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Poertner 2005 Bioreactor Design Tissue Culture PDF
Poertner 2005 Bioreactor Design Tissue Culture PDF
REVIEW
Bioreactor Design for Tissue Engineering
Ralf Prtner,1* Stephanie Nagel-Heyer,1 Christiane Goepfert,1
Peter Adamietz,2 and Norbert M. Meenen3
Technische Universitt Hamburg-Harburg, Bioprozess- und Bioverfahrenstechnik, Denickestr. 15, 21071 Hamburg,
Germany,1 Universittsklinikum Eppendorf, Institut fr Biochemie und Molekularbiologie II, Martinistr. 52,
20246 Hamburg, Germany,2 and Universittsklinikum Eppendorf, Unfall-, Hand- und
Wiederherstellungschirurgie, Martinistr. 52, 20246 Hamburg, Germany3
Received 7 March 2005/Accepted 31 May 2005
Bioreactor systems play an important role in tissue engineering, as they enable reproducible and
controlled changes in specific environmental factors. They can provide technical means to perform controlled studies aimed at understanding specific biological, chemical or physical effects.
Furthermore, bioreactors allow for a safe and reproducible production of tissue constructs. For
later clinical applications, the bioreactor system should be an advantageous method in terms of
low contamination risk, ease of handling and scalability. To date the goals and expectations of
bioreactor development have been fulfilled only to some extent, as bioreactor design in tissue engineering is very complex and still at an early stage of development. In this review we summarize
important aspects for bioreactor design and provide an overview on existing concepts. The generation of three dimensional cartilage-carrier constructs is described to demonstrate how the properties of engineered tissues can be improved significantly by combining biological and engineering
knowledge. In the future, a very intimate collaboration between engineers and biologists will lead
to an increased fundamental understanding of complex issues that can have an impact on tissue
formation in bioreactors.
[Key words: tissue engineering, bioreactor, design considerations, cartilage]
mensional (3D) biohybrid structure of a scaffold and cultured cells or tissue. Furthermore, non implantable tissue
structures can be applied as external support devices (e.g.,
an extracorporal liver support when a compatible donor organ is not readily available [4, 5]) or engineered tissues can
be used as in vitro physiological models for studying disease pathogenesis and developing new molecular therapeutics (e.g., drug screening [5, 6]).
The generation of 3D tissue substitutes in vitro requires
not only a biological model (e.g., an adequate source for
proliferable cells with appropriate biological functions, a
protocol for proliferating cells while maintaining the tissuespecific phenotype), but also the further development of new
culture strategies including bioreactor concepts (5, 7, 8).
Bioreactors are well established for the cultivation of microbes or mammalian cells under monitored and controlled
environmental and operational conditions (e.g., pH, temperature, oxygen tension, and nutrient supply) up to an industrial scale. However, as individual cells are mostly applied,
these concepts are inapplicable to 3D tissue constructs. Furthermore, each type of tissue construct (e.g., skin, bone,
blood vessel, and cartilage) will likely require an individualized bioreactor design (7). Therefore, tissue-specific bioreactors should be designed on the basis of comprehensive
understanding of biological and engineering aspects. Addi-
The loss and damage of tissues cause serious health problems (1). In the US, almost one-half of the costs for medical
treatments are spent on implant devices annually (2). Worldwide, 350 billion USD are expended for substitute of organs
(3). The substitution of tissues (such as bone or cartilage) or
joints with allograft materials includes the risk of infections
by viruses (such as HIV, hepatitis C) or a graft rejection. Artificial implants such as those used in knee or hip replacement, have limitations due to their limited lifespan, insufficient bonding to the bone, and allergic reactions caused by
material abrasion. New therapy concepts for practical medical applications are required. To this end, tissue engineered
substitutes generated in vitro could open new strategies for
the restoration of damaged tissues. The goal of tissue engineering can be defined as the development of cell-based
substitutes to restore, maintain or improve tissue function.
These substitutes should have organ-specific properties with
respect to biochemical activity, microstructure, mechanical
integrity and biostability (2). Cell-based concepts include the
(i) direct transplantation of isolated cells, (ii) implantation
of a bioactive scaffold for the stimulation of cell growth
within the original tissue and (iii) implantation of a three di* Corresponding author. e-mail: poertner@tuhh.de
phone: +49-40-42878-2886 fax: +49-40-42878-2909
235
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PRTNER ET AL.
tionally, typical engineering aspects such as reliability, reproducibility, scalability and safety should be addressed (5, 8).
Several bioreactor systems have been developed and usually
the expectations are very high (5, 6). The question is, however, whether bioreactors can indeed fulfil these expectations.
In this review, key technical challenges are identified and
an overview of existing culture systems and bioreactors used
for tissue engineering is provided. These topics have been
addressed to some extent by several authors (4, 5, 716).
Therefore, they will be discussed only briefly. Particular
focus will be given to the interaction between biological and
engineering aspects. Using cartilage tissue formation as an
example, it will be shown how an increased fundamental
understanding of biological, biochemical, and engineering
aspects can significantly improve the properties of 3D tissue
constructs.
IMPORTANT ASPECTS FOR
BIOREACTOR DESIGN
With respect to tissue engineering, bioreactors are used
for different purposes such as (i) cell proliferation on a
small scale (e.g., for individual patients) and on a large scale
(e.g., for allogenic therapy concepts), (ii) generation of 3D
tissue constructs from isolated and proliferated cells in vitro
and (iii) direct organ support devices (15). These bioreactors should enable the control of environmental conditions
such as oxygen tension, pH, temperature, and shear stress
and allow aseptic operation (e.g., feeding and sampling).
Furthermore, a bioreactor system should allow for automated processing steps. This is essential not only for controlled, reproducible, statistically relevant basic studies but
also for the future routine manufacturing of tissues for clinical application (5, 17). Besides these global requirements,
specific key criteria for 3D tissue constructs based on cells
and scaffolds have to be met, including the proliferation of
cells, seeding of cells on macroporous scaffolds, nutrient
(particularly oxygen) supply within the resulting tissue, and
mechanical stimulation of the developing tissues (5).
Proliferation of cells is the first step in establishing a tissue culture. Usually, only a small number of cells can be obtained from a biopsy specimen and expansion up to several
orders of magnitude is required. The proliferation of cells is
quite often accompanied by the dedifferentiation of cells.
For example, proliferating chondrocytes show a decreased
expression level of collagen type II and an increased expression level of collagen type I (18, 19). Small culture dishes
(e.g., petri dishes, 12-well plates, and T-flasks) are generally
used for cell expansion. As these devices allow only an increase in cell number by a factor of approximately 10, several subcultivations are required. These are considered as a
major cause for the dedifferentiation of cells. Recent studies
have shown that microcarrier cultures performed in wellmixed bioreactor systems can significantly improve cell expansion (2022).
The cell seeding of scaffolds is an important step in establishing a 3D culture in a macroporous scaffold. Not only
seeding at high cell densities, but also a homogenious distribution of cells within the scaffold is essential. High initial
J. BIOSCI. BIOENG.,
237
FIG. 1. Set-up of flow-chamber-bioreactor system consisting of flow chamber with inserts for tissue constructs, conditioning vessel, peristaltic
pump, humidifier, exhaust flask, and medium exchange bottle.
binant drugs including tPA, and EPO) or recombinant retroviruses for gene therapy (4143). With respect to tissue engineering they have been investigated for several applications including the cultivation of liver cells as an extracorporal liver device (4, 10, 37), proliferation of stem cells
(4446), cultivation of cardiovascular cells (11) and cartilage cells (9). As most of the scaffolds have large, interconnected pores, during seeding, cells are distributed quite
uniformly. During cultivation, medium flow through a construct enhances the mass transfer of substrates, particularly
oxygen to immobilized cells when interconnected cell free
pores are available.
In membrane bioreactors including hollow-fiber reactors
(47), the miniPerm system (48) or the tecnomouse (49),
cells are cultivated at tissuelike densities in a compartment,
which contains one or several types of membrane for nutrient and oxygen supply and removal of toxic metabolites.
Hollow-fiber systems are widely used in the production of
biopharmaceuticals including monoclonal antibodies. Several
examples of modified membrane bioreactors exist for the
3D culture of tissue cells including hepatocytes (4, 5054),
skin cells (55) or other human cells (49, 56).
Most of the culture systems and bioreactors discussed so
far were first developed for the cultivation of mammalian
cells and adapted to the engineering of 3D tissue constructs.
However, besides some exceptions, they can hardly be used
in the generation of implantable tissue constructs. Each type
of tissue intended for implantation (e.g., skin, heart valve,
blood vessel, and cartilage) requires a different geometric
structure and a specific bioreactor design, particularly when
mechanical stimulation has to be provided. Finally, engineered constructs have to be transferred from the culture
system to the patient after cultivation. Therefore, several tissue-specific culture systems and bioreactors have been suggested.
One of the most prominent culture systems is the rotat-
238
PRTNER ET AL.
J. BIOSCI. BIOENG.,
19, 58, 59, 69, 70), recently, a new concept involving the
use of a bone substitute carrier covered with a layer of
tissue-engineered cartilage without scaffolds has been presented (17). This concept consists of four steps: (i) explanted articular chondrocytes are expanded in a monolayer
culture and then seeded on a solid carrier to form a primer
layer; (ii) simultaneously, chondrocytes are suspended in
alginate gel and cultivated for two weeks to allow the formation of a pericellular matrix; (iii) redifferentiated chondrocytes are then eluted out of the alginate gel and sedimented on the primer layer; (iv) the biphasic constructs are
further cultivated for three weeks. Below, mainly different
aspects of step iv are addressed. A similar concept was suggested by Waldman et al. (71).
A critical parameter during the cultivation of cartilage
tissue is oxygen supply. The effect of oxygen during the in
vitro cultivation of chondrocytes is poorly understood, and
therefore it is presently a controversial issue (72). In several
studies, chondrocytes were immobilized in alginate beads
and cultivated under different oxygen concentrations in the
gas phase (for review see Ref. 72). ODriscoll et al. (73)
observed a limited collagen type II production at very high
(90% O2) and very low (15% O2) oxygen concentrations.
Domm et al. (19) showed a stimulatory effect of a decreased
oxygen tension (5% O2) on matrix production. In a recent
study of Malda et al. (18), the pellets of chondrocytes were
suspended in a stirred bioreactor under different oxygen
concentrations. They observed an increased production of
glycosaminoglycan at 5% and 1% O2 (v/v) in comparison
with aeration at 21% O2 (air). The increased glycosaminoglycan production is accompanied by a decrease in collagen type I level. On the other hand, several studies of
chondrocytes embedded in a 3D matrix or a scaffold have
demonstrated an enhanced matrix formation, particularly
proteoglycan synthesis under more aerobic conditions (74,
75).
The main difference between the applied methodologies
(alginate and pellet culture vs. cartilage generation in 3D
scaffolds) can be observed in oxygen gradients in the vicinity and within the formed cartilage (38, 76). In the case of
alginate and pellet culture, oxygen gradients at the surface
of constructs can be neglected, and only oxygen limitations
within the constructs are likely. For the cultivation of 3D
scaffolds, even more significant oxygen gradients within the
formed matrix are expected.
Another attempt to improve tissue-engineered cartilage is
the application of mechanical force during the cultivation to
produce a phenotypically appropriate tissue. Four main types
of force are currently used in cartilage cultivation: hydrostatic pressure, direct compression, and high- and low-shear
fluid environments. All these forces have been integrated
into culturing devices used as bioreactors for articular cartilage. The individual effects have been reviewed by Darling
and Athansiou (9).
Our work started from an engineering view point, that is,
we invastigated the oxygen transport in culture systems for
the generation of cartilage pellets and within the pellet itself
theoretically. Two culture strategies were modelled, a static
culture system with the supply of oxygen through the medium by diffusion and a perfused culture system with an
239
240
PRTNER ET AL.
J. BIOSCI. BIOENG.,
FIG. 3. Modelling of oxygen profiles within tissue-engineered cartilage. (A) Calculated oxygen profile in static culture system for cultivation
of artificial cartilage pellets. (B) Calculated oxygen profile in perfusion culture system for cultivation of artificial cartilage pellets.
cartilage (approximately 1 mm). For both oxygen concentrations the content of glycosaminoglycan (GAG) was similar, but still significantly lower than that in the native cartilage (data not shown). Qualitatively, the most stable attachment of the cartilage on top of the carrier was found for 10%
O2 (v/v). Furthermore, the cultivated cartilage contained a
large content of collagen type II (data not shown). From
these observations, a lower oxygen concentration in the gas
phase is recommended.
Experiments in the flow-chamber bioreactor showed a
significantly higher matrix thickness but a lower content of
GAG then cultures in 12-well plates as the control. The appearance of the cartilage obtained in the bioreactor seemed
to be closer to the native cartilage with respect to the shape
of cells, distribution of cells within the matrix, and smoothness of the surface among others. The cartilage obtained
from 12-well plates showed an inhomogeneous distribution
of cells, a more uneven surface and holes within the matrix.
Another important requirement for a successful implantation is the consistency of the cartilage. In the case of bioreactor cultures, the compaction of the cartilage and the attachment between the cartilage and carrier were very good,
indicating that the construct is appropriate for implantation. In contrast, the cartilage-carrier constructs cultivated
in 12-well plates were soft and the attachment between the
cartilage and carrier was not sufficient. In this case, the cartilage tended to slip off the carrier with only a slight mechanical impact. Furthermore, the cultivated cartilage should
241
FIG. 4. Culture systems used for generation of three-dimensional cartilage carrier constructs.
TABLE 1. Biochemical data of cartilage-carrier constructs cultivated in 12-well plates, in flow-chamber bioreactor,
under intermittent hydrostatic pressure, and for native cartilage (glycosaminoglycane, GAG)
Matrix
GAG
thickness
(g)
(mm)
12-well plate
4
1.2 0.7
162 103.5
12-well plate
4
1.3 0.6
155.9 74.4
Flow-chamber bioreactor
6
1.6 0.2
142.7 9.7
12-well plate (control)
6
1.1 0.1
238.9 22.1
Static culture under intermittent hydrostatic pressure
3
1.1 0.7
466.53 7.1
12-well plate (control)
3
0.7 0.0
268.8 74
Native cartilage
1.0 0.0
1680.1 211.8
The cultivation principle consists of the following steps (17): (i) chondrocytes are isolated from the articular cartilage of an adult mini-pig,
expanded in a monolayer culture and then seeded on a solid ceramic carrier (diameter 4.5 mm) to form a primer layer; (ii) simultaneously, chondrocytes are suspended in alginate gel and cultivated for two weeks to allow the formation of a pericellular matrix; (iii) redifferentiated chondrocytes
are then recovered from the alginate gel and sedimented on the primer layer; (iv) the biphasic constructs are further cultivated for three weeks to
allow free-swelling of the cartilage matrix. This step was performed either in a 12-well plate, a flow-chamber bioreactor (flow rate, 0.5 ml/min) or
under intermittent hydrostatic pressure (total pressure of 3 105 Pa, 82.5 min per hour, applied hourly during the last week of cultivation). Three
sets of experiments were performed: (i) cultivation of cells in passage 4 in 12-well plates at 21% and 10% oxygen tensions in the headspace;
(ii) cultivation of cells in passage 6 in a flow-chamber bioreactor and in 12-well plates (control) at 21% oxygen tension; (iii) cultivation of cells in
passage 3 in a bioreactor under intermittent hydrostatic pressure and in 12-well plates (control) at 10 % oxygen tension. Native cartilage from an
adult mini-pig; diameter, 4.5 mm; average of five samples.
Culture system
Passage
no.
O2-tension in
the gas phase
(%)
21
10
21
21
10
10
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J. BIOSCI. BIOENG.,
PRTNER ET AL.
NOMENCLATURE
c0 : oxygen concentration in the flowing medium layer
at xm = 0 m, mol l1
cm : oxygen concentration in flowing medium layer,
mol l1
cR : oxygen concentration in static medium layer,
mol l1
cP : oxygen concentration in cartilage matrix, mol l1
D : diffusion coefficient in medium, m2 s1
Deff : diffusion coefficient in cartilage matrix, m2 s1
dP : pellet diameter, m
kS : Monod constant, mol l1
qmax : maximal cell-specific oxygen uptake rate,
mol cell1 h1
u : flow velocity, m2 s1
V : volume of cartilage, l
X : cell density, cells l1
x : length, m
xm : length in medium, m
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