Bioreactors in tissue engineering
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1 Bioreactors in tissue engineering S. Swaminathan Director Centre for Nanotechnology & Advanced Biomaterials School of Chemical & Biotechnology SASTRA University Thanjavur Tamil Nadu Joint Initiative of IITs and IISc Funded by MHRD Page 1
2 Table of Contents 1. BIOREACTORS IN TISSUE ENGINEERING Establish spatially uniform cell distributions on 3D scaffolds Maintain desired nutrient and gas concentrations in medium: Expose the developing tissue to physical stimuli: TYPES OF BIOREACTORS FOR TISSUE ENGINEERING APPLICATIONS BIOREACTORS IN TISSUE ENGINEERING Bone tissue engineering Cartilage tissue engineering Cardio vascular tissue engineering:... 6 Joint Initiative of IITs and IISc Funded by MHRD Page 2
3 1. Bioreactors in tissue engineering Bioreactor is a device used to carry out all biological or any biochemical process under controlled environment like controlled ph, temperature, pressure and also nutrient supply with the waste removal. The major use of bioreactors in tissue engineering applications is to establish spatially uniform cell distributions on 3D scaffolds, to maintain desired nutrient and gas concentrations in medium, to provide efficient mass transfer and also to expose the developing tissue to physical stimuli. 1.1 Establish spatially uniform cell distributions on 3D scaffolds: Tissue engineering mainly require the development of 3-dimensional porous scaffold along with cells and growth factors for the construction of three dimensional tissue structures. However, developing 3-D cell-scaffold construct is really challenging, since initial high seeding density is required on the scaffold for possible optimal efficiency in tissue progression. In addition, initial distribution of cells onto the scaffold also play vital role in the uniform tissue progression. Hence, the first and foremost work is to seed cells homogenously with high density in the three-dimensional scaffold for establishing the uniform tissue progression. There are different cell seeding procedures available to date such as static cell seeding, dynamic cell seeding, and magnetic cell seeding. Static cell seeding is a very simple and most often used technique. However, poor seeding efficiency of 10-25% and cell penetration inside the matrix limits its potential towards tissue engineering. This problem can be overcome by dynamic cell seeding method where bioreactor systems are used to seed the cells. Using this technique, the seeding efficiency has been improved to 90%. Hence seeding requirement for tissue engineering applications such as high yield to maximize the utilization of cells; high kinetic rate to minimize the time in suspension and also homogenous distribution of cells all over the scaffold has been achieved by the use of bioreactors. Joint Initiative of IITs and IISc Funded by MHRD Page 3
4 1.2 Maintain desired nutrient and gas concentrations in medium The next critical challenge in the in vitro culture of 3D tissues is to supply the nutrients and oxygen throughout the scaffolds. This is because, when we do static culture, the only mechanism for the nutrient delivery to and waste removal from the scaffold is diffusion. When the scaffold size increased, process of diffusion is not so easy to the centre of the scaffold. Further, as the culture time increases, the cells present on the scaffold surface grow well and secrete the ECM contents due to exposure of medium can hinder the movement of fluid towards interior of the scaffolds. 1.3 Expose the developing tissue to physical stimuli: There are various scientific reports available to explain the primary role of mechanical forces in the improvement of biosynthetic activity in 3-D scaffold thereby enhance the in vitro tissue progression. It was found that the cyclical mechanical stretch has improved the proliferation of human heart cells on to the gelatin based scaffolds; improved the mechanical strength of tissues developed using muscle cells entrapped in collagen or matrigel; enhanced the elastin expression and tissue organization in smooth muscle cells loaded polymeric scaffolds. Even reports are evident that the mechanical stimulations promoted the differentiation of specific phenotype from stem cells. 2. Types of bioreactors for tissue engineering applications: (a) Spinner flask bioreactor: In this bioreactor, scaffolds should be fixed at the end of the needles in reactor containing culture media. A magnetic stirrer is used to mix the contents of media. When media flow across the scaffold surface, it promotes the formation of small turbulent eddies (clump of fluid particles) onto the pores of the scaffolds. The dilute cell suspension has to mix around the static scaffolds which could transport the cells through convection. However, convection of cells into the scaffold interior is insufficient due to presence of turbulent inside the pores which could lead to low seeding efficiency and also non-uniform distributions of cells. In addition, mass transfer is also not sufficient to promote the homogenous cell distribution. Joint Initiative of IITs and IISc Funded by MHRD Page 4
5 (b) (c) (d) (e) (f) Rotating wall bioreactor: Unlike spinner flask, here scaffolds are free to move in the culture media. Culture vessel wall is rotating by which it balancing upward hydrodynamic drag force, centrifugal force and downward gravitational force. This type of rotation facilitates the fluid transport all over the scaffolds thereby contributing homogenous cell distribution. As the tissue grows in the bioreactor, the rotational speed of the reactor should be adjusted to balance the gravity force. Care should be taken to ensure whether the scaffolds remain in suspension. Direct perfusion bioreactors: This type of reactor consists of a pump and a scaffold chamber which are joined together by tubing along with the media reservoir. In this bioreactor, a scaffold should be kept across the flow path of device, thereby enhance the fluid transport all through the scaffold. This type of bioreactor is found to be more efficient to maintain the highly metabolically active cells like hepatocytes by enhance the mass transport of nutrients and gas. Hollow fiber bioreactor: In this type, cells will be loaded within a gel which should be kept in the lumen of the permeable hollow fiber. When the medium perfused above the external surface of the fiber, permeable membrane enhance the mass transfer of the nutrients and oxygen thereby maintaining the metabolically active cells such as hepatocytes. Hydrostatic pressure bioreactors: This type of bioreactors is extensively used to apply physical stimuli to the cell-seeded constructs. Media filled chamber is subjected to pressure using a piston. Hence the chamber of this bioreactor should be designed to withstand the pressures. Biomimetic bioreactors: This type of bioreactors is designed to combine different types of above mentioned bioreactors in order to mimic the native environment. This is very complex design of bioreactors used to allow the nutrient exchange throughout the scaffold by perfusion in order to get a homogenous distribution of cells all over the scaffold and also provide physical stimuli by which one can stimulate the culture. 3. Bioreactors in tissue engineering: Joint Initiative of IITs and IISc Funded by MHRD Page 5
6 3.1 Bone tissue engineering: The major challenge in bone regeneration is that the development of mechanically strong scaffold in order to withstand the load during and after implantation. At the same time, the pore size and porosity should require the cellular infiltration as well as vascularization. However, the porous characteristics of the scaffold interfere with the mechanical strength. Hence it is very difficult to bring a balance between mechanical strength and the porosity which could be essential for successful bone regeneration. Thus the use of bioreactor with bone substitutes could promote the homogenous distribution of cells with the increased expression of bone formation of markers. It was proved that the flow perfusion bioreactor improved the expression of bone formation markers, alkaline phosphatase at the end and 14 days as compared to spinner flasks or rotating wall flasks. 3.2 Cartilage tissue engineering: Another tissue which is very prone to mechanical damage is cartilage. Regenerating cartilage is critical since this tissue is alympatic, aneural and avascular tissue. It was found that in static culture, tissue engineered cartilage has shown its modulus of around 179 ± 9 kpa at the end of twenty weeks. Whereas the aggregate modulus was drastically increased six fold at the end of day of cultures with increased expression of glycosaminoglycans. 28 th 3.3 Cardio vascular tissue engineering: Mainly tissues like heart valve tissue, cardiac muscle and also vascular tissues are mainly exposed to mechanical environments. Hence bioreactor can be used to generate such tissues by attempting physical stimuli. The major problem associated with the vascular tissue engineering is that the development of three layers of tissue. i.e inner layer is called tunica intima which is made up of endothelial cells; middle layer tunica media composed of smooth muscle cells; outer layer of blood vessels are tunica externa which are made up of connective tissue and collagen fibers. Atheroscelrosis is the main problem persists in the blood vessel which may lead to coronary heart disease or stroke. Hence this can be treated with the artificial vascular graft. However, the major challenge in the development of artificial blood vessels is the development of endothelial lining in inner part of the graft which can reduce the risk of thrombosis during blood flow. At the same time, blood has to flow at a particular flow rate through this artificial Joint Initiative of IITs and IISc Funded by MHRD Page 6
7 graft. Hence it is mandatory to develop a graft which can withstand the pressure exerted by the blood flow. This can be fulfilled by the smooth muscle cell lining on the outer side of the graft. In order to create such complex structure of native blood vessel, co-cultures of different cell types like endothelial cells, smooth muscle cells can be used. Static cultures are not enough to create homogenous distributions of two different cell types in the graft. Therefore researchers have made an attempt to culture the vascular tissue in the biomimetic bioreactors which are using both strain as well as perfusion simultaneously. In this bioreactor, polymeric tubular scaffold should be fixed with silicone tubing into the inner side of the tube to mainly avoid the smooth muscle cell adhesion in inner lining of the polymeric tube. Then smooth muscle cells seeded construct was maintained in the stirred culture media with the pulsed rate of 165 beat/min, which contribute 5% radial strain to the scaffold for eight weeks. After this the silicone tube should be removed, and the construct is subjected to endothelial cultures with the perfusion of culture medium via the centre of the construct. This type of attempt has promoted the development of blood vessel analogue in terms of arrangement of layers and also with rupture strength which is not possible in static cultures. Fig 1: Types of bioreactors in tissue engineering Joint Initiative of IITs and IISc Funded by MHRD Page 7
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