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-4.1.2-08/1/A-2009-0011

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-4.1.2-08/1/A-2009-0011 Dr. Judit Pongrácz Three dimensional tissue cultures and tissue engineering – Lecture 5 Bioreactors (1)

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

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

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

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

Shear forces in dynamic fluids t 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

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

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

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

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.)

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

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 Spinner flask bioreactor

Rotating wall bioreactors I Fd Fc Fg

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

Compression bioreactors I Head for dispensing pressure Scaffold constructs

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

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-4.1.2-08/1/A-2009-0011 Dr. Judit Pongrácz Three dimensional tissue cultures and tissue engineering – Lecture 6 Bioreactors (2)

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

Flow perfusion bioreactors I Scaffold constructs with seeded cells

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

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

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

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

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

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

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

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

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