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

BIOREACTORS (1)

Dr. Judit PongráczThree dimensional tissue cultures and tissue engineering – Lecture 5

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

TÁMOP-4.1.2-08/1/A-2009-0011

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 easyDrawbacks: lower cell density, lower metabolism rate

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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

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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

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Mass transport challenges in 3D tissue culturesDiffusion of oxigen and nutrients:• From the static medium to the surface cells• From the surface cells to the deeper

structuresImortant parameters of the cultured cell/tissue

construct:• Porosity• TortuosityTissue thickness under static conditions should not exceed 100 mm

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Shear forces in dynamic fluidsShear 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.

t

l

Dx

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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

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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

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Bioreactor design requirements IThe 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:

1. Need to maintain desired nutrient and gas concentration in 3D constructs

2. Need to facilitate mass transport into 3D cultures3. Need to improve even cellular distribution in 3D

constructs4. Need to expose the construct to physical stimuli5. Need to provide information about the formation of

3D tissue

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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.)

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Structure of an industrial bioreactor

Peristaltic pumps

Drive

Water in

Water out

Air

Counterpressure

valve

Electromagnetic

valve for cooling

Pump

Safetyvalve

Process controller

Heater vessel

Acid BaseAntifoam Substrate

Q

Q valve

FoamT

pHpO2

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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

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Rotating wall bioreactors I

Fc Fd

Fg

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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

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Compression bioreactors I

Head for dispensing pressure

Scaffold constructs

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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 PongráczThree dimensional tissue cultures and tissue engineering – Lecture 6

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

TÁMOP-4.1.2-08/1/A-2009-0011

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

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Flow perfusion bioreactors I

Scaffold constructs with seeded cells

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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

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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

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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

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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

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

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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

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

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Current achievements in bioreactor designTE 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

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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