate/5% CO2 OI' 20 mmol/l HEPES, and contained 30 mg/l benzylpenicillin and 50 mg/l streptomycin (Cristamycin, Glaxo). The medium was usually supplemented with 10% autologous serum or 10% inactiv ated foetal calf serum. It was changed every other day and the gas phase was renewed daily. Specimens were usually examined en face after silver nitrate impregnation and counterstaining with haematoxylin, either as fresh wet preparations or after making celloidin " hautchen " (Poole, Sanders and Florey, 1958). Shrinkage during fixation tends to distort and fragment the endothelium of arterial segments. This can be partially avoided by formol saline fixation under pressure (de Bono, 1972). Conventional paraffin sections were cut after fixation in formol saline and stained with haematoxylin and eosin. Specimens for electron microscopy ws ere fixed in glutaraldehyde/osmium fixative. RESULTS
Intact sheets of endothelium have been maintained in culture by this technique for up to 10 days. Viability of the cells
EN FACE ORGAN CULTURE OF VASCULAR ENDOTHELIUM
was assessed by light microscopy, electron microscopy, Trypan blue exclusion and growth in culture. Successful specimens present an intact sheet of endothelium with narrow cement lines (Fig. 1) and close intercellular apposition (Fig. 2). Mitosis
9
was rarely seen in intact endothelial sheets. However, after 4 or 5 days endothelium tends to grow out in continuity over the edges of the specimen, over fibrin strands, and over areas accidentally or purposelv denuded of endothelium when
FIG. 1.-Rabbit vena cava, 8-day culture. " Hautchen " preparation, silver nitrate and haematoxylin. x 240. FIG. 2. Rabbit vena cava, 6-day culture. Electron micrograph to show close apposition of cells and numerous small vesicles. x 9000.
10
D. DE BONO
FIG. 3.-Pig jugular vein, 3-day culture. The endothelium to the left was deliberately scraped away while setting up the culture and a more basophilic zone of regenerating endothelium separates it from the undamaged endothelium to the right. " Hautchen " x 135. FIG. 4.-Pig jugular vein, 4-day culture. Mitosis in regenerating endothelium. " Hautchen " x 510.
EN FACE ORGAN CULTURE OF VASCULAR ENDOTHELIUM
II
the culture was set up. The appearance of ing and bubbling of cement lines (Mcthe spreading endothelium resembles that Govern, 1955) and the endothelial nuclei described in vivo by Poole et al. (1958) become pyknotic. Eventually the cells peel off. Areas of unhealthy endothelium (Fig. 3, 4). Deteriorating specimens show thicken- have a brownish tinge after silver nitrate
FIG. 5. Pig carotid artery, 6-day culture. Osmiophilic inclusions and thickened " glycocalyx " Electron micrograph x 9000. FIG. 6.-Pig jugular vein, endothelium adjacent to a clot in the medium. The cells are contracted and the apparent nucleocytoplasmic ratio increased, while the endothelial sheet is beginning to fragment. " Hautchen " x 200.
12
D. DE BONO
staining-this appears to be due to increased endothelial permeability, allowing the access of silver to subendothelial tissues, as these areas stain heavily when incubated before fixation with Evans blue (0.05% in 10% albumin solution) Distinct from this is a tendency for cells in culture for some days to acquire a faintly granular, greyish appearance after silver nitrate staining, usually accompanied by an accentuation of the cement lines. This appearance is parallelled at electron microscope level by a thickening of the " glycocalyx" on the luminal side of the cells. The thickening may be partially accounted for by contraction of the specimen, but it may also represent slower removal of the glycocalyx in the absence of flowing blood, or increased secretion (Fig. 5). The maintenance of viable endothelium in culture requires an adequate oxygen tension In the centres of large thick specimens, or in cultures gassed with air instead of oxygen, endothelium is rarely preserved for more than 24 h. Provided the medium is well buffered, a protein supplement does not seem to be essential though most reliable results were obtained with medium containing 10% inactivated foetal calf serum Autologous serum, heparinized plasma or albumin solution were usually well tolerated at a concentration of 10%. Endothelium cultured for 6 days in lipaemic serum developed osmiophilic inclusions, presumably representing fat droplets (Fig. 5). Some samples of fresh serum are toxic to endothelium. This is particularly the case with serum separated before coagulation is quite complete, so that a small clot forms in the culture vessel. The toxicity could be inhibited by adding heparin (10 u/ml) to the serum, and hitherto non-toxic serum could be made toxic by adding an excess of thrombin (10 N.I.H. u/ml). This would accord with the evidence of Rafelson et al. (1973) for a direct action of thrombin on endothelium. Endothelium damaged in this way tends to contract and fragment, forming sheets or rafts with closely packed nuclei and
little visible cytoplasm when viewed en face (Fig. 6). The contraction loosens the attachment of endothelium to the vessel wall and the rafts may float away in the medium. Similar rafts have been described in the blood stream of guinea-pigs subjected to anaphylactic shock (PaylingWright and Giacometti, 1972). The damage to endothelium in the vicinity of a clot may not be permanent and it may be very localized. Specimens have been seen with viable endothelium in one part of the culture and fibrin strands attached to the intima only a millimetre away. Occasionally the endothelium grows out over the fibrin, in a manner similar perhaps to that by which intravascular webs are formed in vivo. Presumably such fibrin is plasminogen depleted as endothelium is known to contain a plasminogen activator and its persistence for up to 3 weeks in vein organ cultures has been reported (Pandolfi, 1970). On the basis of these observations, a simplified scheme for the interaction of endothelium and the blood clotting mechanism would be as follows: intravascular clotting damages endothelium by a pathway involving thrombin. Contraction of the endothelium allows adherence of the thrombus to the vessel wall, and the damaged cells encourage platelet aggregation (Rafelson et al, 1973). Once the clot has formed, endothelial plasminogen activator co-operates with its circulating counterpart in breaking down the clot. If the clot is plasminogen deficient, or if its breakdown is delayed until its plasminogen content is ineffective, endothelium will grow over the fibrin, which will be organized as part of the vessel wall or as in intravascular web. I am grateful to Professor R. Y. Calne for advice and encouragement, to Dame Honor Fell for introducing me to tissue culture and to the Medical Research Council of Great Britain for a Junior Research Fellowship. The electron microscopy was performed by Mrs J. Wardle.
EN FACE ORGAN CULTURE OF VASCULAR ENDOTHELIUM
REFERENCES DE BONO, D. P. (1972) Host Repopulation of Endothelium in Human Kidney Transplants. Transplantation, 14, 438. JAFFE, E. A., NACHMAN, R. L., BECKER, C. G. & MINICK, C. R. (1973) Culture of Human Endothelial Cells derived from Umbilical Vein. J. clin. Invest., 52, 2745. MCGOVERN, V. J. (1955) Endothelial Response to Injury. J. Path. Bact., 69, 283. PANDOLFI, M. (1970) Persistence of Fibrinolytic Activity in Fragments of Human Vein Cultured in vitro. Throm. Diath. Haem., 24, 43. PAYLING WRIGHT, H. & GIACOMETTI, W. (1972) Circulating Endothelial Cells and Arterial Endo-
13
thelial Mitosis in Anaphylactic Shock. Br. J. exp. Path., 53, 1. POOLE, J. C. F., SANDERS, A. G. & FLOREY, H. W. (1958) The Regeneration of Aortic Endothelium. J. Path. Bact., 75, 133. RAFELSON, M. E., HOVEKE., T. P. & BOOYSE, F. M. (1973) The Molecular Biology of Platelet-Platelet and Platelet-Endothelial Interactions. Series Haematologica, Ul, 3, 367. TROWELL, 0. A. (1959) The Culture of Mature Organs in a Synthetic Medium. Expl Cell Res., 16, 118. TSUTSUMI, H. & GORE, I. (1969) Isolation of Living Endothelial Cells by Gelatin Film Stripping of Vascular Walls. Stain Technol., 44, 139.
EN FACE ORGAN CULTURE OF VASCULAR ENDOTHELIUM D. DE BONO From the D)epartment of Surgery, University of Cambridge, Douglas House, Trumpington Road, Cambridge, England Received for publication 5 August 1974
Summary.-A simple technique is described for maintaining in organ culture segments of blood vessel in such a way that the endothelium can be studied en face. The behaviour of such cultures throws further light on the interaction between endothelium and intravascular clotting.
\'ASCULAR endothelium is involved in thrombosis, atherogenesis, allograft rejection and other interesting pathological processes. A simple technique is described for maintaining intact sheets of endothelium in vitro for up to 10 days as part of an " organ culture " of vessel wall. The technique is applicable to a wide variety of vessels and species, and produces virtually an " instant " monolayer of endothelium. It is intended to be complementary to technique3 already described for making cell cultures of endothelium (Tsutsumi and Gore, 1969; Jaffe et al., 1973). MATERIALS AND METHODS
Vessels which have been successfully cultured include human umbilical artery and v-ein, saphenous and mesenteric vein: pig carotid and iliac arteries, jtugular and iliac veins, and inferior vena cava; rabbit vena cava and aorta and rat aorta. Care is needed in dissecting out the vessel. Thiis is best done by sharp dissection and a no-touch technique after tying ligatures at the ends of the segment to be used and injecting a s;nall quantity of heparin (1000 u/ml) into the lumen. Attempts at stripping the adventitia almost always damage the endothelium. The culture technique is based on that of Trow ell (1959). The vessel is slit open with scissors and placed endothelial side upward on a sterile Millipore filter (0-8 pore size). The adventitia adheres to the filter, which helps to support and flatten the vessel wall. Vessel and filter are cut into pieces 5 mm square w;ith a scalpel blade and these are laid endothelial side downwards over 4 mm diameter holes punched
in stainless steel " expanded metal " grids. The grids are 12 mm square with the edges turned down so as to support the culture 3 mm above the floor of the culture dishes. These are 30 mm diameter glass petri dishes filled with culture medium to the level of the grid (2-25 ml). Two or 4 of these are placed on moist filter paper in a 90 mm diameter petri dish, and the whole is incubated in an atmosphere of 100% 02 or 95% 02/5% CO2 depending on the medium used. This was either BGJ 5 medium, kindly supplied by Strangeways Research Laboratory, Cambridge, or Waymouth's medium (Burroughs Wellcome). The medium was buffered to pH 7-3 with either bicarbonate/5% CO2 OI' 20 mmol/l HEPES, and contained 30 mg/l benzylpenicillin and 50 mg/l streptomycin (Cristamycin, Glaxo). The medium was usually supplemented with 10% autologous serum or 10% inactiv ated foetal calf serum. It was changed every other day and the gas phase was renewed daily. Specimens were usually examined en face after silver nitrate impregnation and counterstaining with haematoxylin, either as fresh wet preparations or after making celloidin " hautchen " (Poole, Sanders and Florey, 1958). Shrinkage during fixation tends to distort and fragment the endothelium of arterial segments. This can be partially avoided by formol saline fixation under pressure (de Bono, 1972). Conventional paraffin sections were cut after fixation in formol saline and stained with haematoxylin and eosin. Specimens for electron microscopy ws ere fixed in glutaraldehyde/osmium fixative. RESULTS
Intact sheets of endothelium have been maintained in culture by this technique for up to 10 days. Viability of the cells
EN FACE ORGAN CULTURE OF VASCULAR ENDOTHELIUM
was assessed by light microscopy, electron microscopy, Trypan blue exclusion and growth in culture. Successful specimens present an intact sheet of endothelium with narrow cement lines (Fig. 1) and close intercellular apposition (Fig. 2). Mitosis
9
was rarely seen in intact endothelial sheets. However, after 4 or 5 days endothelium tends to grow out in continuity over the edges of the specimen, over fibrin strands, and over areas accidentally or purposelv denuded of endothelium when
FIG. 1.-Rabbit vena cava, 8-day culture. " Hautchen " preparation, silver nitrate and haematoxylin. x 240. FIG. 2. Rabbit vena cava, 6-day culture. Electron micrograph to show close apposition of cells and numerous small vesicles. x 9000.
10
D. DE BONO
FIG. 3.-Pig jugular vein, 3-day culture. The endothelium to the left was deliberately scraped away while setting up the culture and a more basophilic zone of regenerating endothelium separates it from the undamaged endothelium to the right. " Hautchen " x 135. FIG. 4.-Pig jugular vein, 4-day culture. Mitosis in regenerating endothelium. " Hautchen " x 510.
EN FACE ORGAN CULTURE OF VASCULAR ENDOTHELIUM
II
the culture was set up. The appearance of ing and bubbling of cement lines (Mcthe spreading endothelium resembles that Govern, 1955) and the endothelial nuclei described in vivo by Poole et al. (1958) become pyknotic. Eventually the cells peel off. Areas of unhealthy endothelium (Fig. 3, 4). Deteriorating specimens show thicken- have a brownish tinge after silver nitrate
FIG. 5. Pig carotid artery, 6-day culture. Osmiophilic inclusions and thickened " glycocalyx " Electron micrograph x 9000. FIG. 6.-Pig jugular vein, endothelium adjacent to a clot in the medium. The cells are contracted and the apparent nucleocytoplasmic ratio increased, while the endothelial sheet is beginning to fragment. " Hautchen " x 200.
12
D. DE BONO
staining-this appears to be due to increased endothelial permeability, allowing the access of silver to subendothelial tissues, as these areas stain heavily when incubated before fixation with Evans blue (0.05% in 10% albumin solution) Distinct from this is a tendency for cells in culture for some days to acquire a faintly granular, greyish appearance after silver nitrate staining, usually accompanied by an accentuation of the cement lines. This appearance is parallelled at electron microscope level by a thickening of the " glycocalyx" on the luminal side of the cells. The thickening may be partially accounted for by contraction of the specimen, but it may also represent slower removal of the glycocalyx in the absence of flowing blood, or increased secretion (Fig. 5). The maintenance of viable endothelium in culture requires an adequate oxygen tension In the centres of large thick specimens, or in cultures gassed with air instead of oxygen, endothelium is rarely preserved for more than 24 h. Provided the medium is well buffered, a protein supplement does not seem to be essential though most reliable results were obtained with medium containing 10% inactivated foetal calf serum Autologous serum, heparinized plasma or albumin solution were usually well tolerated at a concentration of 10%. Endothelium cultured for 6 days in lipaemic serum developed osmiophilic inclusions, presumably representing fat droplets (Fig. 5). Some samples of fresh serum are toxic to endothelium. This is particularly the case with serum separated before coagulation is quite complete, so that a small clot forms in the culture vessel. The toxicity could be inhibited by adding heparin (10 u/ml) to the serum, and hitherto non-toxic serum could be made toxic by adding an excess of thrombin (10 N.I.H. u/ml). This would accord with the evidence of Rafelson et al. (1973) for a direct action of thrombin on endothelium. Endothelium damaged in this way tends to contract and fragment, forming sheets or rafts with closely packed nuclei and
little visible cytoplasm when viewed en face (Fig. 6). The contraction loosens the attachment of endothelium to the vessel wall and the rafts may float away in the medium. Similar rafts have been described in the blood stream of guinea-pigs subjected to anaphylactic shock (PaylingWright and Giacometti, 1972). The damage to endothelium in the vicinity of a clot may not be permanent and it may be very localized. Specimens have been seen with viable endothelium in one part of the culture and fibrin strands attached to the intima only a millimetre away. Occasionally the endothelium grows out over the fibrin, in a manner similar perhaps to that by which intravascular webs are formed in vivo. Presumably such fibrin is plasminogen depleted as endothelium is known to contain a plasminogen activator and its persistence for up to 3 weeks in vein organ cultures has been reported (Pandolfi, 1970). On the basis of these observations, a simplified scheme for the interaction of endothelium and the blood clotting mechanism would be as follows: intravascular clotting damages endothelium by a pathway involving thrombin. Contraction of the endothelium allows adherence of the thrombus to the vessel wall, and the damaged cells encourage platelet aggregation (Rafelson et al, 1973). Once the clot has formed, endothelial plasminogen activator co-operates with its circulating counterpart in breaking down the clot. If the clot is plasminogen deficient, or if its breakdown is delayed until its plasminogen content is ineffective, endothelium will grow over the fibrin, which will be organized as part of the vessel wall or as in intravascular web. I am grateful to Professor R. Y. Calne for advice and encouragement, to Dame Honor Fell for introducing me to tissue culture and to the Medical Research Council of Great Britain for a Junior Research Fellowship. The electron microscopy was performed by Mrs J. Wardle.
EN FACE ORGAN CULTURE OF VASCULAR ENDOTHELIUM
REFERENCES DE BONO, D. P. (1972) Host Repopulation of Endothelium in Human Kidney Transplants. Transplantation, 14, 438. JAFFE, E. A., NACHMAN, R. L., BECKER, C. G. & MINICK, C. R. (1973) Culture of Human Endothelial Cells derived from Umbilical Vein. J. clin. Invest., 52, 2745. MCGOVERN, V. J. (1955) Endothelial Response to Injury. J. Path. Bact., 69, 283. PANDOLFI, M. (1970) Persistence of Fibrinolytic Activity in Fragments of Human Vein Cultured in vitro. Throm. Diath. Haem., 24, 43. PAYLING WRIGHT, H. & GIACOMETTI, W. (1972) Circulating Endothelial Cells and Arterial Endo-
13
thelial Mitosis in Anaphylactic Shock. Br. J. exp. Path., 53, 1. POOLE, J. C. F., SANDERS, A. G. & FLOREY, H. W. (1958) The Regeneration of Aortic Endothelium. J. Path. Bact., 75, 133. RAFELSON, M. E., HOVEKE., T. P. & BOOYSE, F. M. (1973) The Molecular Biology of Platelet-Platelet and Platelet-Endothelial Interactions. Series Haematologica, Ul, 3, 367. TROWELL, 0. A. (1959) The Culture of Mature Organs in a Synthetic Medium. Expl Cell Res., 16, 118. TSUTSUMI, H. & GORE, I. (1969) Isolation of Living Endothelial Cells by Gelatin Film Stripping of Vascular Walls. Stain Technol., 44, 139.
