The Rotating Wall Vessel Bioreactor Biology Essay

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Recently there are significant amount of research work undergoing about tissue engineering and bioreactor designing. Therefore there are so may research paper published around the world's. It may use embryonic stem cell, mesenchymal stem cell, tissue graft or other animal spaces tissues or cell for development of human and animal medicine treatment. In that case there should be some ethic and law to control the usage of the tissue or cell in the medical treatment. Some government organization and private sector by independently or by joining do some research work about the tissue engineering and bioreactor designing. The cardiovascular system is the major disease problem in the human and animal medicine treatment. In recent decade there are cell and tissue engineering and the bioreactor designing involving treating the cardiovascular disease condition. Researchers may try to developed heart valve, wall and blood vessel etc. Hole in the heart is complex congenital heart diseases, in new born babies and leading causes of mortality. The treatment of this kind of the cardiovascular disease only performed surgery correction, the very painful after the surgery at tolerate by baby. When correcting the hole, that has to closed properly otherwise it lead to another problem to the young one, but measurement of the diameter of the hole is very difficult and also correction also very difficult. In the recent decade there is stem cell therapy and the tissue engineering has rapidly developed. By using stem cell and tissue culture there are so many researches and development of the treatment about cardiovascular system. Myocardial tissue engineering developed the heart tissue by using the stem cells in three-dimensional matrices of biodegradable polymers scaffold is the new innovation of the myocardial constructs and cardiovascular treatment.

Introduction

The heart is the most important organs in the human body. It transports blood to the organs, tissues, and cells of the body. Blood delivers oxygen and nutrients to every cell and removes the carbon dioxide and waste products excreted by those cells. A "Holes in the Heart" is an opening in the septum between atria or ventricles of heart, this is congenital condition. 8-10/1000 live born babies has congenital defects in the heart. This condition occurred during the baby's heart does not develop inside the womb; no specific cause for this condition, but some increase risk of being born. If mother had German measles or toxoplasmosis during pregnancy, or if she has diabetes, or if someone else in her family was born with a heart complaint. A hole in the heart may be noticed in the first few months of life - or even before the baby is born, sometimes a hole is not found until a person is much older. This often happens when the hole is between the upper chambers of heart. It may notice person are feeling a bit short of breath and don't know why. But sometimes there are no complaints at all. Because of the hole, the flow of blood through the heart is abnormal. This makes noises in the heart, so a doctor can find the hole by listening to the heart with a stethoscope. If the doctor hears a murmur, this tells the doctor there could be a hole. If the doctor thinks there is a hole, person will have an echocardiogram ultrasound test of heart. Sometimes the hole isn't found until a person is much older - when they notice they are feeling tired and breathless, and can't find a reason for it. Some holes are so small that they cause no problem, and are left alone. Some holes in small babies may close by themselves: if the cardiologist thinks this is likely, he will not close it immediately, but wait for some time to see if it has closed by itself, by repeating an echo. Other holes must be closed, either because they are already a problem, or because they will cause a problem in the future. 1, 2, 6 and 7

There are three different types:

Atrial Septal Defect (ASD): this is a hole in the wall between the atria ( interatrial septum) . This causes more blood to flow to the lungs, and may not have any symptoms; the excess flow can damage the lungs. If the hole is small, and doesn't affect the function of heart, there's no need to fix it. 5

Ventricular Septal Defect (VSD): this is a hole in the interventricular septum or wall between two lower chambers (RV and LV). If it's large, can change the mechanics in heart. This makes the heart work harder than it should, and can enlarge it. If the hole is small, and doesn't affect the function of heart, there's no need to fix it. 8

Atrioventricular Septal Defect: this is a large hole in the middle of heart between the atria and ventricles. Some people with this condition only have one valve between the atria and ventricles - instead of two. This defect can also damage the lungs by iallowing too much blood to flow to the lungs. Although this condition is uncommon, t can be found in babies born with Down's syndrome. 3, 5 and 8

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VSDs are the commonest lesion about 25-30% of all congenital heart defects whereas ASD are about 5-8% of them. Another point to remember is that all of us are born with small ASD. However, VSD is never found in normal heart. The only treatment available was surgical closure. Though the ultimate outcome was good, these children had to inevitably suffer the pain, scar and long hospital stay. There are two ways to do this. The first way is via an operation called catheterisation. This is when a cardiologist puts a tube into leg that goes up towards heart.  Then put a device through that tube so that it fits into the hole. When it's in the right place, the device opens like a little umbrella, and blocks the hole. The device stays inside forever. This is not possible, because of the size, shape or position of the hole. In these more complicated situations, a surgeon will perform an operation where he puts a patch over the hole directly. If holes have between the two pump chambers of the heart that stay open, will need antibiotic treatment at certain times. This might be before having other operations or serious treatment at the dentist. Most patients who have ASD/VSD corrections go on to lead perfectly normal lives. Person will be followed-up for a short period, but if everything's OK after a year, won't need to worry about it ever again. It also doesn't increase the chances of having any other heart-related issues in the future, but should take regular exercise and aim for a healthy diet. After correction of the hole in the heart there are low risk for structural degeneration, thrombo-embolism and endocarditis and growth potential for pediatric patient. From 1970s onwards, a group of cardiologists started thinking differently. They experimented on animals by creating holes in their hearts and then tried closing them without surgery. Gradually they replicated the whole procedure on humans. For the last twenty years, nonsurgical closure or device closure has been the normal. 3, 4, 7 and 8

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Adult life heart muscle cells do not proliferate, if there is damage or injuries happened to the heart, functional tissue try to form the non-functional scar tissue. In 1996, 98 klug et al. Suggest that development of human Embryonic stem cell derived cardiomyocytes help for therapy of cardiac disease.

There are some experiments done by using stem cells. Stem cells are the cell ability for self-renewal and the potential for differentiating into mature cell types. The embryonic stem cells can give rise to almost every mature cell type, while adult stem cells are classified as restricted to differentiation into only few types of mature cells. The mesenchymal stem cell can only differentiate to one specific mature cell type, are referred to as precursor cells. First clinical applications of stem cells for cardiac regeneration comprised cell transplantation trials. These trials were less successful than promising preclinical studies; these efforts initiated intense research activities providing new insight into the mechanisms of tissue growth and differentiation. Cardiac tissue engineering is focused on three different organ subunits: the myocardium, valves, and vessels. These three compounds of the heart can already be replaced by artificial or biological transplant constructs with their respective limitations, like assist devices, commercial heart valves, autologous coronary bypasses, and etc. When developing the heart tissue have to consider produced cardiomyocytes, vascular endothelial cell and the smooth muscles cell. Engineering these tissues must compete with the durability, efficiency and safety of existing substitutes and be affordable at the same time. Tissue engineering is the development of biological substitutes that restore, maintain, or improve tissue function or a whole organ, includes an in vitro. 9 and 20

During designing of bioreactor, physiochemical environment maintain is very impotent, that will help to kept high quality of the stem cells and high degree of reproducibility of the cells. But have to make sure cell culture has developed under sterile environment and sufficient nutrition and waste product exchange throughout the medium and clean and maintain the medium. After that, design the some mechanical and hydrodynamic force to compression or expansion of the developing tissue, like shear stress to the tissue. Then maintain steady flow of media in pulsatile manner and reduce the excessive turbulence in the fluid flow rate. Other than that have to provide the low volume capacity for effective use for growth factors and medium, also select the fabricate material compatible with the heart tissue or stem cell. The bioreactor designing for heart tissue development has to determined some specific design and functional requirement. Both biomechanical and biochemical factors affect the growth of the cell therefore essential to create some control mechanism by stimulate the physiological environment for heart cell growth, like pulsatile forces, pressure, flow rate, compression, expansion, shear stress, frequency, stroke rate and stroke volume. Other than that when creating heart tissue have to consider cardiac flow rate and pressure. When consider the design the bioreactor, there are nutrition, oxygen, carbon dioxide, waste product, pH, temperature and humidity are main important biochemical controls affect the growth of the cell. There than the flow rate, volume, shear stress, pressure, resistance and compliance like biomechanical controls also involved in the cell growth. Therefore specific bioreactors are need for the growth of the stem cell. Because inside the body, cells are always stimulated by mechanical, electrical and chemical signals, these influencing their behavior. In fact, biological tissues adapt their structure and composition to surrounding specific and functional demands. By putting cells alone or only in contact with materials in culture medium is not enough to obtain a functional tissue. In vivo, the heart valves are subject to a unique combination of mechanical stimuli, including flexure, shear stress, and tension (Vesely and Boughner 1989). In growth of the embryonic stem cells require temperature, partial pressure of oxygen, partial pressure of carbon dioxide, pH, and shear force and biochemical conditions of their micro- and macro-environment. Then try to find homogeneous and constant conditions for micro- and macro-environment for the entire cells population. By uneven cell distribution, lack of nutrition and oxygen and insufficient extracellular matrix production cause the some limitation to bioreactor scaffold in stem cell culture. Therefore get rid of those have to make to stem cell onto polymers, which will increase the mechanical strength of the heart tissue construction and also develop subsequent tissue formation.20

To develop a bioreactor to provide cyclic flexural stimulation, to demonstrate the operation of the bioreactor and sterility maintenance and to evaluate the effects of unidirectional flexure on the effective stiffness of bioresorbable polymeric scaffolds which have been used extensively in the tissue engineering of the heart tissue. Therefore have to design the devices for closed controlled environment in which biological and/or biochemical processes are developed maintained pH, temperature, pressure, nutrient supply and waste removal, with high degree of reproducibility of the heart valve. Therefore, bioreactors are particularly crucial for the regeneration of complex 3D tissues. The bioreactor was designed using 3D software. The structural element of the device were machined from polysulfone; chosen for its excellent thermal and chemical stability; and abrasion-resistant acrylic; which provides good optical transparency. Culture medium was Dulbecco's Modified Eagle's Medium with 4.5 g/L glucose and L-glutamine supplemented with 10% fetal bovine serum. Antibiotics were excluded in order to assess the intrinsic ability of the bioreactor to maintain sterility. When developing scaffold use the degradable material and permanent materials as in artificial implants and in use of cells. Then preparing scaffold has to test in vitro and in vivo how they hypotheses of scaffold and cell interaction, scaffold effect on tissue growth and 3D environment effect on stem cell differentiation. Scaffold materials consisted of a non-woven mesh of polyglycolic acid(PGA) fibers dip-coated with poly-4-hydroxybutyrate (P4HB), and a non- woven, 50:50 blend, mesh of PGA and poly-L-lactic acid (PLLA) fibers dip-coated with P4HB. The PGA and PGA/PLLa scaffold had an approximate fiber diameter of 0.012-0.015 mm and desity of 69mg/ml. Rectangular scaffold sample were cut to size (approximately 25x7.5x2mm) and dipped briefly into a solution of P4HB in tetrahydrofuran (1% wt/vol), resulting in a P4HB coating following solvent evaporation. P4HB is a bioresorbable thermoplastic that allows for scaffold to be molded into any shape. Scaffold were cold gas sterilized with ethylene oxide prior to use. 10, 11, 12, 13, 14, 15 and 25

The use of 'bioreactors', chambers which provide the flow of nutrient media for the development and culture of heart valves construct, to provide an environment which as closely as possible mimics the natural in vivo conditions. These bioreactors have been designed for pulsatile flow, driven by a pulsatile pump, which leads to the exertion of only a positive pressure. This is not the case in vivo, as during the cardiac cycle the positive pressure exerted by fluid force is slightly counterbalanced by a little vacuum. Stem cells grow in vitro under bioreactor conditions have to provide the nutrient and they produced the nitrogen contain waste product, but they sensitive to the nitrogenated waste product. This will be varying with the tissue and that will change the shear stresses effecting on the tissue. The oxygen pressure is maintained at set constant value with calculated volume of solution added every time to the medium. Other way round maintained the carbon dioxide pressure at set constant value with calculated volume of waste product removed from the medium. Oxygen is most important nutrients for cells in all aerobic metabolic cycles. It is the limiting nutrient in successful tissue growth in vitro, sufficient amounts of oxygen to the surface of the cells mainly because of the poor solubility of oxygen in culture media. In that case hypo-oxygen or hyper-oxygen stresses will be concern the stem cell culture causes of programmed cell death or apoptosis. Therefore adjust the stem cell for the anaerobic cell metabolism with low oxygen tension (40 mmHg) and low pH or for aerobic cell metabolism with higher oxygen tension (80mmHg) and high pH. Then living tissue is sensitive to pH changes in the medium, during maintain of the oxygen level has to maintain the pH also. Other than that glucose and lactate are providing to cell metabolic process. Therefore they act as the indicator for cells activity.13, 14 and 24

In the bioreactor environment stem cell proliferates and increased the mass that leads the limitation of the final size of the tissue grow. Other than that there are spaces to pass oxygen and nutrient throughout the scaffold otherwise this also leads to limitation of the tissue growth. Therefore bioreactor has to design to proper diffusion of oxygen and the nutrient and also mass proliferation; cell will survive and proliferate within 150-200µm distance.13 and 24

Shear stress will affect the tissue culture growth, most of the stem cell responds to it. They are proliferating according the orientation of the flow direction. In that case stem cell can aggregates by using higher shear stress that can be used for tissue function and viability. If design the rotating bioreactor that can decrease the shear stress and avoids the contact between the cells and the wall of the bioreactor, chamber has to permanently rotate with one direction and control to forming uniformed growth of the tissue. But if design the non-rotating bioreactor then have to create the specific mechanical stress applied on the cell culture, by perfusion solution can passed through the cell tissues by flow through the culture chamber. Some experiments were demonstrated that the shears stress 0.1 dyn/cm2 was ideal for stem cell to growth. If that exceeds the shears stress 1 dyn/cm2 were damaged the cells and the shears 0.01 dyn/cm2 were insufficient to promote the growth. 12, 13, 14 and 24

Bioreactors have developed functional heart tissue in vitro environment over specific biochemical and physical signals known to regulate cell differentiation, by improving the formation of the heart tissue by proving uniformed mixing pattern, transported the nutrient to enhance the cell growth and hydrodynamic or mechanical stimulation for stem cell to develop. Simple static flasks or a magnetically stirred flask is not suitable environment for 3 dimensional heart tissue scaffolds to develop. To develop the lowest possible homogeneous cell number for heart tissue, have to grown the cell with uniform and efficient of porous scaffolds. When compare the cells seeding into mixed petri dishes yield with the static loading of the cell into the scaffolds has thicker constructs and more spatially uniformed distribution of cells. By seeding in rotating vessels or mixed flask has to maintain a uniformed suspension of isolated cells and provide a relative velocity between cells and the scaffold during seeding. Dynamic seeding using mixed flasks will show to achieve seeding efficiencies approaching 100%, but led to cell densities higher at the scaffold periphery. Therefore when design bioreactor; have to provide the scaffold perfusion with a cell suspension in alternate directions, which lead to the more homogenous seeding on a variety of scaffold with potential yield. Once the cells are associated with the scaffold, cell-polymer constructs can be cultured in bioreactors applying specific regimes of fluid flow. 14 and 24

Selecting rotation wall vessels bioreactor

The bioreactors are used for proliferation of cells on a small or large scale, in order to generate 3D tissue constructs, a certain process must occur. That case the cells are proliferated in a bioreactor to provide the quantity of cells needed. The cell lose their specialized characteristics during the process of proliferation is the problem. Therefore microcarrier culture used for improves cell expansion significantly and that mixed the bioreactor system well. After the cell proliferation they have to associate with enhanced heart tissue formation. In above process cells have to receive proper nutrition and a stable environment. There for controlled the temperature, optimum pH, sufficient substrate, water, salts, vitamins, and oxygen. The Rotating-wall vessel culture is the best bioreactor for culturing constructs stained intensely, and homogeneously for scaffold for their cross-sectional area. Inside the bioreactor a dynamic flow generated by a rotating fluid environment is an alternative and efficient way to reduce diffusion limitations of nutrients and wastes. The rotation produced the low level of the shear stress to the cells, creating mechanical stimulation. Other than that there are other mechanical forces that affect the cells during growth, like mechanical compression, hydrodynamic pressure, and fluid flow. They will affect the magnitude, frequency, and duty of the bioreactor cycle. 16

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To control the free falling state adjusted the rotation speed, it protect the fragile tissue by decreasing the shear stress and avoiding the contact between cells and the walls of the bioreactor. During 1990s NASA scientist did some research about the microgravity involved in to the cell tissue of the mammals. They used the closed tubular cylinder forms the system's cell culture chamber, which filled with a liquid medium where the cell grow on micron-size beads. The chamber has rotated along the horizontal axis; in that case they allowed the cell to develop in an environment similar to the free fall of microgravity. They supply oxygen and nutrition through a porous wall in the chamber, as same way they removed the waste product and the carbon dioxide. The rotating wall vessel bioreactor is providing the conditions of weightlessness for microbes by growing them inside of a slowly rotating liquid-filled chamber. The process of the rotation liquid has counteracts with slow sedimentation of the cell by creating a constant free fall of the cells through the culture medium. While rotation cell get a slight sheer stress from liquid, lead to avoid the flattened on the bottom of the container. The scientist used the clear shell for allowed to check growth and cylindrical filter holds on the centre for supply the oxygen and nutrition and removed the carbon dioxide and waste products. And also they insure the fluid rotation without shear stress would leads to destroy the cells. They noticed rotation vessels did not cancelled the gravity, but that maintain the cells in continual free fall environment inside the shell. 17, 18 and 19

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"Bioreactors for the application of physical forces to engineered cartilage tissues. In the rotating wall vessel system (A), the rotational speed is adjusted so that the drag force of the medium (Fd) is balanced by the centrifugal (Fc) and gravitational (Fg) forces. The constructs are thus maintained in a tumble-slide regime and the resulting dynamic laminar flow enhances the production and accumulation of cartilaginous extracellular matrix. Specific culture chambers (B) have been developed for the application of direct deformation to engineered constructs. Chambers include wells to allocate tissue constructs (I), a magnetic bar for medium stirring (II), an inlet/outlet port for medium change (III), a cover lid to maintain sterility (IV), and micrometer screws to accurately establish the contact position between the plungers and each specimen (V)." 18

The cell seeding is effects of shaking speed and initial cell concentration in suspension on cell culture medium, therefore cell seeding has to do in efficiency. In that case initial seeding density and cell distribution within the scaffolds has to understand. Initially cell concentration is low , in that time seeding efficiency and initial density will decreased with increasing shaking speed. But high initial cell concentration that will reverse the result. All the different cell concentration uniformity of the cell distribution decreased with increased shaking speed. But under the same shaking intensity were observed with on significant differences in uniformity between cells with different initial concentration. In vitro the tissue engineering of heart tissue structures is to develop combined cell seeding and perfusion system. Cell seeding is consist of whole system, that incorporated into the perfusion system and air-driven respirator pump connected to the bioreactor. Therefore cell culture medium is closed-loop system that will continuously circulate. Therefore scientist developed a cell seeding device for static and dynamic seeding of vascular cells onto a polymeric vascular scaffold and a closed-loop perfuse bioreactor for long term vascular conditioning. By using cell seeding chamber can be easily connected to the bioreactor, which have combines continuous pulsatile perfusion and mechanical stimulation to the tissue -engineered conduct. In that scientist adjust the stroke volume, the stroke rate, and inspiration/ expiration time of the ventilator allow various pulstile flows and different levels of pressure. 21, 22 and 23

Discussion

When selecting of the scaffold consider the biocompatibility, reproducibility, biodegradability, ability to be processed to complex shapes, ability to support cell growth and proliferation and mechanical properties of materials. Other than that scaffolds has to have similar electrical and functional activity with create systolic force. The limited availability of the incubator space; the place where the multiple bioreactors place, in this space multiple bioreactors have to be places. Development of the stem cell is temperature depended process, any cells grow at body temperature in optimal level therefore temperature has to maintain in that level as possible. The bioreactor design has to set the temperature parameter to monitor the temperature. If inside temperature changes by increased or decreased then that has to alarm on, then it can adjust manually.14 and 24

Sterility is very important throughout the development of the heart tissue. We used flask and glass vessels with threaded fitting, which is cheap and proved to maintain perfect sterility. To reduce the risk of contamination, make sure all connections before sterilisation and sterilize bottles with correction solutions connected to the vessel, by using either alcohol or stem. The tubing can be placed into the pump head easily after the sterilization. Because contamination of the medium lead to the growth of the heart tissue. Therefore bioreactor has to develop as a semi-closed system. 14 and 24

Maintain the small cell culture medium all the time, easy replace the balance amount of the cell culture medium for requirement created by cell seeded as soon as possible. If require in addition to that easy seeding of the additional cells. Maintain the oxygen level in the medium is very essential; therefore reassure the amount of the oxygen in the medium is enough for the development of the stem cells. When we maintain the pH level in the medium that passively adjust oxygen level in the medium, by enrich the medium with CO2 level up to 5%. The biocompatible substances have to use when the designing process of the bioreactor, those substance will not kill the stem cell during the tissue growth. There are many analytic parameters, those have to monitor regularly with some sensory methods to alarm if there are any changes occur in the media and correct it manually. Any design bioreactor can have ability to experiment several times with longer period of time. If there are any alternations, like change the cell culture medium with ingredients needed or changer scaffold materials change those and can perform the process easily. By using roller pump can sucks the cell culture medium from the bioreactor, which leads to stress of the scaffold. This help to stem cell growth towards the heart tissue. 20 and 24

This bioreactor has to use inside the hospital, for treat each of the hole in the heart patient therefore this has to produce low cost heart tissue for the patient. Other than that there should be very low laboratory involvement and convince for patient and the surgeon. When using this kind of tissue engineering think some social highlight that affect the both quality and quantity of the life. Some religious background this technology is some bad for the life, ethical concern there are some extent to do those kind of experiment. But medical point of view this is the good solution for treatment of the patient without suffering. In that case be careful of handling with stem cell and other, that will leads to caused critical threat to handler. 20 and 24

Conclusions

The developed bioreactor has set sterility at least week, with working tool for conducting experiments regarding heart tissue growth. The growth of the heart tissue helps to develop entire heart, which can helpful to many heart diseases. Nutrition concentration has to keep in mind when performing the bioreactor process. When the time of the replacing the medium nutrition concentration has to maintain, also try to minimize the number of time replacement the medium.

Acknowledgment

I would like to thank Professor Alicia El Haj, Dr. Nicholas R. Forsyth and Dr. Ying Yang for their support and guidance in completing this study.

I would like to special thank Dr. Sun Tao for his support and guidance in completing this study.

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