IL107212A - Method and device for the cultivation of cells - Google Patents

Description

METHOD AND DEVICE FOR THE CULTIVATION OF CELLS Ο·>ΝΠ m:n>n!> ιρηηι πο>κ» 1 107,212/2 The present invention relates to a method for the cultivation of cells of higher organisms in communicating chamber systems, in which the cells to be cultivated are introduced into the chamber system and bred, said method being characterized in that the conditions required for cultivations are obtained through uniform surface gassing by means of a microporous gas supply line introduced into the chamber system. Preferably, the method is carried out within a closed system in which the gas mixture circulates. The invention also relates to a device for the cultivation of cells of higher organisms in communicating chamber systems, said device being characterized in that the device contains a microporous gas supply line, which is preferably in the form of a tube or rod.

The rapid development of biotechnology calls for the simplest and most economic methods possible for the cultivation of the cells of higher organisms, for the purpose of acquiring special products from these cell cultures. Examples of these products are such therapeutically and diagnostically important substances as interferon, TP A, insulin, monoclonal antibodies, etc. These substances have already found a wide range of applications in diagnosis and therapy.

There are various techniques for cultivating the cells which produce such substances. In addition to suspension cultures and microcarrier cultures in the bioreactor, conventional techniques using so-called stationary cultures have a special significance. In this connection, it has been found that the use of chamber systems having a number of compartments which can be filled or emptied at the same time, offers advantages over using plain T-flasks or roller flasks. Chamber systems enable a large surface area of growth to be prepared in a technically simple way, particularly in the case of surface-adherent cell lines. Owing to their simple construction and manageability, these systems also offer an alternative, within certain limits, to the substantially more complex and expensive bioreactors. These systems are already known, e.g. from DE-PS 26 24 047. Such systems are more economical than the T-flasks or roller flasks mentioned earlier, as their logical design makes them much easier to work with.

One disadvantage of these chamber systems, however, lies in the fact that the cultivating conditions can only be controlled within rather narrow limits, and so the product yield of the particular substance desired must necessarily be quite small.

The principal problem is with the oxygen supply and the over-acidification of the growth medium by carbonic acid arising from the carbon dioxide of the cell metabolism process. This is particularly evident with rapidly growing cultures with vigorous metabolism.

It has been found in practice that growth and protein synthesis in a chamber system is limited by the available oxygen diffusing over the liquid surface into the growth medium. Simple devices designed to improve the supply of oxygen by pumping in an appropriate mixture of gases have the disadvantage that the individual culture chambers are not uniformly provided with oxygen, which leads to an uneven growth of cells and results in reduced yields. It is also possible that products of inferior quality may be formed, which are of little economic value.

The applicant has now found that the above-mentioned disadvantages may be overcome by introducing a mixture of gases designed to promote optimum cell growth into this type of chamber system by means of a microporous supply line. This supply line could, for example, be a hollow membrane in the shape of a tube or rod. The important point is that it should give off gas along its entire length within the chamber system when pressure is applied. Commercially available materials suitable for such a supply line are e.g. microporous membrane tubes made from material such as "Accurel" (Registered) or "Gore-Tex-T bes" . The essential factor is that resistance is offered to the free exchange of gases between the interior of the supply line and the chamber system through the microporous membrane material, and that this resistance can be partly overcome by raising the internal pressure of the supply line. The volume of gas passing into the chamber system can be regulated by the selection of pore size, the diameter of the tube and the pressure gradient.

The object of the invention is a procedure for breeding cells of higher organisms in communicating chamber systems, in which the cells to be cultivated are brought into the chamber system and bred, characterized in that the requisite cultivating conditions are attained by means of uniformly gassing tne surface of the culture medium with gas through a microporous gas supply line introduced into the chamber system.

A further object of the present invention is a device for cultivating the cells of higher organisms in communicating chamber systems, characterized in that the device contains a microporous gas supply line.

By means of the procedure in accordance with the invention, it is possible to compensate for the consumption of oxygen required for cell metabolism as well as for the over-acidification of the growth medium arising from the carbon dioxide given off by the cells. The use of a gas mixture containing between 10% and 50% by volume carbon dioxide will be found advantageous. The balance of the volume can be made up of nitrogen, which is largely physiologically inert.

The concentration gradient between the liquid phase and the gas phase leads to processes of diffusion in which molecules move from a location with a higher concentration of particles to one with a lower concentration. This is due to the energy of motion of the molecule. For example, part of the released carbonic acid, following the concentration gradient, passes over into the gas phase. On the other hand, oxygen molecules move away from the area of higher concentration, the gas phase, into the area of lower concentration, the liquid phase. By precisely metering the carbon dioxide and the oxygen content in the gas mixture, the acidity (pH) and oxygen saturation (ρθ2) of the growth medium can be maintained in the range which is most favourable for the cells. Additional parameters to be considered are the relevant coefficients of diffusion and the length of the path which the gas has to cover. while diffusion is of primary importance as a transport mechanism where transition from one phase to the other is concerned, further movement of the gases is essentially carried out by thermal convection processes. Microgradients may build up at the surface boundaries of gas and liquids and reduce the concentration gradient and consequently the exchange of gases. By raising the flow of gas volume when necessary and therefore the convection, the formation of such microgr dients can be prevented, thus improving the exchange of gases at surface boundaries. In addition, the amounts of gas to be carried over are specifically dependent on the oxygen efficiency of the particular cell line being cultivated, e.g. for diploid fibroblasts, they are in the order of magnitude of 0.05 mmol 02 /(109 cells · h) , although the range is considerably higher in the case of transformed cells.

For optimum configuration of a system, the pore width and the diameter of the microporous tube must be selected in such a way that while a sufficient rate of flow of the gas volume is ensured, the required counter-pressure still builds up on the inner side of the tube. In practice, pore widths between 0.2 μια and 4.0 μη. and a tube diameter between 2mm and 10 mm have been found satisfactory. However, different pore widths and diameters may also be found advantageous, depending on the given system application or tube material. The difference in pressure between the interior of the tube and the chamber system can in principle be set as high as desired, although a difference as low as 0.1 to 0.5 bar has been found adequate in practice. The relationship between the internal pressure and the rate of gas flow using a commercial type of tube is shown in Fig. 2.

The closed system type of construction has proved to be particularly advantageous with respect to precisely controlling the oxygen saturation and acidity of the growth medium, in addition to being economical with the gases used. The gas mixture is circulated and only the oxygen used is replaced and the surplus carbon dioxide removed, with the aid of an appropriate measuring and regulating unit. An example of this type of closed system is diagrammatically illustrated in Fig. 3. By introducing measuring probes e.g. for measuring the released oxygen and the acidity (pH value) in the cell growth medium of one or more sample chambers of the system, limiting values can be determined and monitored, so that when these either fall short or are exceeded, the composition of the gas mixture and, if required, the volume of flow may be regulated as required. Typical diagrams showing values found in an ungassed, as well as a gassed and regulated chamber system are illustrated in Example 4, Figs. 4 and 5.

Should the gassing system be subsequently installed in a regular commercial multi-chamber system, the mounting of a microporous tube can be considerably facilitated by using a macroporous or half-shell type of jacket serving to stiffen the flexible tube. This would not be necessary if a microporous hollow rod were used, as this is sufficiently stiff in itself.

Experience has shown that cell production yield can be substantially increased by using a chamber system using the device in accordance with the invention when compared with a conventional chamber system, as illustrated in Example No. 1 below and in Fig. 1. These examples are intended to iHustrate invention, without restricting it in any way.

Example 1 This example compares the yield of the desired protein when using a chamber system with the device in accordance with the invention, a chamber system using simple gassing and an ordinary chamber system. In a commercially available tank, stacked with over 40 communicating chambers, a microporous tube, closed at one end, was inserted into one of the two filling ducts and connected to a gas-mixture device at its open end. By means of a suitable pump, a defined quantity of gas was then pumped through the tube into the cultivating system. The gas passed out through the second filling duct of the tank ("A"). The second step involved the use of tank ("B") identical with tank ("A") described above, but without the device in accordance with the invention. The gas mixture was simply pumped in through the filling opening. No gas at all was used in the third tank ("C") , which acted as a control.

A culture of genetically altered cells was grown in all three tanks, with the quantity of protein synthesized being measured at certain predetermined intervals. The results are shown in Fig.l. As these results show, tank ("A") , using the device in accordance with the invention, produced a quantity of protein approximately twice as great within the same period of time as did the gassed tank ( "D" ) , without the device in accordance with the invention.

When compared with the control ank ("C") where no gas was used, the yield of ("A") was about three times as great.

Example 2 For this example, a commercially available microporous tube was used, with an internal diameter of 2mm, a wall thickness of 0.4 mm and a pore width of approximately 1.2 /xm, sealed at one end and attached to an air-pressure source at the other. The amount of air seeping through the pores of the tube was quantified using a gas flow meter, and is represented graphically in Fig. 2.

Example 3 Fig. 3 represents in diagrammatic form the lay-out of a sealed gassing system in which the gas mixture, monitored by a measuring and control unit, circulates through the chamber system, replacing only the used oxygen, the excess carbon dioxide being removed.

For this purpose, the chamber system is provided with two measuring probes, namely one for the oxygen dissolved in the medium (2) and one for the acidity (1) . The measured data are sent to a commercial control unit, which meters out doses of oxygen (02) , nitrogen (N∑) and carbon dioxide (C02) , in accordance with pre-programmed limiting values via magnetic valves (3,4,5). In the event of extremely high oxygen consumption or of a high level of acidification of the culture, the regulating unit can increase the effect of the membrane pump (9) by raising the flow of gas which is registered by a sensor (6), thus causing an improvement of the exchange of gases. The microporous tube in accordance with the invention is inserted into one of the two filling ducts and directly connected to the circulation system. The overpressure in the system brought about by the metered doses is limited by an overpressure valve (10) set at 0.2 bar. Since a substantially more rapid filling and emptying of the chamber system with the cell culture is required than could take place using the microporous tube system, a supplementary bypass (7) is provided, permitting rapid equalization of pressure when required by means of a three-way valve.

By use of the arrangement described above, not only the oxygen saturation but also the acidity of the medium in the cultivation of various cell lines can be maintained within narrow limits, as explained in more detail in Example 4.

Example 4 In the closed system described in Example 3, the concentration of dissolved oxygen and the acidity of the growth medium (pfi value) are continually monitored via measuring probes 1 and 2. Fig. 4 compares the behaviour pattern of oxygen saturation in a closed and gassed system with that occurring in an open ungassed system. Fig. 5 shows the corresponding behaviour of the pH values. It may be readily seen that when using the procedure in accordance with the invention, a high degree of uniformity of oxygen concentration is achieved, along with an elevation of the pH factor in the area desired, while unfavourable levels of oxygen concentration and pH value are observed -in the ungassed open system.

Example 5 An acid-neutral cell culture medium containing the pH indicator phenol ".red* was placed in a tank stacked with 40 cultivating chambers which was equipped with the gassing system in accordance with the invention, and then gassed with pure carbon dioxide gas. At the same time, a second tank, without the device in accordance with the invention, was flooded with the same quantity of carbon dioxide gas. Due to the partial absorption of the carbon dioxide by the medium, the level of acidity increased, marked by a change in the colour of the indicator from red to yellow. After the tank without the device in accordance with the invention had been exposed to the gas for 20 minutes, yellowing was observed only in the two upper chambers, while the other tank, using the device in accordance with the invention, showed uniform colour in all chambers.

Claims (6)

11 107,212/2 WHAT IS CLAIMED IS:

1. A method for the cultivation of cells of higher organisms in communicating chamber systems, in which the cells to be cultivated are introduced into the chamber system and bred, characterised in that the conditions required for cultivation are attained through uniform surface gassing by means of a microporous gas supply line introduced into the chamber system.

2. A method in accordance with claim 1, wherein said gassing is carried out within a closed system in which the gas mixture circulates.

3. A device for the cultivation of cells of higher organisms in communicating chamber systems, characterized in that the device contains a microporous gas supply line.

4. A device according to claim 3, characterized in that said microporous gas supply line is in the form of a tube or rod.

5. A device according to claim 4, characterized in that said microporous gas supply line comprises a microporous polypropylene or teflon membrane.

6. A device according to claim 3 or 5, characterized in that said microporous gas supply line is provided with a bypass for the purpose of pressure equilization, for the rapid emptying or filling of the chamber system. for the Applicant: WOLFF, BREGMAN AND GOLLER by: . A λ M