Bioreactors for animal
cell culture
Cell and tissue engineering
Professor Claudia Lobalto da Silva
Claudia Bartolucci
Claudia Siverino
72240
72322
What is a bioreactor?
• An apparatus for growing organisms (yeast, bacteria, or animal cells) under
controlled conditions.
• Used in industrial processes to produce pharmaceuticals, vaccines, or
antibodies
• Also used to convert raw materials into useful byproducts such as in the
bioconversion of corn into ethanol.
• Bioreactors supply a homogeneous (same throughout) environment by
constantly stirring the contents.
• Bioreactors give the cells a controlled environment by ensuring the same
temperature, pH, and oxygen levels.
Required properties of bioreactors
•
Simplicity of design
•
Large number of organisms per unit volume
•
Uniform distribution of micro-organisms
•
Simple and effective oxygen supply
•
Low energy requirement
•
Uniform distribution of energy
•
providing information about the formation of 3D tissue
Types of bioreactors
You can classify bioreactors based on three different parameters.
1. Sterility of the container :
Sterile : used for the production of antibiotics or vitamins.
Not sterile : used for example in conventional fermentations
such as in the production of beer, or more modern
as the treatment of water
2. Conditions imposed by the bioprocess
Organisms growing in bioreactors may be:
• Suspended
• Immobilized. A simple method, where cells are immobilized, is
a Petri dish with agar gel.
Large scale immobilized cell bioreactors are:
•
moving media, also known as
Moving Bed Biofilm Reactor (MBBR);
•
Packed bed;
•
Fibrous bed;
•
Membrane.
3. Methods of cultivation of micro-organisms
BATCH culture
A typical batch reactor consists of a tank with
an agitator and integral heating/cooling system. These
vessels may vary in size from less than 1 liter to more than
15,000 liters. The advantages of the batch reactor lie with
its versatility. A single vessel can carry out a sequence of
different operations without the need to break containment.
FED-BATCH culture
In a fed-batch reactor, fresh media is continuous or
sometimes periodically added but there is no continuous
removal. The fermenter is emptied or partially emptied
when reactor is full or fermentation is finished. It is
possible to achieve high productivities due to the fact
that controlling the flow rate of the feed entering the
reactor can optimize the growth rate of the cells.
Continuous PERFUSION culture
Perfusion bioreactors involve continuous culture,
feeding, and withdrawal (harvesting) of spent media
for long periods. Perfusion systems accumulate no
waste products. Once established, bioprocessing with
perfusion bioreactors can in many cases be simpler
and experience fewer failures.
CONTINUOUS-FLOW (chemostat) culture
A chemostat (from Chemical environment is static) is
a bioreactor in which fresh medium is continuously
added, while culture liquid is continuously removed to
keep the culture volume constant. By changing the rate
with which medium is added to the bioreactor
the growth rate of the microorganism can be easily
controlled.
Others Bioreactors
-
Spinner flask
Used in tissue ingeneering bioprocessing, in
particular for cartilage grown in static medium,
even if it is still too thin for clinical use.
-
Rotating wall bioreactor
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. As
tissue grows in the bioreactor, the rotational speed
must be increased
- Rotary Perfusion bioreactors
System allows a continuous feeding of the cell
chamber from external media bottle; cells
are retained in the cell chamber by molecular
weight cutoff membrane.
-
Compression Bioreactor
It provides a controllable mechanical and
physiological environment for simulating in
vivo conditions in vitro. This class of
bioreactor is generally used in cartilage
engineering and can be designed so that both
static and dynamic loading can be applied
Case study 1
Introduction
• Rotating cell culture system (RCCS) is a cell culture device made by NASA to
simulate microgravity condition.
It is also a 3D dynamic culture system for cell growth.
• The rotational motion can prevent sedimentation, and create a suspension culture
environment and enhance cell-cell interactions. Several researches showed that
RCCS contribute to cellular aggregation, intercellular adhesion and formation of 3D
cell clumps.
• 2 different condition static and with RCCS.
Methods
• Human foreskin samples were derived from voluntary circumcisions(with
informed consents and the protocol was approved by the Ethical Committee of
the Institute of Zoology, Chinese Academy of Sciences).
Skin from children’ foreskin aged 1 to 5 years were isolated based on their rapid
adherence to collagen type IV and their small cell size.
• cytodex 3 beads cover of a thin layer of denatured
collagen IV chemically coupled to a matrix of
cross-linked dextran
Culture
56103 cells/ml EpSCs + 1 mg/ml micro-carriers
inoculated to 10 ml culture vessel of RCCS
1° day : 12 rpm
after 24h cells are adhered
to micro-carries
2 groups static culture group
RCCS group
cells cultured in
6-well plates
22 rpm until the end
of the 15 days
Bioreactor- NASA
NASA in the 1980s developed the bioreactor, a can-like vessel equipped with a
membrane for gas exchange and ports for media exchange and sampling. As the
bioreactor turns, the cells continually fall through the medium yet never hit bottom.
Under these quiet conditions, the cells "self assemble" to form clusters that sometimes
grow and differentiate much as they would in the body.
Bioreactor- NASA
Eventually, on Earth, the clusters become too large to fall slowly and research has
to be continued in the true weightlessness of space.
It has been well established that a
number of cell types grow in the
bioreactor on Earth for extended periods
in ways that resemble tissue-like
behavior. For this reason, the bioreactor
also provides cell culture studies with a
new tool for the study of 3-dimensional
cell growth and differentiation.
Bioreactors have been used aboard the Mir space station to grow larger cultures
than even terrestrial Bioreactors can support. Several cancer types, including
breast and colon cancer cells, have been studied in this manner. Continued
research using the NASA Bioreactor is planned aboard the International Space
Station.
Bioreactor- HARV (High aspect ratio vessel)
Diameter culture chamber = 9 cm
Volume culture chamber = 50 ml
motor-driven rotator
which rotates the
chamber slowly about a
horizontal axis during
the culture period
5% CO2 in air is pumped across the membrane to
ensure adequate oxygen supply and gas exchange
Results
Indentification of hEpSCs by colony forming efficiency, proliferative
capacity and marker expression
Immunofluorescence staining results revealed that almost all of the isolated cells expressed high
level of b1-integrin and p63 protein (Fig.1 D, E), in accord with their high expression in the
epidermal basal layer of skin (Fig.1A, B).
These results indicated that putative epidermal stem cell isolated from the human
foreskin could be successfully propagated in our culture system for maintaining
hEpSCs marker and supporting highly proliferative ability.
Results
Generation of three-dimensional epidermis-like tissue in RCCS.
monolayer sheet structure
cluster of cells or 3D aggregates
forming 3D tissue-like
epidermis structure
Single layer
H&E staining
RCCS : 3D
multicellular
spheroids, which
were similar to
the 3D structure
of epidermis in
vivo.
Multi-layers
Results
Promotion of proliferation and inhibition of differentiation in 3D cell
culture of RCCS
The effect of rotation culture on proliferation of hEpSCs was investigated through MTS
assay.
Ki67 is a marker of
proliferation
RCCS support the
proliferation of hEpSCs
under a feeder free
culture condition
Results
Promotion of proliferation and inhibition of differentiation in threedimensional cell culture of RCCS.
Involucrin is a marker of
terminal differentiation of
hEpSCs
These results demonstrate that RCCS
may provide a condition to promote
cells proliferation and maintain the
low differentiation state forming a
mutilamellar of epidermis.
Discussion
hEpSCs isolated from the human foreskin could be successfully propagated in
vitro, maintaining hEpSCs marker and supporting highly proliferative ability.
RCCS seemed to offer several advantages for hEpSCs growth, particularly for the
generation of 3D epidermal aggregates under feeder-free culture condition
hEpSCs in RCCS proliferated at day 5 and 10 of culture, while cells in static culture
condition exhibited insignificant changes on the surface of micro-carriers
cytodex 3 provides the possibility for the quick expansion of cells on the large
surface of spherical carriers thereby avoiding further enzymatic treatment before
transplantation.
The results demonstrate that RCCS may provide an ideal physical and chemical
environment to guide hEpSCs proliferation and provides an acceptable culture
model to assemble 3D multilayer epidermis tissue.
Case study 2
This study aimed to create a bioreactor that can simulate urinary bladder
mechanical properties, and to investigate the effects of a mechanically stimulated
culture on urothelial cells and bladder smooth muscle cells.
Introduction
The function of the urinary
bladder is to store and empty
urine.
The mechanical forces within
the bladder change during the
physiological process.
The mechanical properties of
the bladder wall in vivo are
essentially visco-elastic.
This study was made dividing the mechanical properties of the bladder from its
complex internal environment.
Step-wise work plan
•
Bioreactor design
• Evaluate the mechanical properties of the bioreactor with a
Pressure-record system
• Test the biocompatibility of the bioreactor, viabilities of urothelial cells
and smooth muscle cells
• Evaluate the effect of mechanical simulations
• Observation and comparison with cells cultured in non-mechanical
stimulated condition
Bioreactor design
Diagram of the disassembled bioreactor
The four parts of the culture chambers
P1
P2
The assembled culture chambers
Cell culture
Human bladder smooth muscle cells (SMC) and urothelium cells (UC)
were cultured under cell culture condition (37°C, 95% air, 5% carbon dioxide).
Two different elastic membranes :
• silastic
• natural rubber (NR)
Maximum deformation of 20% supplied by the silastic membrane
Maximum deformation of 100% supplied by NR membrane.
The tensile test was repeated 8 times in 8 hours.
The cell-seeded membranes were placed in the culture chamber with a culture
medium and stored at 37°C in a static environment for 12–24 hours.
In both the silastic group and the NR group, an identical cell-seeded membrane
without mechanical stimulation was used as a control.
Mechanical evaluation
A pressure-record system was used to evaluate the bioreactor by measuring
the pressure in culture chambers.
The pressure sensor of the pressure-record system recorded the culture chambers
pressure data and plotted it on a real-time chart, which describes the simulated
mechanical environment of the bioreactor in relation to different elastic
membranes.
Biological evaluation
UCs and MSCs from the human bladder were used for the biological evaluation.
In the bioreactor group, a cell-seeded membrane was held in an interlock ring
and cultured in the bioreactor. In the control group, a cell-seeded membrane
was held in an interlock ring and cultured in a 6-well tissue culture plate.
Experimental cell-seeded natural rubber membranes (100% deformation) and
silastic membranes (20% deformation) were divided into two groups:
(1) Static state culture
(2) Stimulation culture:
(I) Natural rubber membranes (NR)
(100% deformation)
(II) Silastic membranes
(20% deformation)
Results
Bioreactor
The bioreactor system was successful in generating pressure waveform
similar to the intended programmed model while maintaining a cell-seeded
elastic membrane between the chambers.
Bioreactor pressure data were sampled every 10 seconds and plotted on a
real-time chart display. These data showed that the bioreactor generated the
desired waveforms successfully.
The two different elastic membranes were deformed as designed yielding a
maximum deformation of 20% for the silastic membrane and 100% for the
NR membrane. The tensile curve showed that both materials could return to
their original shapes from the designed maximum deformation in a timely
manner when the tensile force was removed.
Cell morphology
In the control group, the growing cultures of UCs
retained the differentiated features of epithelial cells;
the monolayer reached confluence with a typical
cobble-like appearance after 7 days.
In the silastic group (20% deformation) the cultured Ucs
showed overlaps at the cell edges after 7 days, while the
cobble-like appearance was only observed in the control
group. In the sylastic group (20% deformation), after
culturing for 7 days, the SMCs exhibited whirlpool-like
shape after cell confluence.
In the control groups, SMCs were present at 9-12
whirlpool-like shapes per 200 fields.
The number of whirlpool-like shapes in the silastic
group were 3-5 per 200 fields.
Conclusion
• This bioreactor system successfully generated waveforms similar to the intended
programmed model while maintaining a cell-seeded elastic membrane between
the chambers.
• The bioreactor can change the growth behavior of urothelial cells and bladder
smooth muscle cells, resulting in the cells undergoing adaptive changes in
mechanically-stimulated environment.
• The biocompatibility of the bioreactor was confirmed by cell viability tests. The
viability tests of static cultured cells (UCs and MSCs) showed no statistically
significant differences between the silastic group and the control group.
• The mechanical simulation was the only factor affecting cells in the bioreactor and
improved the arrangement of cells on silastic membrane.
• The results showed that Ucs and MSCs cannot survive 100% deformations.
Future
Improvement of the bioreactor, with a continuous mechanical stimulation.
An FTE bladder must have structures in place to serve as a buffer between
the cells and the organ when the bladder is deformed.
Utilization of biodegradable materials.
Suitable use of this bioreactor for urinary bladder tissue regeneration and
ex vivo tissue reconstitution.
Case study 3
The present article focuses on the design and development of a bioreactor for long
tubular construct engineering that allows double seeding and culturing on both the inner
and the outer surface of the matrix.
Introduction
A clinically applicable tracheal substitute must meet numerous requirements:
o external hyaline cartilage framework
o internal epithelial covering
Although there are reports of small volumes tracheal cartilage generation and
clinical application, progress with long segment grafts has been limited by
lack of an ideal scaffold, well-established epithelial and chondrocyte culture
techniques, and an appropriate bioreactor environment.
Requirements of a tracheal bioreactor
•
•
the provision of different culture conditions on either side of the organ-wall
the need for adequate mass transport of gases and nutrients within a construct
that has to be more than 4 cm long to be clinically useful.
The step-wise work plan consisting of :
o
o
o
o
o
o
design of a bioreactor
development of predictive analytical models
in vitro testing
in vivo trials in animal model
application of human cells
performance of a first-time-in-man transplantation of the resultant re-cellularized
construct.
Rotating double-chamber bioreactor design
to facilitate cell seeding procedures on both sides of a 3D tubular
matrix, ensuring homogeneous plating
to allow seeding and culturing of different cell types on either side
of the tubular scaffold
to enhance oxygenation of the culture medium and mass transport
(oxygen, nutrients and catabolites) between the medium and the
adhering cells
To stimulate the cells with hydrodynamic stimuli, favoring the
metabolic activity and the differentiation process
to allow the achievement and maintenance of sterility and other
criteria of Good Laboratory Practice (GLP), simplicity and
convenience
to permit the possibility of automation and scale-up/-out.
Rotating double-chamber bioreactor design
The device allows seeding and culturing on both surfaces of a tubular matrix and includes
rotatory movement of the scaffold around its longitudinal axis providing oxygenation to the 3D
structure and improves mass transport between the culture media and the adhering cells.
Rotating double-chamber bioreactor design
T= 37 °C
Pp= 5% CO2
3 main components:
• culture chamber,
• motion
• control units
o polymeric culture chamber biologic sample and the medium
o cylindrical scaffold holders working ends 10-25 mm in diameter
o central portion expose the luminal surface of the matrix for seeding and culturing
o chamber closed by a Petri-like cover permit oxygenation and sterility of the
culture environment
o system can be autoclaved reduction of contamination risks
Bone marrow stem cell (BMSC) culture
Mesenchymal BMSCs were isolated and cultured.
Isolation
Proliferation
Differentiation
Chondrocytes
The multi-lineage differentiation potential of BMSCs was assessed
by examining their osteogenic, adipogenic and chondrogenic capacities.
Anthony Hollander (Bristol) group found a new and sure method to allow the
differentiation of mesenchymal in chondrocytes.
Respiratory epithelial cells culture
Respiratory epithelial cells were isolated from airways and trachea and cultured.
Biopsy
Culture
Differentiation
Human tracheal decellularization
Scaffold = support structure that allows the cell growth, in order to create
a tissue with some specific biologic, functional and morphologic
characteristics.
Decellularization of the biological scaffold:
A 7 cm tracheal segment was retrieved from a transplant donor,
having removed all loose connective tissue.
After 25 cycles of decellularization, epithelial and glandular cells
were completely removed from the tracheal matrix, while only
a few chondrocytes were still visible.
Matrix inside the bioreactor during seeding
Cell seeding was performed inside the bioreactor, avoiding construct
manipulation and thus limiting the risk of cell construct contamination.
Bioreactor cultivation of the trachea construct
Ethical permission
was obtained from
the Spanish
Transplantation
Authority and the
Ethics Committee
of the Hospital
Clinic, Barcelona.
At the end of the culture period, the bioreactor rotation was turned off, both chambers were
emptied and completely refilled with fresh media and the bioreactor was delivered to the operating
room.
The graft was then cut to shape and implanted into the patient as a replacement for her left main
bronchus.
Bioreactor cultivation of the trachea construct
Re-cellularization
Matrix after seeding
Results
At two months, graft biopsy showed vigorous angiogenesis and remodeling
gradients of oxygen and nutrients exist in engineered tissue, due to the balance between
transport and rates of cellular consumption
Immunofluorescence histology
confirmed the presence of
angiogenesis and showed
reconstitution of epithelium, the
continued presence of viable
chondrocytes, and a reappearance of
the mucosal lymphoid cells that
typically densely populate normal
tracheal mucosa
Advantages vs Disadvantages
low level of automation of the system : an automatic medium conditioning and exchange
system is desirable in order to minimize contamination risks and protect homeostasis,
permitting intermittent, sterile evaluation of pH, nutrient or waste concentration.
a system controller for tissue-engineering process : monitoring the data provided by
Sensors, will allow more control and, thereby, reproducibility of expansion, differentiation and
migration of cells within the scaffold.
re-personalization of a donor trachea and successfully airway transplantation
without the need of any immunosuppressive therapies.
no sign of rejection at one year post-implantation.
vigorous angiogenesis
viable chondrocytes, and a layer of viable epithelial cells at two months surgery.
However, the epithelial layer, was microscopically discontinuous.
Future
• improve epithelial cell coverage of the internal surface of the graft pre-operatively
• application of flow stimuli to the internal compartment of the bioreactor will
encourage appropriate alignment and function of cilia prior to implantation,
thereby initiating appropriate clearance of mucus from the first post-operative day.
• refinements to permit scale-up and full clinical trials, as well to explore
hypothetical ways of improving graft production, such as encouraging angiogenesis
and orientated ciliary function.
References
• “NASA-Approved Rotary Bioreactor Enhances Proliferation of Human Epidermal
Stem Cells and Supports Formation of 3D Epidermis-Like Structure” , Xiao-hua Lei1.,
Li-na Ning1., Yu-jing Cao1, Shuang Liu1,2, Shou-bing Zhang1,2, Zhi-fang Qiu1, Hui-min,
Hu1,2, Hui-shan Zhang1,2, Shu Liu1,2, En-kui Duan1*
• “A novel bioreactor to simulate urinary bladder mechanical properties and
compliance for bladder functional tissue engineering”, WEI Xin, LI Dao-bing, XU Feng,
WANG Yan, ZHU Yu-chun, LI Hong and WANG Kun-jie, Chin Med J 2011;124(4):568-573
• “A double-chamber rotating bioreactor for the development of tissue-engineered
hollow organs: From concept to clinical trial” , M. Adelaide Asnaghi a,*, Philipp
Jungebluth b, Manuela T. Raimondi c,d, Sally C. Dickinson e, Louisa E.N. Rees f, Tetsuhiko Go
b, Tristan A. Cogan f, Amanda Dodson f, Pier Paolo Parnigotto g, Anthony P. Hollander e,
Martin A. Birchall h, Maria Teresa Conconi g, Paolo Macchiarini b, Sara Mantero. Biomaterials
30 (2009) 5260–5269
“Clinical transplantation of a tissue-engineered airway”, Paolo Macchiarini, Philipp
Jungebluth, Tetsuhiko Go, M Adelaide Asnaghi, Louisa E Rees, Tristan A Cogan, Amanda
Dodson, Jaume Martorell, Silvia Bellini, Pier Paolo Parnigotto, Sally C Dickinson, Anthony P
Hollander, Sara Mantero, Maria Teresa Conconi, Martin A Birchall, Lancet 2008; 372: 2023–
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