The growth of animal cells in a fermenter is promoted by enriching
the liquid nutrient medium or broth with oxygen diffused into the
liquid through a permeable membrane, such as one made of silicone
rubber or polytetrafluoroethylene (Teflon). Superior cell growth
in larger volumes is achieved by feeding in the oxygen in this way
instead of bubbling it in.
What is claimed is:
1. In a fermentation vessel for propagating animal cells in suspension
cultures and monolayer cultures in which oxygen must be supplied
to the cells in a liquid nutrient medium in the vessel for cell
metabolism and multiplication, the improvement comprising a permeable
membrane partially defining a chamber or volume in the fermentation
vessel and made of a polymeric material on which the cells do not
grow to a significant extent but through which oxygen can diffuse
directly, without bubbling, into at least a 4 liter volume of the
liquid nutrient medium containing the cells;
the membrane being of a size and shape so that a sufficient amount
of oxygen is diffused into the liquid so as to enable cell propagation;
conduit means communicating with the chamber from outside the vessel
for supplying oxygen to the chamber for diffusion through the membrane;
mechanical liquid-agitation means located within the fermentation
2. An improved fermentation vessel according to claim 1 in which
the polymeric material is silicone rubber or polytetrafluoroethylene.
3. An improved fermentation vessel according to claim 1 in which
the membrane is a tube.
4. An improved fermentation vessel according to claim 3 in which
the tube wall is about 0.6 to 1.2 mm thick.
5. An improved fermentation vessel according to claim 3 in which
the tube is on a rigid support.
6. An improved fermentation vessel according to claim 5 in which
the rigid support is a heat exchanger.
7. A method comprising growing animal cells in suspension cultures
and monolayer cultures in a fermentation vessel containing at least
a 4 liter volume of a liquid nutrient medium, and supplying the
cells with oxygen through a permeable membrane partially defining
a chamber or volume in the fermentation vessel and made of a polymeric
material on which almost no cell growth takes place, so that the
oxygen diffuses directly without bubbling into the liquid medium;
the oxygen being supplied to the chamber from outside the vessel;
the membrane being of a size and shape so that a sufficient amount
of oxygen is diffused into the liquid so as to enable cell propagation.
8. A method according to claim 7 in which the membrane is in the
form of a tube.
9. A method according to claim 8 in which the tube has a wall thickness
of about 0.6 to 1.2 mm.
10. A method according to claim 8 in which the tube is silicone
11. A method according to claim 7 in which the membrane is a tube
spirally wound on a rigid support.
12. A method according to claim 11 in which the rigid support is
a heat exchanger.
13. A method according to claim 7 in which the animal cells are
from a non-vertebrate.
14. A method according to claim 13 in which the animal cells are
15. A method according to claim 7 in which cell growth continues
until cell multiplication increases the cell population to at least
20 times the cell population at the start of fermentation.
16. A method according to claim 7 in which the polymeric material
is silicone rubber or polytetrafluoroethylene.
This invention is concerned with a method and apparatus for propagating
animal cells in culture suspension or monolayer culture in a fermentation
BACKGROUND OF THE INVENTION
In the last few years or so, research projects have been started
directed to the production of viruses which, perhaps, can be used
as biological insecticides because of their insect specie's specific
activity. The production of viruses, active against specific insects,
by culture propagation would permit the manufacture at any time
under controlled and reproducible conditions of standardized virus
preparations for use in pest control. However, the mass production
of viruses active against insects requires that large amounts of
insect cells be produced. This is because the insect cells are used
as a necessary substrate for growing the viruses.
The propagation or multiplication of insect cells, as well as cells
of vertebrae animals, in culture suspensions can be effected in
shaken containers, such as the roller flasks, spinner flasks, or
similar containers. In most cases, however, mass production is limited
by (1) the size of the containers, which must be handled manually
and (2) the oxygen partial pressure in the culture liquid nutrient
medium which becomes relatively poorer with increasing culture medium
volume. Usually, the air over a culture medium in a closed container
is only sufficient for a limited time to replenish the oxygen consumed
by the cells multiplying in the culture medium. The resulting oxygen
depletion in the culture medium causes a slow down in the multiplication
of cells and increased cell mortality. Because of this, as well
as for the mechanical circulation of the culture medium which takes
place, it has become necessary and practical to blow sterile, filtered
air, in the form of finely distributed air bubbles, into the larger
volumes of culture medium. In this way, the oxygen content of the
culture medium is enriched.
The bubbling of germ free air into the culture medium, as currently
practiced in the fermentation art to increase oxygen diffusion,
is unsatisfactory for several reasons. In this method, air bubbles
out of one or more openings, below the liquid level, in an air supply
pipe and the bubbles rise to the surface. As the number of bubbles
formed from one liter of air increases, so does the airliquid phase
interface area. However, the size of the bubbles and the number
of bubbles can only be varied within limits. If the openings, and
thus the bubbles, are too big, they may unite before reaching the
liquid surface. If the openings are made very small to generate
many small bubbles, there is a danger that the openings will become
plugged shut or reduced in size. Besides, the manufacture of many
very small openings presents a technical difficulty.
The factors discussed above lead to a compromise in which medium
sized bubbles of medium number are produced which, as a consequence,
leads to an unsatisfactory phase interface area.
The phase interface area is afterwards brought to the required
size by dividing the bubbles by means of a stirring apparatus and
distributing them throughout the volume of culture solution in the
fermenter. The pressure and pulling forces (shearing stress and
tangential strain) involved in this often may damage animal cells
so much that they may die.
Furthermore, additional shearing stress occurs when the gas bubbles
break at the interface area (culture liquid/gas area). These forces,
as well, damage the cells so that the ratio of intact cells to damaged
and dead cells becomes less and less favorable although the total
number may still increase.
In case it becomes necessary to increase the air (oxygen) supply
by increasing the number of air bubbles, the number of damaged and
dying cells increases, which is not only contrary to the desired
aim of cell multiplication, but also leads increasingly to the accumulation
of toxic cell decay products. These toxic products can additionally
hamper cell production.
Prior to now the growth of animal cells in containers with a volume
larger than three liters was hampered by the problem of oxygen supply.
Regardless of whether the cells are suspended in a culture solution
in the fermenter or whether they grow on surfaces in the fermenter
container, after a certain ratio A/V, the growth becomes stagnant
(wherein A=the area for the oxygen diffusion from the gaseous phase
into the dissolved phase and V=the fermentation volume). Measurements
in spinner containers of several liters showed that the O.sub.2
content in a 3 liter or larger volume of nutrient medium could not
be maintained, even by increased blowingin of air, on a level necessary
for normally multiplying cells.
If the surface on which animal cells of certain cell groups preferably
grow is artificially increased by filling the fermenter with small
synthetic balls, which then are fluidized or suspended in the culture
solution, the problems become even worse when air is blown in and
the solution is stirred because then the balls bump against the
stirring vanes and/or against one another so that damaging shearing
forces and foam formation occur.
Culture liquids generally contain minimum amounts of calf-serum
or other albumen-containing nutrients which promote excessive foaming
when air is bubbled in. This can extremely hamper and inhibit the
Although stabilization of the pH value, the culture medium temperature,
and the nutrient quality (by adding fresh nutrients) are important,
the factor limiting the maximum volume of the fermentation vessel
or container is the amount of oxygen dissolved in the nutrient medium.
It should also be emphasized that while mechanical rotation of
a spinner container has to be accomplished by means of a standardized
agitating apparatus, such as at 70 revolutions per minute, the mechanical
rotation does not abolish oxygen depletion in the nutrient medium
An object of the invention is to provide an adequate supply of
oxygen to cells being propagated in a liquid nutrient medium without
bubbling or blowing in air, to thereby avoid the inherent disadvantages
The invention also has as an object maintaining the necessary micro-mixture
and macro-mixture of the fermenter liquid culture nutrient medium.
This means that each volume from the liter scale to the microliter
scale will contain essentially the same amounts of cells, nutrients
and oxygen. Micro-mixing pertains to mixing in an area of 1 to 10
cell diameters, while macro-mixing means mixing of the entire liquid
volume in the container.
According to one aspect of the subject invention there is provided
an improved fermentation vessel for propagating animal cells in
suspension cultures and monolayer cultures in which oxygen must
be supplied to the cells in a liquid nutrient medium in the vessel
for cell metabolism and multiplication, with the improvement comprising
a permeable membrane, in the fermentation vessel, through which
oxygen can diffuse directly into the liquid nutrient medium containing
According to a second aspect of the invention, there is provided
an improved method of supplying oxygen to animal cells growing in
suspension cultures and monolayer cultures in a fermentation vessel
containing a liquid nutrient medium, comprising passing the oxygen
through a permeable membrane in the vessel so that it diffuses directly
into the liquid medium and thereby enriches it for the benefit of
It was found, surprisingly, according to the invention that all
of the oxygen needed for propagating animal cells in culture suspensions,
or monolayer cultures, in fermentation vessels can be supplied by
having the oxygen pass through a permeable membrane into the liquid.
No additional supply of oxygen is needed and, in particular, the
bubbling in of air is unnecessary and, thus, the disadvantages associated
with that method are avoided. The method of the invention avoids
the shearing forces caused by bubbling in a stream of air or oxygen,
eliminates or substantially reduces foam formation, and avoids the
prior art problem of correctly sizing the holes through which the
A very important advantage of the invention is that now, for the
first time, propagation of animal cells, and particularly insect
cells, is possible in culture suspensions and monolayer cultures
in fermentation vessels in much larger volumes than was previously
possible or customary. Thus, because of the invention is to possible
to produce or ferment culture suspensions of ten liters or more
with the cells multiplying at a maximum rate. Fermentation volumes
can now be produced of 15 to 20 liters or more, which were not previously
possible, and volumes of 50 and even 100 liters are not considered
The material used for the permeable membrane must be one which
permits an adequate amount of oxygen to pass through without bubbling.
However, the material selected should also be one on which the cells
do not grow, or grow only slightly, for otherwise passage of oxygen
through the membrane would be impaired and flow possibly reduced
thereby leading to an insufficient, or undesirably low, oxygen supply
in the liquid medium.
Permeable membranes useful in the invention can be made of any
synthetic inert solid polymeric material. Particularly useful are
membranes made of silicone rubber, laminated silicone rubber products,
and polytetrafluoroethylene (Teflon). Other synthetic polymers can
be used provided the animal cells do not adhere to or grow on them.
Silicone foil and silicone tubes provide a surface on which the
cells do not adhere, or adhere to it only with great difficulty.
Therefore, synthetic silicone polymers are preferred. Regardless
of the material use, the membrane should be thick enough to provide
the necessary mechanical strength but thin enough to permit oxygen
to pass through readily. The membrane must, of course, prevent reverse
flow through it of liquid from the fermenting broth.
The permeable membrane positioned in the fermentation vessel can
have any suitable size or geometric shape but one must be selected
so that there is sufficient oxygen diffusion to supply the amount
needed for cell metabolism. In addiiton, the permeable membrane
size and shape should not interfere with cell propagation in the
fermentation vessel. Those skilled in the art will be able to adapt
these features, and the material of which the membrane is made,
to the customary bio-technological requirements. While the permeable
membrane can, itself, completely enclose a space or volume and thus
constitute a hollow member, such as when in the form of a tube,
sphere or closed pouch, it is also within the scope of the invention
to employ a membrane which constitutes only a portion of a chamber
wall or surface surrounding a space. In all instances, however,
a gas supply conduit means is provided to feed an oxygen-containing
gas under pressure so that it can pass from one side of the membrane,
through it, and into the liquid nutrient medium on the other side.
The gas supply conduit means can comprise a tube extending from
outside to inside of the fermentation vessel. In addition, a gas
withdrawal conduit means can be included extending from inside to
outside of the fermentation vessel. Both conduit tubes should, of
course, communicate with a space on the same side of the membrane
or with a common chamber or volume wholly or partially defined by
A tube or hose, having a wall about 0.6 to 1.2 mm thick and preferably
about 1.0 mm thick, wound around a suitable support in the fermentation
vessel is particularly useful. The support, for example, can be
a heat exchanger such as is customarily used in a fermentation vessel
to keep the nutrient medium and culture broth at optimum temperature.
Representative oxygen sources for the fermentation are air, a mixture
of air and oxygen, or a mixture of oxygen and nitrogen.
The permeable membrane used for supplying oxygen to the culture
growing in the nutrient medium also serves as a filter to remove
any microorganisms in the oxygen gas supply stream, particularly
when air is used, and keeps them out of the culture broth. This
is a distinct advantage since all of the oxygen can be supplied
through the membrane.
It should be obvious that, when the fermentation process is carried
out according to the invention, the entire apparatus and the nutrient
medium used must be sterile.
Providing oxygen by means of a permeable membrane according to
the invention results in basically improved environmental conditions
for cell culture so it is expected that improved cell multiplication
rates will be obtained with a wide variety of vertebrae and nonvertebrae
culture suspensions and monolayer cultures.
Vertebrae cell lines of primary importance for propagation according
to the invention are those which are used in the mass production
of biological products such as immunity factors, hormones, enzymes,
anti-viral agents, virus preparations, and vaccines. These include
the Psylla (plant lice) cell lines BHK 21, NAMALWA and 1301 cell
line (from the leukemia line CCRF-CEMT).
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a vertical sectional view through a fermentation vessel
particularly useful for cultivating animal cells suspended in a
nutrient media and shows one form of permeable membrane; and
FIG. 2 is a vertical sectional view through a fermentation vessel
for cultivating vertebrae cells in suspended culture as well as
in monolayer culture.
DETAILED DESCRIPTION OF THE INVENTION
The same numbers will be used to identify the same or similar elements
in the various views of the drawings.
With reference to FIG. 1, the fermentation vessel comprises a glass
tube 1, circular in horizontal section, of borosilicate glass, a
bottom 7 and a cover 2 of high quality steel. The bottom 7 and cover
2 are joined liquid and air tight to glass tube 1 by sealing rings
8. A U-shaped steel heat exchanger tube 3 penetrates, and is suspended
by, cover 2. A tube 4 of silicone rubber is wound around that portion
of the heat exchanger tube 3 which will normally be submerged in
the liquid contents in the fermentation vessel. The wall thickness
of tube 4 will usually be in the range of about 0.6 to 1.2 mm and
will vary about .+-.0.05 mm. A wall thickness of about 1.0 mm is
usually preferred. The spirals of tube 4 are not placed tightly
together and desirably are spaced slightly apart. The ends of tube
4 are connected to fittings 5 which penetrate cover 2. Compressed
air is supplied to tube 4 by the fittings 5. A heat exchange fluid,
generally a liquid, is supplied to, and removed from, the heat exchanger
tube 3 by means of fittings 6.
Motor 9 drives shaft 10 which penetrates the bottom 7. Shaft 10
contains propeller 11 inside of the fermentation vessel. Propeller
11 rotates at about 60 RPM and, by its design and low speed, assures
gentle micro-mixing of the vessel contents 12. Micro-mixing is effected
by the turbulent action at and adjacent to the spirally wound silicone
tube 4. The oxygen, which penetrates through the wall of tube 4
and diffuses into the liquid, is predistributed by means of this
micro-mixing and then it is distributed through out the vessel contents
by the propeller induced circulation. The turbulence caused by gas
flowing out of the spiral tube 4, and the entire liquid flow itself,
assures even distribution of the nutrients and growing cells throughout
the fermenting liquid volume.
With reference to the embodiment illustrated by FIG. 2, the fermentation
vessel comprises a glass cylinder 1, circular in horizontal section,
made of borosilicate glass. The cylinder 1 is closed by a bottom
7 and a cover 2 of high quality steel. A sealing ring 8 is positioned
between the bottom 7 and the lower end of cylinder 1. Similarly,
a sealing ring 8 is positioned between the cover 2 and the upper
end of cylinder 1.
A heat exchanger 3 is supported by pipes 20 and 21 to cover 2.
The upper ends of the pipes 20 and 21 communicate with fittings
and holes in cover 2 to which the hose connections 6 are joined.
Double-walled heat exchanger 3 is made of two axially arranged and
vertically positioned steel cylindrical shells 23 and 24, circular
in horizontal section, with cylindrical shell 23 slightly larger
than cylindrical shell 24. The area between the ends of the shells
23 and 24 are closed, thereby forming an annulus with which pipes
20 and 21 are in fluid communication. A heat exchanger fluid, usually
a liquid and generally water, can be supplied to, and be removed
from, the heat exchanger 3 by connections 6.
Silicone rubber tube 4 is spirally wound around the outer cylindrical
shell 23 of the heat exchanger in such a manner that adjacent windings
do not lie tightly together. The ends of tube 4 extend to connections
5 to which high pressure air hoses can be attached. Tube 4 constitutes
a semi-permeable membrane through which gas can flow.
Stirring propeller 11 is mounted inside of the fermentation vessel
on vertical shaft 10 which extends through bottom 7 to motor 9.
The propeller is rotated at a speed of about 50 to 200 RPM selected
to be adequate to achieve gentle macro-mixing of the fermenter culture
liquid 12. The culture liquid and nutrients flow downwardly in the
annular space 13 between cylinder 1 and shell 23 and become enriched
with oxygen flowing through the walls of tube 4. The culture liquid
12 flows upwardly through sieve 14 and circulates around the small
balls 15 on which the cells grow. By suitable sizing of sieve 14,
the selection of the size and specific density of the balls 15,
and by the speed of propeller 11, fluidization or suspension of
the balls 15 can be achieved so that packing of the balls by settling
is avoided. Fluidization of the balls eliminates static ball to
ball, ball to sieve, and ball to heat exchanger contact which would
create zones poor in nutrients and oxygen where cells could not
live or grow, and would die.
Micro-mixing is achieved by flow of gas from the wall of spirally
wound tube 4, and by the nonhomogeneous flow through the bed of
balls 15. The circulation time of the liquid flow through the annular
space 13 and through the bed of balls 15 can be adjusted so that
the oxygen concentration within the bed of balls from the bottom
to the top does not decrease enough to be considered.
When fermentation runs were carried out according to the invention,
the bubbling in of air was completely avoided. Oxygen was supplied
solely through the silicone tube spirally wound around the heat
exchanger. The oxygen content of the culture liquid, measured continuously
by means of an oxygen electrode, could be maintained uniform over
several days when supplied in this manner. This indicated that the
oxygen transfers from the flowing air in the silicone tube into
the liquid nutrient medium and was always available in sufficient
amount, even with increasing cell population and increasing culture
volume. By increasing the flow rate of air in the silicone tube,
or by increasing the air pressure, it is possible to increase oxygen
transfer into the nutrient medium in the event the culture consumes
increased amounts of oxygen.
By using the described method and apparatus, a constant increase
of Mamestra brassicae cells could be achieved in cell culture suspensions
of four and ten liters. It was possible for the first time, when
compared to all previously used culture methods, to not only multiply
cells but to also achieve a cell propagation of thirty times in
a ten liter culture volume. Previously, the cell propagation increase
was four to five times in culture volumes up to three liters. Furthermore,
the cells produced according to the invention has an excellent morphological
appearance, with at the most 10% of dead cells. In smaller prior
art suspension cultures, such as of one to three liters, the percentage
of dead cells often is substantially higher.
The use of tubes, hoses or similar hollow bands of oxygen permeable
materials, such as silicone rubber, polytetrafluoroethylene (Teflon),
or equivalently permeable material, permits oxygen enrichment of
a nutrient medium to an extent which makes possible maximum propagation
of insect cells in suspension volumes of ten liters or more. The
cells are not prone to settle on, or attach to, the permeable tube
4 and thus do not slowly block oxygen flow, because the material
of which the tubes are made provides a surface not suitable for
cell adherence. For the same reason, cells do not adhere between
the tube spirals, die there, decay and contaminate the nutrient
medium with toxic decay products.
The following examples are presented to further illustrate the
A suspension culture was started from the insect cell line IZD-Mb
0503 (IZD=Institute for Zoology Darmstadt; Mb=Mamestre brassicae=a
type of butterfly; 0503=code number of the cell line; Lepidoptern-cell-line
ATCC #CRL 8003) in a so-called spinner container, to which a standard
liquid nutrient medium (pH 6.6) was added, with constant stirring
by means of a magnetic stirrer. The nutrient medium used was published
by T. D. Grace in Nature, 195, 788-789 (1962). The cells were permitted
to multiply freely suspended in the nutrient medium. A starting
population of 2.times.10.sup.5 cells/ml of nutrient medium is necessary
for multiplication. After three days, as a rule, a cell population
of about 6 to 10.times.10.sup.5 cells/ml is obtained. This is a
population increase of 3 to 5 times the original amount. Because
of nutrient depletion and "aging" of this culture, even
with a longer fermentation time, a higher cell population cannot
be achieved. This "parent culture" provides the cell preparation
used to start a cell culture in a fermenter.
The oxygen and pH measuring electrodes of a fermenter like that
shown in FIG. 1, were calibrated, autoclaved and recalibrated. After
that they were inserted in the fermenter cover. A tube of silicone
rubber having a 1.0 mm wall thickness was used as the membrane and
wound on the heat exchanger. The cover (with the electrodes) and
all parts of the fermenter which contact the cell suspension and
the nutrient medium (same as above) flowing back and forth, as well
as the systems supplying and removing the airstream, are sterilized
in an autoclave.
The fermenter vessel was put together observing all conditions
necessary to maintain the equipment sterile. The desired nutrient
medium volume was filled through openings in the fermenter vessel
cover provided for this purpose. Two liters of nutrient medium,
to which cells were added to provide a population of 10.sup.5 cells/ml,
were added to the 12 liter capacity fermenter vessel.
The fermenter was put into operation with stirring at 60 to 70
RPM, and a temperature of 28.degree. C. in the suspension. The initial
oxygen value (7.5 mg/l) and pH value were set via the measuring
In the first 16 to 24 hrs. after the start of the cultivation,
an oxygen enrichment of the nutrient was not absolutely necessary.
However, oxygen enrichment is needed when the cell culture enters
into its logarithmic growth phase and when the oxygen in the nutrient
is used up due to the increase of the cell population and increased
The oxygen is supplied via the silicone tube functioning as a permeable
membrane through which oxygen from atmospheric air, or from an oxygen-air
mixture, diffuses into the nutrient medium. To enrich the nutrient
medium after the first 16 to 24 hrs., compressed air at a pressure
of 0.5 to 1.0 atmosphere (gauge) was introduced via the silicone
tube. If necessary, the pressure can be increased up to 2.0 atmospheres.
With an initial cell concentration of 10.sup.5 ml, the oxygen concentration
decreases from 7.5 mg/l within 24 hours during the logarithmic cell
increase (cell proliferation) to below 1% of the initial value.
However, by means of oxygen (air) supplied through the silicone
tube during the logarithmic cell increase, the oxygen concentration
can be maintained at 10 to 30% of the initial concentration, which
guarantees very good cell proliferation.
By means of the oxygen diffusion method according to the invention
the number of cells per ml of nutrient medium was increased from
10.sup.5 up to 2 to 3.times.10.sup.6 in four days.
After 2 to 3 days, maximum cell multiplication has been surpassed
and the cell culture has entered the stationary phase in which the
cells gradually stop dividing. Each ml then contains 2 to 3.times.10.sup.6
cells. Then 6 to 7 liters of fresh nutrient medium was added under
sterile conditions. The cell concentration was correspondingly reduced.
The cells then change over from the stationary phase into a multiplying
phase. After 2 to 3 days fermentation a cell concentration of 2
to 3.times.10.sup.6 ml was again obtained. From the original 3.times.10.sup.8
cells/3 liters, up to about 10.sup.11 cells have developed in a
total volume of 10 liters.
Using the bubbling air method of the prior art, a volume increase
of more than 4 liters would not have been possible. Four prior art
cell propagations of 2.5 liters each would have resulted, at the
most, in about 4.times.10.sup.9 cells. The membrane method according
to the invention yields a 20 times higher cell population.
About 5 liters of the cell suspension was removed from the 10 liter
volume when the stationary phase was reached (2nd to 3rd day). Then
5 liters of fresh nutrient medium was added under sterile conditions
to the fermenter. Thus, the remaining suspension was diluted and
the cells, due to the new nutrient supply, again started their multiplying
phase. Repetitions of these diluting-multiplying phases, when sterile
conditions are maintained in the fermenter vessel, can be continued
as long as permitted by the total condition of the cells. In stabilized
cell lines, this can lead to a continuous operation.
The following cell lines can be fermented using the method described
in example 1:
IZD-Mb 2006=Mamestra brassicae
IZD-Mb 1203=Mamestra brassicae
IZD-Mb 0504=Mamestra brassicae
IZD-Ld 1307=Lymantria dispar
IZD-Ld 1407=Lymantria dispar
A multiplication rate, equally as good as with IZD-Mb 0503, can
be expected for all of the listed insect cell lines. Other cell
lines in addition to those just listed, which grow as suspension
cultures, can be propagated or multiplied by the membrane oxygen
diffusion method of the invention.
Recently it has become possible to propagate cells, not previously
multipliable as a culture suspension, in a fermenter. Oxygen was
supplied by bubbling air. By the use of so-called micro-carriers
which are, as a rule, small balls having a diameter of less than
1 mm, cells that normally only grow on a solid base in the form
of a cell "meadow" can be multiplied on the surface of
balls. Polystyrene balls are particularly suitable.
About 5 g of the balls/liter of nutrient medium is introduced into
a fermenter. That quantity of balls provides a surface of up to
30,000 cm.sup.2. Tests with non-diploid Psylla (plant lice) cells
in a suspension culture with micro-carrier balls have resulting
in cell population figures of 4.times.10.sup.6 /ml of culture broth.
Because high cell populations can result from the use of micro-carriers,
the oxygen consumption is correspondingly high. With the air bubble
method of the prior art the increased oxygen requirement is much
harder to satisfy than with the membrane oxygen diffusion method
according to the invention. With the membrane method, the metabolism
efficiency of cells growing on the micro-carrier balls is improved,
resulting in faster and increased cell division. The cell population
increases faster per unit of time so that fermenter preparations
from micro-carrier cultures supply more cells.
The foregoing detailed description has been given for clearness
of understanding only, and no unnecessary limitations should be
understood therefrom, as modifications will be obvious to those
skilled in the art.