Section Materials TechnologyDepartment of Tissue-engineering and BiomechanicsEindhoven University of Technology

Tissue-engineering of anarterial tunica media equivalent

Ruud DasJanuary 2002BMTE 02.14

Supervised by:Dr. Ir. M.C.M.RuttenIr. L.H. van den Heuvel

Table of contents

CHAPTER 1 INTRODUCTION ......................................................................................................3

1.1 OBJECTIVE.................................................................................................................................31.2 GENERAL ANATOMY OF BLOOD VESSELS .....................................................................................31.3 THE DIFFERENT TYPES OF BLOOD VESSELS...................................................................................4

CHAPTER 2 MATERIALS AND METHODS ................................................................................6

2.1 MOULD......................................................................................................................................62.2 CELL TYPES ...............................................................................................................................72.3 COLLAGEN SCAFFOLD ................................................................................................................72.4 STAINING AND VISUALIZATION ...................................................................................................82.5 EXPERIMENT..............................................................................................................................82.6 CONFOCAL LASER SCANNING MICROSCOPY .................................................................................8

CHAPTER 3 RESULTS .................................................................................................................10

3.1 CONSTRUCT MORPHOLOGY.......................................................................................................103.2 VISUALIZATION .......................................................................................................................11

CHAPTER 4 DISCUSSION ...........................................................................................................13

CHAPTER 5 CONCLUSION.........................................................................................................14

REFERENCES ...............................................................................................................................15

APPENDIX I CONSTRUCTION DRAWINGS.............................................................................16

APPENDIX II GROWING H9C2 CELLS.....................................................................................19

APPENDIX III COUNTING CELLS.............................................................................................20

APPENDIX IV MAKING A COLLAGEN SOLUTION ...............................................................21

APPENDIX V DUO-STAINING ....................................................................................................22

APPENDIX VI PROTOCOL FOR TISSUE ENGINEERING AN ARTERIAL TUNICA MEDIA.........................................................................................................................................................23

APPENDIX VII SETTINGS OF THE CLSM................................................................................27

Chapter 1 Introduction

1.1 ObjectiveThe field of tissue engineering opens a whole new scope of possibilities for research andtreatment of a wide variety of clinical conditions. Among these diseases is cardiovasculardisease (CVD), the leading cause of death in the Western world. A common procedure in thetreatment of cardiovascular disease is bypass surgery, which typically involves the use of ablood vessel from the patient. However, sometimes these blood vessels are not available,either because they have been used in previous surgery or the quality of those vessels is poor.Tissue engineering could solve this problem by providing a virtually inexhaustible source ofusable vessels. In research into the effect of treatment of cardiovascular disease, animalarteries are used, but these typically have a highly irregular geometry. Research might benefitfrom tissue engineering, for it could provide vessels in all shapes and sizes and withreproducible geometry.

At the Eindhoven University of Technology, an attempt in constructing tissue-engineeredblood vessels was made (Kortsmit, 2001). These vessels could then be used for research intothe effects of surgery in cardiovascular disease. A protocol was made, derived from thestudies of Hirai et al. (1994), but it did not yield a construct that could be used for furtherresearch. The dimensions of the vessel were too small and the construct contained many deadcells. The goal of this study was to optimize the protocol and create tissue-engineered arterialequivalents that can be used for research of CVD treatment. To do so, the anatomy of arteriesmust be matched as closely as possible.

1.2 General anatomy of blood vesselsThe structure of blood vessels is one of the simplest tissue structures in the body (Rubin,1999). The vessel wall consists of only two types of cells, endothelial cells and smoothmuscle cells. Generally, most vascular diseases result from malfunction of these cell types, orarise from leukocyte interaction with them. The cells in the surrounding connective tissueusually do not play a role in the pathogenesis of most vascular disease processes. The simpletwo-cell structure of the vessel wall is made more complex by the organization into threelayers called “tunicae”. This organization is found in all vessels, expect for the capillaries andthe small veins, where the wall consists only of endothelial cells (Junquira, 1996).

Tunica intimaThe first layer is in direct contact with the blood and is called the tunica intima. This layerconsists of endothelium and connective tissue. The sub-endothelial tissue of larger vessels cancontain collagen, proteoglycans and small amounts of elastin. In larger vessels, smoothmuscle cells can be found in this layer. The internal elastic lamina, an elastic membrane,separates the intima from the tunica media. Holes in this membrane provide a pathway formigration of smooth muscle cells from the media, metabolites and nutrients.

Tunica mediaThe second layer, the tunica media, displays layers of smooth muscle cells. These cells have acircular orientation. Varying amounts of collagen and elastic fibers (also with a circularorientation) are interposed between the smooth muscle cells. For cells in larger vessel wallsthat are situated too far from the lumen, nutrient deficiency or the inability for exchangewastes with the circulating blood might be a problem. This is resolved by providing thesecells with a vasculature of their own, the vasa vasorum. The vasa vasorum penetrate theexterior of the vessel wall and provide blood for the tunica media. Outside the media, in theborder area with the adventitia, is the external elastic lamina, which has a fiber-likeappearance and has no distinct edges. In addition to this external vascular supply, the tunicamedia also contains autonomic nerve fibers that influence vascular contractility.

Tunica adventitiaThe outermost layer of the vessel wall is the tunica adventitia. It contains mostly longitudinalcollagen and sometimes smooth muscle cells can be found. In addition, the tunica adventitiaalso contains small vessels that are the beginning of the vasa vasorum, nerves and lymphaticvessels.Figure 1.1 gives an illustration of this three-layer organization (Rubin,1999 and Junqueira,1996).

Figure 1.1: The organization of a vessel wall (Junqueira, 1996).

1.3 The different types of blood vesselsThere are three types of blood vessels, all differing in length, diameter and wall structure.These types are: arteries, capillaries and veins.

ArteriesWe distinguish three types of arteries according to size and function. The aorta and her mainbranches are called elastic arteries. In these arteries, elastic fibers have a high elastinconcentration in the media in order to provide a means of minimizing energy loss duringpressure changes. The sub-endothelial layer is thick and the connective tissue fibers have alongitudinal orientation.The main branches of the aorta form muscular arteries upon further branching. These vesselsare a few millimeters in diameter. They also have a distinct three-layer structure. The borderbetween the intima and media in clearly marked by the internal elastic lamina. Characteristicfor muscular arteries is the thick muscle layer (up to 40 muscle fiber layers), formed by themedia. Among the muscle fibers lie collagen fibers in a glycosaminoglycan-rich substance,elastin fibers and membranes. The inner layer of the adventitia consists mainly oflongitudinally oriented fibers and, outward, loose fibers.Arterioles are the smallest elements of the arterial system. They have a small diameter (up to0,3 mm) and consist of an endothelial layer with one or two layers of smooth muscle cells.They have no adventitia and no elastic layers can be seen. Arterioles provide the dynamicregulation of blood flow.

CapillariesCapillaries are the smallest blood vessels (5 to 9 µm diameter) in the body and consist only ofendothelial cells. Their length in normal capillary beds varies from 0.25 mm to severalmillimeters. The endothelial layer is usually surrounded by a lamina basalis, which is formedby the endothelial cells. This lamina is surrounded by a layer of type III collagen fibers that isconnected to the surrounding connective tissue.

VeinsThe venous system consist of venules and veins of varying size that return blood to the heartfrom the capillary beds. The venules are the first vessels that collect blood from thecapillaries. They have a thin media, because they don not have to withstand high pressure likearteries. The venules merge into small- and medium-sized veins, which in turn form largeveins. The walls of these veins do not have the characteristic elastic lamellae of the elasticarteries. Only the largest veins have a well-developed internal elastic lamina.(Rubin,1999 and Junqueira, 1996)

Figure 1.2 shows the global organization of these vessels in the body.

Figure 1.2: The organization of the blood vessels in the body. On the right side are the arteries, startingwith the large elastic arteries like the aorta. Followed by the muscular arteries and the small arteries.Finally the capillary beds spring from the arterioles and eventually come together to form venules. Thevenules transport the blood back to the heart, forming medium and finally large veins. The windowboxes at each side give a cross-section of the different types of blood vessels (Rubin, 1999).

As explained earlier, tissue engineered blood vessels could play a part in the treatment ofcardiovascular disease. Many of these diseases are caused by poor functioning of coronaryarteries. To replace such an artery with a tissue engineered vessel, this construct should matchof this type of vessel as closely as possible. Coronary arteries are muscular arteries, with athick tunica media consisting mostly of collagen and smooth muscle cells. Therefore, in thisstudy a tissue engineered tunica media equivalent is made, consisting of cells and collagen.

Chapter 2 Materials and methods

Preliminary research for tissue engineering of a vascular construct has been done atEindhoven University of Technology by J. Kortsmit (2001). His work was based on thestudies of Hirai et al.(1994) on constructing tubular vascular tissue. Their method of addingcells to a collagen solution was adapted and led to a protocol for the development of a tissue-engineered tunica media equivalent.This study is based on the protocol that was made by Kortsmit. Cells are grown until there areat least enough for 2.5 ml of cell suspension. This suspension is added to a collagen solutionand the mixture is poured into a mould. The construct is allowed to shrink. When shrinkage isno longer observed, the construct is tested for cell viability, this is tested with fluorescencemicroscopy. Reproducibility of the results is tested by repetition of the protocol.

2.1 MouldTo ensure the desired geometry of the construct, a tubular form, a mixture of collagen andcells is poured into a mould. The mould consists of a PMMA pedestal, with a glass rod (3 mmdiameter and 126.5 mm long) sticking out of the center of the pedestal. Two glass tubes areplaced concentrically around the glass rod. The inner diameters of these tubes are 10 mm and15 mm respectively, the lengths of the tubes are 102 mm and 80 mm. Silicon O-rings are usedto prevent leakage. Construction drawings of the mould are given in appendix IV. The innerglass tube and the glass rod form an inner chamber into which the cell- and collagen mixturecan be poured. The volume of this compartment is 5 cm3. The second compartment (formedby the two glass tubes) can be filled with culture medium. Finally, a cap is placed over theglass rod, which can close the inner compartment and cover the outer compartment. Picturesof the individual parts and the assembled mould are given in figure 2.1.

Figure 2.1: Left: the individual parts of the mould. Right: the assembled mould.

Sterilization of the mouldSterilization of the mould is done as follows. First, all the individual parts are sonified in anultrsone bath to get rid of dirt particles. After that, the parts are kept in 70% alcohol for atleast 1 day (figure 2.2). Before the mould can be assembled, the alcohol must be completelyevaporated. This is done in the LAF-cabinet. The mould is assembled while wearing sterilegloves and it is then kept at –20oC until use. This is necessary to avoid early hardening of theconstruct when it is poured into the mould.

Figure 2.2: The parts of the mould are stored in 70% alcohol for sterilisation.

For the first experiment, a sterile stainless steel bracket and a sterile glass Petri dish wereneeded. The bracket is kept in the alcohol along with the mould. Both top and bottom of thePetri dish are filled with 70% alcohol and kept in the chemical safety cabinet. Both must becompletely dry before use.

2.2 Cell typesIn this study H9C2 cells are used, which are embryonic cardiomyoblasts from mice. The factthat cardiomyoblasts are used instead of smooth muscle cells should not be a problem,because vascular tissue and heart tissue are identical in the embryonic phase (Kortsmit, 2001).Because the cell line is immortalized, continuous cell proliferation is ensured.

Growing of cellsThe volume of the inner compartment of the mould is 5 cm3, this volume is filled with amixture of collagen and cell suspension in equal amounts. This means 5*106 are needed forone construct (assuming a cell suspension with a concentration of 2*106 cells/ml). To acquirethis amount, cells are grown in culture flasks. The culture medium used is Dulbecco’sminimum essential medium (DMEM, Biochrom) with 10% fetal bovine serum (FBS), 2%HEPES and 1% PenStrep (see appendix I). The cells are kept in a standard humidifiedincubator at 37 oC and 5% CO2.At 70-80% confluency the cells have to be transferred to new culture flasks. The number ofpassages indicates the number of time a cell culture has been transferred. Cells with a highpassage number are avoided, as they tend to grow slower.All actions must be performed in a sterile environment to avoid contamination of the cellculture. For this purpose, a Laminar Air Flow (LAF) cabinet is used. Since the LAF cabinetprovides a sterile workspace, all materials that are used for culturing (i.e. pipettes, PBS)should be sterile as well. Gloves are worn while handling the cells.The complete protocol for growing H9C2 cells is given in appendix II.

Counting of cellsThe required amount of cells (5*106) is usually reached at 3 fully confluent culture flasks of175 cm3. To determine the amount of cells, the Improved Neubauer haemocytometer is used,according to the protocol in appendix III.

2.3 Collagen scaffoldThe collagen scaffold consists of rat tail collagen (Sigma type VII c-8897), a type I collagenequivalent derived from the tail of rats. This type is used because it is the predominant type ofcollagen in the human body. Other types of collagen, like collagen G, might be used but thesedo not produce the desired shrinking effect (Kortsmit, 2001).The collagen comes in small jars containing 10 mg of collagen. The collagen is dissolved in 4ml of acetic acid to a final concentration of 2.5 mg/ml. In order to avoid early hardening, theacetic acid must be ice cold (4 oC at most). Also, all materials that are used with collagenmust be cooled at –20 oC a day before use and all actions must be performed on ice. It takes48 hours for the collagen to completely dissolve in the acetic acid, so the solution should bemade two days before the construct is made. See appendix IV for the protocol.

Because of the acetic acid, the collagen solution is acidic. Cells cannot survive in anenvironment with a pH of less than 6.8, so 50 µl of a 1M NaOH solution is added to raise thepH. However, the NaOH is hard to mix with the viscous collagen solution, so always checkthe pH before adding the cell solution.

2.4 Staining and visualizationViability of the cells in the construct is tested with a duo-staining method. In this methodCelltracker Green (CTG, Microprobes) and propidium iodide (PI, Microprobes) are used asfluorescent labels to visualize both living and dead cells with fluorescence microscopy. CTGstains the cytosol of living cells, so living cells will appear green under a confocal laserscanning microscope. Propidium iodide binds to DNA and RNA by intercalating between thebases, increasing fluorescence 20 to 30-fold. Because PI is membrane impermeant, generallyonly dead nuclei are stained. This way, the nuclei of dead cells appear red when visualizedwith a CLSM. Caution must be taken when handling PI, because it is highly toxic andpossibly mutagen.To be able to visualize the cells in the vessel wall, a small piece is cut from the edge with asurgical blade. Generally, the thinner the sample, the better the results from the CLSM.The complete protocol for staining and visualization of the cells is given in appendix V.

2.5 ExperimentThe biggest problem with the protocol for tissue engineering of an artery of Kortsmit was thelarge percentage of dead cells (see also chapter 3). Also, the center of the construct wall wasvoid of any cells, both living and dead. Kortsmit suggested that the cell death might be causedby insufficient diffusion through the vessel wall. Our hypothesis was that cell death occurreddue to high stress during shrinkage. Also, the lack of cells in the center of the wall could bethe result of cell migration to the edges. If the cell death is indeed caused by the high stress, asolution could be free shrinkage of the construct instead of shrinkage around the inner glassrod. This way, the cells will not experience such high stresses. A convenient extra is that themedium will be in direct contact with the inner edge of the wall, thus accommodating forsome diffusion problems there might be.Testing of the hypothesis involved two experiments. In the first experiment the results ofKortsmit were validated by repetition of his protocol. In the second experiment, a tissue-engineered vessel was created that was allowed to shrink without any constrictions, so noextra internal stresses were generated. Following the instructions of the tissue-engineeringprotocol (appendix VI), a cell/collagen-suspension is made and poured into the mould. Afterone hour of incubation, the outer chamber was filled with medium and the inner tube wasremoved. For the first experiment, the construct was left on the glass rod for seven days(transferring the rod and construct to a Petri dish after 2 days), according to the protocol ofKortsmit (2001). To be sure that the shrinkage had ceased after seven days, the length of theconstruct was measured daily. When no further shrinkage was apparent after seven days, theconstruct was stained and visualized with a confocal laser scanning microscope (CLSM) totest for cell viability. In the second experiment, the mould was kept in the incubatorovernight. The following day, the inner rod with the construct attached to it was placed in aPetri dish. There the rod was removed and the construct was allowed to shrink withoutconstriction for another six days. Again, shrinkage was monitored daily in this experimentand the construct was visualized with the CLSM when no further shrinkage was apparent.Multiple experiments will indicate the reproducibility of the results.

2.6 Confocal laser scanning microscopyConfocal Laser Scanning Microscopy (CLSM) is a valuable tool for obtaining high-resolutionimages and 3-D reconstructions of biological specimens. Figure 2.3 shows the principle ofconfocal laser scanning microscopy.

Figure 2.3: A simple arrangement of a CLSM illustrating the confocal principle(www.cs.ubc.ca/spider/ladic).

In CLSM, a laser light beam is expanded through a x-y deflection mechanism. This beam isused as a scanning beam, focussed to a small spot by an objective lens. The spot illuminates afluorescent specimen and a mixture of reflected and fluorescent light is sent back. Thismixture is captured by the same objective and is converted into a static beam by the x-yscanner device. The static beam is focused onto a photodetector via a dichroic mirror (beamsplitter). The reflected light is deviated by the dichroic mirror while the emitted fluorescentlight passes through in the direction of the photodetector (or vice versa, depending on the set-up). A pinhole is placed in front of the photodetector, such that the fluorescent light frompoints on the specimen that are not within the focal plane will be largely obstructed. In thisway, out-of-focus information is greatly reduced. This becomes especially important whendealing with thick specimens. Ideally, the pinhole is very small, but this reduces the intensity,so an optimum between intensity and out-of-focus information needs to be found.A 2-D image of a small partial volume of the specimen centered around the focal is generatedby performing a raster sweep of the specimen at that focal plane. A 3-D reconstruction of aspecimen can be generated by stacking 2-D optical sections collected in series. (Sheppard,1997)

For this study a dual beam CLSM was used, with an Argon-laser and a HeNe-laser withwavelengths of 488 and 543 nm, respectively. Two wavelengths are needed to individuallyvisualize cells with PI (543 nm) and CTG (488 nm).

Chapter 3 Results

3.1 Construct morphologyAfter one hour of incubation of the cell-collagen mixture, a yellowish construct is visible inthe inner chamber of the mould. The inner glass tube is removed easily as the outer chamberis filled with medium. The construct has shrunk to a length of 5 centimeters.

First experimentFor our first experiment, the construct is kept on the glass rod for an entire week. To ensuresufficient diffusion during this time, the tissue-engineered vessel is transferred to a Petri dish,filled with medium. The rod is supported by the stainless steel bracket. Because the constructis still weak the first day, this is delayed until the second day of shrinkage. The two situationsare given in figure 3.1. After seven days the construct has reached its final length,approximately 1.6 cm. This is shrinkage of 68%. The diameter of the construct obviously isthe same as the diameter of the glass rod, 3mm. The vessel appears white and has a toughstructure. The construct is removed from the glass rod using the blunt end of a surgical bladeand gently pushing it into the medium. Finally, the construct is stained and visualized with aCLSM. A repetition of the experiment yielded a similar construct, with a final length of 1.7cm (shrinkage of 66%) and an inner diameter of 3 mm. The construct has similar shape andappearance.

Figure 3.1: (Left) After 1 day of shrinkage. The construct is visible in the upper part of the mould.(Right) The tissue-engineered vessel takes shape and is allowed to shrink further in a Petri dish.

Second experimentFor our second experiment, a new tissue-engineered vessel is carefully transferred to a Petridish the second day after construction, and at the same time, the inner glass rod was removedto allow free shrinkage. The cylindrical shape of the construct remained during shrinkage.Free shrinkage resulted in a length of 0.9 centimeters, a shrinkage percentage of 82%, and aninner diameter of less than 1 millimeter. The vessel has the same appearance as the vesselfrom the first experiment. This construct is also visualized the seventh day.

3.2 VisualizationFirst experimentThe seventh day, slices of the construct are cut manually with a surgical blade. Preferably,these slices are very thin, because signals from out of focus planes disturb the image.Unfortunately, this method of cutting of the vessel does not allow for very thin slices. Duo-staining is carried out on three slices. The slices are visualized with a CLSM.Following the results of Kortsmit, a large percentage of dead cells can be expected. Also, thecenter of the vessel will not contain any cells. The left image of figure 3.2 shows the splitimage of the CLSM, with part of the ring visible. The upper left image of figure 3.2 shows theresult of the PI staining: a lot of dead cells at both edges of the vessel wall, and no cells in thecenter. Staining with cell tracker green (upper right image) revealed only living cells at theouter edge of the vessel wall. The combined image (lower left image) gives a good indicationof the ratio of living to dead cells. It is apparent that the construct contains mostly dead cells.The right image of figure 3.2 shows the results from the repetition experiment. This constructobviously also contains many dead cells (upper left image). While some more living cells arevisible (lower left image) the construct still contains much more dead cells than living (lowerright image). The right image also shows some more cells in the center, but cell accumulationis still visible at the inner wall. The settings for the CLSM are given in appendix VII.

Figure 3.2: (Left) The CLSM image of a slice of tissue-engineered blood vessel, after constrictedshrinkage around a glass rod. Dead cell accumulation at both walls is visible and only a few living cellsare found. The center of the construct contains no cells. (Right) Repetition of the protocol gave similarresults. Notice that the vessel wall is thinner in this image than in the left image. More cells appear inthe center.

Second experimentThe same procedure as in the first experiment is used regarding the preparation and stainingof the samples. The results of duo-staining are given in figure 3.3. The magnification in figure3.3 is larger than the magnification in figure 3.2, because no clear image could be taken with alarger magnification, due to large disturbances from signals from out of focus areas. Theupper left image again shows the results of PI staining. Dead cell accumulation at the inneredge is not seen and dead cells are visible in the center of the construct. Living cells arevisible in the lower left image of figure 3.3. These too can be found throughout the construct.Finally, the overlap image in the lower right corner reveals a greatly improved dead/livingcell ratio. The setting for this image are also given in appendix VII.

Figure 3.3: The CLSM image of a slice of tissue-engineered blood vessel, after free shrinkage. Fewerdead cells are observed. Both living and dead cells appear throughout the construct. The ratio ofdead/living cells is much lower.

Table 3.1 Overview of the resultsExperiment 1 Experiment 2

Tubular geometry Yes YesPercentage shrinkage (axial) 68% 82%Inner diameter (mm) 3 <1Cells in center No/Few YesRatio of dead/living cells(after seven days)

High Low

Chapter 4 Discussion

• The results in this report show a clear difference in construct morphology and cellviability between the two experiments. However, it must be noted that the secondexperiment was only performed once. The first experiment was performed twice withmore or less the same results. These results also corresponded with those found byKortsmit. Limited time prevented the repetition of the second experiment so furtherresearch should verify these findings.

• Cell viability was only determined on the last day of shrinkage. After the initial cutting ofthe samples there was not enough left of the construct to perform another visualization.Although Kortsmit found that the construct still contained living cells after 22 days,further experiments should determine if that is true for the new construct as well.

• The construct consists only of the most important building blocks of the tunica media,cells and collagen. Other components of an artery, like elastin and endothelial cells (of theintima) are not present.

• It is uncertain to what extend the used cells are a good substitute for smooth muscle cellsof an arterial wall. Besides the fact that the cells are embryonic cardiomyoblasts insteadof smooth muscle cells, the cells come from an immortalized cell line. It would be betterto start a new cell line, using cells from animal arteries. The drawback of this method isthat there is a limit to the number of times that the cell line can be transferred(approximately 10 times).

• Scaling of the mould could produce larger constructs. However, it is unknown if scalingof the mould dimensions produces an equally scaled construct. Research should revealwhich mould dimensions produce a usable vessel. The problem with scaling of the mouldis that much more cells are needed for one construct. Approximately 25*106 cells areneeded for a construct with a length of 5 cm and an inner diameter of 3 mm.

Chapter 5 Conclusion

Optimization of the tissue engineering protocol to create usable constructs for CVD research,is not an easy task. Early experiments by Kortsmit resulted in a construct with many deadcells and almost no cells in the center of the vessel wall. Also, the mould made handling ofthe weak construct very difficult. The changes to the previous protocol, described in thisreport, are significant improvements. First, results from this report show that free shrinkage ofa tissue engineered arterial tunica media equivalent is beneficial to cell survival. Duo-stainingof the construct reveals a substantial decrease in dead cell as well as an increase in livingcells. Second, cells can be found throughout the construct, instead of at the edges, which isclearly an improvement to the previous protocol. And third, the new mould allows easierhandling of the construct while it is still very weak. The new protocol looks promising forcreating a construct that can be used for research of cardiovascular disease. Although theconstruct that was made with the protocol described in this report cannot yet be used forresearch due to its dimensions, scaling of the mould might yield larger and usable constructs.

Recommendations for future research

The results presented in this report clear the way for further research of arterial equivalents.Once the results are verified with repetition of the second experiment, there are some follow-up experiments to consider.• Scaling of the mould can yield a wide variety of construct dimensions. Larger constructs

might be tested for mechanical strength. The construct could be placed inside abioreactor, where it can be loaded mechanically. This might enhance differentiation andorientation of the cells.

• New staining experiments can reveal the presence of living cells after a prolonged period.• Seeding of endothelial cells on the inside of the construct could provide an intimal layer.

The construct will then resemble an arterial wall more closely.• A new cell line (smooth muscle cells instead of H9C2 cells), taken from animal arteries,

could be used for a better tissue-engineered blood vessel.

References

Junqueira, L.C., Carneiro, J., Kelley, R.O., Functionele histologie, 7th edition, Bunge, 1996.

Rubin, E., Farber, J.L., Pathology, 3rd edition, Lippincot-Raven, 1999.

Sheppard, C.J.R., Shotton, D.M., Confocal laser scanning microscopy, 1st edition, Biosscientific publishing, 1997.

Freshney, R.I., Culture of animal cells: a manual of basic techniques, 3rd edition, Wiley-Liss,Inc., New York, 1994.

Kortsmit, J., Tissue-engineering van een bloedvat, internship report BMT01.013, SectionMaterials Technology, Eindhoven University of Technology, 2001.

Hirai, J., Kanda, K., Oka, T., Matsuda, T., Highly oriented tubular hybrid vascular tissue for alow pressure circulatory system, ASAIO Journal 40: M383-388, 1994

www.microprobes.com

Appendix I Construction drawings

The pedestal.

The top of the mould.

The complete mould.

1. Pedestal2. Inner glass tube3. Outer glass tube4. Top5. Glass rod6. Silicon O-ring7. Silicon O-ring8. Silicon O-ring

Appendix II Growing H9C2 cells

H9C2 is a commercial cell-line that is available at ECACC. H9C2 are cardiomyoblasts fromrats. They can be grown in H9C2 medium and have to be transferred at 80% confluency.

Materials

• H9C2 cells in liquid nitrogen• Dulbecco’s minimum essential medium (DMEM, Biochrom)• Fetal bovine serum (FBS, Biochrom)• Hepes (1M, 2% v/v)• PenStrep (10000 U/10000 µg/ml, Biochrom)• Phosphate buffer solution (PBS)• Trypsin/EDTA solution (0,05%/ 0,02% (w/v) in PBS w/o Ca2+, Mg2+, Biochrom)• Culture flasks• Sterile pipettes

Preparing culture medium

H9C2 medium consists of the following:• 500 ml Dulbecco’s Modified Essential Medium (DMEM, Biochrom) (fridge)• 50 ml Fetal Bovine Serum (FBS) (freezer)• 10 ml (1M, 2% v/v) HEPES• 5 ml PenStrep (penicillin/ streptomyocin) (freezer)

Setting up a new cell culture

• Quickly thaw the cells from the liquid nitrogen by placing the vial in a glass with coldwater. Make sure that the top does not make contact with the water. Warm the vial furtherby taking it in your hand.

• After thawing, add the cells to 8 ml medium in a 10 ml tube.• Centrifuge at 1000 rpm during 5-10 minutes.• Suspend the cells in new medium, typically 5 ml.• Transfer the cells to a 25 cm3 culture flask.

Transferring cells

• The cell cultures have to be transferred at 70-80% confluency. Normally this will beevery 3-4 days. If the cells are not yet confluent, refresh the medium.

• Remove the medium from the flasks.• Rinse the cells twice with PBS at room temperature. Do this by adding approximately 5

ml of PBS to the flask and moving it horizontally. Remove the PBS and repeat theprocedure.

• Add 3 ml trypsin (or whatever amount is appropriate for the flask) and directly remove itagain.

• Incubate for 2 minutes at 37 oC, this will allow the cells to be releases from the bottom.Check this with a (light)microscope, the cells should appear round.

• Completely loosen the cells by tapping the side of the flask.• Suspend the cells in new medium (depending on the amount of cells).• Divide the cells among the new culture flasks.

Appendix III Counting cells

A mixture of trypan blue with cells in medium is added to the counting chamber of ahaemocytometer. Trypan blue is added so the cells are better visible under the microscope.Also, trypan blue colors the dead cells blue while the living cells remain colorless. This waythe percentage of dead cells can be determined. The chamber has a known depth and whenviewed under a light microscope, reveals a grid which covers a known area. Counting of thecells under the grid and calculation of the volume under the grid yields the cell concentrationaccording to:

V

NC =

With C the cell concentration in cells/ml, N the number of cells under the grid, V the volumeunder the grid (ml).

The Improved Neubauer haemocytometer has two counting chambers. The concentration isdetermined for both chambers, the concentration of the cell solution is the average of bothresults. The two chambers both have a depth of 0.1 mm and the grids cover an area of 1 mm2,the volume under the grid then is equal to 0.1 mm3 or 1*10-4 ml. Therefore, the concentrationof the cell solution is equal to N*104.

Materials

• Mixture of cells in medium and trypan blue• Improved Neubauer haemocytometer• Q-tips• Alcohol (70%)• Pipette boy with pipette tips• Cell counter

Method

• Clean the counting chambers and cover glass with 70% alcohol and wait until they havedried.

• Moisturize the q-tips with 70% alcohol.• Use the q-tips to apply alcohol to the higher region next to the counting chamber.• Cover the chambers with the cover glass. The cover glass should adhere to the higher

regions because of the alcohol.• Use a pipette to take up a small amount of the cell/trypan blue mixture.• Slowly fill the chambers with the mixture, capillary action should fill the whole chamber.• Place the haemocytometer under a light microscope and focus on the grid.• Count the cells under both grids with a cell counter. Determine the number of both living

and dead cells and calculate the cell concentration.• Clean the haemocytometer and the cover glass with 70% alcohol.

Appendix IV Making a collagen solution

Preparation of a collagen solution with 2.5 mg collagen/ml entails the following procedures.• Remove the sticker from the jar so the collagen becomes visible.• Gently tap the jar on the table so the collagen is at the bottom of the jar.• Slowly add 4,0 ml ice-cold acetic acid with a cooled 5 ml pipette.• Store the solution at 4 oC.• Turn the jar up side down after 24 hours.The collagen will be completely dissolved after 48 hours.

Appendix V Duo-staining

Materials• Celltracker Green (C-2925, Molecular Probes)• Propidium Iodide (P-1304, Molecular Probes)• Phosphate buffer solution (PBS)• Culture medium• Confocal laser scanning microscope

Celltracker green is available in a stocksolution of 1mM (dissolved in DMSO) in the freezer.Propidium iodide (1.5 mM) can be found in the refrigerator.

• Take a vial of Celltracker Green out of the –20 oC and allow it to thaw, this takes about 10minutes.

• Rinse the sample with PBS.• Dissolve 10 µl of the stocksolution Celltracker green (1 mM) in 1 ml medium to obtain a

final concentration of 10 µM.• Add this final solution to the sample and incubate for 15-30 minutes at 37 oC.• Rinse the sample twice with PBS.• Add fresh medium to the sample.• Leave the sample in the incubator for at least 30 minutes before visualizing the cells with

the CLSM.• Meanwhile, dissolve 10 µl of the stocksolution PI (1.5 mM) in 1 ml medium to obtain a

final concentration of 15 µM.• Add this solution to the sample after the resting period for Celltracker Green and incubate

for 10 minutes at 37 oC.• Rinse the cells twice with PBS.• Add fresh medium to the cells.• Visualise directly after the incubation period for PI!

Appendix VI Protocol for tissue engineering an arterial tunica media

Materials

-Dulbecco’s Minimum Essential Medium (DMEM, FG 0415, Biochrom)-Fetal Bovine Serum (FBS, S 0113, Biochrom)-HEPES buffer (1M, L 1613, Biochrom)-Penicillin/Streptomyocin (10000 U/10000 µg/ml, A 2212, Biochrom)-Trypsin EDTA 0,05%/0,02% (w/v) in PBS w/o Ca2+, Mg2+ (L2143, Biochrom)-Trypan blue

-Sterile pipettes. 2, 5 and 10 ml. Cooled and non-cooled-Pipette tips. 50 and 100 µl. Cooled and non-cooled-Eppendorf tubes-Falcon tubes-Syringes (2 and 5 ml) and needles. Cooled and non-cooled.-pH indicator-Ice-Improved Neubauer heamocytometer-Glass Petri dish (15 cm diameter)-Stainless steel bracket-Sterile scalpel-Small Petri dish (5 cm diameter)

Preparations

The following solutions should be readily available.1. PBS solution. Dissolve 2 tablets of PBS in 400 ml demi-water. Autoclave the solution

using the liquid program.2. 2% acetic acid solution. Add 200 µl pure acetic acid to 100 ml demi-water. Autoclave the

solution using the liquid program.3. NaOH 1,0M. Dissolve 4.0 g NaOH in 100 ml and autoclave with liquid program.4. 70% alcohol. Add 175 ml ethanol to 75 ml demi-water.5. H9C2 culture medium. Combine the following:

-500 ml DMEM-50 ml FBS-10 ml HEPES-5 ml PenStrep

Time schedule

Day 0 is the day the cell and collagen solutions are mixed and poured into the mould.

Day –9:Starting a new cell culture. Or continue growing cells from a previous experiment(recommended!). On day 0 three 175 cm3 culture flasks should be fully confluent.

Day –2:Sterilisation of the mould.Making a collagen solution.

Day –1: Assembly and cooling of the mould (store at -20 oC).Sterilisation of the stainless steel bracket and glass Petri dish.Cooling of all materials, i.e. pipettes, falcon tubes, needles and pipette tips.

Day 0: Preparation of the cell (2.0*106 cells/ml) and collagen solution (2.5 mg/ml)Mixing of the solutionsAdding the mixture to the mould

Incubate and remove the inner glass tube after one hour.Day 1 or 2: Depending on the type of experiment, transfer the construct to a Petri dish and

place it on the bracket or remove it from the inner glass rod.Day 7: The construct has taken its final shape.

Day –9

Start a new cell culture or continue growing the cell from a previous experiment as explainedin the appendix “Growing H9C2 cells”. Three fully confluent 175 cm3 culture flasks areneeded for one construct. It is recommended to have four confluent flasks so one flask can beused for further culturing of the cells for the next experiment.

Day -2

When the three 175 cm3 flasks are about 60% confluent, the collagen solution with 2.5 mgcollagen/ml should be made, this entails the following procedures.• Remove the sticker from the jar so the collagen becomes visible.• Gently tap the jar on the table so the collagen is at the bottom of the jar.• Slowly add 4.0 ml ice-cold acetic acid with a cooled 5 ml pipette.• Store the solution at 4 oC.• Turn the jar up side down after 24 hours.The collagen will be completely dissolved after 48 hours.

At the same day that the collagen solution is made, the mould is sterilized.• Take the mould apart and clean it in an ultrasone bath for 10 minutes. Store the parts in

70% alcohol.

Day -1

Sterilize the bracket and Petri dish.• Clean the bracket in an ultrasone bath for 10 minutes and store it in 70% alcohol.• Fill the top and bottom of the Petri dish with 70% alcohol and keep it in the chemical

safety cabinet.

Assemble the mould.• Take the parts out of the alcohol and leave them to dry in the LAF cabinet.• When the mould is completely dry, assemble it wearing sterile gloves.• Put a falcon tube over the top of the mould.• Store the assembled mould at –20 oC.

Make sure all needed materials are placed in the freezer. These materials include:-Pipettes of 5 and 10 ml.-Syringes and needles.-Pipette tips.-Falcon tubes.

Day 0

1. Preparation of the cell solution

• Remove the medium from the three 175 cm3 culture flasks.• Rinse the flasks twice with 10 ml of PBS at room temperature.• Add 5 ml trypsin to all flasks, make sure it has totally covered the bottom and remove it

again.• Place the flasks in the incubator for 2 minutes.• Check the flasks under a light microscope. The cells should be round.• Tap the flasks against the table to loosen them completely.• Add 10 ml of medium to all flasks to suspend the cells.• Add the cell solutions to a 100 ml falcon tube and centrifuge for 7,5 minutes at 1000 rpm.• Remove the medium from the tube.• Resuspend the cells in 2 ml medium.• Take 50 µl of the solution and add it to an Eppendorf tube, place the rest in the incubator

during counting.• Add 50 µl of fresh medium to the 50 µl in the Eppendorf tube.• Take another Eppendorf tube and fill it with 50 µl trypan blue.• Add 50 µl of the cell solution to the trypan blue and mix thoroughly.• Count the cells with the Improved Neubauer Heamocytometer, as explained in appendix

II.• Count the living and dead cells, dead cells should not exceed 10% of the total amount.

The total amount of living cells in 1,95 ml of solution:Living cells= 1,95/2,00*(counted amount of cells)*2*2*20.000.

• Because there should be at least 5.0*106 cells in the remaining 1,95 ml, the countedamount of cells must exceed 64.

• To obtain a final solution of 2.0*106 cells/ml add:

95.1100.2

000.8000.2/95.16

−⋅

⋅⋅ x ml of cold medium to the solution in the incubator.

• Store the solution at 4 oC.

2. Preparation of the collagen solution

• Place a cooled falcon tube on ice.• Fill the tube with 2.5 ml of the ice cold collagen solution using a cooled syringe and

needle.• Add 50 µl of cold NaOH (1M) with a cooled pipette tip.• Mix thoroughly. The following method may be employed to avoid air bubbles.

-Before putting the pipette in the solution, push the pipette beyond the point of resistance.-Take up the solution.-Release the solution without pushing the pipette beyond the resistance.-Repeat as many times as necessary.-When completely emptying the tip, keep it above the solution and slowly empty it.

• Check the pH with pH indicator paper. Make sure the pH is around 7.• Store at 4 oC.

3. Making the cell/collagen mixture

• Place a new, cooled falcon tube in a tray filled with ice.• Fill the falcon tube with 2.5 ml of the collagen solution using a cooled 5 ml pipette.• Add 2.5 ml of the cell solution, also with a cooled 5 ml pipette.• Mix the two solutions using a cooled 5 ml syringe, carefully avoiding air bubbles.• Use the syringe to fill the inner chamber of the cooled and sterilized mould.• Seal the mould with its top and place a falcon tube over the mould.• Place the mould in the incubator for 1 hour.

4. Removing the inner glass tube

The following procedure should be done by two persons.Person 1 wear sterile surgical gloves, while person 2 can make do with normal latex gloves.• Person 1 removes the top of the mould from the inner glass tube.• Person 2 fills the outer chamber of the mould with fresh medium using a 5 ml syringe.• Person 1 removes the inner glass tube by gently pulling and turning. Stop at the point

where the tube would slide over the final o-ring.• Person 1 slides the tube over the construct. This should be done as straight as possible. At

the same time, person 2 keeps adding medium.• When the tube is removed, person 1 puts the top back on the mould, covering the outer

tube, but leaving some space for diffusion.• Place the falcon tube back over the mould and place it in the incubator.

Day 1 and 2

For experiment 1:• On day one, replace the medium. Do this with two syringes. One empty and one filled

with fresh medium. Use the empty syringe to simultaneously(!) remove medium from thetop of the mould, use the full syringe to add fresh medium at the bottom.

• On day two, dry the stainless steel bracket and the large glass Petri dish in the LAFcabinet.

• When both are completely dry, place the bracket in the Petri dish. Fill the Petri dish withfresh medium, completely covering the bracket.

• Take the mould of the incubator.• Remove the inner rod with the construct attached from the mould and place the rod on the

bracket (wear sterile gloves!). Make sure the construct is covered with medium.• Place the Petri dish in the incubator. Refresh the medium every other day.

For experiment 2:• On day 1, fill a small Petri dish with fresh medium.• Take the mould out of the cabinet.• Remove the inner rod from the mould and hold the part of the rod with the construct

attached in the medium of the Petri dish. Gently push the construct in the medium withthe blunt end of a surgical blade.

• Place the Petri dish in the incubator. Refresh the medium every other day.

Day 7

The construct will not shrink any further and has taken its final shape. In experiment 1,remove the construct with the blunt end of a surgical blade. Cut thin slices of the construct forvisualization.

Appendix VII Settings of the CLSM

Figure 3.2 (left)

Scan Mode: Plane

Scaling: X:2.54 µmY:2.54 µm

Stack Size: X:1302.7 µmY:1302.7 µm

Scan Zoom: 0.7

Objective: Plan-Neofluar 10x/0.3

Average: 1

Pinhole: Ch1 -1: 89 µmCh2 -2: 110 µm

Filters: Ch1 -1: LP585Ch2 -2: BP 505-530

Beam Splitters: MBS: HFT 488/543DBS1: NFT 570DBS2:DBS3: Plate

Wavelength: Track1543 nm, 24%Track2488 nm, 50%

Figure 3.2 (right)

Scan Mode: Plane

Scaling: X:1.80 µmY:1.80 µm

Stack Size: X:921.3 µmY:921.3 µm

Scan Zoom: 1.0

Objective: Plan-Neofluar 10x/0.3

Average: 1

Pinhole: Ch1 -1: 80 µmCh2 -2: 110 µm

Filters: Ch1 -1: LP585

Ch2 -2: BP 505-530

Beam Splitters: MBS: HFT 488/543DBS1: NFT 570DBS2:DBS3: Plate

Wavelength: Track1543 nm, 24%Track2488 nm, 50%

Figure 3.3

Scan Mode: Plane

Scaling: X:0.90 µmY:0.90 µm

Stack Size: X:460.6 µmY:460.6 µm

Scan Zoom: 1.0

Objective: Plan-Neofluar 20x/0.5 Ph2

Average: Line 2

Pinhole: Ch1 -1: 98 µmChD -1: 0 µmCh2 -2: 84 µm

Filters: Ch1 -1: LP 585ChD -1:Ch2 -2: BP 505-530

Beam Splitters: MBS: HFT 488/543DBS1: NFT 570DBS2:DBS3: PlateNDD_NT2:FW1: NoneNDD_NT1:

Wavelength: Track1543 nm, 24%Track2488 nm, 50%