U.S. patent application number 09/956172 was filed with the patent office on 2002-12-26 for cell culture tube and multiple roller tube cell culture system using the same.
Invention is credited to Choi, Wan Kyu, Shin, Sung Ho, Yoo, Kwang Hyun.
Application Number | 20020197710 09/956172 |
Document ID | / |
Family ID | 19709729 |
Filed Date | 2002-12-26 |
United States Patent
Application |
20020197710 |
Kind Code |
A1 |
Yoo, Kwang Hyun ; et
al. |
December 26, 2002 |
CELL CULTURE TUBE AND MULTIPLE ROLLER TUBE CELL CULTURE SYSTEM
USING THE SAME
Abstract
A cell tube for ex vivo culturing animal cells and a multiple
roller tube cell culture system are disclosed. Suitable for use in
culturing cells, the tube has at its opposite end walls two
openings through which culture media can come in and out. The
openings are eccentrically located at corresponding positions in
contact with the edge sides of the end walls. The system has a
plurality of roller drums on which a multitude of cell tubes are
assembled. As the roller drums are rotated, cells adhering to each
tube experience a nutrient-rich state and aerobic starvation,
repeatedly. In the nutrient-rich state, the cells are grown
flourishingly. When subjected to starvation, cells select
metabolism pathways for utilizing carbon sources effectively,
produce lactate at a low rate and can maintain a constant pH,
because they are in direct contact with air. The system makes cells
adhere to the wall of cell tubes and provides air directly to cell
surfaces. Adherent cells can be grown with normal morphology at
high yield for a long time in the cell culture system. The cell
culture can be easily scaled up simply by increasing the number of
the roller drums. The system can exchange media and feed a gas
mixture of oxygen and carbon dioxide easily, thus providing optimal
environments suitable for small to large scale cell culture.
Inventors: |
Yoo, Kwang Hyun;
(Yongin-city, KR) ; Choi, Wan Kyu; (Yongin-city,
KR) ; Shin, Sung Ho; (Cheongju-city, KR) |
Correspondence
Address: |
MADSON & METCALF
GATEWAY TOWER WEST
SUITE 900
15 WEST SOUTH TEMPLE
SALT LAKE CITY
UT
84101
|
Family ID: |
19709729 |
Appl. No.: |
09/956172 |
Filed: |
September 19, 2001 |
Current U.S.
Class: |
435/298.2 ;
435/394 |
Current CPC
Class: |
C12M 27/12 20130101;
Y10S 435/809 20130101; C12M 25/12 20130101 |
Class at
Publication: |
435/298.2 ;
435/394 |
International
Class: |
C12M 003/00 |
Foreign Application Data
Date |
Code |
Application Number |
May 21, 2001 |
KR |
2001-27831 |
Claims
What is claimed is:
1. A cell tube for use in culturing cells, having two openings
through which culture media can come in and out, said openings
being eccentrically located on opposite end walls at corresponding
positions in contact with the edge sides of the end walls.
2. The cell tube as set forth in claim 1, wherein the cell tube has
a cross sectional shape selected from the group consisting of
circles, ellipses, and polygons.
3. The cell tube as set forth in claim 1, wherein the cell tube
ranges, in length, from 10 to 300 mm and, in diameter, from 5 to
110 mm.
4. The cell tube as set forth in claim 1, wherein the openings have
a shape selected from the group consisting of circles, ellipses,
and polygons and occupy an area of 1 to 70% based on the total area
of the end walls.
5. The cell tube as set forth in claim 1, wherein the openings are
means serving as an inlet and an outlet of culture media.
6. A multiple roller tube cell culture system, comprising: one or
more cell culture tube bundles, each consisting of a plurality of
cell culture tubes axially arranged around a central rotating shaft
within a cylindrical housing such that an eccentric hole formed on
each end wall of each of said tubes is positioned outward in a
radial direction of the housing; an air inlet and an air outlet
formed on said housing for feeding oxygen to the interior of said
housing; a level controller for maintaining a desired level of cell
culture medium inside the housing; a medium inlet for feeding the
cell culture medium to the interior of said housing; a sensor for
sensing the pH and dissolved oxygen level inside the housing; and a
harvest outlet for automatically discharging culture products from
the housing after the cell culturing process is finished.
7. The multiple roller tube cell culture system as set forth in
claim 6, wherein the tubes are arranged inside the housing such
that the holes of the tubes are positioned outward in a centrifugal
direction of the housing, thus allowing the cell culture medium to
smoothly flow into or from the tubes.
8. The multiple roller tube cell culture system as set forth in
claim 6, wherein the cell culture medium flows into or from the
tubes through the holes due to gravity.
9. The multiple roller tube cell culture system as set forth in
claim 7, wherein said cell culture tubes are arranged parallel to
the centrifugal direction or inclined from the centrifugal
direction at an angle of 0 - 10.degree. to accomplish a desired
flow of the medium relative to the tubes.
10. The multiple roller tube cell culture system as set forth in
claim 7, wherein said cell culture tube bundles are arranged
parallel to each other in the system.
11. The multiple roller tube cell culture system as set forth in
claim 6, wherein said cell culture tubes are made of glass or
plastic.
12. A method for culturing adherent animal cells in a mass scale
using the multiple roller tube cell culture system of claim 6,
comprising the steps of: feeding a predetermined amount of a
culture medium into the cell culture system through the inlet by
use of the medium transfer pump under the control of the level
controller; rotating the roller drum on which cell tubes are
assembled, bring a portion of cell tubes into contact with the
influent medium and air, said cell tube assembly being connected to
the axis of the motor; controlling the pH and dissolved oxygen
level of the cell culture with the aid of sensors; and replacing
the cell culture with a fresh medium and recover the cell
culture.
13. The method as set forth in claim 12, wherein the cell tubes are
coated with a gelatin solution at their inside surfaces or contain
fibers or spongy therein to facilitate the attachment of cells.
14. The method as set forth in claim 12, wherein the controlling
step is carried out by feeding sterile moisture gas mixture of
oxygen and carbon dioxide in proportions with 90:10 to 100:0.
Description
BACKGROUND OF THE INVENTION
[0001] 1.Field of the invention
[0002] The present invention relates, in general, to a tube
suitable for use in ex vivo culturing of animal cells and a
multiple roller tube cell culture system using the same and, more
particularly, to a cell tube having at its opposite end walls two
eccentric openings through which culture media can flow in and out.
Also, the present invention is concerned with a multiple roller
tube cell culture system using a multitude of cell tubes, which is
able to culture adherent cells in a continuous or batch type manner
with high efficiency.
[0003] 2.Description of the Prior Art
[0004] Conventionally, it is difficult to grow ex vivo cultures of
animal cells at high yield and in a concentrated level compared to
microorganisms because animal cells which have weak cell membranes
and are apt to undergo shear stress. The necessity for animal cell
cultures has been increased for various reasons. Various efforts
have been made to culture animal cells, but in most such efforts,
culture methods for bacteria were applied to the cultures of animal
cells irrespective of characteristics of animal cells. Thus, the
success of the efforts was not great in spite of much
investment.
[0005] As a result of recent advances in cell characterization,
culture methods have been studied for various cells, especially ES
(embryonic stem) cells, hybridoma cells, and CHO (Chinese hamster
ovary) . Highly concentrated cultures of lymphocyte lineage cells,
of which hybridoma cells are representative, can be grown on a
membrane by virtue of the development of the hollow fiber system of
ASM company. Also, it is possible to culture ES and cancerous cells
can be cultured in tissue slices as the rotary cell culture system
developed by NASA ensures cell's undergoing low shear stress and
provides environment control.
[0006] Cytokines or useful proteins are generally produced from
transformed adherent cells, such as CHO, 3T3 or C127, by gene
manipulation. The accomplishment of the human genome project allows
the expectation that various useful proteins might be produced
using adherent cells.
[0007] However, mass cultures of adherent cells to which
fibroblasts and epithelial-like cells have not yet been developed
completely. In fact, culture methods for ES cells or hybridoma
cells are applied to the adherent cells, so that not only is the
culture yield decreased significantly, but also the adherent cells
cannot be cultured for a long time period.
SUMMARY OF THE INVENTION
[0008] It is an object of the present invention to overcome the
problems, encountered in prior arts, of being unable to culture
adherent cells for a long period of time and at high yield, and to
provide a cell culture system in which adherent cells can be
cultured with ease and high productivity.
[0009] It is another object of the present invention to provide a
method for culturing animal cells on a large scale with high
efficiency.
[0010] In accordance with an aspect of the present invention, there
is provided a cell tube for use in culturing cells, having two
openings through which culture media can flow in and out, the
openings being eccentrically located on opposite end walls at
corresponding positions in contact with the side edges of the end
walls.
[0011] In accordance with another aspect of the present invention,
there is provided a multiple roller tube cell culture system,
comprising: one or more cell culture tube bundles, each consisting
of a plurality of cell culture tubes axially arranged around the
central rotating shaft within a cylindrical housing such that the
eccentric opening formed on each end wall of each of the tubes is
positioned outward in a radial direction of the housing; an air
inlet and an air outlet formed on the housing for feeding oxygen to
the interior of the housing; a level controller for maintaining a
desired level of cell culture medium inside the housing; a medium
inlet for feeding the cell culture medium to the interior of the
housing; a plurality of sensors for sensing the pH and dissolved
oxygen level inside the housing; and a harvest outlet for
automatically discharging culture products from the housing after
the cell culturing process is finished.
[0012] In accordance with a further aspect of the present
invention, there is provided a method for culturing adherent animal
cells on a large scale using the multiple roller tube cell culture
system, comprising the steps of: feeding a predetermined amount of
a culture medium into the cell culture system through the inlet by
use of the medium transfer pump under the control of the level
controller; rotating the roller drum on which cell tubes are
assembled, at a speed of 1 rpm or less to repeatedly bring a
portion of cell tubes into contact with the influent medium and
air, the cell tube assembly being connected to the axis of the
motor; controlling the pH and dissolved oxygen level of the cell
culture with the aid of sensors; and increasing the rotation speed
to 60 rpm or less to exchange the cell culture with a fresh medium
and recover the cell culture.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] FIG. 1 is a diagram showing the structure of the multiple
roller tube cell culture system of the present invention, which is
equipped with a plurality of roller drums, each provided with a
multitude of cell tubes.
[0014] FIG. 2 is a perspective view showing the structure of the
cell tube.
[0015] FIG. 3 is a cross sectional view showing a roller drum on
which cell tubes are assembled.
[0016] FIG. 4 illustrates the inflow and outflow of medium of the
cell tube during cell growth (a), and culture withdrawal (b), as it
revolves around the axis.
[0017] FIG. 5 is a graph in which the pH (-.DELTA.-), consumed
glucose (-.diamond.-), and produced lactate (-.quadrature.-) of a
cell culture are plotted versus culture period when the cell
culture is grown in the multiple roller tube cell culture
system.
[0018] FIG. 6 is a graph in which the pH (-.DELTA.-), consumed
glucose (-.diamond.-), and produced lactate (-.quadrature.-) of a
cell culture are plotted versus culture period when the cell
culture is grown in a tissue culture flask.
[0019] FIG. 7 is a graph in which levels of glucose (-.diamond.-)
and lactate (-.quadrature.-) in the medium are plotted versus
culture period when the cell culture is grown in the multiple
roller tube cell culture system.
[0020] FIG. 8 is a graph in which levels of glucose (-.diamond.-)
and lactate (-.quadrature.-) in the medium are plotted versus
culture period when the cell culture is grown in the tissue culture
flask system.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
[0021] The application of the preferred embodiments of the present
invention is best understood with reference to the accompanying
drawings, wherein like reference numerals are used for like and
corresponding parts, respectively.
Construction of the invention
[0022] FIG. 1 shows the construction of the multiple roller tube
cell culture system 10 according to the present invention. In the
cell culture system 10, a plurality of cell culture tubes 11 are
closely and axially arranged around a central rotating shaft 31
within a cylindrical housing 12, thus forming a cell culture tube
bundle 13. In such an arrangement, the eccentric openings 14 of the
tubes 11 are positioned outward in a radial direction of the
housing 12. One or more cell culture tube bundles 13are integrated
into a single unit to form a desired system 10. The rotating shaft
31 of the tube bundle 13 is connected to the output shaft of a
drive motor 30 by a belt transmission mechanism, and rotated in
conjunction with the motor 30. The housing 12 is provided with an
air inlet 21 and an air outlet 22 for feeding oxygen to the
interior of the housing 12 of the cell culture tube bundle 13. The
system 10 also has a level controller 20 for maintaining a desired
level of cell culture medium inside the housing 12. In addition, a
medium transfer pump 51 feeds a predetermined amount of cell
culture medium from a medium reservoir 50 to the interior of the
housing 12 through a medium inlet 52 of the housing 12. The system
10 also has a plurality of sensors 40 for sensing the pH and
dissolved oxygen level inside the housing 12 during a cell
culturing process of the system 10. The system 10 also has a
harvest outlet 61 on the housing 12 for automatically discharging
the culture products from the housing 12 to a harvest reservoir 60
after the cell culturing process is finished.
[0023] In the system 10, the opposite ends of each cell culture
tube 11 are closed with end walls, but are provided with eccentric
openings 14 at corresponding positions where the openings 14 are
inscribed with the circular edges of the end walls. Therefore, the
cell culture tubes 11 are almost free from interference or
resistance of the medium during the flow of the medium relative to
the tubes 11, thus being less likely to be ill-affected by shear
stress and accomplishing a desired cell culturing process.
[0024] In the cell culture system 10, a predetermined number of
cell culture tubes 11 are closely and axially arranged around the
rotating shaft 31 within a cylindrical housing 12 such that the
eccentric openings 14 of the tubes 11 are positioned outward in a
direction of centrifugal force of the housing 12. A cell culture
tube bundle 13 is thus produced. One or more cell culture tube
bundles 13 are arranged parallel to each other inside the system
10. Such a structure and arrangement of the cell culture tubes 11
is specifically designed to allow the cell culture medium to
smoothly flow into or from the tubes 11, and is included in the
gist of this invention.
[0025] In an operation of the system 10, a predetermined amount of
cell culture medium is pumped from the medium reservoir 50 by the
medium transfer pump 51 under the control of the level controller
20, and fed to the interior of the housing 12 through the medium
inlet 52 of the housing 12, and so a predetermined level of the
medium inside the housing 12 is continuously maintained. After the
cell culturing process, the culture products are automatically
discharged from the housing 12 to the harvest reservoir 60 by the
harvesting pump 61. In addition, the ambient air as well as the pH
and dissolved oxygen level inside the housing 12 are monitored by
sensors 40 with circulation pump 41, and the sensors 40
automatically sense the state of the cultured cells.
[0026] During the cell culturing process of the system 10, a
predetermined amount of humidified and sterilized air is
continuously fed into the housing 12 through the air inlet 21, thus
feeding oxygen to the cells. In such a case, gases are smoothly
discharged from the housing 12 through the air outlet 22, thus
maintaining a target oxygen level for the cells.
[0027] When the cell culture tubes 11 inside the housing 12 are
rotated at a speed of 1/2rpm or less around the central rotating
shaft 31 by the rotating force of the motor 30, the cell culture
tubes 11 are gradually sunk into the medium inside the housing 12.
The medium is thus introduced into the cell culture tubes, and
allows the cells to be stuck to the internal surface of the tubes
and to grow in the tubes. The cells inside the culture tubes are
induced to propagate by exposure to the media.
[0028] As described above, the cell culture tubes having the
eccentric openings on their end walls are arranged around the
rotating shaft inside the housing such that the openings of the
tubes are positioned outward in the radial direction of the housing
as best seen in FIG. 3. Therefore, when the tubes are rotated in a
direction as shown in FIG. 3, the media contained in the housing
smoothly flows into or from the tubes through the openings. In the
present invention, the cell culture tubes may be arranged parallel
to a direction of centrifugal force or inclined from the direction
of centrifugal force at an angle of 0 - 10.degree. to accomplish a
desired flow of the medium relative to the tubes.
[0029] When cell floating liquid is introduced into the cell
culture tube bundle as shown in FIG. 3, the complete attachment of
the cells on the internal surface of the tubes necessarily requires
24 - 72 hours. When the cells are completely stuck to the internal
surface of the tubes, the cell culture medium inside the tubes is
distributed to the cells and changed with new medium as shown in
FIGS. 4aand 4b. In addition, the cells inside the tubes are
cultured with appropriate concentrations of glucose, lactate, and
ammonia at the appropriate pH. In such a case, the nutrient
concentrations and the pH are set at a media changing interval
while considering productivity of the cells. The cell culture media
inside the housing is automatically replaced under the control of
the level controller. The cell culturing process of the system is
performed with an injection of humidified and sterilized gases
including oxygen and carbon dioxide into the housing for
controlling the pH and dissolved oxygen level of the cells. The
system and method of this invention thus automatically performs
desired cell culture and analysis.
[0030] While the multiple roller tube cell culture system is
revolved by the motor, a portion of the bundled cell culture tubes
is brought into contact with externally provided cell culture which
is then flowed into the culture tubes through the hole of each cell
tube. Adhering to the walls of culture tubes, the cells are
uniformly distributed owing to the revolution, so that they can be
cultured on a large scale in aerobic conditions.
[0031] A bundle of the cylindrical culture tubes provide a large
inner surface area onto which adherent cells can be grown, so that
the cell culture system can take maximal advantage of externally
provided oxygen and media to help cells effectively metabolize the
carbon sources and nutrients of the media. Under these conditions,
lactate is produced in a low quantity with maintenance of constant
pH. In addition, with the ability to automatically control the
feeding of media and the withdrawal of cultures, the cell culture
system of the present invention can culture cells at a high density
with high efficiency for a long time. Accordingly, the present
invention provides an automatic cell culture system, which affords
accurate environmental control to produce proteins of interest at
high yield and efficiency.
[0032] While a bundle of culture tubes is revolved slowly, a
portion of the tube bundle is immersed in a medium filling a lower
part of the cell tube housing, so that a culture medium or cell
suspension is flowed into each cell tube via two openings which are
respectively provided on the opposite sides of the cell tube while
being eccentrically located at the corresponding positions in
contact with the wall of the cell tube. In the medium, the cells
are provided with nutrients and metabolize them. As the shaft is
rotated by the motor, the immersed cell tubes emerge from the
medium filling the lower part and the medium filling each cell tube
comes out through the eccentric openings. Under the aerial
condition, the cells remaining in the cell tubes operate their
aerobic metabolism pathways. Accordingly, experiencing a
nutrient-rich state and aerobic starvation, alternately and
repeatedly, the cells adhering to the cell tubes flourish. In the
nutrient-rich state, the cells are grown flourishingly. On the
other hand, the cells, when subjected to starvation, utilize
metabolic pathways for utilizing carbon sources effectively. In
this state, the cells produce lactate at a low rate and thus can
maintain a constant pH because they are in direct contact with
air.
[0033] Culture Principle of the invention
[0034] Operated in such a manner that a medium is dispensed into
culture tubes and exchanged continuously at a predetermined amount
with fresh medium, the cell culture system of the present invention
can grow cultures of cells in a batch type manner, a continuous
batch type manner, or continuous type manner. With the structure in
which air is exchanged through the medium surface within each
culture tube, the culture system of the present invention provides
large air contact surfaces, compared to conventional culture
systems, so that the cells are not under air bubble stress or shear
stress, unlike conventional bioreactors.
[0035] At cell surfaces in contact with air, effective delivery of
air into cells occurs, ensuring smooth glycolysis. In an
oxygen-deficient condition, glucose, serving as an energy source,
is decomposed into lactic acid to lower medium pH, so that cells
are apt to be damaged. On the other hand, in the presence of
plentiful oxygen, cells can obtain a large amount of energy through
the TCA cycle (tricarboxylic acid cycle) without production of
lactic acid which is forced to decrease in pH of the medium. In
addition, since glucose, an energy source, is rapidly exhausted at
the surface with which cells are in direct contact, cells tend to
choose the TCA cycle in order to effectively use carbon
sources.
[0036] When these conditions are sustained for a long period of
time, cells themselves are under stress. Cells can be restored to a
normal state if they are placed in a medium so as to bring the cell
surface into contact with the medium or are provided with a fresh
medium. This principle is used by a roller bottle, which is one of
the most popular systems for culturing adherent cells at present.
In addition to adopting the same principle, the multiple roller
tube of the present invention is improved in air provision and pH
control, has significantly small culture spaces, and cultures cells
in an automatic operation. Therefore, the culture system of the
present invention overcomes the problems that conventional roller
bottles have, that is, incapability of automatic operation, air
exchange and pH control.
[0037] A better understanding of the present invention may be
obtained in light of the following examples which are set forth to
illustrate, but are not to be construed to limit the present
invention.
EXPERIMENTAL EXAMPLE 1
[0038] Properties of Culture Systems for Adherent Animal Cells
[0039] In order to compare the performance and culture properties
of the multiple roller tube cell culture system of the present
invention with those of other cell culture systems, an measurement
was made of cell morphology, percentage of lactate to glucose, cell
viability, and culture yield using gene-manipulated CHO cells in
flask, roller bottle and bioreactor. The results are given in Table
1, below.
1TABLE 1 Culture Properties of Transformed CHO cells According to
Culture System Cell Bioreactor Culture Roller Cell Property Flask
Bottle Microcarrier cluster Cell Monolayer Monolayer Monolayer&
Cell Morphology cluster cluster Lactate/ 70-80 40-60 90 or more 95
or more Glucose (%) Productivity 500-1200 1000-2500 300-600 300 or
less of Cytokine (unit) Cell 3 weeks 4 weeks 1.5 weeks Not
Viability available
EXPERIMENTAL EXAMPLE 2
[0040] Culture Properties of Transformed C127 Cell According to
Culture System
[0041] Transformed C127 cells were cultured in DMEM with a high
content of glucose in a flask, a roller bottle, a bioreactor, and a
hollow fiber under various conditions while observing cell
morphology, growth location, recombinant viral protein, and culture
period. The results are given in Table 2, below.
2TABLE 2 Culture Properties of Transformed C127 Cell According to
Culture System Roller Flask Bottle Bioreactor Hollow fiber Medium
DMEM As left As left As left Cell morphology Multilayer As left
Multilayer Clustered at production fiber fiber/clustered Matrix
Polystyrene Polystyrene Dextran Cell itself Culture Type Batch
Batch Batch (STR) Continuous perfusion Control 5% CO.sub.2 None 4
gases 2 gases controlled exchanged Growth locus Flask bottom Wall,
in & Surface Fiber surface in medium out medium in medium in
medium Lactate/Glucose .apprxeq.70% .apprxeq.0-50% .gtoreq.95%
.apprxeq.100% Productivity 1-2 mg/L 6-12 mg/L 0.1-0.3 mg/L
0.01-0.03 mg/L (viral protein) Cell Viability 1 month 3 months Not
available Not available
[0042] As seen in Tables 1 and 2, the productivity of recombinant
viral proteins varies with culture systems. It is believed that the
increased productivity and extended cell viability of the roller
bottle is attributed to the fact that oxygen is smoothly fed to the
cell membranes from the air within roller bottle, temporary
exhaustion of carbon sources allows the cells to effectively use
the carbon source remaining, and cells are transferred into the
medium to absorb a sufficient amount of nutrients. In the flask,
cells are grown in the medium, adhering to the bottom. This culture
system is efficient while cells grow at a low density. However, it
is not easy for oxygen to penetrate into the medium contained in a
flask. Further, the cycle from supernutrition to innutrition (that
is, medium exchange cycle) is long so that the cells are liable to
be damaged by high cell density, in addition to having difficulty
in efficiently using carbon sources. On the other hand, when
culturing is carried out with microcarriers serving as a matrix or
cells being suspended, dissolved oxygen levels are increased. In
this case, however, the cells undergo changes in cell morphology so
that carbon sources cannot be efficiently utilized, reducing the
productivity. Particularly, the lactate thus produced acidifies the
medium, making the medium exchange cycle shorter. Further, desired
biomaterials produced as a result of gene manipulation are
generally vulnerable to low pH, which results in reducing the
productivity.
EXAMPLE 1
[0043] To test the performance of the multiple roller tube cell
culture system of the present invention, CHO.sup.dhfr-cells, which
are most widely used for the production of cytokines, were used.
Before culturing, inner surfaces of the culture tubes were coated
with a 2% gelatin solution (Sigma G1393) for 2 hours to facilitate
cell adherence. For comparison, a tissue culture flask made of
polystyrene (equipped with a 0.2 .mu.m filter cap having 75
cm.sup.2, Corning #430641) was also used. For use in this
experiment, a culture medium based on Isocove's modified Dulbecco's
medium (Gibco Cat. No. 12200-036) containing 3.024 g/L of
NaHCO.sub.3 was based. In addition, the medium was added with HT
(hypoxantin thymidine supplement, Gibco Cat. No. 11067-030) and
supplemented with fetal bovine serum (FBS, Gibco Cat. No.
26140-079) at an amount of 10 % by volume. The multiple roller tube
cell culture system was composed of 17 cell tubes with a total
inside area of 694 cm.sup.2. CHO cells were suspended at an initial
density of 4.times.10.sup.7 cells in 200 ml of the medium and
cultured on a roller drum within an incubator (37 .degree. C.) with
the culture system being sealed. In the case of the flask, the
cells were suspended at a density of 4.322.times.10.sup.6 cells in
22 ml of the medium and the suspension was added into the flask,
followed by culturing in a 5% CO.sub.2 incubator. 48 hours after
the initial suspending of cells, the medium was exchanged with a
fresh one. Thereafter, medium exchange was carried out every 48
hours while measuring glucose and lactate levels with an assay kit
(Sigma). The drawn media were measured to maintain pH at 7.0 or
higher. The cell culture system was revolved at a speed of
1/3-1/4rpm.
[0044] Within 24 hours from the addition of the cell suspension
into the cell tubes, cells were desirably attached to the inside
walls of the cell tubes. The cells were still found to maintain
their adherence to tube walls even after 1 month of the culturing,
as measured by crystal violet dying. A predetermined volume of a
fresh culture medium was fed into the cell tubes to replace the
same volume of the used medium by the revolution of the culture
system, which was confirmed to be normally operated at up to 30
rpm. When the medium rapidly decreased in pH as the culture period
was extended, feeding fresh air restored pH values to a normal
state with ease. The attachment behavior and morphology of cells
grown in the cell tubes were not significantly different from those
of cells grown in the tissue culture flask. However, the time
period that it took for the cells to adhere completely to the wall
of the cell tubes was measured to be 24 hours, which was 8 hours
longer than the attachment time period in the flask. Cell growth
could be identified directly through a microscope and indirectly by
the analysis of metabolites.
[0045] From these culture results, it was seen that the cell
culture system could be operated in an automatic manner because the
medium continually fed to the lower part of the cell system flowed
into or out of the cell tubes as the cell culture system revolved.
Employment of culture tubes made of plastic (i.e., polystyrene)
brought about an improvement in cell adherence. Even in the case of
cell suspension, carbon sources could be effectively utilized
through environmental control in the cell culture system. When
materials capable of collecting cells within cell tubes were
inserted or attached to the cell tubes, the suspended cells could
be grown.
EXAMPLE 2
[0046] In the automatic multiple roller tube cell culture system of
the present invention, cells pretreated as in Example 1 were
cultured for a long period of time. First, the culture medium was
transferred at a predetermined amount from the reservoir to the
culture system through the inlet with the aid of the pump and the
level controller. The motor, which was connected through the axis
to the cell tube bundle, was revolved at a speed of 1.0 rpm or less
to bring the cell tubes into contact with the medium, thereby
culturing the cells. Culture conditions were controlled with
monitoring of the pH and dissolved oxygen level of the medium by
use of a sensor. After being measured for concentration, the medium
was exchanged with a fresh one while the revolution speed was
increased to 60 rpm or less. Throughout the culture period, the
cells were attached to the wall and grown without difficulty. In
addition, medium exchange was conducted smoothly in the cell
culture system. A pH increase took place upon medium exchange, but
provision of CO.sub.2 returned the pH to a normal state. That is,
the pH of the medium could be controlled by the provision of air
during cultivation. Accordingly, it was seen that two-gas
environment control was possible.
[0047] With reference to FIGS. 5 and 7, there are shown the
relationships between glucose absorption and lactate production and
between medium glucose and lactate levels, respectively, in the
culture system of the present invention. Able to utilize carbon
sources of the culture medium as proved by a decrease in the
production of lactate in comparison to the amount of glucose
consumed in FIGS. 5 and 7. These results indirectly indicate that
cell culture system could produce cytokines at high yields.
Therefore, increasing the number of culture tubes allows cells to
be cultured in a mass scale.
[0048] Cells were firmly attached to the inside walls of the cell
tubes as in Example 1 and this attachment was still maintained even
after 1 month of cultivation. The revolution of the cell culture
system fed a predetermined volume of a fresh medium into the cell
tubes while drawing the same volume of the used medium. The pH of
the medium was decreased as cells were grown to produce lactate.
However, the pH of the medium could be normalized easily by
providing air.
[0049] From the initiation, the culture was monitored for 27 days.
When being cultured in the cell culture system, the cells were
observed to be improved in metabolism until the 19.sup.th day as
measured for glucose uptake ratio (FIGS. 5 and 7). On the other
hand, when the cells were cultured in the flask, the cell
metabolism was not further improved from the 9.sup.th day with
glucose level remaining at about 100 mg/dl (FIGS. 6 and 8). Lactate
was produced at increasing amounts until the 13.sup.th day after
providing fresh air at the 9.sup.th day in the cell culture system.
Compared with the glucose consumed, however, the lactate production
can be said to be decreased rather than increased. In fact, the
ratio of produced lactate to consumed glucose was reduced to as low
as about 22.9 % (FIGS. 5 and 7). In contrast, the lactate
production was measured to remain at 300 mg/dl from the 9.sup.th
day in the flask culture, which corresponded to 90 % of the amount
of absorbed glucose (FIGS. 6 and 8). Since the 19 .sup.th day, the
revolution speed was reduced to 1/4rpm from 1/3rpm in the cell
culture system. As a result, the ratio of produced lactate to
consumed glucose was significantly lowered, indirectly
demonstrating the starvation effect of utilizing carbon sources
with efficiency.
[0050] As for the pH of the medium, it was reduced to 6.98 in the
cell culture system before feeding fresh air in the early stage and
maintained at 7.0 or higher after feeding fresh air. When the cell
culture system was revolved at 1/3rpm to utilize carbon sources
effectively, the medium was measured to have pH 7.00-7.07. At
1/4rpm, the pH value was increased to 7.20-7.32, providing an
optimal condition for the growth of animal cells (FIG. 5). By
contrast, no starvation effects could be observed from the culture
in the flask. From the 5 .sup.th day, the medium of the flask was
measured to range, in pH, from 6.50 to 6.90 (FIG. 6), which
negatively affects cell culture, especially the production of
cytokines of interest.
[0051] As described hereinbefore, the cell culture system of the
present invention makes cells adhere to the wall of cell tubes and
provides air directly to cell surfaces. Accordingly, adherent cells
can be grown with normal morphology at high yield for a long period
of time in the cell culture system. Additionally, cell culture can
be easily scaled up simply by increasing the number of the roller
drums or enlarging the assembly of cell tubes. Further, the cell
culture system of the present invention enjoys the advantage of
being capable of exchanging media and feeding a gas mixture of
oxygen and carbon dioxide easily, thereby providing optimal
environments suitable for small to large scale cell culture.
[0052] The present invention has been described in an illustrative
manner, and it is to be understood that the terminology used is
intended to be in the nature of description rather than of
limitation. Many modifications and variations of the present
invention are possible in light of the above teachings. Therefore,
it is to be understood that within the scope of the appended
claims, the invention may be practiced otherwise than as
specifically described.
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