Bioreactors (1)

1. Manifestation of Novel Social Challenges of the European Unionin the Teaching Material ofMedical Biotechnology Master’s Programmesat theUniversity of Pécs and at the University of Debrecen Identificationnumber: TÁMOP-4.1.2-08/1/A-2009-0011

2. Manifestation of Novel Social Challenges of the European Unionin the Teaching Material ofMedical Biotechnology Master’s Programmesat theUniversity of Pécs and at the University of Debrecen Identification number: TÁMOP-4.1.2-08/1/A-2009-0011 Dr. Judit Pongrácz Threedimensionaltissuecultures and tissueengineering – Lecture 5 Bioreactors(1)

3. Static cell cultures • Most frequently applied cell culture method • Petri dishes or tissue culture flasks • Adherent cells: monolayer cultures • Suspension cells: relatively lower densities • Advantages:no special equipment required, relatively cheap and easy • Drawbacks: lower cell density, lower metabolism rate

4. Problems concerning staticcell cultures • Lack of vascularization • Nutrient supply is limited • Oxygen supply is limited • Metabolic end-product removal is problematic • Frequent and regular passage required • Periodic medium change needed • In vivo dynamic tissue and cellular environment isphysiological

5. Bioreactors: dynamic cell environment • Dynamic and continuous nutrient and oxygen supply • Possibility of formation 3D tissue structure • Increasing cell-cell contact possibilities • Mechanical stimulation of cell cultures • May promote cellular differentiation in the desired direction • Markedly higher cell densities can be achieved • Higher cell density allows large scale industrial application of cell cultures

6. Mass transport challenges in3Dtissue cultures • Diffusion of oxigen and nutrients: • From the static medium to the surface cells • From the surface cells to the deeper structures • Imortant parameters of the cultured cell/tissue construct: • Porosity • Tortuosity • Tissue thickness under static conditions should not exceed 100 mm

7. Shear forces in dynamic fluids t Dx • Shearstressmeasure unit: • dyn/cm2 • 1 dyn = 10mN • A shear stress,t is applied to the top of the square while thebottom is held in place. l

8. Shearstressinbioreactors • Shear stress distribution is uneven in bioreactors • Highest stress is located around edges and sides of the moving vessel • Design of bioreactors must aim evenly low shear stress in the vessel • Uneven shear stress distribution affect cell survival, density, proliferation, etc • Maximum shear stress for mammalian cells are 2.8 dyn/cm2

9. Cell distribution in dynamic environment • Uneven cell distribution in 3D constructs • Gradually decreasing cell density towards the central area • Cell seeding problems • Diffusion problems • Challenges creating viable 3D tissues

10. Bioreactor design requirementsI The aim of using bioreactors for TE is to overcome the hinders of static culture conditions. Bioreactors need to fulfill at least one of the following requirements: • Need to maintain desired nutrient and gasconcentration in 3D constructs • Need to facilitate mass transport into 3D cultures • Need to improve even cellular distribution in 3D constructs • Need to expose the construct to physical stimuli • Need to provide information about the formation of 3D tissue

11. Bioreactor design requirementsII • Design should be as clear and simple as possible • Avoid structural recesses (risk of infection, cleaning difficulty) • Simple and quick assembly and disassembly • Use of biocompatible or bioinert materials (no chromium alloys or stainless steel) • Withstand heat or alcohol sterilization and humid atmosphere • Proper embedding of instruments (e.g. thermometer, pH meter, pump, rotator motor, etc.)

12. Structure of an industrialbioreactor Acid Antifoam Base Substrate Peristalticpumps Foam Heatervessel T Processcontroller Pump pH Waterin pO2 Counterpressure valve Safety valve Water out Q valve Electromagnetic valveforcooling Air Drive Q

13. Spinner flask bioreactors • Stirred fluid, suspended cells, fixed scaffolds • Eddy around the edges of scaffolds • These small, turbulent flows enhance cell seeding and mass transport into the scaffolds • Typically, stirring speed is 60-80 rpm, volume 120-8000 ml, 50% medium change in every two days • TE cartilage grown to 0.5mm thick under these conditions Spinnerflaskbioreactor

14. Rotating wall bioreactorsI Fd Fc Fg

15. Rotating wall bioreactorsII • Originally developed by NASA to study cell cultures at high g accelerations during space flight • Widespread application in Earth surface • Scaffolds are free to move in the media • Constant angular speed of rotation is ensured • The hydrodynamic drag force balances the gravity ensuring the constant suspension of the scaffold in the medium • Medium change may be either constant or intermittent • RWV provides similar fluid transport and homogenous cell distribution like thoseinthe spinning flask

16. Compression bioreactorsI Head fordispensing pressure Scaffoldconstructs

17. Compression bioreactorsII • Main use for cartilage TE • Static or dynamic pressure can be applied • Motor generating linear motion force • Linear displacement sensors • Load is transferred to the cell seeded constructs via flat platens • Even load distribution is critical • A special method of transferring pressure to cartilage constructs is to use hydrostatic pressure bioreactors

18. Manifestation of Novel Social Challenges of the European Unionin the Teaching Material ofMedical Biotechnology Master’s Programmesat theUniversity of Pécs and at the University of Debrecen Identification number: TÁMOP-4.1.2-08/1/A-2009-0011 Dr. Judit Pongrácz Threedimensionaltissuecultures and tissueengineering – Lecture 6 Bioreactors(2)

19. Strain bioreactors • Tendon, ligament, bone, cartilage and cardiovascular tissue • Constant or pulsatingpower transfer • Tissueconstructsareanchoredtothepowertransferapparatuson an elasticbasis • Strain is appliedtotheelasticbasis and transferredtothetissueconstruct

20. Flow perfusion bioreactorsI Scaffoldconstructs withseededcells

21. Flow perfusion bioreactorsII • Mass transport and nutrient delivery to cells is similar to that of in vivo • Because of perfusion pressure, not only diffusion but also convection contributes to oxigen delivery • Mass transport distance is increased in flow perfusion bioreactors • Medium perfusion can be used for cell seedingtoo

22. Cartilage: tissue featuresand injury repair • Cartilage is an ECM-rich tissue, chondrocytes secreting chondroitin-sulphate, collagen, elastic fibers, etc. • Avascular tissue, nutrition is available through diffusiononly • Chondrocytes exert low metabolic activity and severe damage can not be restored in avascular tissue. • Cartilage repair results in fibrous cartilage of poor mechanical properties • In vivo body weight and joint motion exerts dynamic loadon hyaline cartilage covering joint surfaces

23. Compression bioreactorsfor cartillage TEI • Cartilage tissue aggregate modulus is no more than 40% of native tissue in static cultures • Dynamic load can increase modulus of TE cartilage near to the physiological value • Dynamic load increases ECM production of chondrocytes • Addition of growth factors (TGF-b) also helps chondrocyte differentiation • Compression loading is much more effective promoting chondrocyte differentiation than TGF-b

24. Compression bioreactorsfor cartillage TEII • Forbioengineeringfunctionalload-bearingtissues, likecartilageorbone, mechanicalload has to be appliedinthebioreactor. • Theseforcesareneededtoexpressmechanosensitive Ca2+channels, rearrangement of thecytoskeleton, and also MSC needmechanicalstraintodirectthedifferentiation • Problems: mechanicalpartsareproneforleakage, infection • Scaffoldshavetowithstandmechanicalstimulation, sostrongscaffoldsareneeded, whichmayhavelongerdegradationtime, which is notpreferred

25. TissueEngineeringinbonerepair • Bonedefect and non-unionhealing • Speedinguptheprocess • Autologousorallogenicbonegrafts • Xenografttrials • Thesemethodsareassociatedwith donor site morbidity, chronicpain, diseasetransmission, infetions

26. Flow perfusion bioreactors in bone engineering • Flow perfusionbioreactorsprovedto be superiorcomparedtorotatingwallorspinnerflaskbioreactors • ALP, Osteocalcin and Runx2 expression is higher • Scaffoldmineralization is higher • Carefulsetting of flow ratebecause of disadvantageouseffectsofhighshearstress • Intermittentdynamic flow is more favourablethansteadyspeed flow

27. Two chamber bioreactor • Two separate chambers • Simultaneous culture of different cell types • Application: generation of human trachea

28. Current achievements in bioreactor design • TE human trachea for implantation: • Decellularized donor trachea were seeded with autologouschondrocytes and airway epithelium • Two separate „chambers” allowed simultaneous culture of different cells • Surgical replacement of the narrowed trachea in a TB patient

29. Limitations of current bioreactors • Labor intensive methods • Current bioreactors are specialized devices • Difficult assembly and disassembly • Low cell output • Real-time monitoring of tissue structure and organization is not available