NO134062B - - Google Patents

Description

The present invention relates to a device for the cultivation of living cells, in particular cells of animal origin, in a single cell layer as well as of eumycetes, schizomycetes and unicellular algae.

Production of vaccines to prevent viral disease-

more is characterized by different possibilities for culturing viruses. Viruses can be produced on large, infected livestock (cattle or horses), on small laboratory animals, on incubated eggs and cell cultures (either cell layers or cell suspensions). Virus production from tissue culture cell layers is obviously both a technical and scientific advance over the other production methods. Furthermore, it is known that large quantities of living cells are necessary for technical

production of several vaccines (inoculants), and that these cells cannot be cultured in cell suspensions, and that also fermentation vessels which in the biosynthetic production of antibiotics, meldry alkaloids, etc. are not immediately suitable.

In fact, the production of vaccines and biological agents for use in humans is limited to normal, non-cancerous tissue cells, such as primary cells and cell strains from tissue cultures.

As is known, one of the typical tissue culture preparation methods for culturing inoculum is carried out in "Roux" or "Brockway" flasks. The total capacity of a Roux flask is approx. 1 litre, and the inner surface for growing cell layers is about 250 cm<2>.

The nutrient medium containing the cell suspension is introduced into the sterilized flask and incubated in a horizontal position, so that the cell growth forms a uniform cell layer. The culture medium is removed and replaced with a preservation medium containing the virus, which penetrates the cells and multiplies. When the virus has reached the desired concentration, the contents of the flask are collected and used to produce the vaccine.

The manufacturing method with stationary Roux flasks was significantly improved by means of the so-called "manufacturing method with rotating bottles" which in particular could occasionally be carried out in baskets of stainless steel wire containing approx. 20 round flasks. With the help of this method, a significant increase in production capacity for vaccine was obtained, corresponding to the larger available volume.

However, both methods show a significant disadvantage in that they have a limited technical production capacity, as the use of many bottles that are necessary is too complicated and detailed. A significant number of skilled workers are therefore needed, and the probability of contamination of the biological material is high due to the fact that each flask must be opened, filled, emptied and closed.

A further disadvantage of tissue culture in flasks is that it is impossible to control, monitor and examine the metabolism in the tissue culture by removing carbon dioxide, introducing oxygen or additional nutrient medium, or removing cells, as the flasks are a closed unit that excludes automatic work under sterile conditions. All these above-mentioned disadvantages will naturally influence the price of the final product.

In the Canadian patent document 787,391, a so-called "tissue culture propagator" has recently been described.

This "tissue culture grower" consists of a certain number of glass or suitable plastic plates which are mounted at equal distances below each other in a corresponding fastening device. The whole thing is inserted into a suitable leg holder which is equipped at one end with an airtight end piece. The end piece is equipped with a supply pipe, a gas line, an extraction pipe and a tube.

Although this "tissue culture grower" produced some technical progress over tissue culture in rotary flasks, it also shows significant disadvantages. It does not allow the cycle louse's "closed" work to be carried out in a completely automatic way, nor does it allow for thorough monitoring of all steps without the risk of contamination from the outside.

British patent document 1,119,654 describes a device for extracting cell cultures, which consists of a rotatable, drum-shaped holder in which parallel, side-by-side culture vessels are arranged in the horizontal plane.

In this arrangement, the cultivation vessels are designed as rudders, which are provided with valves at both ends through lines

connected with each other. However, this device also exhibits significant disadvantages, as placing the rudder bundle gives very poor accessibility to the individual rudders, because the rudder bundle cannot lie freely. According to the British patent document, it is the outer drum that is supported so that it can be rotated about the axis of the rudder bundle.

The present invention makes it possible for the column bundle to lie freely accessible, while at the same time it can rotate about its longitudinal axis and be tilted about an axis perpendicular to the longitudinal axis.

The present invention relates to a device for the cultivation of living cells, in particular cells of animal origin, as well as of eumycetes, scnizomycetes and unicellular algae, which device is surrounded by a temperature-regulated room and comprises a large number of tube-shaped columns consisting of a transparent material which at the ends are connected to each other through manifolds, and where the column bundle is both horizontally and vertically rotatably stored, which device is characterized by the fact that the column bundle at both ends is fixed in disk-shaped holders, each equipped with a pivot, which pivots in the central axis of the column bundle are rotatably supported to a frame rotatably stored perpendicular to the center axis.

The means and precautions to achieve the desired goals and inventive advantages are further illustrated by the drawings. There, Fig. 1 schematically shows the rotatable and tiltable device with the carrying or holding device in a horizontal position. Fig. 2 shows the device in a vertical ("tilted") position. The columns 1 that form the apparatus end by means of headers in a single unit. These headers connected to the columns are interrupted by taps 2, 3 which allow working with all or part of them.

All necessary work for the corresponding number of columns is done by opening and closing the taps in the main collector tubes 2. The column bundle, which is held together by a plate 6, is mounted on a common holder 7 and can, with the help of special devices 4, 5, rotate with adjustable speeds about the longitudinal axis and switch between horizontal and vertical position.

The apparatus is mounted on a fixed bench 8 and placed in a room, or surrounded by a heat- and cold-regulating mantle with automatic temperature regulation, so that the temperature of the tissue culture medium is kept within a specific range.

By means of the taps in the main collecting tubes, the apparatus can be connected to various other containers 9 containing cleaning agent, tap water and deionized water, water vapor, and also for the supply and removal of the liquids and gases which are necessary for cell growth.

Corresponding use of the main and sub-collector pipes with the corresponding taps 2, 3 enables the connection of the column bundle as a whole or a single column with not only the above-mentioned storage containers or lines, but also in a closed and sterile state with specific devices for the distribution of carefully measured , automatically calculated liquids, such as nutrient media, buffer solutions, suspended cells, trypsin or other enzymes, viruses, and also the supply of pure or mixed gases such as oxygen, air or carbon dioxide.

Furthermore, a connection with a special venting pipe for removing the carbon dioxide formed during cell growth, and a suction pipe for removing moisture, is possible. Optionally, these devices can carry out the measurement and the automatic or semi-automatic registration of the various parameters. These measuring and recording means can normally be connected to automatic devices for correcting and maintaining the parameters of the selected areas.

Furthermore, each column or column bundle can be equipped with a means to measure and record the internal pH value, whereby an automatic device can keep the tissue culture's pH values within a desired range, either by varying the ratio of oxygen or air to carbon dioxide, or by to add buffer solutions.

When the "rotating columns" are used for growing cells in the cell layer, a microscope with different magnifications along the entire length of the column is used to observe the cells in the cell layers on the column surfaces.

The manufacturing cycle consists of the following steps: washing the device with a cleaning agent solution using vacuum and subsequent rinsing with deionized water, sterilizing the device with steam, introducing cell suspension into the device in a suitable growth medium, and cultivating the introduced cells on the surface of the length of the horizontally rotating columns.

Cell growth on the surface can be followed with a microscope and is characterized by the formation of a thin, translucent layer on the surface of the column. After growth, the Talir cells, when they are to be used in cell layers for further cultivation, are prepared by treatment in the enzymatic cleavage step using trypsin or other suitable enzymes, or they can be used for the production of viruses or other biological products. Due to the simple handling of the equipment, both working methods can be easily carried out. After the cells or other biological products have been collected, the cycle begins again with washing the device.

The above description shows the following advantages of "rotating column" equipment: By opening and closing the taps in the main collection pipes, the necessary filling and emptying of the determined number of columns is achieved in one operation in a sterile manner.

Refilling liquids is done by vacuum and pressure.

The equipment can be quickly washed using this method, while other methods require specific washing devices.

Due to sterilization with steam, the equipment is ready in a short time.

The amount of work is reduced, and the work processes take place safely under sterile conditions, whereby any contamination of the treated products or the risk of infection of workers is avoided.

The equipment can be used with all columns, with part of their number - or stepwise use of individual parts.

If the equipment is connected, it can still be used for other work within a short time, it does not need to be moved, and can be connected to other devices in other rooms by means of a rudder.

The cell growth phase and the cytopathic effect of the virus can be immediately observed using a microscope.

By microscopic observation of the cell layers, it is possible to simply trypsinize cells to be used for other inoculation.

It is possible to use automatic and/or semi-automatic temperature setting.

You can use different amounts of nutrient media and preservation media that are necessary for the culture.

During the various production steps, control and rapid automatic or semi-automatic setting of the pH values is possible.

When using the apparatus for cell cultivation in cell layers on the inner surface of the columns, the growth does not depend on the length, diameter and number of the columns.

It is emphasized that the ratio between the cell culture surface in the cell layer and the necessary volume medium for the growing cells when using the present device is significantly improved compared to conventional devices. Thus, significant scientific advantages are achieved, such as e.g. reduction of the necessary medium, enzymes, even such an expensive substance as trypsin. Furthermore, the liquid, virus-containing medium has such a concentration that it can be used in the second step:-ti 1 iremsti Iling of inoculants without further concentration.

The following table 1 shows the comparison between Soux columns by the "tissue culture grower" and the rotary columns according to the invention.

Table 1 shows that, under the same conditions, the utilization of the medium and the concentration of the biological product at the end of the production line are higher the higher the ratio between the surface area and the volume of the medium. The following examples illustrate the present invention.

Example

Cell layers of primary cell cultures.

Organs from mammals and other animals, which have been removed in optimal condition, were cut into small pieces of 2-5 mm, washed in buffer solution and trypsinized in the usual way. After examining the cells, the pooled cells were suspended in the culture medium to a desired concentration for culture. Introduction into the corresponding, washed and sterilized apparatus with rotating columns followed, connecting a tube between the vessel with the cell suspension and the apparatus itself in a vertical position, and carrying out the filling using vacuum, pressure or a peristaltic pump. The various taps were closed, and the introduction of the sterile gas mixture (air-carbon dioxide) was adjusted to optimal oxidation and pH conditions. After the apparatus had been brought into a horizontal position, the introduced cells were incubated at 36 - 37°C The uniform cell layer,

which is formed on the column surface, can be observed under a microscope at any stage and is finished in a similar time as is required for other cultures in stationary vessels.

Table 2 shows some examples of the time required to achieve a complete cell layer in "rotating columns" and Roux flasks.

Example 2

Cell layers of cell strains and cell lines.

The new device with rotating columns allows the technical production of cell strains and cell lines.

After the formation of the complete cell layers, various tasks must be carried out at carefully defined time intervals, bringing the apparatus several times in different positions (vertical, horizontal, rotating, etc.) to carry out the numerous trypsinization and collection steps for the cells.

The simple handling of the apparatus according to the invention enables an easy and accurate execution of the trypsinization, which in the case of the Roux flask requires manual work. This can be carried out by one or two people at the same time on all or part of the equipment. The size and number of columns can be varied.

Table 3 shows some data on cultivation of BHKg^ cells in the apparatus with rotating columns. The cells used were kept in the apparatus after trypsinization of the cell layer.

The subsequent table k shows the time required to achieve a complete cell layer in rotating columns and stationary Roux flasks from individual cell lines and cell threads.

Example 3

Production of foot-and-mouth disease virus C (F.M.D.V.).

By working in accordance with example 1, and using a pig kidney, a uniform cell layer was obtained after 5-6 days of incubation."

The culture medium was then removed by washing the cell layer, and replacing this medium with a preservation medium for the cells during virus formation in a closed system under sterile conditions, by the fully automatic method described above. Together with the aforementioned preservation medium, a foot-and-mouth disease virus st amme Co. was introduced

After virus inoculation, the apparatus was brought to optimal aeration, pH and temperature conditions, and the virus began to multiply. Closed samples were taken at regular time intervals

under sterile conditions from the virus growth. The maximum production was reached after approx. 15 - 24 hours cultivation. Tissue Culture Infection Dose 50 ("Tissue Culture Infection Dose 50") T.C.I.D.h» x 0.1 = 6 5 7.0

IO °* 3 - 10 ' .

Analogous results were obtained using calf kidneys or BHK21 cells using a pig kidney as a source of primary cells or line cells.

Example 4

Preparation of New Castle Disease Virus (N.C.D.V.).

By working in accordance with Example 3 using an N.C.D. virus strain, an infectious dose of 50 (EID-p.) was obtained on incubated eggs x 0.1 = 10 ' D - 10 ' , after 28 - 72 hours of incubation.

Analogous results were obtained using a calf kidney or B0H.K. 21 cells using pig kidney as a source of primary cells and line cells.

Example 5

By working in accordance with the examples described above, and using other virus strains from human

and veterinary medicine, the following viruses were produced: distemper virus, infectious cow distemper virus, mouse distemper virus, parainfluenza virus, canine rabies virus, infectious canine hepatitis virus, measles virus, etc.

Example 6

By working under suitable conditions, eumycete cultures such as Penicillium cnrysogenum, schizonycete cultures such as Bacillus subtilis, and cultures of unicellular algae such as Chlorella were produced.

Claims (1)

Device for the cultivation of living cells, in particular cells of animal origin, as well as of eumycetes, schizomycetes and unicellular algae, which device is surrounded by a temperature-regulated room and comprises a large number of tube-shaped columns (1) consisting of a transparent material which at the ends are connected to each other through headers (2), and where the column bundle is both horizontally and vertically rotatably stored, characterized by the fact that the column bundle at both ends is fixed in disc-shaped holders (6), each equipped with an axle pin (4), which axle pins (4) in the central axis of the column bundle is rotatably supported to a rotatably supported frame (7) perpendicular to the central axis.