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Ann Thorac Surg 2010;89:512-520. doi:10.1016/j.athoracsur.2009.10.037
© 2010 The Society of Thoracic Surgeons
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Original Articles: Adult Cardiac

Impact of Normothermic Perfusion and Protein Supplementation on Human Endothelial Cell Function During Organ Preservation

Thomas Puehler, MDa,*, Otto Gleich, PhDb, Simon Schopka, MDa, Leopold Rupprecht, MDa, Stephan Hirt, MDa, Christof Schmid, MDa, Karla Lehle, PhDa

a Department of Cardiothoracic Surgery, University Medical Center Regensburg, Regensburg, Germany
b Ear, Nose, and Throat Department, University Medical Center Regensburg, Regensburg, Germany

Accepted for publication October 14, 2009.

Abbreviations and Acronyms ATP = adenosine triphosphate; BSA = bovine serum albumine; CMS = culture medium with 10% human serum; DAPI = 4,5-diamidino-2-phenylindole; DMSO = dimethylsulfoxid; EC = endothelial cells; EDTA = ethylendiamin-tetraacetat; HS = human serum; HSVEC = human saphenous vein endothelial cells; HTK = histidine-tryptophan-ketoglutarate; HTX = heart transplantation; MTS = 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4 sulfophenyl)-2H-tetrazolium, inner salt; NaCl = sodium chloride; PBMC = peripheral blood mononuclear cells; PBS = phosphare buffered sialine; SD = standard deviation; TNF = tumor necrosis factor; UW = University of Wisconsin solution

* Address correspondence to Dr Puehler, Department of Cardiothoracic Surgery, University Medical Center, Regensburg, D-93042, Germany (Email: thomas.puehler{at}klinik.uni-regensburg.de).


   Abstract
Top
 Abstract
Introduction
 Material and Methods
 Results
 Comment
 Acknowledgments
 References
 
Background: Hypothermia-induced changes in endothelial cell (EC) morphology and function after organ storage may influence the initial outcome and development of transplant-associated coronary artery disease.

Methods: Human saphenous vein ECs were incubated with saline (NaCl), University of Wisconsin (UW), and histidine-tryptophan-ketoglutarate (HTK) solution, with and without protein additives, at 4°C and 37°C. After 6 hours, ECs were recultivated for 24 and 48 hours with culture medium (reperfusion). Mitochondrial activity, adenosine triphosphate concentration, cell count, and inflammatory responses were analyzed.

Results: Cold preservation did not affect the mitochondrial activity of ECs and allowed a complete regeneration of the metabolic turnover after reperfusion. However, under normothermic conditions the metabolism of the cells was influenced by time and type of preservation solution. While both the mitochondrial activity and cell count did not change after treatment with NaCl and culture medium, the metabolic turnover of cells treated with HTK and UW solution significantly increased (twofold) and decreased (twofold, p < 0.05), respectively, after reperfusion. The endothelial reactivity remained unchanged after treatment with NaCl and HTK. The addition of serum proteins significantly improved mitochondrial activity of cells treated with warm NaCl and HTK (p < 0.05). The UW-treated cells burned out through a significant up-regulation of the ATP concentration resulting in a complete metabolic regression after reperfusion and induction of apoptosis.

Conclusions: Normothermic preservation in UW prevented regeneration of ECs, while treatment with HKT solution did not irreversibly affect mitochondrial activity of ECs and allowed complete regeneration of metabolism and function. Serum proteins improved the preservation effect of HTK and NaCl.


   Introduction
Top
 Abstract
 Introduction
Material and Methods
 Results
 Comment
 Acknowledgments
 References
 
Cold preservation is the standard procedure for organ transplantation to minimize hypoxic injury during ischemia time [1]. However, hypothermia causes direct deleterious cellular effects and injury through a number of pathways that occur upon reperfusion and increase with as the duration of cold preservation increases [2, 3]. The cellular compartment most sensitive to preservation injury is the endothelial cell (EC) [4, 5], which is also the first target during reperfusion. As a consequence, cellular swelling, platelet accumulation, impairment of procoagulant and anticoagulant properties, and leukocyte adherence [3] might cause worsened initial outcome after heart transplantation (HTx) [6]. In this context, improvements of preservation solutions are one opportunity to minimize changes in cellular homeostasis, membrane barrier function, and cell volume during cold storage [1, 2, 7–9]. Cold University of Wisconsin (UW) [10] and Bretschneider's histidine-tryptophan-ketoglutarate [11] solutions are commonly used for cardiac preservation. While in most studies UW was rated superior for EC preservation during hypoxic storage [6, 12, 13], the subsequent rewarming-reoxygenation exhibited an increase in the expression of inflammatory and stress proteins [13], leading to cold-induced cell volume changes and imbalances in cellular ion homeostasis [14]. Furthermore, cold storage in HTK also disturbed the EC integrity [15].

A "new paradigm for organ preservation" [16] favored warm preservation to maintain physiologic temperature and organ function during preservation for a successful transplantation. Brockmann and colleagues used a pig liver transplant model to show that warm perfusion under physiologic pressure and flow enabled prolonged preservation and successful transplantation. This model is based on the preservation of the liver, but it is likely to be applicable to other organs [16]. Other novel preservation techniques include the optimization of the solution composition with additives [8, 9, 17], and the introduction of normothermic blood perfusion preservation, permitting the maintenance of normal cellular metabolism [18]. So far, the benefit of normothermic preservation was only documented in experimental studies [17, 18] and has not achieved widespread clinical applicability [18].

In our experimental study, we optimized standard cardiac preservation solutions by the addition of plasma proteins and used normothermic preservation conditions to avoid cold shock-induced injury of EC during standard organ preservation. The primary outcome of our study was the preservation of EC metabolism, morphology, and function.


   Material and Methods
Top
 Abstract
 Introduction
 Material and Methods
Results
 Comment
 Acknowledgments
 References
 
Cell Culture and Experimental Protocol
We obtained saline (NaCl) from Baxter (Unterschleißheim, Germany), UW from DuPont (Bad Homburg, Germany), and HTK from Dr. Köhler Chemie (Alsbach, Germany). After informed consent of the patients and agreement by the local ethics committee, human ECs were derived from saphenous veins (HSVEC) using standard isolation procedures [19, 20]. Cells were cultured in CMS (culture medium with 10% human serum; Cat.No. 2010111, Provitro, Berlin, Germany) on 0.1% gelatin-coated (Sigma, Munich, Germany) tissue culture flasks (Falcon, Heidelberg, Germany). Cells in passage 1 were deep-frozen in 10% dimethylsulfoxide (Gibco, Karlsruhe, Germany) and recultured before the experiment started. The HSVEC were characterized by their "cobblestone morphology" and factor VIII-related antigen expression [19]. The HSVEC (4.000 cells/cm2) were grown for 5 days in 96-well plates (Falcon). For preservation conditions, HSVEC were carefully washed and incubated in UW, HTK, NaCl (0.9 %), and CMS (= control) for 2 to 6 hours at 4°C and 37°C, respectively. After a 6 hour preservation, prewarmed CMS was added for another 24 (= 6 + 24) and 48 (= 6 + 48) hours under standard culture conditions (= reperfusion). The latter was defined as the regeneration of the metabolic turnover. At each time point, the level of mitochondrial activity [21], the absolute cell count, and the adenosine triphosphate (ATP) content [22] were measured (see Appendix to Materials and Methods).

Furthermore, the impact of different additives (bovine serum albumin [BSA], Sigma; HS, human serum) to the preservation solutions was evaluated analyzing the mitochondrial activity. In addition, the influence of selected preservation solutions on the endothelial function (expression of cellular adhesion molecules, adhesion of peripheral mononuclear blood cells) and the apoptosis induction (apoptotic bodies) was checked. Details on the test systems and the experimental protocol were presented in the Appendix.

Statistics
Data are expressed as mean ± standard deviation (SD). The statistical analysis was performed using the software packages SPSS15 (SPSS Inc, Chicago, IL) and SigmaStat 2.03 (Systat Software, San Jose, CA). To analyze the different factors a 2-way and 3-way analysis of variance (ANOVA) and post-hoc pairwise Bonferroni t test was used. A p value less than 0.05 was considered significant. We report and indicate only relevant significant findings in the figures. The details on statistical analysis are shown in the Appendix.


   Results
Top
 Abstract
 Introduction
 Material and Methods
 Results
Comment
 Acknowledgments
 References
 
Cold Preservation
After cold preservation, the mitochondrial activity as a function of time was very similar for all the media tested (Fig 1A). The activity remained constant over a time period of 6 hours independent of the different preservation techniques. However, after reperfusion at 37°C the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4 sulfophenyl)-2H-tetrazolium (MTS) absorbance increased significantly. The MTS value doubled from a mean of 0.68 at 2 hours (preservation) to 1.26 at 48 hours reperfusion, independent of the preservation conditions. Hypothermic preservation did not affect the absolute cell count of the EC monolayers (data not shown).


Figure 1
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  		Fig 1. Impact of cold preservation on the mitochondrial activity (A) and the ATP content (B) in HSVEC. The HSVEC were pretreated with CMS (= control), NaCl, UW, and HTK for 2 and 6 hours (preservation). After 6 hours solutions were replaced with CMS and cells were cultivated under standard culture conditions (24 and 48 hours reperfusion). The MTS absorbance and ATP content was determined as described in the Appendix. (*p < 0.05 comparing different incubation times; # comparing different solutions.)

 
In contrast, the composition of the preservation solutions affected the adenosine triphosphate (ATP) synthesis of the cells under hypothermic preservation. After 6 hours the ATP value was highest in UW (mean 12,817) and significantly different from CMS (mean 57,511) and HTK (mean 79,609; p < 0.05). After reperfusion the ATP concentration remained unchanged in cells treated with CMS, NaCl, and HTK, while the synthesis of ATP in UW-treated cells doubled significantly (Fig 1B).

Effect of Normothermic Preservation on Mitochondrial Activity, Cell Count, and ATP Content
Under normothermic conditions the preservation solutions showed quite different effects on the mitochondrial activity, the cell count in the monolayer, and the ATP content of preserved HSVEC (p < 0.001) (Figs 2; 3). As shown in Figure 2A, the ANOVA revealed no significant effect of incubation time on the parameters for cells incubated in CMS. In addition, the mean cellular volume of the cells in CMS remained constant over the whole test period (6 hour preservation: 4,700 ± 208 femtoliters [fL]; 48 hours regeneration: 4,660 ± 115 fL).


Figure 2
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  		Fig 2. Impact of warm preservation on the mitochondrial activity and cell count. The HSVEC were preserved with CMS (A), NaCl (B), HTK (C), and UW (D). After 6 hours solutions were replaced with CMS and cells were cultivated under standard culture conditions (24 and 48 hours reperfusion). The MTS absorbance (open symbols) and cell count per 96-well (filled symbols) were determined as described in the Appendix. (*p < 0.05 comparing different incubation times.)

 

Figure 3
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  		Fig 3. Impact of warm preservation on the intracellular ATP content. The HSVEC were preserved for 6 hours with CMS, NaCl, HTK, and UW and reperfused with CMS for an additional 24 and 48 hours. For experimental details see the Appendix. (*p < 0.05 comparing 6 hour preservation and 24 hour reperfusion after UW-treatment; # comparing different solutions.)

 
Incubation of HSVEC in NaCl for 6 hours did not significantly affect the mitochondrial activity over time (Fig 2B). However, MTS activity in NaCl was significantly lower than in cells treated with CMS at each incubation time. Medium exchange with CMS after NaCl led to a significant increase of cell number at 24 and 48 hour reperfusion compared with 6 hour preservation (Fig 2B). Consistent with the MTS activity data, cell number was significantly lower in NaCl compared with CMS. In addition, NaCl treatment significantly reduced mean cellular volume compared with CMS (3,760 ± 348 fL) during preservation while the subsequent reperfusion balanced the value to control conditions (4,800 ± 31 fL). After preservation in NaCl the ATP content was, by a factor of 2 to 3, significantly lower than after CMS treatment (p < 0.05). In addition, reperfusion of NaCl-treated cells inhibited the ATP synthesis (222 ± 126).

In HTK the mitochondrial activity as well as the cell count increased significantly (Fig 2C) from preservation to reperfusion at 48 hours. In addition, the mean cellular volume of treated cells increased significantly after reperfusion (6 hour preservation: 4,160 ± 114 fL; 48 hour reperfusion: 4,630 ± 69 fL). After preservation in HTK the ATP content was by a factor of 2 significantly lower than after CMS treatment (p < 0.05). Subsequent regeneration did not affect the ATP concentration in the cells (Fig 3).

The UW-treated HSVEC showed a completely divergent time response (Fig 2D) as compared with HTK. In UW reperfusion significantly decreased the MTS values as well as the cell count compared with preservation. The mean cellular volume of UW-treated cells was very high during preservation (5,940 ± 329 fL) while after reperfusion the values returned to control levels (4,460 ± 198 fL). The ATP concentration (Fig 3) was highest in UW-preserved cells and significantly different to all other media tested during preservation. After reperfusion, the ATP content decreased significantly for UW-treated HSVEC.

Effect of Preservation on Endothelial Function
To check the continuance of the endothelial reactivity after preservation-reperfusion of HSVEC in HTK and NaCl, the inflammatory response was analyzed. Cold preservation did not affect the basal expression of cellular adhesion molecules (E-selectin) and adhesion of peripheral blood mononuclear cells (PBMC) (Figs 4A and 4B). After stimulation with TNF the amount of E-selectin significantly increased by a factor of 23 ± 5, 28 ± 4, and 31 ± 7 for control cells, HTK-treated cells, and NaCl-treated cells, respectively. Simultaneously, TNF significantly enhanced the adhesion of PBMC by a factor of 1.9 ± 0.4, 2.9 ± 1.0, and 2.9 ± 1.1 for preservation in CMS, HTK, and NaCl, respectively. Thus, inflammatory responses of treated EC were independent of the type of preservation solution. As shown in Figures 4C and 4D a similar response was documented after normothermic preservation. Preservation in warm HTK and NaCl did not influence the basal and TNF-induced expression of E-selectin and adhesion of PBMC. The tumor necrosis factor (TNF) increased the expression of E-selectin (CMS: 24 ± 3; HTK: 26 ± 4; NaCl: 17 ± 10) and the adhesion of PBMC (CMS: 1.9 ± 0.9; HTK: 4.7 ± 6.8; NaCl: 2.4 ± 1.8) independent of the type of preservation solution.


Figure 4
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  		Fig 4. Impact of cold (A) (B), and warm (C) (D) preservation on the expression of E-selectin (A) (C) and adhesion of PBMC (B) (D). The HSVEC were preserved with CMS, HTK, and NaCl (6 hours), reperfused with CMS for an additional 48 hours, and treated with TNF or PBS. (#p < 0.05 comparing basal and TNF-induced responses; *comparing with maximal TNF response of control cells.)

 
Exceptional Position of UW-treatment
As shown in Figure 2D, normothermic preservation of HSVEC in UW prevented regeneration of the mitochondrial activity. The amount of apoptotic cells increased after warm preservation in UW (Fig 5A). While the nuclei of control cells remained unchanged (Figs 5C and 5E) the chromatin in nuclei of cells treated with warm UW was fragmented (Figs 5D and 5F). The total amount of cells decreased during reperfusion in CMS. In contrast, cold UW did not affect the nuclear morphology of the cells and the amount of viable cells remained unchanged up to 48 hours of reperfusion (Fig 5B).


Figure 5
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  		Fig 5. Apoptose-induced activity of UW-treated cells. The HSVEC were preserved (6 hours) with warm (A) and cold (B) CMS (circles) and UW (diamonds), reperfused with CMS (48 hours), fixed, and DAPI stained. (Filled symbols = amount of viable cells; closed symbols = amount of apoptotic nuclei.) (C) Six hours in warm CMS; (D) 6 hours in warm UW; (E) reperfusion after warm CMS preservation; (F) reperfusion after warm UW treatment. (Magnification [40x]).

 
Serum Protein Supplementation Under Normothermic Preservation
Because cold preservation did not affect the mitochondrial activity of the cells (see above) an improvement of the preservation conditions under normothermic conditions was required. As shown in Figure 6, the addition of serum proteins to preservation solutions affected the mitochondrial activity of HSVEC differently suggesting complex interactions. The variation of MTS absorbance for CMS and the other media without additives closely resembles the data presented in Figure 2 at each time point. These were independent sets of measurements and the similarity of data over time for the different media, shown in Figures 2 and 6, demonstrates the reproducibility of the data.


Figure 6
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  		Fig 6. Mitochondrial activity (MTS absorbance) in HSVEC after normothermic (37°C) preservation (A), (D), (G) in solutions with serum proteins and reperfusion in culture medium at 37°C for 24 hours (B), (E), (H) and 48 hours (C), (F), (I). NaCl (A), (B), (C), HTK (D), (E), (F), and UW (G), (H), (I) were spiked with 4% and 10% bovine serum albumin (BSA) and human serum (HS). The ECs were stored in CMS (black bars) and spiked preservation solutions (white and patterned bars) for 6 hours; medium was exchanged and cells were incubated in CMS at 37°C (reperfusion) for 24 and 48 hours.

 
During 6 hour preservation (Figs 6A,D,G), there was no significant interaction between medium and additive condition. Over all, MTS absorption in the absence of an additive was significantly lower compared with the other four conditions, while 10% HS was significantly higher compared with both BSA concentrations.

The MTS values increased over time in NaCl with all additives (Figs 6A–C). At both reperfusion times MTS values in the presence of an additive were significantly higher compared with the condition without additive. At 24 hours, 10% HS was significantly more effective than 10% BSA and 4% HS, while there was no significant difference among the four additives at 48 hours.

In HTK (Fig 6D–F) the MTS absorbance significantly increased for all conditions of the additive from preservation to reperfusion. During both reperfusion times there was no significant effect of different additive conditions.

In contrast to preservation in NaCl and HTK, incubation of HSVEC in UW significantly decreased the mitochondrial activity over time (Fig 6G–I). The addition of serum proteins to UW during preservation did not improve the MTS activity of the cells. Especially for both concentrations of HS the MTS absorbance was significantly decreased from preservation to 24 hours, and also from 24 to 48 hours of reperfusion.


   Comment
Top
 Abstract
 Introduction
 Material and Methods
 Results
 Comment
Acknowledgments
 References
 
We were interested in the direct effects of cardiac preservation solutions, UW, and HTK solutions on EC for periods similar to those employed in current clinical practice. To exclude hypothermic cellular damage we focused on normothermic storage of EC and the addition of serum proteins. The experiments were performed with patient-derived saphenous vein EC, an endothelial model, previously employed to evaluate cytotoxicity of cryoprotective solutions [23], immunosuppressive drugs [24], and different substrate material for cardiovascular tissue engineering [25, 26]. Furthermore, a functional similarity of HSVEC and arterial EC was documented by Tan and colleagues [27]. Independent methods were employed; mitochondrial activity, cell counting, ATP production, and inflammatory responses.

In our experimental setup cold preservation of EC in HTK, UW, and also NaCl performed reasonably well. Six hour incubation in cold HTK and UW did not affect the mitochondrial activity and the membrane integrity. Reperfusion in warm nutrient solution enabled a complete regeneration of the metabolic turnover. Not even incubation in saline solution at 4°C inhibited regeneration with culture medium. These findings are supported by experimental and clinical studies, which suggest a superiority of UW in preservation of liver, pancreas, and small bowel as well as cardiac and lung allografts [28, 29]. The superior protective potential of UW for EC was verified by unchanged morphologic and bioenergetic evaluation [12, 13]. However, after reperfusion of UW-treated human umbilical vein endothelial cells (HUVEC) the expression of membrane-bound adhesion molecules and stress proteins was considerably upregulated [13]. In addition, the usage of HTK interrupted the integrity of the EC in the monolayer [12, 13]. Nevertheless, as shown in our present study, reperfusion of cells treated with cold preservation solutions at 4°C with nutrient solution allowed a complete regeneration of the mitochondrial activity after 48 hours. The same effect was presented by Alamanni and colleagues [30]. Only a 6 hour and not a 24 hour cold storage of HSVEC with preservation solutions allowed a regeneration of EC [30]. However, despite this reversibility of endothelial function the initial injury after cold storage impairs microcirculation by leukocyte adherence during reperfusion [31] and finally ends in an enhanced rate of complications and unfavorable outcomes after HTx [32, 33].

To exclude cold shock we focused on the effect of normothermic storage on endothelial metabolic activity. While a 6 hour warm incubation in HTK did not affect the mitochondrial activity and the proliferative activity of the cells (Fig 2C), saline and UW showed a significant interaction of time and temperature. Warm perfusion with saline impaired metabolic turnover and the regeneration after reperfusion with nutrient solution (Fig 2B). This was also described after long-term incubation (24 to 72 hours) of HUVEC with cold saline causing a significant reduction in viable cell counts and severe alteration of morphology [13]. The effect on the viability of the cells was dramatic after incubation in warm UW. After 6 hours in warm UW, the mitochondrial activity as well as the absolute cell count decreased significantly by about 40% and 80%, respectively. The HSVEC failed to regenerate after reperfusion with culture medium (Fig 2D). The high concentration of adenosine in the UW and the high temperature stimulated the ATP synthesis of HSVEC (Fig 3). One consequence was the disproportionate energy consumption of the cells which finally failed to regenerate after rewarming. Our data clearly demonstrated that treatment of HSVEC with warm UW induced apoptosis and inhibited cell regeneration (Fig 5). Already 3 hours after addition of UW half of the cells showed condensation of chromatin in the nuclei resulting in a decreasing cell density. In contrast, short-term cold preservation in UW did not affect cell survival. Regarding all tested preservation solutions, only warm storage in HTK allowed a complete regeneration after reperfusion. Obviously neither the mitochondrial activity nor the integrity of the monolayer was changed. In addition, treatment of HSVEC with warm HTK had no stimulatory effect; the basal expression of cellular adhesion molecules as well as the adhesion of PBMC remained unchanged. Furthermore, preservation in warm HTK did not affect the response of HSVEC to exogenic stimuli such as TNF. Even storage in warm NaCl allowed regeneration of the mitochondrial activity and the maintenance of the inflammatory reactivity of the cells. However, there was a high interindividual variability regarding different HSVEC cultures (Fig 2B). Overall, the efficiency of warm preservation is viewed as controversial [18]. Already in 1981, Carpentier and colleagues [34] evaluated the toxicity of several cardioplegic solutions, documenting that 3 hour incubation of human ECs and fetal fibroblasts with several cardioplegic solutions at 19°C followed by reperfusion with culture medium negatively affects cell viability. They concluded that temperature and addition of blood to the various solutions significantly influenced cytotoxicity.

Addition of either blood or albumin to cold crystalloid cardioplegic solutions improves preservation of endothelium-dependent relaxation [17], and cold as well as warm blood cardioplegia provides better protection of endothelium-dependent relaxation than crystalloid cardioplegia [35–37]. Kruman and colleagues [38] demonstrated that the sensitivity to cold shock-induced cell death was critically dependent on the serum concentration in the medium and limited to serum-deficient medium, whereas cells in the complete growth medium with 10% serum were completely resistant. In our present study, the addition of serum albumin and human serum to warm saline prevented extensive mitochondrial injury and allowed a complete regeneration in culture medium. Obviously, an increase in the plasma protein concentration or addition of albumin to crystalloid cardioplegic solutions adjusted the colloid osmotic pressure (COP) of the preservation solutions and prevented the swelling-destruction of treated cells [39]. The protective effect of protein addition to HTK on EC survival was marginal (Figs 4D–F). The HTK, per se, performed an endothelial protection. The formula of the HTK is based on a high histidine concentration, which results in a potent buffer. We speculated that HTK preserved the COP. The protective effect of protein addition seems to depend on the underlying preservation solution. Thus, neither high concentrations of serum albumin nor 10% human serum changed the negative effect of warm storage in UW (Figs 6G–I). Obviously, the combination of serum proteins and type of cardioplegic solution may preserve endothelium-dependent microvascular responses [17]. In the clinical routine normothermic surgery with warm blood cardioplegia is becoming more and more frequently used [36, 40]. The critical components in the blood cardioplegia are still unknown. Further studies may optimize the efficiency of organ preservation solutions and dissolve the mystery of blood perfusion.


   Appendix
 
Preservation Solutions
We obtained NaCl solution (0.9%) from Baxter (Unterschleißheim, Germany), UW solution from DuPont (Bad Homburg, Germany), and HTK from Dr. Köhler Chemie (Alsbach, Germany).

Cell Culture
Human EC were derived from saphenous veins (HSVEC) of 7 patients undergoing coronary artery bypass surgery, according to a protocol approved by the local human ethical committee. The ECs were isolated with collagenase treatment (Roche, Basel, Switzerland) [19, 20]. Cells were resuspended in endothelial medium (Provitro, Berlin, Germany) containing 10% pooled heat-inactivated human serum (CMS, culture medium with serum), seeded on a 0.1% gelatin-coated (Sigma, Munich, Germany) tissue culture flask (T25; Falcon, Heidelberg, Germany). After 7 to 10 days, ECs were washed with PBS/BSA (phosphate-buffered saline, 0.02% bovine serum albumin), detached, and recultured in CMS. Cells in passage 1 were deep-frozen in 10% dimethylsulfoxide (Gibco, Karlsruhe, Germany) and recultured 2 days before the experiment started. The HSVEC were characterized by their "cobblestone morphology" and factor VIII-related antigen expression [21].

Experimental Protocol
The HSVEC (4,000 cells/cm2) were grown for 3 days in gelatin-coated 96-well plates (Falcon). Supernatant was exchanged for an additional 2 days (90% confluence). For preservation conditions, HSVEC were carefully washed with prewarmed PBS/BSA and incubated in UW, HTK, NaCl, and CMS (= control) for 2 to 6 hours at 4°C and 37°C, respectively. After a 6 hour preservation, prewarmed CMS was added for another 24 (= 6 + 24) and 48 (= 6 + 48) hours under standard culture conditions (= reperfusion). The latter was defined as the regeneration of the metabolic turnover. At each time point, the level of mitochondrial activity, the absolute cell count, and the adenosine triphosphate (ATP) content was measured (see below). In another set of experiments the impact of different additives (BSA, Sigma; HS, human serum) to the preservation solutions was evaluated analyzing the mitochondrial activity (n = 6).

Test Systems
The mitochondrial activity of 5 different HSVEC cultures and the ATP content of 3 different HSVEC cultures (see below) specified the effect of preservation solutions, time of preservation-reperfusion, and storage temperature on cell viability. In addition, the quantification of the cell count (5 different HSVEC cultures) after treatment with different solutions reflected the integrity of the cells in a monolayer. Each parameter was measured in quadruplicate on separate 96-well plates that were exclusively prepared for the respective interval of preservation and reperfusion (experimental protocol).

Mitochondrial dehydrogenase activity was assessed by using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTS) test (Promega, Madison, WI), which reflects the capability of mitochondria to reduce MTS [Corey and colleagues 1991]. After preservation and reperfusion, cells were washed and incubated with serum-free endothelial medium + MTS substrate (1 hour). After addition of 1% Triton X-100 (Merck, Darmstadt, Germany) the absorbance was measured at 570 nm. Higher absorbance corresponds to higher enzymatic activity and a better metabolic turnover of the cells.

The cell count per 96-well (= 0.3 cm2) was calculated after proteolytic digestion and resuspension in CMS (150 µL). An aliquot was diluted in isotonic buffer solution (Innovatis, Reutlingen, Germany). Cell count and the mean volume of single cells (in femtoliter = 10–15 L) were measured using an automated cell counter (CASY-TTC, Innovatis).

The HSVEC preservation injury was characterized by evaluation of the intracellular content of adenosine triphosphate (ATP) [22]. Cells were seeded in the same way as described above on 96-well plates (White, TopCount; Packard Bioscience, Dreieich, Germany). The ATP was measured by a luminescence ATP detection assay after 6 hours of preservation and an additional 24 hours of regeneration in CMS (CellTiter-Glo, Promega), according to the manufacturer's instructions.

Endothelial Function
According to the experimental protocol, HSVEC (n = 7) were seeded on 96-well plates. Confluent monolayers were incubated with CMS (= control), HTK, and NaCl for 6 hours at 4°C and 37°C (= preservation), respectively. After preservation, prewarmed CMS was added for another 48 hours under standard culture conditions (= reperfusion-regeneration). To analyze the inflammatory response of ECs, 10 µL tumor-necrosis factor (10 ng/mL; Merck, Darmstadt, Germany) or PBS (= nonactivated) was added for another 4 hours.

In one set of experiments, the surface-expression of E-selectin was analyzed using a cellular enzyme-linked immunosorbent assay (ELISA) [20]. The HSVEC were fixed in a mixture of acetone-methanol (1:1) at –20°C for 10 minutes, rehydrated in PBS/0.2% bovine serum albumin (Sigma) and incubated for 1 hour at 37°C with 50 µL of the primary antibody (mouse antihuman E-selectin; Ancell, Bayport, MN) diluted 1:1,000 with PBS/0.02% BSA. After two washing procedures, a biotinylated goat antimouse IgG (50 µL/well, 1:1,000; Vector, Burlingame, CA) was incubated for 30 minutes at 37°C, followed by repeated washing and further incubation (30 minutes) with alkaline phosphatase streptavidin (50 µL/well, 1:1,000; Vector). The substrate p-nitrophenylphosphate (1 mg/mL, Sigma-Aldrich) in 0.1M diethanolamine pH 10.3 (Sigma-Aldrich) was added, and after 30 minutes the absorption was measured at 405 nm with an ELISA-reader (MWG-Biotech, Ebersberg, Germany).

In another set of experiments, the adhesion of peripheral mononuclear cells on treated cells was analyzed. Blood from male healthy adult volunteers was collected into ethylendiamine-tetraacetate monovettes. Peripheral blood mononuclear cells (PBMC) were isolated by Ficoll density gradient separation (1.073 g/mL; GE Healthcare, Uppsala, Sweden) (900g, 15 minutes, room temperature). The PBMC were isolated from the buffy coat layer, washed two times in PBS (300g, 10 minutes), loaded with calcein-acetomethylester (calcein-AM; stock solution: 1 mg/mL in dimethyl sulfoxide; working concentration: 5 µg/mL; Molecular Probes, Eugene, OR), and counted using a Neubauer hemocytometer. For the adhesion assay, treated HSVEC were incubated with 2 x 105 PBMC per 0.3 cm2 culture area (37°C, 5% CO2, 30 minutes). Nonadherent PBMC were removed by rinsing three times with PBS. Adherent PBMC were lysed in 1% triton X-100 (Biorad, Hercules, CA) and frozen overnight. After thawing lysates were transferred into new 96-well plates. Fluorescence intensity (385/535 nm) was measured using the Wallac 1420 Victor 3TM Plate Reader (PerkinElmer, Boston, MA). The amount of adherent PBMC was estimated using a standard curve. Definite amounts of labeled PBMC were correlated with respective fluorescence intensity.

Apoptosis-inducing Activity of Preservation Solutions
The HSVEC (n = 2) (3,000 cells/cm2) were seeded on gelatin-coated Permanox chamber slides (10 cm2; Nunc, Langenselbold, Germany) for 5 days. The confluent monolayers were incubated in 2 mL UW and CMS (= control) for 6 hours at 4°C and 37°C (= preservation), respectively. After preservation, prewarmed CMS was added for another 48 hours under standard culture conditions (= reperfusion). Treated cells were fixed in 4% paraformaldehyde, stained with 4,6-diamidino-2-phenylindole (DAPI; 0.5 µg/mL; Sigma) and fluorescent nuclei were counted under a fluorescent microscope. Apoptosis is characterized by a specific sequence of nuclear changes culminating in chromatin condensation and nuclear fragmentation (apoptotic bodies) [41]. Quantitative analysis comprised counting the number of apoptotic cells in relation to all identifiable cells from 10 microscopic fields. Results were determined as percentage of apoptotic cells.

Statistics
Data are expressed as mean ± standard deviation (SD). The statistical analysis was performed using the software packages SPSS15 (SPSS Inc, Chicago, IL) and SigmaStat 2.03 (Systat Software, San Jose, CA). For the analysis of MTS absorbance a 3-way ANOVA was performed with the fixed factors being temperature (4°C, 37°C), time (2, 6, 6 + 24, 6 + 48 hours), and preservation solution (CMS, NaCl, HTK, UW). The dependent variable was the mean MTS value of the 4 assays performed for each combination of the different test conditions. Only for significant main effects, subsequent post-hoc pairwise Bonferroni t tests were used. For the analysis of the dependent variables cell number and cell volume, a 2-way ANOVA with the factors medium (CMS, NaCl, HTK, UW) and time (6, 6 + 24, 6 + 48 hours) was performed. For the analysis of intracellular ATP we used a 3-way ANOVA with the fixed factors time (6, 6 + 24 hours), temperature (4°C, 37°C), and medium (CMS, UW, HTK, NaCl) with the ATP value as dependent variable. To evaluate the potential effect of different additives, a 3-way ANOVA was performed with medium (NaCl, HTK, UW), additive (none, 4% BSA, 10% BSA, 4% HS, 10% HS), and time (6, 6 + 24, 6 + 48 hours) as factors and MTS absorbance as the dependent variable. The CMS data without an additive are shown as a reference in Figure 4 but were not included in the statistical evaluation of different additives. A p value less than 0.05 was considered significant. For the sake of clarity we report and indicate only relevant significant findings in the figures.


   Acknowledgments
Top
 Abstract
 Introduction
 Material and Methods
 Results
 Comment
 Acknowledgments
References
 
The authors gratefully acknowledge the excellent technical assistance of Sara Bergmann and Christina Leykauf.


   References
Top
 Abstract
 Introduction
 Material and Methods
 Results
 Comment
 Acknowledgments
 References
 

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Louis M. Chu and Frank W. Sellke
Ann. Thorac. Surg. 2010 89: 520-521. [Extract] [Full Text] [PDF]



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