United States Patent [191
`Miltenburger et a1.
`
`4,649,114
`[11] Patent Number:
`[45] Date of Patent: Mar. 10, 1987
`
`[54] OXYGEN PERMEABLE MEMBRANE IN
`ggngggNTriEn FOR OXYGEN ENRICHMENT
`
`[56]
`
`References Cited
`U_S_ PATENT DOCUMENTS
`
`[75] lnvem°rs= Herb?" G- Mlltenb‘u'ge" Darmstadt;
`slg?'led Hessbera, Mclsungen, both
`of Fed. Rep. of Germany
`
`_
`
`2,522,947 9/1950 Hatch et a1. ...................... .. 435/314
`
`3,850,748 11/1974 Cook et a1. . . . . . . .
`
`. . . . . .. 435/241
`
`..... .. 435/284
`3,927,981 12/1975 Viannay 61 al. ..
`3,997,396 12/1976 Delente ................. .. 435/240
`4,242,460 12/1980 Chick et al. .... ..
`435/284
`4,259,449 3/1981 Katinger et a1.
`435/241
`4,391,912 7/1983 Yoshida et al. ..
`435/241
`4,416,993 "/1983 McKeown
`435/313
`4,537,860 8/1985 Tolbert eta]. .................... .. 435/313
`
`FOREIGN PATENT DOCUMENTS
`1530705 11/1978 United Kingdom .
`OTHER PUBLICATIONS
`Jakoby, W. B. & Pastan, I. H., “Cell Culture”, Methods
`in Enzymology, vol. 58; Academic Press, 1979; pp.
`450455'
`Primary Examiner-Thomas G. Wiseman
`Assistant Examiner-Elizabeth C. Weimar
`Attorney, Agent, or Firm--Marshall, O’Toole, Gerstein,
`Murray & Bicknell
`ABSTRACT
`[57]
`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 poly
`tetra?uoroethylene (Te?on). Superior cell growth in
`larger volumes is achieved by feeding in the oxygen in
`this way instead of bubbling it in.
`
`16 Claims, 2 Drawing Figures
`
`-
`
`_
`
`[73] Assignee. lsntermeihcflt GmbH, Emmenbrucke,
`M w ‘"1
`
`-
`
`--
`
`[21] APPL No’, 446,644
`
`[22] Filed‘
`
`Dec-391982
`
`Related US, Application Data
`Continuation of Ser. No. 185,668, Sep. 10, 1980.
`
`Foreign Application Priority Data
`
`[63]
`
`[30]
`
`Oct. 5, 1979 [DE] Fed. Rep. of Germany ..... .. 2940446
`
`[51] Int. Cl.4 ....................... .. C12N 5/00; c12N 5/02;
`C12M 3/00; C12M 3/02; C12M 1/06; B01D
`13/00; B01D 29/42; B01D 29/48
`[52] US. Cl. .................................. .. 435/240; 435/241;
`435/284; 435/286; 435/315; 210/220;
`210/321.1; 210/433.2; 210/497.1
`[58] Field of Search .......
`435/240, 241, 280, 284,
`435/285, 286, 287, 313, 315, 2, 314; 422/48;
`210/497.1, 433.2, 220, 150, 321.1, 322.2;
`261/93, 122
`
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`U._S. Patent Mar; 10; 1987
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`GE-1028.002
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`

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`US. Patent Mar. 10,1987
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`GE-1028.003
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`

`
`1
`
`OXYGEN PERMEABLE MEMBRANE IN
`FERMENTER FOR OXYGEN ENRICHMENT OF
`BROTH
`
`This is a continuation, of application Ser. No.
`185,668, ?led Sept. 10, 1980.
`This invention is concerned with a method and appa
`ratus for propagating animal cells in culture suspension
`or monolayer culture in a fermentation vessel.
`
`10
`
`25
`
`35
`
`40
`
`45
`
`20
`
`4,649,114
`2
`volume of culture solution in the fermenter. The pres
`sure 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 li
`quid/ 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 (oxy
`gen) 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 multiplica
`tion, 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 fer
`menter or whether they grow on surfaces in the fer
`menter 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
`02 content in a 3 liter or larger volume of nutrient me
`dium could not be maintained, even by increased blo
`wingin of air, on a level necessary for normally multi
`plying cells.
`If the surface on which animal cells of certain cell
`groups preferably grow is arti?cially increased by ?ll
`ing the fermenter with small synthetic balls, which then
`are ?uidized 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 oc
`cur.
`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 process.
`Although stabilization of the pH value, the culture
`medium temperature, and the nutrient quality (by add
`ing fresh nutrients) are important, the factor limiting the
`maximum volume of the fermentation vessel or con
`tainer 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 me
`dium containing spaces.
`THE INVENTION
`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 described
`above.
`The invention also has as an object maintaining the
`necessary micro-mixture and macro-mixture of the fer
`menter 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, nutri
`
`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 speci?c activity. The
`production of viruses, active against speci?c insects, by
`culture propagation would permit the manufacture at
`any time under controlled and reproducible conditions
`of standardized virus preparations for use in pest con
`trol. 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
`?asks, spinner ?asks, or similar containers. In most
`cases, however, mass production is limited by (l) 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
`suf?cient for a limited time to replenish the oxygen
`consumed by the cells multiplying in the culture me
`dium. 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, ?ltered air, in the form of
`?nely 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 me
`dium, 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 bub
`bles, 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 dif?culty.
`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 unsatis
`factory 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
`
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`20
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`4,649,114
`3
`ents and oxygen. Micro-mixing pertains to mixing in an
`area of l to 10 cell diameters, while macro-mixing
`means mixing of the entire liquid volume in the con
`tainer.
`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 improve
`ment comprising a permeable membrane, in the fermen
`tation vessel, through which oxygen can diffuse directly
`into the liquid nutrient medium containing the cells.
`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 mono
`layer 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 bene?t of cell multiplication.
`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 oxy
`gen pass through a permeable membrane into the liquid.
`No additional supply of oxygen is needed and, in partic
`ular, 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, elimi
`nates or substantially reduces foam formation, and
`avoids the prior art problem of correctly sizing the
`holes through which the air bubbled.
`A very important advantage of the invention is that
`now, for the ?rst time, propagation of animal cells, and
`particularly insect cells, is possible in culture suspen
`sions 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
`45
`volumes of 50 and even 100 liters are not considered
`impossible.
`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 me
`dium.
`'
`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 polytet
`ra?uoroethylene (Te?on). Other synthetic polymers
`can be used provided the animal cells do not adhere to
`or grow on them. Silicone foil and silicone tubes pro
`vide a surface on which the cells do not adhere, or
`adhere to it only with great dif?culty. Therefore, syn
`thetic 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
`
`4
`membrane must, of course, prevent reverse ?ow
`through it of liquid from the fermenting broth.
`The permeable membrane positioned in the fermenta
`tion vessel can have any suitable size or geometric shape
`but one must be selected so that there is suf?cient oxy
`gen diffusion to supply the amount needed for cell me
`tabolism. 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, it
`self, 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 con
`duit 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 com
`mon chamber or volume wholly or partially de?ned by
`the membrane.
`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 particu
`larly 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 ?lter 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 dis
`tinct 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 mem
`brane 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 non
`vertebrae culture suspensions and monolayer cultures.
`Vertebrae cell lines of primary importance for propa
`gation 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).
`
`55
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`GE-1028.005
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`

`
`5
`BRIEF DESCRIPTION OF THE DRAWINGS
`FIG. 1 is a vertical sectional view through a fermen
`tation 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 fermen
`tation vessel for cultivating vertebrae cells in suspended
`culture as well as in monolayer culture.
`
`10
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`4,649,114
`6
`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 to
`gether. 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 fermen
`tation 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 down
`wardly 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 ?ows
`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,
`?uidization or suspension of the balls 15 can be achieved
`so that packing of the balls by settling is avoided. Fluid
`ization 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 nonhomoge
`neous 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 con
`sidered.
`When fermentation runs were carried out according
`to the invention, the bubbling in of air was completely
`avoided. Oxygen was supplied solely through the sili
`cone 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 ?owing air in the silicone tube into the liquid
`nutrient medium and was always available in suf?cient
`amount, even with increasing cell population and in
`creasing culture volume. By increasing the ?ow 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 con
`stant increase of Mamestra brassicae cells could be
`achieved in cell culture suspensions of four and ten
`liters. It was possible for the ?rst time, when compared
`to all previously used culture methods, to not only mul
`tiply 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 pro
`duced according to the invention has an excellent mor
`phological 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,
`
`35
`
`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 con
`tents in the fermentation vessel. The wall thickness of
`25
`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 ?t
`tings 5 which penetrate cover 2. Compressed air is sup
`plied to tube 4 by the ?ttings 5. A heat exchange ?uid,
`generally a liquid, is supplied to, and removed from, the
`heat exchanger tube 3 by means of ?ttings 6. _
`Motor 9 drives shaft 10 which penetrates the bottom
`7. Shaft 10 contains propeller 11 inside of the fermenta
`tion 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
`40
`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 ?owing out of the spiral tube 4, and the entire
`liquid ?ow itself, assures even distribution of the nutri
`ents 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 commu
`nicate with ?ttings and holes in cover 2 to which the
`hose connections 6 are joined. Double-walled heat ex
`changer 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 ?uid com
`munication. A heat exchanger ?uid, usually a liquid and
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`GE-1028.006
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`

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`4,649,114
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`7
`diffuses into the nutrient medium. To enrich the nutri
`polytetrafluoroethylene (Te?on), or equivalently per
`ent medium after the ?rst 16 to 24 hrs., compressed air
`meable material, permits oxygen enrichment of a nutri
`at a pressure of 0.5 to 1.0 atmosphere (gauge) was intro
`ent medium to an extent which makes possible maxi
`duced via the silicone tube. If necessary, the pressure
`mum propagation of insect cells in suspension volumes
`can be increased up to 2.0 atmospheres.
`of ten liters or more. The cells are not prone to settle on,
`With an initial cell concentration of 105 ml, the oxy
`or attach to, the permeable tube 4 and thus do not
`gen concentration decreases from 7.5 mg/l within 24
`slowly block oxygen ?ow, because the material of
`hours during the logarithmic cell increase (cell prolifer
`which the tubes are made provides a surface not suitable
`ation) to below 1% of the initial value. However, by
`for cell adherence. For the same reason, cells do not
`means of oxygen (air) supplied through the silicone tube
`adhere between the tube spirals, die there, decay and
`during the logarithmic cell increase, the oxygen con
`contaminate the nutrient medium with toxic decay
`products.
`centration can be maintained at 10 to 30% of the initial
`concentration, which guarantees very good cell prolif
`The following examples are presented to further illus
`eration.
`trate the invention.
`By means of the oxygen diffusion method according
`to the invention the number of cells per ml of nutrient
`medium was increased from 105 up to 2 to 3 X l06in 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 divid
`ing. Each ml then contains 2 to 3X106 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 fer
`mentation a cell concentration of 2 to 3X 106 ml was
`again obtained. From the original 3X 108 cells/3 liters,
`up to about 1011 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
`4x109 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 fer
`menter. 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 main
`tained in the fermenter vessel, can be continued as long
`as permitted by the total condition of the cells. In stabi
`lized cell lines, this can lead to a continuous operation.
`
`15
`
`20
`
`25
`
`EXAMPLE 1
`A suspension culture was started from the insect cell
`line IZD-Mb 0503 (IZD=Institute for Zoology Darm
`stadt; Mb=Mamestre brassicae=a type of butter?y;
`0503=code number of the cell line; Lepidoptern-cell
`line ATCC #CRL 8003) in a so-called spinner con
`tainer, 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 pub
`lished 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 2X 105
`cells/ml of nutrient medium is necessary for multiplica
`tion. After three days, as a rule, a cell population of
`about 6 to 10X 105 cells/ml is obtained. This is a popula
`30
`tion increase of 3 to 5 times the original amount. Be
`cause 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 fer
`menter like that shown in FIG. 1, were calibrated, auto
`claved and recalibrated. After that they were inserted in
`the fermenter cover. A tube of silicone rubber having a
`40
`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) ?owing 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 ?lled 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 105 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° C. in the sus
`pension. The initial oxygen value (7.5 mg/l) and pH
`value were set via the measuring electrodes.
`In the ?rst 16 to 24 hrs. after the start of the cultiva
`tion, 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 metabolism.
`The oxygen is supplied via the silicone tube function
`ing as a permeable membrane through which oxygen
`from atmospheric air, or from an oxygen-air mixture,
`
`35
`
`50
`
`55
`
`65
`
`EXAMPLE 2
`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 l407=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.
`
`EXAMPLE 3
`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,
`
`GE-1028.007
`
`

`
`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 pro
`vides a surface of up to 30,000 cmz. Tests with non
`diploid Psylla (plant lice) cells in a suspension culture
`with micro-carrier balls have resulting in cell popula
`tion ?gures of 4 X 106 /ml of culture broth. Because high
`cell populations can result from the use of micro-carri
`ers, the oxygen consumption is correspondingly high.
`With the air bubble method of the prior art the in
`creased oxygen requirement is much harder to satisfy
`than with the membrane oxygen diffusion method ac
`cording to the invention. With the membrane method,
`the metabolism ef?ciency of cells growing on the mi
`cro-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 modi?
`cations will be obvious to those skilled in the art.
`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 permea
`ble membrane partially de?ning a chamber or volume in
`the fermentation vessel and made of a polymeric mate
`rial on which the cells do not grow to a signi?cant
`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
`suf?cient 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
`45
`chamber for diffusion through the membrane; and .
`