Optimization of an immunohistochemical method for detection of hypoxia in tissue engineered constructs

L.M. Kock s020730 Supervision: R.A.A. Pullens C.V.C. Bouten M.J. Post Eindhoven, 17 March 2006

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Contents

1 Introduction 3 2 Materials and methods 5

2.1 Cell culture………………………………………………………………………………….... 5 2.2 Constructs……………………………………………………………………………………… 5

2.3 Histology……………………………………………………………………………………….. 5 2.4 Hypoxyprobe-1 immunohistochemistry…………………………………………... 5 2.5 Hypoxia through mineral oil layer…………………………………………………… 6

2.5.1 Experiment A: Effect of different incubation times………………. 6

2.5.2 Experiment B: Effect of adding pimonidazole at different time points ………………………………………………………..... 6

2.6 Hypoxia through infusion of gaseous N2………………………………………… 7 3 Results 8

3.1 Hypoxia through mineral oil layer………………………………………………….. 8 3.1.1 Effect of different incubation times…………………………………….. 8 3.1.2 Effect of adding pimonidazole at different time points……….... 8 3.2 Hypoxia through infusion of gaseous N2……………………………………….... 8 3.2.1 Effect of different incubation times…………………………………….. 8 3.2.2 Effect of re-oxygenation……………………………………………………… 9 3.3 Hypoxyprobe-1 Mab labeling in constructs……………………………………… 10

4 Discussion 11 Bibliography 14 A Protocols 15 A.1 Hypoxia staining on a monolayer…………………………………………………… 15 A.2 Hypoxia staining on coupes of constructs……………………………………….. 15

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Chapter 1: Introduction

Tissue engineering aims to develop biological substitutes for implantation into the body and/or to tissue regeneration and remodeling, with the purpose being to replace, repair, maintain, or enhance function. The engineered tissue, or construct, usually consists of three-dimensional scaffolds and living cells with the desired structure and functionality. One of the important challenges in tissue engineering at the moment is creating thicker (cardiovascular) constructs. The thickness of a tissue engineered construct is currently limited to the diffusion of nutrients to the cells in the center of the construct. When the cells in the center do not get enough nutrients, they will not stay alive. In order to create thicker constructs in which cell viability throughout the construct is maintained, vascularization in vitro is necessary [1]. That is why tissue engineering of blood vessel substitutes is another important goal of tissue engineering. Three basic structural constituents make up the walls of normal blood vessels: endothelium, smooth muscle and connective tissue, including elastic elements (figure 1). The endothelium consists of endothelial cells (ECs) which form a monolayer that lines the entire vascular system. ECs are polygonal, elongated cells that have many pinocytotic vesicles and form junctional complexes with their neighbors. Vascular endothelium is a versatile multifunctional tissue having many synthetic and metabolic properties and is an active participant in the interaction between blood and tissues. The second layer of cells are the smooth muscle cells (SMCs). These are capable of many functions, including vasoconstriction and dilatation in response to normal or pharmacologic stimuli; synthesis of various types of collagen, elastin, and proteoglycans; elaboration of growth factors and cytokines; migration and proliferation. In

thicker blood vessels a vascular network is present in the wall to provide all the cells with nutrients [2]. Several methods are used for tissue engineering of cardiovascular constructs [3]. A

method which seems to work well for heart valves is a PGA/P4HB scaffold seeded with human vena saphena myofibroblasts (MFs) [4]. This scaffold combines the high porosity of PGA with the thermoplastic properties of P4HB. With this method attempts are made to create tissue engineered blood vessels. In spite of MFs also ECs are necessary to create a functioning blood vessel substitute. Recent attempts are made to coculture ECs on top of MFs in such a PGA/P4HB scaffold [5]. During visualization, with fluorescent markers, of the top layers of such a scaffold with a confocal

scanning laser microscope (CSLM), it was observed that the ECs formed tube like structures (Ulex, UEA-1 lectin, green) in direction of the center of the

Figure 1: Composition of a blood vessel [2]

Figure 2: CLSM image with EC tube

like structures (green) and fibers of

PGA (red) [5]

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construct (figure 2). Prior research showed that ECs can be triggered to form tube like structures; this process can be seen as a form of physiological angiogenesis, the formation of new blood vessels. A mounting body of evidence suggests that hypoxia is a major trigger for both physiological and pathological angiogenesis [6]. With respect to this present research, it is hypothesized that the MFs in the center of the construct are experiencing a low oxygen concentration and thus are producing growth factors which trigger the ECs to form the tube like structures. Therefore it will be interesting to verify hypoxia in the tissue engineered cardiovascular construct. There are several methods available to verify hypoxia in tissues or cells. First there are oxygen electrodes available for direct measurement of the partial pressure of oxygen, in the gas phase or dissolved solution. These electrodes give very precise results, but are very expensive. The disadvantage of these electrodes is that it is not possible to measure partial pressure of oxygen on µm scale, which is the scale that was used in this research. Also it is difficult to measure partial pressure of oxygen in tissue engineered constructs with such an electrode, without damaging the constructs [7]. Spectroscopy of redox-sensitive markers for hypoxia is another method to determine hypoxia. Oxygen related changes in the spectroscopic properties of pyridine nucleotides, cytochromes and other proteins have been extensively investigated as indicators of the oxygenation status of tissues. Nevertheless these redox-related changes are reversible by re-oxygenation on exposure to air and are therefore not applicable in this present research. Another group of hypoxia markers are metabolically-activated chemical markers. The bioreductive metabolism of nitroaromatic compounds involves the generation of an initial free radical which is reactive enough towards oxygen that further metabolism is effectively inhibited in oxygenated cells. In hypoxia, reactive metabolites that bind to cellular macromolecules are generated, thus labeling hypoxic cells even if they are subsequently re-oxygenated [8]. Many types of nitroaromatic compounds have shown promise as probes for hypoxic cells in in vitro and in vivo test systems. Numerous isotopically-labeled 2-nitromidazoles have been proposed as hypoxia markers (figure 3). More recently, 2-nitroimidazoles with immunohistochemically detectable side-chains have been investigated as potential hypoxia markers and using such compounds, hypoxia-specific staining can be demonstrated. These compounds are already used in some animal and clinical studies, to detect hypoxia in tissues in vivo [9, 13]. Pimonidazole is such a 2-nitroimidazoles and in previous research was investigated that this is a good marker for hypoxia [9]. Pimonidazole is a good, easy and relatively cheap compound for detecting hypoxia in tissues and individual cells, so therefore it is used in this present research. The aim of this study was to verify if the myofibroblasts in the center of tissue engineered constructs were hypoxic. This was done by using the Hypoxyprobe-1 Kit for the immunohistochemical detection of tissue hypoxia. The working of this kit was verified on monolayers of 3T3 mouse fibroblasts and human vena saphena myofibroblasts under hypoxic conditions. Two ways to cause hypoxia in a monolayer were analyzed; with a mineral oil layer and by infusion of 100% gaseous N2. Hypoxia was shown in monolayers of cells that where flushed with gaseous N2. Furthermore sections of the tissue engineered constructs were cut and also analyzed with the Hypoxyprobe-1 Kit.

Figure 3: pimonidazole, a 2-nitroimidazole [9]

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Chapter 2: Materials and Methods

2.1 Cell culture For the performed experiments two types of cells were used: 3T3 mouse fibroblasts (passages 6-12) and human vena saphena myofibroblasts (MFs) (passages 8-9). Unless stated otherwise, the cells were grown and incubated at 37°C in 5% CO2. The 3T3 mouse fibroblasts were cultured in a 1:1 mixture of HAM’s F12 and DMEM medium. DMEM medium consists of DMEM low glucose, 10% FBS, 1% glutamine and 1% penstrep. HAM’s F12 medium consists of HAM’s F12, 10% FBS and 1% penstrep. Human vena saphena MFs were cultured in 2-D growth medium. This medium consists of DMEM Advanced, 10% FBS, 1% glutamax and 0.1% gentamycin. All media constituents were purchased from Cambrex (Belgium). Unless stated otherwise, the cells were cultured in a monolayer at about 80-90% confluency. 2.2 Constructs Rectangular PGA/P4HB scaffolds (20x7x1 mm) were glued into wells of a 12 wells plate using 20% polyurethane solved in THF (figure 4). The PGA/P4HB scaffolds were seeded with human vena saphena myofibroblasts (MFs) using fibrin as cell carrier. After 4 days of culture, human vena saphena endothelial cells (ECs) were seeded on top of the MFs. The constructs were cultured for 11 or 15 days in EGM endothelial cell growth media, supplemented with EGM-2 Bulletkit (Clonetics). 2.3 Histology Samples were fixed in formalin and embedded in paraffin. Sections were cut at 10 µm thickness. Some sections were stained with haematoxylin and eosin (H&E) for general tissue morphology and others were stained with an immunostaining for hypoxic cells. 2.4 Hypoxyprobe-1 immunohistochemistry For the detection of hypoxia in cell monolayers and histological sections, hypoxyprobe-1 Mab1 Kit (Chemicon, USA) was used. This kit contains pimonidazole hydrochloride and a primary antibody: Hypoxyprobe-1 Mab1. More information about the kit can be found in the manual. Pimonidazole is reductively activated in an oxygen-dependent manner and is covalently bound to thiol-containing proteins in hypoxic cells. The reductive activation of pimonidazole under hypoxic conditions happens in several steps (figure 5). The first step is the addition of an electron from the cell’s transport system to form a nitro radical anion. This step in the reductive process is totally inhibited at oxygen concentrations ≥ 14 µM. The activated intermediate that binds to the hypoxic cells is believed to be a hydroxylamine derivative resulting from the sequential addition of four electrons to pimonidazole. The hydroxylamine intermediate reacts with thiol-

Figure 4: PGA/P4HB

scaffold glued in wells

plate [5]

Figure 5: Scheme for the reductive activation of

pimonidazole under hypoxic conditions [9]

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containing peptides and proteins to form stable adducts that can be detected with an immunochemical assay [9]. This immunochemical assay involves a series of incubations, including incubation with

Hypoxyprobe-1 Mab1, the primary antibody for the marker pimonidazole. Pilot studies showed that a dilution of 1:100 gave good results. Afterwards there was incubation with a secondary biotinylated antibody (Vectastain ABC Kit, Vector Laboratories, USA), incubation with biotin-streptavidin-peroxidase complex, and, finally, incubation with chromogens: 3,3’-diaminobenzidine (DAB) which imparts a clear brown color to the marker-antibody complex around the nucleus of the hypoxic cells (figure 6). A more detailed protocol can be found in Appendix A.

Finally the cells and histological sections were viewed with a microscope (Zeiss, Germany) and photographed with a digital camera

(Canon, Japan) to investigate whether they were stained brown and thus were hypoxic.

2.5 Hypoxia through mineral oil layer To test and optimize the Hypoxyprobe kit, the goal of the first experiment was to cause and show hypoxia on monolayers of 3T3 mouse fibroblasts and human vena saphena MFs, which were cultured in 24-wells plates. Pilot studies showed that the wells on the edges of a 24-wells plate showed more evaporation than the wells in the middle of the plate. Hypoxia was created by placing a 1 cm (2 mL) mineral oil (Sigma, USA) layer on the monolayers with 0.5 mL medium containing 200 µM pimonidazole (pimo). The oil layer should restrict exposure to oxygen of the cells [10]. After incubation with mineral oil, the oil and the medium with pimonidazole were removed and the cells were fixated with a 4% formaldehyde solution. Several experiments were performed to create and evaluate hypoxia in cell monolayers. Unless stated otherwise, two wells were used per group and the entire test groups were tested double. 2.5.1 Effect of different incubation times With this experiment it was investigated when cells under a layer of oil showed signs of hypoxia. Therefore different incubation times under oil were chosen: 1, 6 and 24 hour. This experiment was performed with 3T3 mouse fibroblasts. There were 6 test groups, including 3 control groups: without oil, without pimonidazole (pimo) and without primary antibody (1e AB) (table 1).

Groups Incubation time Cells with oil 1, 6 and 24 h Control Iwithout oil 1, 6 and 24 h Control IIwithout pimo 24 h Control IIIwithout 1e AB 24 h Table 1: Test groups: effect of different incubation times

2.5.2 Effect of adding pimonidazole at different time points With this experiment it was investigated whether it matters at which time point pimonidazole was added to a monolayer of human vena saphena MFs. In the previous experiment, medium with pimonidazole was added to the monolayer from the beginning, at t=0, together with the oil layer. This method was compared with a second method were

Figure 6: Example of hypoxyprobe-1 Mab1

staining in tissue section [14]

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pimonidazole was added to the medium with a pipette below the mineral oil layer, one hour before the end of the experiment. With this experiment it was also investigated when human vena saphena MFs showed signs of hypoxia, therefore different incubation times were tested: 1, 6 and 24 hour. The same test and control groups as in the experiment were used, but per time point there was a well were pimonidazole was added at t=0 and a well were pimonidazole was added 1 hour before the end of the test.

2.6 Hypoxia through infusion of gaseous N2 A second manner to cause hypoxia on a cell monolayer is flushing the air above the cells, with 100% gaseous N2 where pimonidazole is added from the beginning [11]. In this experiment a 24-wells plate with different test groups was placed in a plastic reservoir. Hypoxic conditions were created by infusion of 100% gaseous N2 (figure 6). Another 24-wells plate was placed in a plastic reservoir, without infusion of gaseous N2, acting as control group (Control I). Pilot studies with 3T3 mouse fibroblasts showed that there was hypoxia after 3 hours of N2 infusion, so flush periods of 1, 2 and 3 hours were chosen.

Figure 6: Experimental setup flushing with gaseous N2

This experiment was performed on monolayers of human vena saphena MFs. There were 6 different test groups with 4 wells per group, and three control groups (table 2). Some of the test groups were brought in normoxic conditions with fresh medium without pimonidazole for 24 hours after the experiment, to test whether the hypoxia of cells was reversible. From each test group, 1 well was used to do a viability test with tryptane blue (Sigma, USA). The principle of this staining agent is that it only penetrates dead cells, colouring them blue. Tryptane blue is not capable of passing an intact cell membrane and therefore the viable cells remain unstained.

Groups Flush period Re-oxygenation Cells with N2 1, 2 and 3 h - Cells with N2 1, 2 and 3 h Y Control Iwithout N2 1, 2 and 3 h - Control IIwithout pimo 3 h - Control IIIwithout 1e AB 3 h - Table 2: Test groups N2 experiment

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Chapter 3: Results 3.1 Hypoxia through mineral oil layer 3.1.1. Effect of different incubation times The cells with and without oil did not show any staining after 1 hour of incubation. After 6 hours of incubation both the cells with and without oil showed light brown staining. The cells were totally stained, the nucleus as well as the cytoplasm were equally colored. After 24 hours, the cells with oil and the control without oil were stained dark brown. Around the nucleus, the staining was darker than in the cytoplasm. There was no significant staining visible in the control groups without pimonidazole and without primary antibody, after 1, 6 and 24 hours. An overview of these results can be found in table 3.

Groups 1 h 6 h 24 h Cells with oil - + ++

Control Iwithout oil - + ++ Control IIwithout pimo - - -

Control IIIwithout 1e AB - - - Table 3: Staining results. (-): no staining; (+): light brown staining; (++): dark brown staining

3.1.2. Effect of adding pimonidazole at different time points This experiment was performed with human vena saphena myofibroblasts. After 1 and 6 hours of incubation no staining or very light staining was found in the experimental and control groups. No difference was found between the different moments of adding pimonidazole. After 24 hours light brown staining can be seen in the wells were pimonidazole was added at t=0 and in the negative control without oil. In the group where pimonidazole was added one hour before the end and in the control groups, there was no staining. An overview of these results can be found in table 4.

Group Incubation time Add pimonidazole From beginning 1 hour before end

1 h - - 6 h -/+ -

Cells with oil 24 h + -

1 h - - 6 h - -

Control I without oil

24 h + - Control IIwithout pimo 24 h - - Control IIIwithout 1e AB 24 h - -

Table 4: Staining results. (-): no staining; (-/+): almost no staining; (+): light brown staining; 3.2 Hypoxia through infusion of gaseous N2 First it was observed that the human vena saphena MFs did not die during flushing with gaseous N2 for 1, 2 and 3 hours. The cells were not stained blue after incubation with tryptane blue, indicating cell death did not occur. Nevertheless pilot studies with 3T3 mouse fibroblasts showed that these cells looked quite strange, after flushing with gaseous N2 for 3 hours. The 3T3 cells did not have their original shape, but they had a

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more spherical structure with long and small bulging. This effect was not really observable in the human vena saphena cells that were flushed with gaseous N2. 3.2.1. Effect of different incubation times The negative control, without N2 did not stain at all after 1 hour (figure 6A) and light brown staining was found after 2 and 3 hours (figure 6B, C). After infusion of gaseous N2 for 1 hour, the cells were stained light brown (figure 6D) with the same intensity as the negative control groups after 2 and 3 hours (figure 6B, C). After 2 hours of flushing with N2, the cells were stained darker brown (figure 6E) than after flushing for 1 hour. Around the nucleus the staining was more intense than in the cytoplasm. The cells that were brought under hypoxic conditions for 3 hours were stained dark brown (figure 6F) with a more intense staining around the nucleus. After 3 hours the control groups without pimonidazole and without primary antibody were not stained at all. 3.2.2. Effect of re-oxygenation After flushing with gaseous N2, one well from each time point was brought under normoxic conditions for 24 hours. After 24 hours of re-oxygenation quite the same staining intensity was found in every group compared to directly after flushing with N2.

1 h 2 h 3 h - N2

A B C + N2

D E F

Figure 6: Staining after flushing with gaseous N2 for different periods. (A, B and C) Negative controls of incubation without gaseous N2 for 1, 2 and 3 hours. (D, E and F) Stained MFs after 1,2 and 3 hours of flushing with gaseous N2. (Magnifications 100x).

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1 h 2 h 3 h A B C 3.3 Hypoxyprobe-1 Mab1 labeling in constructs Sections of 10 µm thickness were made of constructs that were cultured for 11 or 15 days. One hour before the sections were cut, pimonidazole was added to the construct. For general tissue morphology, the sections of the constructs were stained with H&E. There were parts of the scaffold visible, tube-shaped, in the middle of the construct, as well as cut artifacts. A quite broad band of spherical cells could be seen on the bottom of the sections with H&E staining (figure 8A and B). On the top there were also cells visible, but these seemed to be smoother. Two phenomena could be distinguished during this experiment. Some of the sections had a light H&E staining of the band of spherical cells, which seems to be background staining (figure 8A). In the sections of the same sample that where immuno stained, no hypoxyprobe-1 Mab1 staining was visible in the spherical cells (figure 8D). On the other hand, there were also sections that had a very intense H&E stained band of spherical cells on the bottom (figure 8B). In the sections of the same sample that were stained with the immuno staining, many cells in the broad band on the bottom were colored dark brown. H&E

A A B Immuno C D E

Figure 7: Staining after re-oxygenation for 24 hours after (A) 1 hour, (B) 2 hours, (C) 3 hours of flushing with gaseous N2. (Magnifications: 100x).

Figure 8: Immunostaining and H&E staining of sections. (C) Control group without pimonidazole. (A, B) Sections with H&E staining. (D, E) Sections with immunostaining. (Magnifications 100x).

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Chapter 4: Discussion The aim of this study was to verify if the myofibroblasts in the center of tissue engineered constructs were hypoxic. This was done by using the Hypoxyprobe-1 Kit for the immunohistochemical detection of tissue hypoxia. Measurement of hypoxia in small tissue samples, in vitro as well as in vivo, has been reliably achieved by labeling hypoxic cells with pimonidazole, a compound of the Hypoxyprobe-1 Kit [12]. The working of this kit was verified on monolayers of 3T3 cells and human vena saphena myofibroblasts under hypoxic conditions. Two ways to cause hypoxia in a monolayer were analyzed; with a mineral oil layer and by infusion of 100% gaseous N2. Hypoxia was shown in cell monolayers that where flushed with gaseous N2. Furthermore sections of the tissue engineered constructs were cut and also analyzed with the Hypoxyprobe-1 Kit. The Hypoxyprobe-1 Kit for the detection of tissue hypoxia is used and tested in previous research, but mainly on tumor tissue in vivo [9, 10, 13]. That is why we tested the method first on a monolayer in vitro. It was shown that causing hypoxia to monolayers of cells with a mineral oil layer does not work very well. Although Covington et al. showed that hypoxia can be created on a monolayer of cells through incubation with a mineral oil layer [9]; this method did not work in this research. There was staining visible in the cells after incubation under oil for 6 and 24 hours, but cells that were incubated without an oil layer, were also stained. There seemed to be two problems, first it seemed that pimonidazole gave background staining to the cells, independent of the critical hypoxic oxygen tension in the medium. Second, it is questioned whether the oil layer did create hypoxia. On the one hand there was a low number of cells which might not consume enough oxygen to lower the concentration, on the other hand it might be that the oil layer was not thick enough to prevent diffusion of oxygen in the medium. To exclude this assumption, a quantitative test should be done with different heights of oil layers on medium and different incubation times of medium under oil, while measuring the oxygen tension in the medium with an oxygen electrode on different time points. With the results of such a test, the best height of the oil layer can be determined, to be certain that hypoxic conditions will arise. After incubation of a monolayer under oil for 1 hour, no staining was visible, in none of the wells. Two possible explanations could be given for this phenomenon; firstly it is possible that there was no hypoxia after 1 hour, so pimonidazole was not able to bind. It was shown that the moment of adding pimonidazole to the cells is of importance. To prove this, an experiment was performed where pimonidazole was added from the beginning of the incubation or one hour before the end of the experiment. There was no staining visible in the cells that received pimonidazole one hour before the end and cells that had pimonidazole from the beginning were stained. Because there was even no background staining visible in the cells that were incubated for only 1 hour with pimonidazole, this is probably too short for pimonidazole to bind. To know exactly how much time is necessary for pimonidazole to bind under hypoxic conditions, supplying experiments need to be done, in which cells have to be incubated with pimonidazole under hypoxic conditions, for different time periods. Second it was shown that infusion of 100% gaseous N2 is a better manner to cause hypoxia in monolayers of cells [11, 12]. The longer the cells were flushed with gaseous N2, the more intense was the immuno staining of the cells. From previous research it was founded that infusion of gaseous N2 for 3 hours is certainly long enough to reach an oxygen level of 0% in the plastic reservoir and thus long enough to cause hypoxia in the monolayer [12]. It was observed that especially the 3T3 cells had a strange, retracted shape with some long and small offshoots, but this effect was not really observable in the

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human vena saphena MFs. so probably the environment made the cells to feel not quite well. Several environmental conditions could be changed due to the infusion of gaseous N2, so there were several aspects that could be responsible for the retracted shape of the cells. First the CO2-level dropped to near zero, normal air level, during the period of flushing, while this is normally held constant on 5%. At a CO2-level of 5% the pH buffer will remain constant, therefore it is likely that the pH changed during the infusion of gaseous N2 and that maybe caused the retracted shape of the cells. This effect could be eliminated through flushing with a mixture of 95% gaseous N2 and 5% CO2. To get more insight in the effect of pH change, the pH of the medium should be measured during flushing with gaseous N2. A decrease in temperature could also be a reason for the cells to retract. The optimum temperature for the cells is 37°C, but the infusion of gaseous N2 was performed at room temperature. This temperature effect can also be eliminated through flushing with gaseous N2 in an incubator which has a constant temperature of 37°C. The retracted shape of the cells is a point that certainly needs to be investigated, but it had no negative influence on our results. The goal was to investigate the effect of hypoxia and therefore the cells needed to stay alive during the experiment. It was proved with a viability staining that the cells were not death after the flushing period and that was enough for this research. It was also shown that the binding of pimonidazole is not reversible. When a monolayer, incubated with pimonidazole, was brought in normoxic conditions with fresh medium without pimonidazole, for 24 hours after the flushing period, the same staining intensity was visible than directly after the flushing period. Finally it was shown that there was probably hypoxia in one side of some of the tissue engineered constructs. With H&E staining for tissue morphology it became clear that on the bottom of the construct there was a broad band of many spherical shaped cells. The cell density in this region seemed to be higher than in the rest of the section. It is possible that this can be explained with the fact that the gel of the matrix first leaked out of the bottom of the section, due to gravity, so that the cells in the bottom of the construct became denser packed. On the top of the sections, the cells were smoother and more stretched. The assumption is that the spherical cells were endothelial cells and the smoother cells were myofibroblasts (smooth muscle like cells). It has to be proven which cells are endothelial cells, to get more insight in the cellular composition of the tissue engineered constructs. Therefore there are endothelial cell markers commercially available. Immuno staining of coupes that were cultured for 11 or 15 days and were incubated with pimonidazole showed staining of the spherical cells on the bottom of some of the constructs. In other sections, no staining was visible in the spherical cells. The smoother cells on the other side seemed not to be stained. There is a possible reason imaginable for the phenomenon that some sections where stained and other sections from the same sample were not stained. It could be bad diffusion of the pimonidazole through some of the constructs. It seemed to be that 1 hour of incubation with pimonidazole was probably not long enough for pimonidazole to diffuse through all the constructs. To investigate how much time it takes for pimonidazole to diffuse through the whole construct, an experiment has to be performed, that functions as positive control. After immuno staining with hypoxyprobe-1 Mab1 of sections of this construct, it could be determined what the best incubation time is for pimonidazole with tissue engineered cardiovascular constructs, so that it diffuses through the whole construct. In this experiment constructs have to be incubated with pimonidazole and flushed with gaseous N2 for different periods. On the other hand, if we assume that the diffusion of pimonidazole was adequate enough, then it is possible that only in some of the constructs the spherical shaped cells were hypoxic and in other constructs not. It is possible that the hypoxic cells trigger the formation of tube like structures, but further research, for example with markers for different growth factors that are produced during hypoxia, is needed to prove this assumption.

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There was no significant staining difference visible between constructs that were cultured for 11 days or constructs that were cultured for 15 days. This could mean that there was hypoxia in some constructs before the 11th day, which triggered the cells to form tube like structures that are visible after 11 and certainly after 15 days of culturing. Further research is needed to get more insight in the development of hypoxia in the tissue engineered constructs. For example sections of the constructs need to be cut each day, from day 1 till day 15. With such a kind of test, it can be monitored when the cells start to become hypoxic. Nevertheless it is very difficult to make coupes from constructs, younger than 11 days. Taken together more research needs to be done to prove hypoxia in the tissue engineered constructs. First the different cell types have to be identified and than hypoxia have to be detected on different time points. In this report it was shown that hypoxia in a monolayer can be caused through flushing with 100% gaseous N2 for about 3 hours. It is also proved that the Hypoxyprobe-1 Kit works well. Finally a start is made with the detection of hypoxia in the tissue engineered cardiovascular constructs. More research on this subject is necessary.

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Appendix A Protocols A.1 Hypoxia staining on a monolayer

1. Dip and blot 10 times in deionized water + 0.2% Tween-20 (2 minutes, RT) 2. Incubate samples in 0.3% H2O2 in deionized water (30 minutes, RT) 3. Antigen retrieval1 (8 minutes, 37°C) 4. Wash with PBS (5 minutes, RT) 5. Incubate samples with diluted normal serum2 (Vectastain Kit) (20 minutes, RT) 6. Blot excess serum from samples 7. Incubate samples with primary antibody: Hypoxyprobe-1 Mab1 (1:100) diluted in buffer (40 minutes, RT) 8. Wash with PBS (5 minutes, RT) 9. Incubate samples with diluted biotinylated secondary antibody Solution3 (Vectastain Kit). (30 minutes, RT) 10. Wash samples with buffer (5 minutes, RT) 11. Incubate samples with VECTASTAIN ABC Reagent4 (30 minutes, RT) 12. Wash samples with buffer (5 minutes, RT) 13. Incubate sections in peroxidase substrate solution5 until desired stain intensity develops (10 minutes, RT) 14. Rinse the samples in distilled water (2 minutes, RT)

A.2 Hypoxia staining on coupes of constructs 1. Dewax and rehydrate the sections (22 minutes, RT)

- 2x 5 minutes xylene

- 3x 2 minutes 100% EtOH

- 1x 2 minutes 96% EtOH

- 1x 2 minutes 70% EtOH

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- 1x 2 minutes MilliQ water

2. Incubate samples in 0.3% H2O2 in deionized water (30 minutes, RT) 3. Antigen retrieval1 (8 minutes, 37°C) 4. Wash in PBS buffer3 on shaking plate (5 minutes, RT) 5. Incubate samples with diluted normal serum2 (Vectastain Kit) (20 minutes, RT) 6. Blot excess serum from samples 7. Incubate samples with primary antibody: Hypoxyprobe-1 Mab1 (1:100) diluted in buffer (40 minutes, RT) 8. Wash in buffer on shaking plate (5 minutes, RT) 9. Incubate samples with diluted secondary antibody solution3 (30 minutes, RT) 10. Wash samples in buffer on shaking plate (5 minutes, RT) 11. Incubate samples with VECTASTAIN ABC Reagent4 (30 minutes, RT) 12. Wash samples in buffer on shaking plate (5 minutes, RT) 13. Incubate sections in peroxidase substrate solution5 (10 minutes, RT) 14. Rinse the samples in distilled water (5 minutes, RT) 15. Dehydrate, mount and cover the sections (10 minutes, RT)

- 10 dips in 70% EtOH

- 10 dips in 96% EtOH

- 3x 10 dips in 100% EtOH

- 2x 3 minutes Xylol

- Entellan and coverslip

- Let dry O/N in fumehood

1 Antigen retrieval � 0.06 g Bohringen Milk Powder � 0.0048 g pepsin � 200 µL 6M HCl � 12 mL PBS 2 Blocking serum (normal serum) � add three drops of stock (yellow label) to 10 ml buffer3 in mixing bottle (yellow label) 3 Secondary antibody solution � add one drop of stock (blue label) to 10 mL of buffer in mixing bottle (blue label)

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4 VECTASTAIN ABC Reagent � add two drops of REAGENT A (orange label) to 10 mL of buffer in the ABC Reagent mixing bottle

� add two drops of REAGENT B (brown label) to the same mixing bottle � mix immediately and let it stand for about 30 minutes before use! 5 Peroxidase substrate solution � add 1 tablet DAB to � 15 mL TRIS buffer � add 12 µL fresh H2O2