Bioreactors in tissue engineering

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TissueEngineering-(2021-2022)
Bioreactorsintissueengineering
Introduction:
Bioreactors are devices in which biological or biochemical processes develop under a
closely monitored and tightly controlled environment. Bioreactors have been used in animal
cell culture since the 1980s in order to produce vaccines and other drugs and to culture large
cell populations. Bioreactors for use in tissue engineering have progressed from such devices.
A tissue engineering bioreactor can be defined as a device that uses mechanical means to
influence biological processes. In tissue engineering, this generally means that bioreactors are
used to stimulate cells and encourage them to produce extracellular matrix (ECM). There
are numerous types of bioreactor which can be classified by the means they use to stimulate
cells.
Tissue engineering technologies are based on the biological triad of cells, signalling
mechanisms and extracellular matrix. To simulate the development of tissues in vitro, tissue
engineering aims to optimize cell growth, by providing regulatory signals in the form of growth
factors and a regeneration template in the form of scaffold. Bioreactors may be used as an
alternative to or in conjunction with growth factors in the signalling part of the triangle. Another
important application of bioreactors is in cellular differentiation. Mechanical stimulation can
be used to encourage stem cells down a particular path and hence provide the cell phenotype
required. Bioreactors can provide biochemical and physical regulatory signals that guide
differentiation.
Bioreactor design requirements:
Bioreactors are designed to perform at least one of the following five functions:
1) Providing uniform cell distribution
2) Maintaining the desired concentration of gases and nutrients in the medium
3) Providing mass transport to the tissue
4) Exposing tissue to physical stimuli

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5) Providing information about the formation of 3D tissue.
 The material selection is very important as it is vital to ensure that the materials used to
create the bioreactor do not elicit any adverse reaction from the cultured tissue.
 Any material which is in contact with media must be biocompatible or bioinert. This
eliminates the use of most metals, although stainless steel can be used if it is treated so
that chromium ions do not leach out into the medium.
 Materials must be usable at 37˚C in a humid atmosphere.
 They must be sterilisable if they are to be reused.
 Bioreactor parts can be sterilised by autoclaving or disinfected by submersion in
alcohol. If they are to be autoclaved, materials that can withstand numerous cycles of
high temperature and pressure must be used in bioreactor manufacture.
 Alternatively, some nonsterilisable disposable bioreactor parts may be used which can
be replaced after each use of the bioreactor. Other material choices are between
transparent or opaque and flexible or inflexible materials.
 Simplicity in design should also mean that the bioreactor is quick to assemble and
disassemble. Apart from being more efficient, this ensures that cell seeded constructs
inserted into the bioreactor are out of the incubator for the minimum amount of time
possible. This minimises the risk to the cells and the experiment being undertaken.
various parameters such as pH, nutrient concentration or oxygen levels are to be
monitored, these sensors should be incorporated into the design. If a pump or motor is
to be used, it must be small enough to fit into an incubator and also be usable at 37˚C
and in a humid environment.
1. Spinner flask bioreactor
A Spinner flask is a basic form of a tissue-engineered bioreactor that is ideal for cultures
cultivated under static conditions. In a spinner flask, scaffolds are suspended at the end of
needles in a flask of culture media. A magnetic stirrer mixes the media and the scaffolds are
fixed in place with respect to the moving fluid. Flow across the surface of the scaffolds results
in eddies(Vortexes) in the scaffolds’ superficial pores. Eddies are turbulent instabilities
consisting of clumps of fluid particles that have a rotational structure superimposed on the
mean linear motion of the fluid particles. They are associated with transitional and turbulent

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flow. It is via these eddies that fluid transport to the center of the scaffold is thought to be
enhanced.
spinner flasks are around 120 ml in volume (although much larger flasks of up to 8 litres have
been used), are run at 50–80 rpm and 50%of the medium used in them is changed every two
days. Cartilage constructs have been grown in spinner flasks to thicknesses of 0.5 mm. While
this is an improvement on cartilage grown in static medium, it is still too thin for clinical use.
Figure: A spinner flask bioreactor. Scaffolds are suspended in medium and the medium
stirred using a magnetic stirrer to improve nutrient delivery to the scaffold.
An advantage of the spinner flask design is it maintains a well-mixed environment within the
flask, which therefore reduces the stagnant layer of cells what would form in a poorly mixed
environment.
Spinner flasks are not always ideal since the constant mixing motion causes turbulent flow
within the capsule and the associated high shear stress causes the formation of an outer fibrous
capsule in the cartilaginous tissue. Spinner flasks have been modified (2004) since the original
design to reduce the turbulent flow. Current designs called wavy-walled bioreactors induce
small waves in the mixing instead of the rough, turbulent flow induced from traditional spinner
flasks. Spinner flasks are intended for small scale production and do not appear to be used as
much as other types. They are primarily used for the seeding of cells in 3D scaffolds until they
are ready for more large-scale cell culture procedures Mass transfer in the flasks is not good

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enough to deliver homogeneous cell distribution throughout scaffolds and cells predominantly
reside on the construct periphery.
2. Rotating wall bioreactor
The rotating wall bioreactor was developed by NASA. It was originally designed with a view
to protecting cell culture experiments from high forces during space shuttle take off and
landing. The device has proved useful in tissue engineering here on earth. In a rotating wall
bioreactor, scaffolds are free to move in media in a vessel. The wall of the vessel rotates,
providing an upward hydrodynamic drag force that balances with the downward gravitational
force, resulting in the scaffold remaining suspended in the media.
FIGURE: Two common rotating wall vessels are shown in the picture above: [top] the Slow
Lateral Turning Vessel (SLTV) and [bottom] Rotating Wall Perfused Vessel (RWPV).
Fluid transport is enhanced in a similar fashion to the mechanism in spinner flasks and the
devices also provide more homogeneous cell distribution than static culture. Gas exchange
occurs through a gas exchange membrane and the bioreactor is rotated at speeds of 15–30 rpm.
Cartilage tissue of 5 mm thickness has been grown in this type of bioreactor after seven months
of culture. As tissue grows in the bioreactor, the rotational speed must be increased in order to
balance the gravitational force and ensure the scaffold remains in suspension.

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3. Compression bioreactors
This bioreactor is generally used in cartilage engineering and can be designed so that both static
loading and dynamic loading can be applied. This is because static loading has been found to
have a negative effect on cartilage formation while dynamic loading, which is more
representative of physiological loading, has provided better results than many other stimuli.
FIGURE: A compression bioreactor. Constructs are housed in a petri dish and subjected to
compressive forces exerted on them via a cam and follower mechanism.
In general, compression bioreactors consist of a motor, a system providing linear motion and a
controlling mechanism. A cam-follower system is used to provide displacements of different
magnitudes and frequencies. A signal generator can be used to control the system and load cells
and linear variable differential transformers can be used to measure the load response and
imposed displacement respectively. The load can be transferred to the cell seeded constructs
via flat platens which distribute the load evenly, however in a device for stimulating multiple
scaffolds simultaneously, care must be taken that the constructs are of similar height or the
compressive strain applied will vary as the scaffold height does. Mass transfer is improved in
dynamic compression bioreactors over static culture (as compression causes fluid flow in the
scaffold) and dynamic compression can also improve the aggregate modulus of the resulting
cartilage tissue to levels approaching those of native articular cartilage.
4. Strain bioreactors
Tensile strain bioreactors have been used in an attempt to engineer a number of different types
of tissue including tendon, ligament, bone, cartilage and cardiovascular tissue. Some designs
are very similar to compression bioreactors, only differing in the way the force is transferred
to the construct. Instead of flat platens as in a compression bioreactor, a way of clamping the

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scaffold into the device is needed so that a tensile force can be applied. Tensile strain has been
used to differentiate mesechymal stem cells along the chondrogenic lineage. A multistation
bioreactor was used in which cell seeded collagen glycosaminoglycan scaffolds were clamped
and loaded in uniaxial tension. Alternatively, tensile strain can also be applied to a construct
by attaching the construct to anchors on a rubber membrane and then deforming the membrane.
This system has been used in the culture of bioartificial tendons with a resulting increase in
Young’s modulus over nonleaded controls.
5. Hydrostatic pressure bioreactors
In cartilage tissue engineering, hydrostatic pressure bioreactors can be used to apply
mechanical stimulus to cell seeded constructs. Scaffolds are usually cultured statically and then
moved to a hydrostatic chamber for a specified time for loading. Hydrostatic pressure
bioreactors consist of a chamber which can withstand the pressures applied and a means of
applying that pressure (Fig. 5). For example, a media filled pressure chamber can be pressurised
using a piston controlled by an actuator. For sterility, the piston can apply pressure via an
impermeable membrane so that the piston itself does not come into contact with the culture
media. Variations on this design include a waterfilled pressure chamber which pressurises a
mediafilled chamber via an impermeable film and is controlled using a variable backpressure
valve and an actuator.

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Figure: A hydrostatic pressure bioreactor. Constructs are placed in a chamber which is
subsequently pressurised to the required level
6. Flow perfusion bioreactor
Flow perfusion bioreactors generally consist of a pump and a scaffold chamber joined together
by tubing. A media reservoir may also be present. The scaffold is kept in position across the
flow path of the device. Media is perfused through the scaffold, thus enhancing fluid transport.
Culture using flow perfusion bioreactors has been shown to provide more homogeneous cell
distribution throughout scaffolds. Collagen sponges have been seeded with bone marrow
stromal cells and perfused with flow. This has resulted in greater cellularity throughout the
scaffold in comparison to static controls, implying that better nutrient exchange occurs due to
flow.
Using a biphasic calcium phosphate scaffold, abundant ECM with nodules of CaP was noted
after 19 days in steady flow culture. In comparisons between flow perfusion, spinner flask and
rotating wall bioreactors, flow perfusion bioreactors have proved to be the best for fluid
transport. Using the same flow rate and the same scaffold type, while cell densities remained
the same using all three bioreactors, the distribution of the cells changed dramatically
depending on which bioreactor was used. Histological analysis showed that spinner flask and
static culture resulted in the majority of viable cells being on the periphery of the scaffold. In
contrast, the rotating wall vessel and flow perfusion bioreactor culture resulted in uniform cell
distribution throughout the scaffolds. After 14 days in culture, the perfusion bioreactor had
higher cell density than all other culture methods.

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FIGURE: A flow perfusion bioreactor (A). Media is forced through the scaffold in the scaffold
chamber (B) by the syringe pump.
7. Combined bioreactors
In addition to the bioreactors mentioned thus far, numerous combinations of the different types
of bioreactor have been used in order to better mimic the in vivo environment in vitro. In most
cases, these more complicated bioreactors involve adding a perfusion loop on top of a standard
bioreactor. Examples include compression, tensile strain or hydrostatic bioreactors with added
perfusion. These different bioreactors allow for nutrient exchange to take place due to perfusion
while stimulation occurs due to a different mechanical stimulus. Bioreactors for engineering
very specialised tissues have also been developed. One example of such a bioreactor is a
combined tensile and vibrational bioreactor for engineering vocal fold tissue. This bioreactor
mimics the physiological conditions that human vocal folds experience in an attempt to develop
the tissue in the laboratory. Vocal fold tissue experiences vibrations of 100–1000 Hz at an
amplitude of approximately 1 mm, which is a unique stimulus in the human body.

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FIGURE: Abioreactor for vocal fold tissue engineering. Constructs can be stimulated using
both tensile and vibrational forces.
Tissue Engineered Bioreactor Products
Dermagraft
Artificial skin is one of the well-known tissue engineered products created with the use of
bioreactors. TransCyte and Dermagraft are two artificial skin products that employ the use
of a modified perfusion system to culture the cells. TransCyte and Dermagraft are dermal tissue
replacement solutions developed for use in diabetic foot ulcers and Dermagraft is produced
using roller bottle technology in which the cells adhere to the bottle surface during the initial
expansion phase. During this phase, up to 100 roller bottles can be used over five weeks for
the expansion. Then manifolds with the graft are constructed and the cells and fresh media are
constantly replenished over the scaffold until it is seeded with a high concentration of cells.
AlloSource
AlloSource is a company currently producing allografts or bone graft for damaged bone tissue.
A study performed by Zhang et al illustrated that the RWV bioreactor was the only type of
bioreactor that was successfully able to produce bone grafts from mesenchymal stem cells.
AlloSource holds one of the largest tissue banks in the United States and they are responsible
for creating safe bone and soft tissue replacements throughout the body. Their products range
in allografts for all sections of the body. Some of the bone grafts they produce are shown in the
pictures.
The Future of the Bioreactor
As bioreactor technology develops into a much more prolific technology, researchers aim to
improve the power behind single bioreactor systems. The following diagram is a rendition of
what the bioreactor of the future would look like. The future vision of bioreactor technology is
an all-encompassing closed bioreactor system in which the surgeon can go from biopsy to
implantable graft with one "bioreactor". The central idea is to make an all-encompassing
“bioreactor” that can be available onsite for the surgeons to have access to. The idea behind
this “bioreactor” is to allow the surgeon to perform a biopsy and inject the sample into the
reactor. The media and nutrient supplies should be held on board or fed directly into the
bioreactor.

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FIGURE: The future vision of bioreactor technology is an all encompassing closed bioreactor
system in which the surgeon can go from biopsy to implantable graft with one "bioreactor".
This all-encompassing system will have the ability to automatically isolate and expand the cells
and seed them on a scaffold. Additionally, the system will be able to produce a graft and
perform all required tests on the newly engineered device to output a device ready for
implantation. This closed system design is the vision for the next generation bioreactors and
would eliminate the need for large GMP facilities by minimizing handling time and therefore
minimizing contamination. Currently the costs associated with this design is simply too
expensive to implement.
References:
 Plunkett, Niamh, and Fergal J. O'Brien. "Bioreactors in tissue engineering."
Technology and Health Care 19, no. 1 (2011): 55-69.
 https://openwetware.org/wiki/Bioreactors_for_Tissue_Engineering_by_Varun_Chalu
padi
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