1. Manifestation of Novel Social Challenges of the European Union in the Teaching Material of Medical Biotechnology Master’s Programmes at the University of Pécs and at the University of Debrecen
Identification number: TÁMOP /1/A

2. Bioreactors (1) Dr. Judit Pongrácz
Manifestation of Novel Social Challenges of the European Union in the Teaching Material of Medical Biotechnology Master’s Programmes at the University of Pécs and at the University of Debrecen
Identification number: TÁMOP /1/A
Dr. Judit Pongrácz
Three dimensional tissue cultures and tissue engineering – 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 static cell 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 is physiological

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 in 3D tissue 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 fluidst
Shear stress measure unit: dyn/cm2 1 dyn = 10mN A shear stress, t is applied to the top of the square while the bottom is held in place. Dx l

8. Shear stress in bioreactors
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 requirements I
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 gas concentration 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 requirements II
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 industrial bioreactor
Acid
Antifoam
Base
Substrate
Peristaltic pumps
Foam
Heater vessel T
Process controller
Pump pH
Water in
pO2
Counterpressure
valve
Safety
valve
Water out
Electromagnetic
valve for cooling
Q valve
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 rpm, volume ml, 50% medium change in every two days
TE cartilage grown to 0.5mm thick under these conditions
Spinner flask bioreactor

14. Rotating wall bioreactors IFd Fc Fg

15. Rotating wall bioreactors II
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 those in the spinning flask

16. Compression bioreactors I
Head for dispensing
pressure
Scaffold constructs

17. Compression bioreactors II
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. Bioreactors (2) Dr. Judit Pongrácz
Manifestation of Novel Social Challenges of the European Union in the Teaching Material of Medical Biotechnology Master’s Programmes at the University of Pécs and at the University of Debrecen
Identification number: TÁMOP /1/A
Dr. Judit Pongrácz
Three dimensional tissue cultures and tissue engineering – Lecture 6
Bioreactors (2)

19. Strain bioreactors
Tendon, ligament, bone, cartilage and cardiovascular tissue
Constant or pulsating power transfer
Tissue constructs are anchored to the power transfer apparatus on an elastic basis
Strain is applied to the elastic basis and transferred to the tissue construct

20. Flow perfusion bioreactors I
Scaffold constructs
with seeded cells

21. Flow perfusion bioreactors II
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 seeding too

22. Cartilage: tissue features and injury repair
Cartilage is an ECM-rich tissue, chondrocytes secreting chondroitin- sulphate, collagen, elastic fibers, etc.
Avascular tissue, nutrition is available through diffusion only
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 load on hyaline cartilage covering joint surfaces

23. Compression bioreactors for cartillage TE I
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 bioreactors for cartillage TE II
For bioengineering functional load- bearing tissues, like cartilage or bone, mechanical load has to be applied in the bioreactor.
These forces are needed to express mechanosensitive Ca2+ channels, rearrangement of the cytoskeleton, and also MSC need mechanical strain to direct the differentiation
Problems: mechanical parts are prone for leakage, infection
Scaffolds have to withstand mechanical stimulation, so strong scaffolds are needed, which may have longer degradation time, which is not preferred

25. Tissue Engineering in bone repair
Bone defect and non-union healing
Speeding up the process
Autologous or allogenic bone grafts
Xenograft trials
These methods are associated with donor site morbidity, chronic pain, disease transmission, infetions

26. Flow perfusion bioreactors in bone engineering
Flow perfusion bioreactors proved to be superior compared to rotating wall or spinner flask bioreactors
ALP, Osteocalcin and Runx2 expression is higher
Scaffold mineralization is higher
Careful setting of flow rate because of disadvantageous effects of high shear stress
Intermittent dynamic flow is more favourable than steady speed 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 autologous chondrocytes 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