Note: Descriptions are shown in the official language in which they were submitted.
-- 2 --
.
The present invention relates to processes
for the surface culture of eukaryotic cel~s and to
substances thereby obtained.
It is known that non-transformed nucleated
cells ~eukaryotic cells) can only be successfully
cultured by the surface culture method. Thus eukaryotic
cells held in suspension do not grow. If however
the eukaryotic cells are allowed to settle on a suitable
surface, they can multiply up to contact inhibition.
In its simplest form the surface culture method
may.be effected in a vessel having a flat bottom,
it being necessary to ensure that the bottom of the
vessel is as exactly horizontal as possible. Such
a vessel is preferably made of glass or of a suitable
plastics material, the size of the container being
limited, in the final analysis, only by handling
requirements. In order to obtain as lar~e a surface
area as possible containers also exist in which several
flat faces lie horizontally one above the other,
the faces being connected to one another by appropriate
openings. In accordance with the same principle
a tray stack has also been described which has 10
flat culture faces lying one above the other, each
face having a surface area of 600 cm2. This latter
container may also be connected in parallel with
other containers to form blocks of 4.
Where it is desired to use other cell culture
techniques the entire inner face may be used Eor
~ cell culture by rotation of the container, for example
using so-called roller flasks. Such roller flasks
may be fitted with discs or other bodies so that
the available surface area can be enlarged whilst
ensuring not only the supply of nutrients to the
cells via the medium but also the necessary provision
~5 of oxygen.
These known processes suffer from a number
of disadvantages including the fact that they can
___~_
., ,.
; ~'?,~
'
no longer be handled or can only be handled with
difficulty as a result of the size of the container
used. Furthermore it is difficult to effect the
necessary hose connections/ requirecl for effecting
changes in the culture medium as a result of the
movement of the container.
Appliances comprising a container having several
cell culture faces and a pump have also been described
in the literature. The necessary supply of nutrient
and oxygen to the cells is ensured by the circulation
of the medium as a result oE the pump. Thus, for
exa~ple, an appliance having a volume of 2 litres
has been described in which the flat culture faces --
are replaced by small glass cylinders and the pH
value of the circulating medium is regulated during
operation by gassing. The procedure is the same
as in the known organ perfusion technique.
In the above-mentioned surface culture process
in which small glass cylinders having a length of
about 6 mm, an outside diameter of about 4 mm and
an inside diameter of about 2 mm are used in a container
wherein simultaneous circulation of the medium is
effected during operation by pumping, a covering
of all culture faces is not possible as a result
of the physical effects, such as wetting, capillary
action and other interfacial effects, which arise
especially due to the formation of air bubbles.
For the same reasons, it is not possible to
adequately wash the culture faces covered with cells.
Thus there is no complete interchange of washings
with original medium since part of the original medium
is persistently retained. The physical forces associated
with glass cylinders or balls hitherto used in surface
propagation, e.g. in fibroblast interferon production,
prevent the necessary complete removal~ by washing,
of both the induction medium and foreign protein.
Moreover, the pumps which are conventionally used
in interferon production give rise to shearing forces
,
.
and these forced tend to destroy a substantial part of the interferon which
is produced during culturing.
A further technique termed the microcarrier technique, comprises
the use of suitable balls made for example of dextran, with a diameter of
about 0.15 to 0.~5 mm suspended in a culture medium and held in suspension
by stirring. Cells can be cultured on the surface of these balls using suit-
able techniques ~see Biotechnology and Bioengineering, volume XXI, 433-~2
~1979)). The available surface area given by the medium/volume relation is
especially large in the case of this technique. Thus a surface area of 3,000
cm2 is available with 0.5 g of dextran carriers in 100 ml of medium. However,
the balls on which the cells grow are very sensitive to mechanical influence
and the medium has to be removed by allowing the carriers to settle after
which the supernatant liquid is drawn off. ~gain, no co~plete washing can
consequently be effected.
The present invention is based on the discovery that the ~bove-
mentioned disadvantages in the surface culture method, especially in the
production of cell culture producible substances such as interferon, viruses,
enzymes and antibodies e.g. immunoglobulins, may be overcome, at least in
part by effecting the cell culture according to the surface culture method
on carriers dimensioned to reduce adverse interfacial effects, such as wet-
ting and capillary action, e.g. dimensioned to possess no substantial adverse
interfacial effects thus permitting complete covering and sufficient washing
of the culture face.
Thus according to one feature of the present invention there is
provided an improvement in a process for the surface culture of nucleated
cells in aqueous meclium and for the production of cell culture-dependent
substances from the cells thus obtained which comprises using as culture
face moving bodies comprising saddle-shaped or cylindrical filling bodies
made of metal which after the release of the aqueous culture medium hold
back less than 8 percent of the medium relative to free volume due to their
form and where the ratio of surface of the filling bodies used to free volume
_ ~ _
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7~
is greater than about 5:1~
The culture medium is generally any convenient aqueous medium.
Minimum essential medium, e.g. basal medium, Dulbeco's* medium with Hank's or
Earle salts, Glasgow* medium, medium 199*, Liebowitz medium L15*, Weymonth's
MB 752/1* medium and McCoy's 5A* medium may all for example be used in the
process of the present invention.
The bulk volume of the carrier minus the clisplacement volume is al-
so referred to herein as the "free volume", which term is known in the liter-
ature.
Thus the carrier is for example dimensioned to retain by adhesion
less than 8% of the culture medium based on the free volume of the carrier
after draining off of said medium.
The carriers for use with the present invention convenientLy include
fixed carriers such as for example, the plates or screens e.g bubble plates
used in fractional distillation apparatus where it is also desired to provide
the maximum surface area for phase contact and flowable carriers such as sad-
dle-shaped, spherical or cylindrical carriers which may, if desired be made
from materials such as for example glass; ceramic materials, e.g. clay or por-
celain; plastics, e.g. Duranite*, Azidur* or Teflon*; metals, e.g. titanium,
V2A or V~A steel; or sintered products, e.g. corundum.
The term flowable carriers as used throughout the present specific-
ation means especially rings such as Pall* rings, Raschig* rings, Prym* rings,
porcelain rings, Perfo* rings, expanded metal rings and Intos* rings; coils
and spirals such as glass spirals, Wilson* spirals, spring coils, gLass coils,
wire spirals, spools and rolls; saddle-shaped carrier bodies such as bead
saddles, Super* saddles and Novalox* saddles, Interlox* and Interpack* carri-
ers, beads, balls, startype carriers, twin carriers and solid carriers.
Especially preferred flowable carriers for cell culture include
spirals, coils and saddle-shaped carriers. Coils and wire spirals for example
in expanded form, e.g. made of V2A or V~A steel, in which the individual turns
do not touch one another, and saddles, are particularly preferred since these
forms present a large surface area to the circulating medium whilst giving
*Trade Mark
rise to only a small flow resistance and, during washing, have only a small
retention of the medium. Especially preferred wire spirals are those which
have a total length of 4 to 8 mm, preferably 5 to 7 mm, a total width of 4 to
8 mm, preferably 5 to 7 mm and a wire diameter of 0.4 to 0.8 mm, preferably
0O5 to 0.7 mm, the gap between the individual turns being 0.1 to 0.5 mm, pre-
ferably 0.2 to 0.4 mm.
The following tests may, for example, be employed to determine
whether a given carrier is suitable for use ln the process of the present in-
vention and the following carriers were tested, by way of example, for their
suitability:
A = Glass rods (diameter: 10 mm, length: 30 mm).
B = Teflon rings (outside diameter: 10.5 mm, inside diameter: 5.5 mm, length:
10.2 mm).
C = Duranite cylinders (outside diameter: 10.5 mm, inside diameter: 6.5 mm,
length: 11.5 mm).
D = V4A spirals (outside diameter: 6 mm, wire diameter: 0.6 mm, length: 6mm).
E = Glass balls ~diameter: 10 mm).
F = Duranite cylinders (outside diameter: 8 mm, inside diameter: 5 mm,
length: 9.5 mm).
G = Glass rods (diame~er: 6 mm, length: 10 mm).
H = Saddles (diameter: 6 mm, length: 6 mm).
I = V4A wire gauze cylinders (diameter: 3 mm, length: 5 mm)
and
J = Glass balls (diameter: 3.5 to 5 mm)
in comparison with
K = Glass cylinders (outside diameter: 4 mm, inside diameter: 2 mm, length:
6 mm).
~lethod:
1. The apparatus used for conducting the above-mentioned -tests com-
prises a glass tube (inside diameter: 3 to 5 cm3 tapered downwardly to a
- 6 -
7~
funnel, fitted with a tube having an inside diameter of about 0.5 cm as an
outlet and provided with a stop-cock. A marking is made at the transition
from the funnel to the 0.5 cm tube. With the stop-cock closed the glass tube
is then filled with water up to this mark and the volume determined by weigh-
ing. The tube is subsequently filled with ~ater to this mark again and a
weighed quantity of water (e.g. 200 ml) added. The upper level is marked on
the tube. The water is then drained off down to the lower mark. The quantity
of water which remains adhering to the glass wall is simultaneously determined
by weighing the drained quantity of water and subtracting this quantity from
the fixed nominal volume ~e.g. 200 ml) referred to above. The entire opera-
tion is repeated se-veral times and the average value is fixed as the value
when empty.
Before each test the test tube must be completely dry (e.g. by wash-
ing with acetone and subsequent drying by blowing with air). The carrier bo-
dies to be tested are similarly dried in a drying chamber. ~or testing, the
glass tube is filled up to the upper mark with the carriers. The weight of
the carriers introduced and the quantity are determined. Subsequently~ with
the stop-cock closed the glass tube is filled up to the upper mark with a
weighed quantity of water. The volume of water added is determined by weigh-
ing the quantity of water remaining and subtracting from the previously deter-
mined total. The free volume (i.e. the bulk volume of the carriers minus the
displacement volume of the carriers) may then be calculated using thP above
determined measurements. The stop-cock is subsequently opened to allow the
water to drain out to the lower mark, the water being weighed on a balance
after collection in a tared vessel. By subtracting the drained quantity from
the "free volume", the "remaining quantity" of water left on the carrier bo-
dies may be calculated. The weight of the tube when emptied of the carriers
is also taken into consideration. The surface area of the carrier bodies is
either calculated or taken from the manufacturer's specification.
The experimentally determined data is calculated for the following
values: free volume per litre in cm3, ratio of the surface area of the car-
riers to the free volume and the volume of water remaining on the carriers
after draining as a percentage of the free volume.
2. Using the same glass tube ~dry) the same quantity of carriers is
employed, but the tube is topped up to the upper mark with dyed water (e.g 1%
neutraL red stock solution = 100%). The upper opening is then closed in an
air-tight manner with a hose which is filled with water and clampe~ in posi-
tion by a hose pump. After the stop-cock has been opened water is added via
the hose pump in a measured quantity of 100 ml per minute and the washing
effect per minute is determined by measuring the colour intensity in a photo-
meter. The measured value is related to the initial value and calculated to
give the washing effect as a percentage of residual dye after washing x times
~1
with a free volume quanti-ty. 5 ~inutes after the start of washing the intake
of water is stopped, the column is shaken briefly and the value after 30 se-
conds of pumping is measured. The value is given in the form of residual dye
as a percentage after washing x times with a free volume quantity and after
subsequent shaking. ~lthough water is used in this test the same results are
obtained when using Hank's solution without calcium and magnesium ions at
pH8, instead of water.
The following table contains the results of the above-mentioned
tests:
a) a
a)~~1~3)~ ... . ~ ....
I h O
~ rd 3 X 3 ~1 >
,~ a~
O ~ ';P O O O ~ ~
O . ~ O . O O . .
p~ C::~ O O o o o o ~-1
~1 3 ~--~ r-l O + O ~ O O ~1
~: ~ ~ O O ~ O. O O
a d' O O O O O O
~) ~IJ ~ tJ~
O
~r ~ In ~ ~1 ~ ~ o r~
a~ x ~ ~ ~ ~ o
~1 )-I O O O O
~0 a
~
tn ~ al ~ o~ co ~ ~ ~ ~ I` ~ o ~ a~
trJ ~ ~I VJ 3 . . . ~ .. . . . . ~1
~: 3 ~ J r~ 10 ~ O 0~1 0 ~1
~ . O
r~
rl 4 ~1
S ~ ~1 1
O ~ O
rl ~ ~1 ~
o aJ ~ ) .
S~ Ll E3 t~7 ~ ~ o ~ Q~
a) ~IJ oY~ :~ . . . ., . . , , . . ~
1~ 1--1 r l r-l ~ ~~~r ~~D~r r~CO ~1
~ ~ O
Ir; 1~1 rl C- rl
~_1
a) o ~ ...................... a~
r~ ~ ~1
o a~ L~ O ~ U~ a
U h U~ ~ r~ 1 O~ D ~ Ul ~r ~ OD d~ r
o ~ .
,, ~ u ,, a) In r~ ~ o r` c~ ~ Lr) ~ ~ ~ ~
a) r~ l ~1
t~ ~ ~1 0 Ll a
~ ~0 0~4~ ~
+
a~ ~
I ~ rl
r-l 3
I~ Il') ~ ~D ~ t- ~ ~`1 0 ~) ~ ~a
~ o a~ oo ~ ~ ~ oo s~l ~r 1` f'l
a~ ~ ~ ~ u~ D ~ W a~ ~
~ ~ ~ (a
~ U ~
U~
a) u~
,~ a~
~-rl ~ m c~ Q ~ ~ H 1~ ~C
Ll
~ O
C) ~ E~
. ~P____
~ ~`'~'' ' ' '''' ' .
. ! . , ~ _ .
,~
.
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-- 10 --
On the basis of the above testsr carriers A-
J possess the following favourable characteristics:-
1. after release of the medium the carriers retain
less than 8% of the medium relative to the free volume
(medium retained due to adhesion~, and
2. have a maximum residual dye percentage of 0.15
after washing four times with a free volume quantity
or a maximum residual dye percentage of 0.5% after
washing four times with a free volume quantity and
shaking.
Such carriers enable the suspended eukaryotic
cells, e.g. fibroblast cells or epithelial cells,
to adhere to them within a short timer for example
1 to 3 hours. Furthermore, the nutrients of the
circulating medi~m are also absorbed, so that rapid
growth of the cells is ensured. It is especially
advantageous, however, that the closely grown cultures
are brought into contact with inducers, such as for
example Poly I:C cycloheximide, Mitomycin C, viruses
or other known inducers described in the literature,
within the desired time and that the necessary removal
of the inducers by simple washing subsequently presents
no difficulties. Washing can be effected continuously
or intermittently, for example by replacing the circu-
lating medium with a washing medium or by optionally
draining off the medium several times and subsequent
replacement by a washing medium; in the latter case,
the washing medium is preferably introduced from
the bottom of the culture vessel.
The carriers for use in the process of the
present invention may be cleaned without difficulty
and subsequently used again. Thus for example the
carriers for use in the process of the present invention
are such that cells adhere sufficiently strongly
to their surface to ensure undisturbed cell growth,whilst not adhering so firmly that the cells can
no longer be detached from the carrier upon subsequent
cleanlng .
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Thus whilst it is essential that the carrier
body for cell culture is dimensioned and disposed
to retain by adhesion less than 8~ of the culture
medium, based on the free volume of the carrier body,
after draining off of the said medium, it i5 preferred
that the carrier body should also be such that less
than 0.15~ of the said medium is retained adhered
thereto after four washings of the c:arrier, each
washing being effected using a "free volume" o a
washing medium~ Each washing may for example by
effected by adding a free volume of the washing medium
to the carriers and then allowing the washing medium
to drain away. The washlng medium may for example
be minimum essential medium without serum.
The process of the present invention is advan-
tageously effected using a pump for circulatlng the
culture medium, the pump being adapted to avoid the
introduction of shear forces into the medium. Thus
for example a peristaltic pump or a hose pump, bellows
or siphon pump, diaphragm pump or air-lift pump may
be employed for circulating the culture medium.
Especially preferred carrier bodies for cell
culture, include those in which the ratio of the
surface area of the carrier to the free volume is
20:1 to 2:1, preferably 15:1 to 5:1.
The process according to the invention has
proved especially advantageous in the production
of fibroblast interferon. For this purpose, a reaction
vessel, preferably a pressure-stable reaction vessel,
for example a 25-litre double~jacket reaction vessel
of Duran glass, provided conventionally with an inlet
and outlet, pH electrodes and permeator, is filled
about 3/4 full with carefully washed carriers, preferably
V~A wire spirals of expanded form (length: 6 mm,
width: 6 mm, wire diameter: 0.6 mm, pitch: 0.3 mm)
and is preferably sterilised with superheated steam.
The ratio of the surface area of the carriers to
the free volume is conveniently about 11:1. Subsequently,
.~
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- 12 -
cells preferably obtained by trypsination of surface
cell cultures are suspended at a cell density of
approximately 20,000 cells/cm2 in minimum essential
medium to which or example 10~ foetal calf serum
and preferably an antibiotic such as kanamycin, e.g.
in a concentration of 200 ug/ml are added.
The cell culture suspension introduced conveniently
remains at about 37C for 2 to 4 hours without pump
circulation; subsequently, the medium is circulated
preferably with a peristaltic pump at about 30 revolutions
per minute and the pH is preferably adjusted to from
7.3 to 7.5. 24 to 48 hours after introduction of
the cell culture suspension the medium is conveniently
removed at regular intervals and fresh growth medium
of the same composition is added; altogether, preferably
80 litres of medium are added and removed over the
following 3 to 4 days. Further processing depends
on the substance which i5 to be obtained Erom the
cell culture. With, for example, interferon the
~0 total medium is preferably removed 8 to 10 days after
preparation of the cell culture and induction is
conveniently subsequently effected by known processes,
eOg. with Poly I:C, Mitomycin C, viruses or any other
convenient substance. The entire induction medium
is thereafter preferably removed and the cells washed
several times with minimum essential medium. Subsequently,
additional medium is added, to which a stabiliser,
e.g. serum albumin, suitable for interferon has
been added. This liquid is again circulated by pu~ping
as specified above. After a further 12 to 24 hours
the medium is harvested and the crude interferon
obtained is concentrated and purified by a process
known per se.
According to the invention, a peristaltic pump
is preferably used which has a rate of revolution
of 25 to 60 revolutions per minute and conveniently
a delivery rate of 50 to 400~ of the total volume
per hour. The process according to the invention
_
7~
can therefore be effected with considerably large volumes than hiterto known
processes and is limited only by the requirement for a sufficient supply of
nutrient and oxygen as well as by the need to keep -~he pH within an appro-
priate range for the cells.
The process accorcling to the invention enables cell-culture-
dependent substances, such as for example interferon, formed during the
production phase to be removed from circulation, batchwise or continuously,
and replaced by new medium. The substance, e.g. interferon9 thus removed
can then be purified or concentrated on a purification and/or concentration
column.
Moreover, the process of the present invention enables the addi-
tion of a so-called stimulator substance or a mixture of such substances to
be made at any desired stage in the process J in order to increase the yield
of a cell-culture-dependent substance, and the removal thereof present no
difficulties as may be seen from our European Patent A2-0.005.476 filed
30th April 1979 and published on November 28, 1979.
The following Example illustrates the present invention:
~xample
A 20-litre double-jacket reaction vessel of Duran glass is filled
with 17 litres of carefully washed carriers for cell culture [V4A wire
spirals, expanded formJ length 6 mm, width 6 mm, wire diameter 0.6 mm, pitch
0.3 mm ~ratio of surface area of filling bodies to free volume is about 11:1)~.
The inlet and outlet of the vessel are provided with silicone rubber hoses,
having an inside diameter of 8 to 10 mm, and equipped with p~l electrodes con-
nected to a p~l meter and a permeator and connected to the circuit. The
apparatus is sterilised with superheated steam. Cells obtained by trypsina-
tion of surface cell cultures are suspended in 17 litres of minimum essential
medium, with 10% foetal calf serum and 200 ug/ml of kanamycin,
7~i~7~
in a concentration of 20,000 cells/cm2 and the suspension
is introduced into the reaction vessel. The reaction
vessel is heated to 37C via the double jacket and
a water-bath thermostat and is maintained at this
temperature. The cell culture suspension introduced
is left to stand for 2 to 4 hours in the reaction
vessel without pump circulation. Subsequently, the
silicone hose is inserted into a peristaltic pump
and circulation is effected at a rat:e of 30 revolutions
per minute with a delivery rate oE 40 to 80 litres
per hour. The pH value is adjusted to between 7.3
and 7.5. 24 to 48 hours after addition of the cell
suspension the medium is removed at regular intervals
and fresh growth medium of the same composition is
addedG Altogether, 80 litres of medium are added
and removed over the following 3 to 4 days. 8 to
10 days after introduction of the cell culture the
entire medium is removed and the cells are washed
by the addition of minimum essential medium without
serum and the induction medium is subsequently added
thereto. The induction medium contains 100 ug/ml
of Poly I C and 2.5 ug/ml of cycloheximide. The
medium is left to stand for 3 hours in the reaction
vessel. 2 ug/ml of actinomycin D are subsequently
added. One hour after the addition of the actinomycin
D the entire induction medium is removed and the
cells are washed at least four times with minimum
essential medium without serum. Subsequently, minimum
essential medium containing l,000 ug/ml of serum
albumin is added and circulated by pumping, as described
above. The pH is adjusted to 7.5 to 7.6. l9 to
20 hours later the medium is harvested and is concentrated
and purified by known processes.
The yields of interferon in the crude solution
are 900 to 2,000 units/cm2 of culture face, referring
to the international reference preparation G 023-
902-527 of the National Institute of Health, USA.
Using various carrier bodies for cell culture
~ ~ _
1~
~ . .~
:'
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- 15 -
in various amounts fibroblast interferon was prepared
in a similar manner with the following results:
Carrier body Starting Interferon reference units/cm2
(as herein-quantityoE culture face
beforein litres
defined)
~ 0.2 1,147
A 0.2 1,956
A 1.2 593
B 0.3 83
D 2.2 1,580
D 17.0 1,374
D 108.0 1,103
H 0.2 470
_______ ________________________________________ ._____ _
Comparison (K) 0.2 23
0.~ 19
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