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.