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J Thorac Cardiovasc Surg 1995;110:998-1004
© 1995 Mosby, Inc.

SURGERY FOR ACQUIRED HEART DISEASE

EFFECTS OF CRYOPRESERVATION ON THE PROLIFERATION AND ANTICOAGULANT ACTIVITY OF HUMAN SAPHENOUS VEIN ENDOTHELIAL CELLS

L. S. Bambang, DVM, J. P. Mazzucotelli, MD, M. Moczar, PhD, F. Beaujean, MDa, D. Loisance, MD


Créteil, France

Received for publication Aug. 12, 1994. Accepted for publication Dec. 23, 1994. Address for reprints: D. Loisance, MD, Centre de Recherches Chirurgicales Henri Mondor, Faculté de Médecine, 8 rue du Général Sarrail, 94000 Créteil, France.

Abstract

Human saphenous veins were cryopreserved in 4% human albumin and 10% dimethyl sulfoxide.The effect of cryopreservation on endothelial cells was studied in terms of the anticoagulant activity of thrombomodulin and in terms of cell proliferation. After storage for 2 weeks at -150°C, 0.45 ± 0.07 x 10 5endothelialcells/cm2 were detected in cryopreserved veins and 1.03& plusmn; 0.04 x 105 endothelial cells/cm2 in freshveins ( p <0.01). The thrombin-catalyzed activation of protein C decreased after cryopreservation, indicating altered thrombomodulin activity in the endothelial cells. On a cell number basis, the release of soluble thrombomodulin was three times higher from the cryopreserved endothelium than from the fresh endothelium (p < 0.05). The amount of spontaneous release of von Willebrand factor from the endothelial surface was not significantly different between fresh and cryopreserved veins. Endothelial cells were cultured from fresh veins and from their cryopreserved counterparts. On plating of endothelial cells in primary culture, the number of adhered cells was 0.9 ± 0.09 x 103 cells/cm2 from fresh veins and 0.25 ± 0.03 x 103 cells/cm2 from cryopreserved veins ( p <0.01). The positive immunohistochemical stain for von Willebrand factor indicated that the endothelial cell character was maintained after cryopreservation. The endothelial desquamation with loss of anticoagulant function and the slow proliferation of surviving cells in vitro suggest an impaired endothelial healing in vivo. The loss of anticoagulant activity complicates the problems of the exposure of thrombogenic subendothelial matrix to blood in implanted cryopreserved veins. (J THORAC CARDIOVASC SURG1995;110:998-1004)

Cryopreserved blood vessel allografts that retained their native extracellular matrix and cellular integrity with a decreased immunologic reaction on transplantation could be a material of choice to replace autografts in arterial revascularizations. Go Go 1-3 The clinical failures of cryopreserved human venous homografts have been associated with decreased function of endothelial cells in the cryopreserved grafts. Go Go 3-5 The use of dimethyl sulfoxide (DMSO) as cryoprotectant improves cell survival, Go Go 3,6 and the results of functional analysis of cryopreserved venous autografts avoiding immunorejection in a canine model are promising. Go Go 7-9 The aim of our study was to determine the viability and anticoagulant function of endothelial cells in human saphenous veins in vitro after cryopreservation and thawing. We evaluated particularly the thrombomodulin activity, because one mechanism of the regulation of the natural anticoagulant pathway involves thrombomodulin for protein C activation. Go 10 To obtain information on the ability of the injured endothelium to recover after cryopreservation, we investigated the behavior of endothelial cells cultured from cryopreserved veins and from fresh veins. We extended our studies also to cryopreservation of endothelial cells cultured from fresh veins, to differentiate between the effect of the cryopreservant and the effect of cryopreservation of tissue rather than cells.

MATERIALS AND METHODS

Endothelial cells were collected by mechanical scraping from internal saphenous veins from patients undergoing coronary bypass operations. Pooled human AB serum was obtained from Centre de Transfusion Sanguine (Les Ulis, France). M 199 and RPMI 1640 culture medium (Roswell Park Memorial Institute, Buffalo, N.Y.), antibiotics, and additives to cell cultures were purchased from Gibco (Cergy, France) and 2% gelatin solution for cell culture from Sigma Chemical Company (St. Louis, Mo.). Human thrombin was a gift from Biotransfusion (Roissy, France). Purified protein C, recombinant r-hirudin, chromogenic peptide substrate CBS 4246 and CBS 0041 for activated protein C, activated protein C, Asserachrom von Willebrand Factor ELISA Kit, and Asserachrom Thrombomodulin ELISA Kit were obtained from Diagnostica Stago (Asnières, France). Lyophilized human plasma proteins were obtained from Biotransfusion (Roissy, France). DMSO and May-Grünwald–Giemsa reagent were obtained from Merck (Darmstadt, Germany). Optimal cutting temperature (O.C.T.) medium for frozen tissue sections was acquired from Miles (Elkhart, Ind.) and Immu-Mount aqueous mounting medium from Shandon (Pittsburgh, Pa.). Rabbit immunoglobulins to human von Willebrand factor (vWF) and sheep immunoglobulin were purchased from Dakopatts (Dako, Trappes, France). Sheep antibody to human thrombomodulin was a kind gift from Dr. M. C. Boffa (INSERM U 150, Paris, France). An Elite ABC Kit employing the detecting reagent Vectastain, an avidin-biotinylated horseradish peroxidase complex was acquired from Vector (Burlingame, Calif.).

Sampling of veins
Forty veins obtained from patients undergoing coronary artery bypass operations were each divided into two segments of identical length (approximately 3 cm) in the operating room; they were immersed immediately into 25 ml of transport solution containing NaCl (0.15 mol/L) and glucose (0.01 mol/L) and were transported in less than 10 minutes at room temperature into the laboratory. One segment from each vein was submitted to cryopreservation, and cell cultures were propagated from both the fresh samples and the cryopreserved counterparts after thawing, as described in the following sections.

Cryopreservation of veins
The cryopreservation of veins was performed in Centre Départemental de Transfusion Sanguine (CHU Henri Mondor, Créteil, France). The veins were removed from the transport solution and were soaked in 15 ml of a sterile solution of 4% human albumin (Biotransfusion, Lille, France) and 10% DMSO in water for 30 minutes at 4° C. The veins in this solution were transferred into plastic bags and were chilled to -80° C by decreasing the temperature 1° C per minute. The frozen veins were stored in vapors of liquid nitrogen at -150° C for 2 weeks. The cryopreserved veins were allowed to thaw for 2 to 3 minutes in a water bath at 37° C. Then the veins were removed from the preserving solution and were immersed at room temperature in NaCl (0.15 mol/L) and glucose (0.01 mol/L) (25 ml per vein) to eliminate DMSO.

The endothelial cells from cryopreserved veins were cultured, and their biologic activity was studied in terms of their response to stimulation by thrombin, as described in the following sections.

Cryopreservation of cultured endothelial cells
Confluent cultures of second-passage cells propagated from fresh veins were trypsinized, and 7.5 x 105 cells were suspended in 1 ml aqueous 4% human albumin and 10% DMSO. The suspension was refrigerated, frozen, and stored in liquid nitrogen vapors along with the cryopreserved veins.

Histology
Veins.
Cryopreserved vein segments were fixed in 4% formalin in phosphate-buffered saline solution (PBS), embedded in paraffin, and stained routinely with hematoxylin-eosin-saffron. Immunohistochemical detection of thrombomodulin was carried out with sheep immunoglobulins to human thrombomodulin on 5 µm cyrocut sections embedded in O.C.T. medium. The sections on the microscope slides were incubated with 150 µl diluted (1:100) antibody at 4° C overnight. Biotinylated swine multilink secondary antibody complexed with avidin-biotin-horseradish peroxidase and 3-amino-9-ethyl-carbazole were used for staining.

Cells.
The characteristic cobblestone shape of endothelial cells in culture was observed under a phase-contrast microscope (Olympus CK2, Olympus Optical Co., Ltd., Tokyo, Japan) and under brightfield light illumination in a binocular microscope (Olympus SZ 40 PT) after fixation of the cells with 1% formalin in PBS and staining with May-Grünwald–Giemsa reagent. The stained cells were counted under the light microscope. Immunohistochemical detection of factor VIII–related antigen (vWF) was carried out after fixation of cultured cells with 1% formalin in PBS. An avidin-biotinylated horseradish peroxidase complex was the detecting reagent.

Cell cultures
Endothelial cells collected from fresh and cryopreserved human saphenous veins by scraping were cultured routinely as described previously. Go 11 In brief, cells were cultured in 2 ml RPMI 1640/M 199 culture medium containing L-glutamate, 2 mmol/L, penicillin, 100 IU/ml, streptomycin, 100 µg/ml, fungizone, 2.5 µg/ml, and 30% pooled human AB serum and then plated onto 3.5 cm diameter polystyrene culture dishes precoated with 0.5 ml human plasma proteins (3 mg/ml). After 24 hours, the nonadhered cells were aspirated off with the culture medium. Cells were cultured in a humidified atmosphere containing 95% air and 5% carbon dioxide, and the medium of proliferating cells was changed every 2 days. The population doubling time was calculated from the cell numbers, which were obtained by counting the cells under phase-contrast microscopy every 2 days after seeding. The medium in confluent cultures was changed daily. Confluent cells were detached with 0.05% trypsin and 0.02% ethylenediaminetetraacetic acid and seeded with a 1:3 split ratio for subcultivation. In assays for cell adherence, 2 x 104 cells were seeded onto polystyrene culture dishes coated with fibronectin (10 µg/cm2 ).

Biochemical assays
The thrombomodulin activity of endothelial cells in veins
The technique described for cultured endothelial cells Go 12 was applied to the endothelial layers of both fresh and cryopreserved veins. The veins were opened longitudinally, washed with 20 ml PBS, and inserted between two polystyrene plates (6 x 3 x 2 cm), the upper plate bearing two perforated wells. In the upper plate in each well, 0.55 cm2 endothelial surface could be incubated with 200µl of medium. The veins were fixed with Inox pins (Broadwest Corp., New York, N.Y.) (Fig. 1). The cells in one of the wells were used for the blank assay. To remove blood proteins, the endothelial surfaces were incubated in the wells with 200 µl RPMI 1640/M 199 culture medium containing human serum albumin (10 mg/ml) for 20 minutes at 37° C and then rinsed three times with 200 µl of the same medium. The thrombomodulin activity was evaluated by activation of cells with 20 µl human thrombin (0.25 IU/ml) in the presence of 30 µl protein C (65 nmol/L) in 150 µl RPMI 1640/M 199 culture medium for 90 minutes at 37° C. Then the thrombin was blocked by 20 µl hirudin (1.25 antithrombin U/ml). The supernatant was centrifuged, and the amidolytic activity of the generated protein C was measured in 150 µl aliquots on 40 µl chromogenic peptide substrate (CBS 4146 or CBS 0041; 0.05 mmol/L final concentration) by reading optical densities at 405 nm in a Dynatech MR 500 microplate reader (Dynatech Laboratories, Inc., Chantilly, Va.). The amidolytic activity of the reference cell layer, not incubated with thrombin, was used as a blank to correct optical density values. At the end of the reaction, the cells were incubated with crystal violet, and the released nuclei were counted.



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  		Fig. 1. Culture chamber to test endothelial function in veins.

 
Release of soluble thrombomodulin and vWF
The veins were inserted into the polystyrene plates (see Fig. 1) as described for the determination of thrombomodulin activity. The amount of thrombomodulin released spontaneously into the culture medium from the endothelial surface during 48 hours was determined by enzyme-linked immunosorbent-assay (Asserachrom Thrombomodulin ELISA Kit). The secretion of vWF was measured after incubation of cells for 6 hours in 200 µl RPMI 1640/M199 culture medium in the absence and in the presence of a 1 U/ml concentration of thrombin. The Asserachrom von Willebrand Factor ELISA Kit was used for the determination.

Statistical methods
Results are expressed as means ± standard error of the mean and compared by the Student's t test. The p value was calculated by the nonparametric Wilcoxon test.

RESULTS

Histologic characteristics and function of endothelial cells in fresh and cryopreserved veins
Histologic characteristics
The reaction with antithrombomodulin antibody on the histologic sections revealed a continuous endothelial layer in the fresh veins (Fig. 2, A) and partial endothelial desquamation in cryopreserved veins (Fig. 2, B). The immunohistochemical staining of thrombomodulin diffused into the upper medial layers of cryopreserved veins. After release and staining of the cell nuclei with crystal violet, 1.03 ± 0.04 x 105 and 0.45 ± 0.07 x 105 cells were counted per square centimeter of endothelial surface of fresh veins and cryopreserved veins, respectively (p<0.01).



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  		Fig. 2. Immunohistochemical detection of thrombomodulin in fresh vein (A) and in cryopreserved human saphenous vein (B). (Original magnification x 160.) Staining by there action of peroxydase-labeled antibody with 3-amino-9-ethyl carbazole. Counterstaining with Harris hematoxylin.

 
Thrombomodulin activity.
The endothelial cells in the veins in situ were assayed for their ability to activate protein C in the presence of thrombin. On the basis of cell number, the thrombomodulin activity was decreased in cryopreserved veins compared with the fresh endothelium (Fig. 3). The amounts of protein C activated in response to thrombin were 10.52 ± 2.52 and 1.01 ± 0.36 pmol (p < 0.01) per square centimeter of endothelial surface in fresh veins and cryopreserved veins, respectively. These data indicated that the endothelial dysfunction accentuated the effect of endothelial desquamation on the loss of anticoagulant function. The cryopreserved endothelium spontaneously released a higher amount of soluble thrombomodulin into the medium than the fresh cells did (Fig. 4). The assay of protein C activation indicated that this soluble form was devoid of anticoagulant activity.



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  		Fig. 3. Thrombomodulin activity of endothelial cells from fresh and from matched cryopreserved veins, as indicated by the amount of activated protein C generated within 90 minutes after stimulation of endothelial cells by thrombin (p < 0.067 by the Wilcoxon test). F (triangles), Fresh veins; C (circles), cryopreserved veins.

 


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  		Fig. 4. Spontaneous release of soluble thrombomodulin from the endothelial layer of fresh veins (open column) and cryopreserved veins (black column). Results are means ± standard error of the mean from five independent determinations (p < 0.05).

 
Secretion of vWF.
The fresh endothelium and the cryopreserved endothelium secreted 0.36 ± 0.06 and 0.68 ± 0.34 U vWF per 104 cells, respectively, over a period of 6 hours (not significant). Thrombin increased the secretion of vWF by 30% in the fresh cells, but the secretion of vWF was only about 2% higher than basal values in the cryopreserved endothelium.

Culture of endothelial cells from fresh veins and cryopreserved veins
Endothelial cell suspensions from veins were assayed for adherence and proliferation. The number of the adhered cells was 0.90.09 x 103 cells/cm2 from the fresh veins and 0.2 ±0.03 x 103 cells/cm2 from their cryopreserved counterparts (p < 0.01). The cells seeded from fresh veins were grown to confluency in about 2 weeks, whereas the proliferation of cryopreserved cells was retarded. At the sixteenth day of culture, their cell density was approximately one tenth of that of the fresh cells (Fig. 5). After subcultivation in the first passage, fresh cells and cryopreserved cells (n = 11) proliferated with an essentially identical population doubling time: 39.4 ± 2 and 40.0 ± 3 hours, respectively. Both fresh and cryopreserved cells exhibited cobblestone-like structure and positive immunohistochemical staining for vWF.



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  		Fig. 5. Proliferation of endothelial cells harvested from fresh and from cryopreserved human saphenous veins. Results are means from eleven independent determinations ± standard error of the mean. Differences between fresh and cryopreserved cells are significant (p < 0.01) at each time point. Triangles, cells from fresh veins; circles, cells from cryopreserved veins.

 
The cryopreservation process was applied also to cell suspensions from confluent cultures propagated from fresh veins. The proliferation and the thrombomodulin activity of the cryopreserved cells after thawing was compared with that of fresh cells of the same origin and passage. The number of adhered cells at 2 hours after seeding shows that the plating efficiency of fresh cells was significantly higher than that of cryopreserved cells. Nevertheless, the adhered cryopreserved cells proliferated at the same rate as fresh cells (Fig. 6), in contrast to cells cultured from cryopreserved veins (Fig. 5). At day 1 in culture, the thrombomodulin activity was lower by a factor of about 3 in cryopreserved cells compared with fresh cells. At the third day after seeding in dense cultures, the thrombomodulin activity and the secretion of vWF of cryopreserved cells equaled that of fresh cells and were similar to those measured in fresh veins.



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  		Fig. 6. Proliferation of fresh cells (triangles)and cryopreserved cells (squares) cultured from fresh human saphenous veins. Results are means from six independent determinations ± standard error of the mean. Differences between fresh and cryopreserved cells are significant (p < 0.01) at each timepoint.

 
DISCUSSION

Cryopreservation of human saphenous veins in 4% albumin and 10% DMSO resulted in loss of about 50% of the endothelial cells. The decreased thrombomodulin activity and the increased release of soluble thrombomodulin reflect an impaired anticoagulant function of the remaining cells. The endothelial cells harvested after thawing exhibited poor adherence and retarded proliferation in primary culture compared with cells from fresh veins.

In previous works, Go Go Go 3,8,13 the endothelium-dependent contractile responses in cryopreserved veins have been thoroughly investigated. The results of functional analyses of fibrinolytic activity Go 1 and prostacyclin secretion Go Go 5,7 suggest that the modifications in the biologic activity of cryopreserved veins are not limited to the changes in vascular tone. Endothelial cells participate actively in preventing blood coagulation in vivo. Therefore it is of interest to obtain information on the anticoagulant function of endothelium Go Go Go 10,14,15 in cryopreserved veins.

The thrombomodulin expressed on the endothelial cell surface plays a key role in the regulation of the anticoagulant properties of the endothelium. Go Go 14,16 Thrombomodulin is a receptor for thrombin, and the rate of protein C activation increases about 1000-fold when thrombin is complexed by this receptor. Go 10 The activated protein C can cleave the coagulation cofactors Va and VIIIa, preventing thrombin formation. Go 10 The alteration of the macromolecular specificity of thrombin by its binding to thrombomodulin explains the fact that the complexed thrombin loses its procoagulant properties: it no longer catalyzes the formation of fibrin from fibrinogen, Go 17 and its ability to activate platelets is blocked. Go 18

To evaluate the effect of cryopreservation on the endothelial anticoagulant function, we compared thrombomodulin activity of fresh veins with that of their cryopreserved counterparts. We detected a lower thrombomodulin activity (see Fig. 3) and a higher release of soluble thrombomodulin (see Fig. 4) in cryopreserved veins than in the fresh samples. Decreases in thrombomodulin activity on the surface of human umbilical endothelial cells and of cells from the A549 lung cancer cell line, Go 19 in culture, Go 20 have been correlated to the endocytosis of the thrombin-thrombomodulin complex and not to a release of thrombomodulin. Increased levels of soluble thrombomodulin in the human serum have been reported as a marker of endothelial damage Go Go 20-22 in ischemic heart disease Go 23 and in atheromatous arterial disease. Go 24 Thrombomodulin is a transmembrane protein, Go Go 14,16 and its shedding into the medium could be originated by a proteolytic activity provoked by cryopreservation.

Unlike protein C activation, Go 10 the thrombin-mediated release of vWF does not appear to involve high-affinity binding sites on endothelial cells. Go 25 The vWF secreted by the endothelial cells Go 26 promotes platelet adhesion to the damaged vessel wall, Go 27 and it can be a marker of vascular disorders in plasma. Go 21 The great variations in vWF tend to show that the procoagulant function of cryopreserved cells can be altered at least up to 6 hours after thawing.

To evaluate the potential of endothelial recovery after cryopreservation, we cultured endothelial cells from fresh veins and from their cryopreserved counterparts. Cryopreserved cells proliferated more slowly in primary culture than did cells from fresh veins. One possible explanation is alteration of cell membrane proteins by mechanical breakdown or by proteolysis during the freezing and thawing steps. The apparent return to the normal proliferation rate in cryopreserved cells after first passage could reflect either recovery or selection of an endothelial population Go 28 by cryopreservation and subcultivation. When cultured cells from fresh veins were cryopreserved in the form of isolated cells, the cells restored their proliferation rate with 3 days (Fig. 6). Therefore, cryopreserved cell suspensions recover apparently faster than cell cultures propagated from cryopreserved veins. This difference points to the technical difficulties of cryopreservation of cells with their extracellular matrix in a blood vessel wall. The DMSO cryoprotectant appears to be more efficient on isolated cells than on the whole tissue.

The slow proliferation rate of cells harvested from cryopreserved veins suggests an impaired endothelial healing in these veins in the clinical setting. The endothelial desquamation could be greater after an arterial implantation under pulsatile blood flow than after thawing in situ. The cumulative effect of deendothelialization and decreased thrombomodulin activity of surviving cells could result in a persisting exposure of thrombogenic subendothelial matrix to blood. This interpretation of our results in vitro is supported by the poor patency and lack of reendothelialization in distal bypasses with cryopreserved human saphenous vein allografts. Go 29

Acknowledgments

We thank Charles Wildevuur, MD, PhD, for helpful discussion and for reviewing the manuscript and Maryvonne Ginat for technical assistance.

Footnotes

From the Centre de Recherches Chirurgicales Henri Mondor, CNRS URA 1431 (Directeur Professeur D. Loisance), and Centre Départemental de Transfusion Sanguine, a CHU Henri Mondor, Créteil, France. Back

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