Note: Descriptions are shown in the official language in which they were submitted.
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This invention relates to a method and an apparatus
for cellular culture. More particularly, the invention relates
to the production of cells by a method which enables much
improved yields to be obtained within a culture vessel.
Mainly, cellular cultures of animal origin are
derived from chicken embryos,human tissues, monkey tissues and
the like. It is also known that there is a need for massive
cellular culture because of the increasing activity in virology,
cell biology and cancer research~ Also, it is desirable
to substantially improve the present methods of production of
cellular culture.
It is well known that the cultivation of cells is
performed by first preparing by dispersion of an organ a cell
suspension in a suitable medium. The suspension is then
introduced in a culture vessel in which the cells sediment and
adhere to the wall of the vessel when they multiply. As a
result of multiplication which takes place in a few days, a
monolayer of cells is obtained. Simultaneously or subsequently
these cells can be used "in situ" for virus propagation
or can be harvested, diluted and used for seeding other vessels
or used for various purposes~
Presently, the cultivation of cells is performed in
flat bottom flasks such as the Blake bottle, the Roux flask
and plastic flasks which allow growth of the anchored cells on
one surface only. It is also performed in rolling bottles where
the entire inner wall of the bottle serves for anchorage and
growth of the cells. Systems have been developed in which a
number o~ rolling bottles or tubes are assembled and serviced
altogether by means of manifolds (Santero, U.S. 3,732,149,
Mann,U.S. 3,827,943).
Another known system is based on increasing the
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surface by the addition of plates. The plates are made of glass
or titanium and can rotate about an axis. These culture units
are very expensive to construct and have a great number of
moving parts which make them very delicate. In view of the
fact that cells have a normal tendency to descend vertically
in their suspension, these plates must be mounted horizontally.
Thus,in order to obtain adhesion of the cells on both surfaces
of the plates, the unit must be rotated intermittently 180~, for
several hours resulting in a back and forth movement of the
substrate in relation to the sedimenting cells. As a
consequence of such manipulations, uniform distribution of the
cells on the surfaces of the plates is not obtained. Moreover,
proper microscopic examination of the cell sheet is not possible.
Other methods and apparatuses for culturing cells are
also known such as exemplified by the following U.S. Patents:
- 3,821,087 - June 28, 1974 Knazek et al
- 3,873,423 - March 25, 1975 Munder et al
- 3,948,732 - April 6, 1976 Haddad et al
- 3,997,396 - Dec. 14, 1976 Delente.
However, it will be realized that allof these methods
and apparatuses are either too complicated or are unsatisfactory.
In order to overcome the disadvantages of the prior
art and to substantially improve cellular culture, there is
provided a method of cellular culture which comrpises:
a) providing a suspension of anchorage dependent
cells,
b) introducing this suspension of cells in one
culture vessel containing tubes disposed parallel
to and at a constant distance from one another,
c) rotating the culture vessel in such a manner and
at a speed effective to cause adhesion of the
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cells on the inner wall of the culture vessel and
on the inner and outer walls of the tubes, and
d) creating an aerobic condition in the culture
vessel.
In order to carry on this method there is provided an
apparatus which comprises:
a) at least one culture vessel,
b) tubes disposed inside the culture vessel parallel
to and at a constant distance from one another,
c) means to introduce a suspension of ce]ls in the
culture vessel,
d) means creating an aerobic condition in the
culture vessel, and
e~ means to cause a rotation of the culture vessel
and of the tubes about an axis parallel to the
tubes.
Depending on the needs, the suspension of cells is
prepared from tissue of animal, vegetal or protist origin, and
in a suitable culture medium.
When cells of animal origin are selected, they are
preferably selected from primary cultures, non permanent diploid
cell lines, and peL~anent cell lines.
The manner used for introducing the cells into the
culture vessel can be selected at will and it can vary a great
deal. However, the suspension of cells can be poured manually
into the culture vessel, it can be introduced by gravity flow
from a separate reservoir containing this suspension of cells
or it can also be pumped into the cul-ture vessel from a separate
reservoir which contains the suspension of cells.
The so filled vessel is closed at both ends and
rotated about an axis on a mechanically operated rolling device.
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109~478
The rolling phase in the culture process, which is perfor-
med for a few hours only, is to allow adhesion of the cells on
all available surfaces which are passing continuously for several
hours, under the descending cells. As a result, a uniform
distribution of the cells on the substrate is obtained.
In accordance with a preferred embodiment according
to the invention, there is provided a separate supply of liquid
medium, which is in contact with a gaseous phase, and this
separate supply of liquid medium is circulated through the
culture vessel at a rate sufficient to bring nutrients and
dissolved gases necessary for cellular growth, with the proviso
that the rate of circulating does not cause shearing of the
cells from the walls of the culture vessel or of the tubes
mounted inside the culture vessel.
The liquid medium can be monitored in order to adjust
the pH and the pressure of dissolved gases.
Although the rate of circulation of the liquid medium
through the culture vessel can vary to a certain extent provided
there is no shearing of the cells from the walls of the vessel
and of the tubes, it has been found out that better results
will be obtained by adjusting the rate of circulation of the
liquid medium at about 0.5 to about 1.5 ml of liquid medium per
minute per 100 cm2 of growth surface.
The amount of suspension of cells which is introduced
into the culture vessel should preferably be sufficient to
submerge all the tubes which are mounted in the culture vessel
in order to permit cell growth on all surfaces available within
the culture vessel.
The invention will now be illustrated by means of
the annexed drawings which are given only for the purpose of
illustration, and in which
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FIGURE 1 is a flow diagram of an apparatus including
a culture vessel according to the invention
adapted to carry out cellular culture, and
FIGURE 2 is a partial cross-section taken along line
2-2 of FIGURF 1.
Referring to the drawings, it will be seen that the
device according to the invention essentially consists of a
culture vessel 1, a liquid supply 3 to contain a suspension of
cells, a container 5 for the culture medium and a pump 7.
~s shown in FIGURES 1 and 2, the culture vessel 1,
which is preferable made of Pyrex ~trade mark) borosilicate :
glass but which can also be made of an inert plastic allowing
anchorage of the cells, is elongated and cylindrical. The
culture vessel 1 is formed with tapered ends 9 and 11 and ~oth
tapered ends 9, 11 are provided with respective openings 13,15.
It will be realized that opening 13 will serve to introduce the
cells suspension or the culture medium into-the container 1
while the opening 15 will be used for the removal of the cell
suspensions or the liquid medium from the culture vessel 1.
Inside the culture vessel 1 there are a plurality of
small tubes 17 which, as shown, are spaced and parallel to one
another. The length of the tubes inside the culture vessel is
not too critical. However, it will be obvious that better yield
would be obtained by providing tubes which will occupy as much
as the length o~ the culture vessel as possible. For example,
the tube 17 could as shown in FIGURE 1, occupy substantially
the entire interior space of the culture vessel. Of course,
it may be easier for mounting the .tubes:inside the apparatus
to make them of the same len~th, such as to just clear the
tapered ends 9 and 11.
The tubes 17 should be at equal distance to one
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another. For this purpose, a small spacer 19 is mounted near
the end of each tube and the distribution of these spacers 19
could be as shown in FIGURE 1 or in any other arrangement pro-
vided they do not touch one another. It has been found prefera-
ble to use tubes in which the inner diameter is about 7 mm andthe outer diameter is about 9 mm. The spacer 19 should be such
as to provide for the closest distance between each tube to be
about 1 mm.
These spacers 19 should be made of inert material in
order to have no reaction with the cells and the culture medium.
It was mentioned above that the culture vessel should
be made of PYREX or of an inert plastic which would allow
anchorage of the cells. The same is true with respect with the
tubes 19. In practice, PYREX is used because it is more easy
to wor~ with and it is resistant to sterilization temperature
and has good optical properties.
Each of the tapered ends 9, 11 is terminated by a
tubular portion 21, 23 formed with the respective openings 13,
15. The tubular portion 21 and 23 are closed by special types
stoppers 25, 27 enabling rotation of the culture vessel while
the stoppers 25, 27 remain fixed. Stopper 25 is connected to a
duct 29 for introducing the cell suspension or the culture medium
into the culture vessel 1. Stopper 27 is the same as stopper 25
except that it is mounted over tubular portion 23 and is connec-
ted to duct 31 which will be used to remove the culture mediumor cell suspension from the culture vessel 1,
As mentioned above, the cell suspension is contained
in a supply container 3~ The suspension is introduced into the
culture vessel by means of duct 29 along which there is mounted a
stopper valve 33 and a two-way valve 35 the purposes of which
will be discussed later when the operation of the device will be
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` ~0~447~
described.
Duct 31 is used to recirculate the culture medium
from the culture ves~el 1 into the reservoir 5. In order to
create an aerobic condition inside the culture vessel 1, the
culture medium 37 which is allowed to circulate through the
culture vessel 1 is in contact with air 39. This is made
possible by providing air inlets 41 and 43 each of which is
adapted with a respective filter 45, 47 to make sure that no
undesirable impurity is introduced into the culture vessel 1.
Also, a side arm 46 provided with a screwed cap 46a is welded
on the reservoir 5 for sampling, withdrawal or introduction
purposes. As mentioned above, circulation of the culture medium
is made possible by the use of a peristaltic pump 7 which with-
draws the culture medium from reservoir 5 through duct 49 and
forces the latter along duct 51, through stopper valve 53,
two-valve 35 and a portion of duct 29.
In order to determine the amount of cells which is
still to be deposited on the walls of the culture vessel 1 and
the inner and outer walls of the tubes 19, there is provided a
sampling device 55 which is connected to duct 31 by means of a
duct 56 along which there is a stopper valve 56a.
Finally since the culture vessel 1 must be rotated,
it is mounted on motorised rollers 57 which will cause rotatlon
of the vessel at any desired speed.
In operation, the first step consists in introducing
a suspension of cells from container 3 through duct 29 by open-
ing stopper valve 33 and allowing valve 35 to block off any
circulation from duct 51. As soon as the culture vessel 1 is
filled i.e. when the tubes are submerged with the cells suspension
stopper 33 is closed and so are both valves 35 and 31'. After a ro-
tation period on rollers 57,valve 31' is opened,the valve 35 is
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adjusted to connect duct 29 with duct 51, the stopper valve 53
is opened and the pump is put into operation to enable a circula-
tion of the culture medium through the culture vessel 1 in order
to make sure that the nutrients are vehiculed and a aerobic
S condition is maintained in said cultured vessel 1. At the onset
of media circulation the rolling operation is ceased.
Of course, the invention has been illustrated by means
of one (1) single culture vessel. It is obvious that a
plurality of culture vessels could be used whether they be in
series or in parallel. An operation could also be set up by
having a plurality of culture vessels hooked up to independent
circuits to produce three (3) types of culture cells at the same
time
In the examples which follow the experiments were made
using arrangements of one culture vessel or a plurality set in
series.
The following experiments ~re performed as described.
Cell culture was performed with the VER0 cell line
and with a human ambryonic lung diploid line (IMH-P). Both
cell lines were propagated in a media composed of a mixture of
equal parts of l99-Hanks base and MEM-Earle's base, completed
with lOr/~ fetal bovine serum.
For adhesion, the culture vessels are filled with the
cell suspension and closed with standard screw caps and rotated
for a period of 4 hours at a speed of 10 rph on a Rollacell
apparatus (New Brunswick model). The culture vessels of 4.5
and 5.5 cm diameter are rotated. After cell attachment, the
culture vesse~ are set for media circulation by screwing the
modified caps already attached to the tubing leading to the
media reservoir, in replacement of the standard caps, after
which the media is circulated by the peristaltic pump. The
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cell growth is monitored by examination with an inverted
microscope. Results are summarized in the table.
For cell harvesting, the standard caps are screwèd at
each end, from this point the culture vessels are manipulated
as a roller bottle.
The media is discarded and the proper volume of trypsin
is poured in. The trypsin is shaken in the culture vessel and
immediately discarded. Fresh trypsin is put back, shaken and
the culture vessel is allowed to rotate 10 minutes on the roller
apparatus. A volume of serum free media is added to the
culture vessel. After shaking the culture vessel, the cell
suspension is poured out and counted.
For sampling purposes, a sampling device is introduced
in the unit circuit.
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TECHNICAL DATA ON CULTURE VESSEL MODULES
AND CORRESPONDING MEDIA RESERVOIRS, VOLUME AND CIRCULATION
MODULES
DATA Culture Culture Culture Culture
Vessel 1 Vessel 4 Vessel 8 Vessel16
Bottles : diameter 4.5 cm 5.5 cm5~5 cm11 cm
length15 cm 29 cm 58 cm40 cm
wall surface212 cm2 518 cm21036 cm2 1382 cm2
*Glass tubing : number 15 25 25 76
total 2 2 2 2
surface1125 cm 3780 cm7250 cm 15200 cm
Total growth surface 1337 cm 4268 cm8286 cm 16582 cm
Growth surface increase
factor of original bottle 6.3 X 8.2 X8.2 X 12.6 X
Media volume capacity 250 ml 750 ml1500 ml 3000 ml
Preferred total
media volume **650 cc2000 cc 4000 cc8000 cc
Media reservoir capacity 1 1 2 1 4 1 8 1
(surface available for 2 2 2
gas exchange)~80 cm ) (115 cm ) (175 cm2) (300 cm
Working media volume
in reservoir 400 cc 1250 cc 2500 cc5000 cc
Preferred rate range of
media **
circulation (ml/unit) 5-10 20-4050~100 100-200
Recommended volume of
trypsin used for
harvestina, 35-50 ml 100 ml 200 ml400 ml
_
* Prior to first washing cycle
** For single culture vessel arrangement
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GROWTH RESULTS WITH VERO AND IMH-P
(7 DAY CULTURES)
NB. CELLS SEEDED/CM X NB. CELLS HARVESTED/
CM2
.
VERO culture 4 5
vessel 1 1.8 x 10 1.5 x 10 *
culture 4 5
vessel 8 1.8 x 10 3.0 x 10
roller bottle 1.5 x 104 1.1 x 105*
IMH-P culture 4 5
vessel 1 5.2 x 10 X 2.8 x 10
culture 4 5
vessel 4 5.2 x 10 X 2.5 x 10
culture 5.2 x 10 X 2O4 x 105
culture 4 5
vessel 16 3.07 x 10 2.4 x 10
~oller bottle 5.2 x 10 2.1 x 105
N.B.: In all experiments, a 4 hour period was allowed for adhe-
sion at a rotating speed of + 10 RPH. Media ls changed
after 3-4 days of culture. -.,
* No media change.
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