DK141911B - Cell culture system comprising live eucaryotic cells. - Google Patents

Description

Mm show / (11) PUBLICATION REPORT 1 ^ 1911 C 12 il 3/00 DENMARK (· ΐ) [nt.ci.3 c 12 hb / oo '(21) Application No 2261/73 (22) Submit' on 25 · 97 · ΡΓ · ^ 973 (23) Le bed 25 · apr. 1973 (44) The application submitted and the non-disclosure document published on 1 4. CHRISTMAS · you DIRECTORATE FOR __. .

PATENT AND TRADE MARKET (30) Priority requested from it

Apr 26 1972, 19387/72, GB

(71) THE WELLCOME FOUNDATION LIMITED, 183-193 Eueton Road, London N.W.1, GB. “(72) Inventor: Ronald Charles Telling, Furzsholme, Dawneys Hili, Fir = bright, Woking, Surrey, GB: Roy John PassIngham, 31 Lea Wood Road,

Fleet, Hants, GB: Brian Lewis Kitchener, 144 Beta Road, Cove,

Farnborough, Hants, GB: David Tleorge Hopkinson, 12 Windermere Close,

Cove, Farnborough, Hants, GB, (74) Plenipotentiary:

International Patent Office. ________ I II I I I ............— ^ T— "1 —aw— ^ mmmmmmwmm (54) Cell culture system comprising living eucaryotic cells.

The invention relates to a cell culture system comprising live eucaryotic cells, especially those of human or animal origin or those derived from mycophyta. By eucaryotic cells is meant cells with a true chromosome-like nucleus surrounded by a human branch. The cell culture system can be used to grow microorganisms, such as viruses, in, for example, mammalian cells and human cells.

It is known that many different cell cultures, especially of animal or human origin, can be maintained as monolayers on the even surface of a solid support, for example, Petri dishes, glass bottles or tubes. As soon as such cultures become "clashing", ie. forming a total layer of uniform thickness, a virus can be introduced into the liquid medium covering the cells and the culture can be used to grow viruses in the monolayer system. Many types of cells can, however, be advantageously grown in suspension, i.e. substantial dispersion in the nutrient medium, and the same technique can also be used to grow viruses in suspended cells. Apparatus for the purpose usually comprises a sealed container or tank with appropriate agitation means, control of environmental factors, e.g., pH, pC 2 / nutrient supply (cf. British Patent No. 1,090,758).

In the conventionally used system, for example, for growing cell lines in suspension culture, the cells are kept in a medium stirred state in the medium and are sedimented due to gravity when peak concentration is reached. The medium is then decanted and the cells resuspended in a fresh medium which may also contain virus particles. After a period of virus growth determined by observing the cytopathic effect of the cells, the yield is usually passed through a filter and then through a bacterial sterilizing membrane to obtain a solution containing free viruses released by the disrupted cells.

One difficulty with large suspended cultures of mammalian cells is that the flow of air through the medium at high speed can damage the cells, while a certain amount of oxygen supply is constantly needed for optimal growth. Other difficulties are that the sedimentation of the cells is a time-consuming process (ca.

24 hours), the sedimented cells can be exposed to an unfavorable media environment in terms of pH and nutrient factors, and only 95% of the medium used can be drained without the risk of loss of a substantial amount of the cells. In addition, during filtration of the viral yield, the included cellular residues exert a stopping effect on the filter medium and the ratio of filtered volume to filtration area must be small to obtain sterilizing filtration. This can cause severe losses (up to 50%) of viral antigen due to virus adsorption both on the filter medium, especially if it contains asbestos, and on the remaining cell debris. In addition, a large vertical filter press plate is required to provide adequate filtration, which usually causes significant volume loss and, as it is not a closed system, the resulting drip poses a serious risk of disease progression.

The present invention is therefore intended to provide a system where these mentioned disadvantages can be substantially avoided. Preferably, the system should be applicable to cells of both monolayer and suspension type and to the cultivation of viruses for commercial production of vaccines.

3 141911

It has been found that this can be achieved with the cell culture system according to the invention, which is characterized in that the cells are distributed in a porous support body or bed, consisting of particulate material which provides internal cavities or spaces sufficient to retaining the cells and enabling their growth and propagation in the carrier body or bed, as well as passage of liquid substrate in contact with the cells.

The carrier may conveniently be provided in the form of a filter bed, and may consist of natural or synthetic materials, e.g., diatomaceous earth silicates, glass or polymer particles. In particular, SåledeB has various forms of filter aid for diatomaceous earth, and especially diatomaceous earth or diatomite has proved suitable for this purpose. Another useful diatomaceous earth type is infusion soil. Other materials that may be used include spun glass, cellulose pads, nylon or polystyrene beads. All of these materials are virtually insoluble and essentially inert.

The size of the pores or the space available for cell growth in a bed of particulate materials may be suitably selected and adjusted to the requirements. It is often necessary to choose a "retention target" which indicates the filtering ability of the material that would effectively retain the cells but allow rapid flow of the fluids. Thus, according to the invention, the carrier may suitably have a retention target of 0.1 µm to 2.00 µm, preferably between 0.2 µm and 1.2 µm, e.g. 0.4 µm to 0.6 µm. The materials are appropriately processed by removing contaminants and sources of infection and are purified and fractionated according to their particle size and other requirements.

The carrier may comprise a single layer, and this may suitably have a retention target of 0.75 µm to 2.0 µm, preferably 1.2 µm. However, according to the invention, the carrier may comprise more than one layer of porous particulate material. Such a multilayer system may advantageously be used to increase retention. Preferably, the retention target in the layers of carrier is substantially reduced from the input surface layer to the surface layer nearest the exit surface layer, and may, for example, comprise successive layers of material having o, 5, 0.2, 0.5 and 1.2 µm retention dimensions from a basal perforated support plate or filter to the top. The retention dimensions in the layers suitably range from 0/1 to 0.75 μιη, preferably from 0.2 to 0.5 μια, and the starting surface layer preferably has larger retention targets than its neighborhood, e.g. 0.5 μια.

If a single layer is used, this may also have to be provided with an additional low retention target top layer, such as 0.2 μιη, immediately before the final filtration, to increase the retention of smaller particles.

Conveniently, the thickness of a layer in the carrier is from approx. 8 mm to approx. 20 mm.

Any type of cell suitable for wading either in monolayers or in suspension can be incorporated into the culture system of the invention. In this context, the cell types include primary and secondary cell cultures and diploid and heteroploid cell lines or strains of mammalian or human origin. For example, the well-known IBRS2 pig kidney cell line or baby hamster cell line clone 21 (BHK 21) is particularly suitable for this purpose. For cells that can only grow and be used in monolayers, the culture system is highly suitable for all growth stages and also for subsequent culture of microorganisms such as viruses. Other cells that can be effectively grown in suspension cultures can first be grown in this way and then incorporated into the culture system of the invention for virus culture and recovery. Thus, the culture system also includes as a special feature cells infected with microorganisms, such as viruses, for which the cells are susceptible. Of course, other types of eucaryotids, such as mycophyta, such as yeasts, can also be applied to the carrier used in the invention in a suitable nutrient medium.

For example, standard horizontal pressure filters can be used for substrates for the culture system, but it is appropriate to use a cal-mic "high duty" horizontal plate pressure filter, such as Calmic 45-S-9 E-type filter manufactured by Calmic Engineering Ltd., Grewe. Such a filter comprises nine horizontal plate units each with a diameter of 45 cm and with a total filtration area of 1.26 m. Each plate unit comprises a perforated stainless steel plate mounted on a stainless steel plate with recesses. A substrate, such as paper or rayon, can be applied to the perforated plate to support the filter bed.

Preparation of the subject cell culture system is carried out by the following steps: a) preparing a support bed from a slurry of porous or particulate material, b) adding a liquid suspension of eucaryotic cells to the bed, and c) allowing the cells in the bed to settle .

In preparing the culture bed, slurries of suitable types of carrier material can be pumped from a suitable container through the plate filter, thereby retaining the carrier on the plate. For each layer, the slurry is recirculated several times until the filtrate is clear, thereby obtaining the required thickness of the support bed on the substrate or the previous layer, which, as mentioned, for example, may be approx. 8 mm to 20 mm, preferably 10 mm to 14 mm and most preferably 12 mm.

The culture vessel may be of the usual type therein, provided with a stirrer and means for measuring and controlling pH, temperature and for introducing air or oxygen to vent the medium. It may further have an oxygen electrode that measures dissolved oxygen in the culture medium and, as a result, can be used as described below to determine the duration of the virus growth period.

Any culture media known to be suitable for growth of cells and / or microorganisms, e.g., viruses in connection with cells, may be used, e.g., Eagles Basal Medium (Science, 122, 501 (1955)) or modified Eagles Medium (Virology, 16, 147 (1962)). The medium may also contain, for example, 10% v / v bovine serum for growth of BHK21 suspension cell lines and a reduced amount of serum, e.g.

1% serum for growth of foot-and-mouth disease virus on this cell line.

Foot-and-mouth disease (hereafter referred to as FMD) is caused by many antigenically different virus types, several of which have been found in particular areas. For example, types O, A and C occur in Europe and South America, types SAT1, SAT2 and SAT3 in South Africa and types O, A and Asia I and SAT1 in the Near East. The following strains of FMD / virus have so far been found suitable for growth in the cell culture system of the invention, namely A Pando, O BFS 1860, SÅH Pho-5/66, SKU2 Swz. 1/69 and SAT3 Bec 1/65.

The method can be practiced in two ways depending on whether the cell system normally grows in suspension or monolayer culture. In the first case, the cells of a submerged culture are grown in a stirred container in the usual manner and filtered through and retained on the bed when the cells have reached their maximum concentration. Cells which are only suitable for monolayer culture, on the other hand, can be grown advantageously in the culture system. Thus, in the latter case, the appropriate culture medium in the culture vessel can be seeded with live cells and then immediately circulated through the pre-prepared carrier bed whereby the cells are embedded and immobilized in the bed. The culture medium is then continuously circulated throughout the cell culture period.

In either case, medium suitable for virus growth can then be added to the culture vessel and the cells seeded with the virus. The medium is again continuously circulated through the bed so that this virus can multiply in the cells embedded in the carrier bed. When the virus destroys the cells, the resulting residues remain in the carrier bed and the virus is released into the medium.

Although cell growth in the carrier bed cannot be directly observed, growth can easily be measured by glucose utilization. The period of virus growth can be determined indirectly by reading the oxygen electrode giving the dissolved oxygen (PO 2) concentration in the culture medium. In the earlier stages of viral culture, the oxygen uptake of the metabolizing cells is greater than the rate of oxygen dissolution in the culture medium, and consequently, dissolved p02 decreases. so that dissolved pC> 2 increases. This change can therefore be used to measure and control the viral culture step, and it has been found advantageous to recover the viral culture when dissolved p02 in the culture medium is approx. in equilibrium with p02 ~ value for conditions of air saturation.

The general sequence of operations in preparing the cell culture system of the invention e.g. with cells that can be grown in suspension cultures are as follows. Cell culture is started in a stirred container in the usual manner, with the pH of the medium being approx. 7.4 and the temperature approx. 35 ° C in most cases. A carrier bed is then produced by pumping an aqueous slurry of a suitably disposed carrier material, e.g. diatomaceous earth, continuously through a suitable filter, preferably under pressure, and the system is sterilized and kept in this state until used. When the cells have reached their maximum concentration, the cell culture is passed through the bed at a rate of approx. 20 liters per liter. per minute, whereby most of the cells are immobilized in the carrier bed. The culture medium and any non-trapped cells are pumped back into the culture vessel and recycled through the filter until the time when less than 10% of the cells, preferably less than 5%, is found to pass through the system. This can be determined indirectly by cell counting at intervals and when the recirculation step of 7 samples is taken from filtrate immediately after the filter. The filtrate is then pumped to a wastewater compartment, leaving the cells covered with the medium retained in the filter until the virus is washed off.

For viral growth, a new medium of a different composition is usually introduced and passed through the cell culture system. A suitable viral species against which the cells are susceptible can be introduced to infect the culture. After disintegration of the cells by virus propagation, i.e. after the cytopathic effect occurs, a large amount of virus is released and carried away with the medium.

Medium containing the virus particles separated from the cell debris in this way can now be stored or preferably filtered, removing any bacterial contamination from the medium. The viral antigen can then be inactivated with a suitable inactivation medium, such as formaldehyde or especially acetylethylenimine, and processed into a vaccine which preferably comprises an additive such as aluminum hydroxide, advantageously combined with saponin.

The method is particularly suitable for growth of viruses in cell lines and can therefore be used advantageously to produce viral vaccine after appropriate inactivation if necessary.

Use of the cell culture system of the present invention avoids the time-consuming sedimentation step, where, according to the ordinary technique, cells are often kept in an adverse environment for extended periods of time. Removal of used cell media can now be done quickly and more or less completely. Furthermore, switching from the cell culture medium to the virus growth medium can be done easily and quickly, avoiding the cross-contamination of the media commonly found in conventional systems. The cells can be easily washed between the steps if necessary. As cells and cell debris remain in the carrier bed, the pre-filtration which is important to achieve high throughput during the final bacteriological sterilization filtration step can advantageously be incorporated into the virus growth stage, thereby saving several hours.

It is important that the desired nutrients can be added to the recycle medium at any time and the medium is also flowed with air at the maximum useful rate without the risk of physical damage to the cells. In addition, the method can be carried out in a closed system which can, for example, be sterilized, for example by steam injection, and therefore there is no risk of disease contamination from droplets and spilled material, as in the ordinary technique.

The invention will be explained in more detail below with reference to the drawing, which schematically shows the connection between a pressure filter containing a carrier bed and a culture vessel with associated equipment.

In the figure, an ordinary culture container A is provided with an electrode system B, and medium from the container can be pumped via a pump C into a pressure filter D which supports a bearing bed E. Cartridge filters. F and membrane filters G serve to filter the viral yield before leading to an inactivation tank H.

Example 1

Manufacture of diatomaceous earth filter bearings.

Filter bearings used in the cell culture system of the invention were of three kinds a) A single bed was made by pumping an aqueous slurry of 3000 g of "Dicalite" ® 4200 with retention dimensions 1.2 µm through the central aperture of the horizontal plates of a Calmic 45-S-9 pressure filter and down through each plate, (S) forming a layer of "Dicalite" ^ on each plate. The filter system was then sterilized by vapor injection. Immediately prior to filtering the viral yield, 1500 g of sterilized diatomaceous earth filter aid with retention target 0.2 µm was added to the viral yield.

b) A single bed was prepared as under a) by pumping 4000 g of "Dicalite" ® through the pressure filter. 500 g of sterilized diatomaceous earth filter aid was added directly to the support bed immediately prior to sterilization filtration.

(c) A multi-bed was prepared by pumping the following types of diatomaceous earth through the pressure filter in the order indicated: 100 g of the retention product sold under the name "Hyflo Supercel" with retention dimensions 0.5 µm, 100 g and diatomaceous earth filter aid, 2000 g "Hyflo Supercel" and 1000 g of "Dicalite" ^ 4200, which system does not need further addition of diatomaceous earth at the end of the virus cultivation step. The Calmic pressure filter was sterilized by steam injection at 1.37 x 10 Newton / meter and kept under positive pressure with sterile air until use. During use, the temperature of the filter was regulated by circulating water through the jacket of the filter.

161911 9

Example 2

Preparation of FMD virus from BHK21 suspension B cells in the carrier bed.

A culture of 650 liters of medium containing approx. 7 x 10 5 cells per cell. ml of a clone of baby hamster kidney cells (BKH21) was started in a sterile 700 liter closed culture container, which was suitably equipped for pH control, for various nutrients during cell culture and virus growth and with an oxygen electrode to determine the dissolved oxygen concentration (p ) ·

The culture medium used was modified Eagles Medium (virology 16, 147 (1962)) supplemented with 10% v / v bovine serum. The temperature during cell culture was maintained at 35 - 0.25 ° C, and the stirring speed with a reciprocating blade was adjusted to · 36 bpm. An air stream was maintained at a rate of 5 liters / minute over the top of the medium and the pH was adjusted to 7.4 and automatically kept there within the range - 0.03 pH units with a flow of carbon dioxide gas of 10 liters / minute through the medium or by the addition of 4 molar sodium hydroxide solution. An additional current was automatically passed through the culture medium at a rate of 15 liters / minute when dissolution of the oxygen electrode indicated this was necessary. The maximum concentration of cells, approx. 2.5 x 10 celler cells / * 11! ' was obtained after 50 hours and the cell culture was then circulated at 20 liters / minute three times through the multilayer filter, prepared as given in Example 1c). Cell count assays on samples taken from the medium immediately after the filter at 15 minute intervals indicated that this step is approx. 96% of the cells at the maximum concentration were immobilized in the cap bed · The used, essentially cell-free medium was then pumped to a waste container, whereby the carrier bed was covered with medium until the virus culture step.

650 liters of virus culture medium comprising modified Eagles Medium containing 1% v / v bovine serum was added to the culture vessel and set at 35 ° C. The cell culture medium in the filter was drained and virus medium circulation through the filter was started. The medium in the culture vessel was then seeded with 200 ml suspension of a strain of FMD virus called type A Pando, which had been adapted for growth in BHK21 cells. The seeded medium was recycled through the filter for approx. 48 hours at a flow rate of 20 liters / minute, the number of media shifts in the filter tube being approx. 20 / tixne and in the total cultivation volume approx. 2 / hr. The speed at which the medium flowed through the carrier bed was approx. 1.8 cm / minute. The pH of the medium U1911 at pH was 7.4, by automatic injection of air and carbon dioxide gas through the medium.

The virus propagated in the cells immobilized in the carrier bed 7 to a titer of 10 'plaque-forming units (pfu) / ml of culture fluid, with samples for plaque testing being taken from the culture fluid at regular intervals throughout the day. The maximum titer in a complement-fixation test was 1/12.

The end of the virus culture period was determined by measuring pC> 2 in the culture medium and the culture was harvested when the pC 2 value had grown to a value corresponding to the value at air saturation. The medium in the filter was then sent back to the culture vessel by air pressure and the filter isolated from the system. The viral yield in the medium was pumped out through a two-stage bacterial, sterilizing filtration system which had been previously sterilized by vapor injection.

In the first step, the virus culture medium was adjusted to a pH of 7.6 with 2 molar glycine buffer and then filtered at room temperature and at a flow rate of 6.8 l / min through two Balston cartridge depth filters (47.5 cm x 3 cm) of bonded glass fibers. The filter cartridges with a retention measure of 0.35 mm were placed in parallel to provide a filtration area of 1500 cm.

In the second step, medium from the cartridge filters was pumped in the same amount at a pressure of 4.8 to 6.2 x 10 6 N / m 2 through 20 Schleicher and

Schiill membrane filters (20 cm x 20 cm), retention dimensions 0.22 µm. These 2 provide an effective filtration area of 6500 cm or approx. 100 ml 2 filtrate / cm filtration area.

The resulting antigen was inactivated in the pre-sterilized inactivation vessel by treatment with acetylethylenimine (AEI) and processed into a vaccine containing 2 ml of inactivated virus filtrate, 25% by volume 2% w / v aluminum hydroxide and 5 mg saponin per ml. cattle vaccine dose. The vaccine was tested on cattle by inoculation of live virus 21 days after vaccination and was found to have a PD ^ q of 29.7 (PD ^ q is the protective dose at which 50% of the cattle survive the infection with live virus). This shows the relative strength of the vaccine.

Example 3

Preparation of FMD virus from BHK21 suspension cells in the carrier bed.

In accordance with the procedure set forth in Example 2, SAT.2 was Swz. 1/69 strain of FMD grown, filtered, inactive and processed into a vaccine. Tested in cattle, the vaccine had an efficacy value of 29.3 PD50 / dose calculated from the titer level of circulating antibody in the cattle 21 days after vaccination. Example 4 Virus growth by the methods described using both single- and multi-bedding techniques was investigated using several different FMD strains. The following table shows examples of infectivity and maximum complement fixation value obtained from the different strains used:

Scale Diatomite Virus strain Infectivity Complement (liter) bed (log.npfu / ml) fixation (cfu / ml) Single SAT.l-Rho 5/66 6.7 1/16 "" O-BFS 1860 6, 8 1/16 "" "6.4 Vl2 650" A Pando 6.3 / 24 "" "6.8 1/12" Multi "7.5 l / 12" "" 7.3 1/8 "" SAT .l-Rho 5/66 6.4 1/6 "" "7.0 Ve" "SAT.2-SWZ. 1/69 7.1 1/6" "" 6.1 1/8 "" SAT. 3-BEC. 1/65 7.2 λ / 5 "" "6.8 Ve.

Example 5

Preparation of FMD virus from IBRS 2 pig kidney cell line in carrier bed.

Procedures of Examples 2 and 3 were repeated using IBRS 2 cells with an appropriate FMD virus. A vaccine with a satisfactory efficacy value was obtained and tested on live virus cattle 21 days after vaccination.

Example 6 Growth of BHK21 cells in carrier bed in the filter.

500 L of cell culture medium comprising modified Eagles Medium containing 10% v / v bovine serum was added to a 700 L culture vessel and the medium brought to a temperature of 35 ° C and then seeded.