FR2559500A1 - CELL CULTURE PROCESS WITH BARBOTAGE OF A GAS - Google Patents

Abstract

THE INVENTION RELATES TO A PROCESS FOR CULTURE OF NON-WALL CELLS. IT RELATES TO A PROCESS IN WHICH A GAS IS INTRODUCED DIRECTLY INTO THE CULTURE MEDIUM 22 BY A SPRAY HEAD 28, WITH A SUFFICIENT FLOW SO THAT THE CONCENTRATION OF OXYGEN AND CARBON ANHYDRIDE REMAINS CONSTANT IN THE MEDIUM. THE CELLS ARE ENCAPSULATED IN SEMI-PERMEABLE MEMBRANES WHICH PROTECT THEM AGAINST PHYSICAL DAMAGE. APPLICATION TO THE PRODUCTION OF PROTEINS AND ANTIBODIES BY CELLULAR CULTURES.

Description

The present invention relates to a method for culturing fragile cells,

  able to proliferate, and more precisely a process designed to optimize

  gas exchange in cultures of such cells.

  The cells benefiting from the method of the invention are, in particular, animal cell lines such as various myeloma cells, cell lines naturally or artificially transformed from primary culture, associated cells such as hybridomas, transformed malignant cells. and other cells without a wall, for example

spheroplasts.

  Oxygen is known to be essential for animal cell cultures, and the amount of available oxygen that can diffuse into a cell is

  important for the viability and growth of

  Lules. In general, too little oxygen

  can kill a cell or, at best, limit its growth.

  In one example, the growth of hybridoma culture requires at least about 0.12 millimole of oxygen per liter per hour for 109 cells. When the amount of available oxygen is lower, mitosis and protein production are limited. In addition, too much oxygen can cause some toxicity of oxygen that can kill a cell by oxidation of essential proteins. A

  rate of oxygen consumption by cell cultures

  the growth of the culture and, as the density of the cell culture increases, the transfer of suitable amounts of oxygen from the culture surface to its internal volume is

more and more difficult.

  Carbon dioxide, which influences the pH and concentration of bicarbonate, is also necessary for maintenance of animal cell growth; its setting is paramount when large

  quantities of cells must be produced.

  Conventional techniques for culturing mammalian cells on a laboratory scale include suspending cells in a placed medium.

  in a Petri dish. Oxygen and carbohydrate

  bonic diffuse through the middle surface and into the cells. The height of the culture medium - influences the rate of diffusion of oxygen and carbon dioxide to the cells. As the height of a medium increases, the ratio of the area of the medium to the volume of it decreases. At a certain moment, the concentration of oxygen and carbon dioxide in the air above the box and / or the rate of dissolution in the medium are no longer sufficient for the concentration of the gas to be sufficient in all the volume of the medium according to the cell growth criteria. At this moment, availability

  of oxygen becomes a factor that limits growth.

  Thus, the known technique shows that the height of the medium, in these static cultures, must be kept at a value of a few millimeters. In general, the upper limit of cell density in such cultures, when the upper space contains air that provides oxygen, is about 105 cells.

per cubic centimeter of culture.

  When cell culture techniques are implemented on a different scale so that mammalian cells are cultured in production quantities, difficulties arise.

  for the maintenance of the availability of sufficient quantities

  to the criteria set by the cells are met, as described in the work of Glacken et al., Mammalian Cell Culture: Engineering Principles and Scale-up Trends in Biotechnology, Elsevier

  Science Publications, 0166-9430 / 83, p. 102 (1983).

  A solution to this problem is the provision of higher concentrations of oxygen and carbon dioxide in the space bounded above the surface

  middle, when the cell density increases.

  However, in this process, the amount of oxygen present may be too great near the surface of the medium, and it may cause oxygen toxicity, and oxygen sensors and expensive dosing devices must constantly check and adjust the oxygen concentration in the medium so that the optimal proportions of the gases in the medium are maintained. In the case of growth of cells having walls, for example prokaryotic cells, the gases are often introduced directly by bubbling into the medium so that the cells receive the gas at the necessary concentrations. However, direct sparging is not suitable for the structure of cells that have no walls, which are very fragile, and which are physically damaged when the gas bubbles.

  with sufficiently high bitrates so that the concentrations

  Proper gas treatments are maintained in the medium. In addition, the protein-based constituents of the medium that are usually required in all animal cell cultures form a foam on the surface of the medium and this foam can trap cells. The cells trapped in the moss die quickly. Defoaming agents that can be added to the medium to prevent foam formation are potentially toxic to

  cells present in the culture.

  As described in the aforementioned Glacken article, recent advances in the field of cell culture have focused on placing cells in calcium alginate capsules so that cell densities can be elevated. The US patent

  No. 4,352,883 discloses the encapsulation of this

  lules so that they are protected against harmful agents such as bacteria. U.S. Patent No. 4,409,331 discloses a process for producing animal or cellular material of another type.

  harvesting these products from cell cultures

  encapsulated cells, the cells being placed in

  semi-permeable membrane capsules.

  As indicated in the foregoing description,

  it is desirable to ensure an improved exchange of gases when culturing mammalian cells and other fragile cells. Thus, the invention concerns the optimization of the concentration of gases in a medium without deterioration of the cells and without

  implementation of expensive control techniques.

  It also relates to a method of growing large cell cultures, for example from 20 to 30 liters, with high cell densities, for example of 108 cells / cm3. It also relates to the culture of cells having no walls, for example animal cells or spheroplasts, by bubbling gas to give the necessary amounts of gas

  to the cells without these being deteriorated.

  It also concerns the growth of animal cells in crops with densities comparable to those

many normal tissues.

  More specifically, the invention relates to a

  efficient culture process of fragile cells, that is,

  say cells with no walls, at high densities. The method comprises suspending, in a medium, spheroidal capsules containing a seed culture of the cells to be cultured. The capsules have semi-permeable membranes which are sufficient for the protection of the cells against physical deterioration and which are sufficiently permeable so that they allow the passage of gases, ions, vitamins, essential nutrients, etc.,

  necessary for the maintenance of cell growth.

  A gas containing the constituents necessary for the cells is bubbled directly into the medium, among the suspended capsules, continuously or intermittently during cell growth. The composition of

  bubbling gas is sufficient for the oxy-

  gene and / or carbon dioxide remains substantially constant in the medium, this concentration being sufficient for the cells to receive oxygen and / or carbon dioxide in amounts suitable for the maintenance of mitosis cells and their metabolism. The use of conventional media and this technique of gas bubbling allows the growth of cells in the capsules with densities of the order of 5 × 10 8 cells per cubic centimeter of capsules deposited. Where appropriate, the substances

  that are produced by the cells can be harvested

  either inside the capsules or in the

the middle.

  In preferred embodiments, when culturing animal cells, the sparging gas contains carbon dioxide and oxygen in balanced amounts sufficient to obtain

  the desired level of O2 / CO2 in the medium. Preferably

  In contrast, the content of O 2 / CO 2 in the medium is sufficient for maximal cell density in the capsules because optimal cell growth is favored. This means that the oxygen is present in the sparging gas at a value between about 0.2 and 600 millibars, preferably at least 0.2 to 270 millibars, and optimally for

  mammalian cells, between 200 and 230 millibars.

  Mammalian cells that can be cultured according to this method are advantageously

  cells obtained by genetic manipulation,

  lules of myelomas and hybridomas and other associated cells. An essential advantage of the technique according to the invention for the culture of mammalian cells by bubbling ensuring a gas exchange is that the cells have access to abundant gas reserves. Thus, even dense and bulky cultures can receive oxygen and / or carbon dioxide at such concentrations that the exchange of gases does not become a growth limiting factor. This is possible because the cells are encapsulated in membranes

  and are thus protected against physical deterioration

  normally caused by bubbling. The latter is an efficient method of introducing, into the medium, optimal oxygen concentrations and

  of carbon dioxide because a balance can be

  blir with a threshold bubbling rate between the concentration

  of the gases in the splash gas and the concentration

  of gases in the environment. Concentrations of oxygen and carbon dioxide inside the medium can thus be adjusted to optimal values.

  by adjusting the concentrations in the sparging gas.

  The need for the use of expensive equipment

  control and dosing of gas is thus suppressed.

  Other features and benefits of

  the invention will emerge more clearly from the description which

  will follow, made with reference to the accompanying drawings in which: Figure 1 shows schematically mammalian cells coated in a microcapsule, which can be used according to the invention; Figure 2 schematically shows a cell culture apparatus for implementing the invention; Fig. 3 is a graph showing the total number of cells per cubic centimeter of medium as a function of time for various batch cultures, with or without gas bubbling; FIG. 4 is a graph showing the change in the amount of cells per cubic centimeter of cells deposited as a function of time, in the case of an encapsulated, continuous feeding and gas bubbling culture, this graph showing the cell densities which can be obtained using the process according to the invention; and Figure 5 is a graph similar to the

  Figure 4, representing the growth of a food culture

discontinued.

  The present invention is based on the observation that cells having no walls, for example mammalian cells, encapsulated in

  semipermeable membranes, are practically insensitive

  physical deterioration and oxygen toxicity problems due to gas sparging,

  and that gas sparging can be used effectively

  cement for the transmission of oxygen and / or carbon dioxide to such cells. The use of the process according to the invention allows the equilibration of the concentration of oxygen and carbon dioxide so that their values are optimal in a medium, and the yields

  obtained cells can be increased in reci-

  cell culture pients on a large scale. Since the concentrations of 0 2 and CO2 in the medium are proportional to their concentration in the sparging gases, the control of the concentration and the

  corresponding cost are eliminated.

  FIG. 1, which indicates that mammalian cells 11 can be encapsulated in spheroidal capsules 13, for example spherical, by the method described in US Pat. No. 4,352,883. presently preferred encapsulation techniques are described in the patent application filed today.

  by the Applicant under the title "Process of encapsulation

  including cells, in semi-

  For example, an encapsulation method comprises forming a suspension containing about 10 6 cells per cubic centimeter in a solution.

  to 1% (w / v) sodium alginate ("NaG-Kelco LV").

  The density of the cells in this suspension determines the number of cells per capsule in the seed culture finally produced. The suspension is transferred to an apparatus having a spray head formed of a housing having an upper air inlet nozzle and an elongate hollow body frictionally engaged in a plug. A syringe, for example 10 cm3, equipped with a progressive pump, is mounted on the casing, with a needle, for example of 0.25 mm internal diameter, coated with Teflon, passing the entire length of the casing. The interior of the housing is constructed so that the upper part of the needle is subjected to a constant laminar air flow which acts as a pneumatic knife. In use, the syringe full of solution, containing the material to be encapsulated, is mounted on the casing, and the pump

  progressive is ordered so that it hunts progressively

  drops of solution to the tip of the needle. The air stream "tears" each drop which then falls from about 2.5 to 3.5 cm into a 1.2% w / v sodium chloride solution, forming gelled masses that are collected by suction . Gelled masses undergo incubation

  in three renewed volumes of isotonic saline

  the gel, for about 11 minutes in total. A membrane is then formed around the gelled masses by contact with an isotonic saline solution containing 750 mg / l of poly-L-lysine

  (Sigma Chemical Company, molecular weight 65,000 daltons.

  After 12 min of reaction, the resulting capsules are washed for 10 min with a 1.4 g / l CHES (2 cyclohexylaminoethane sulfanic acid) buffer solution containing 0.2% w / v calcium chloride in saline. The capsules are washed for about 8 minutes with 0.3% w / v calcium chloride saline and a second membrane is formed around the capsules by a 10 min reaction with 105 mg / l saline. polyvinyl amine (Polyscience, molecular weight 50,000 to 150,000 daltons). The capsules are washed again with two volumes of isotonic saline solution in 7 min and then coated by immersion for 7 min in a saline solution containing 5.10 2% by weight / volume.

  of NaG. The capsules are washed for an additional 4 minutes.

  The inner volumes of the capsules are then re-liquefied by two immersions in a saline solution of 55 mM sodium citrate, first for 20 minutes and then for 6 minutes. The capsules are washed twice in saline and washed once for 4 min in the medium. The capsules 13 are then ready for incubation in a medium

16 of culture.

  Capsules as shown in Figure 1, prepared by this encapsulation method, have membranes that are substantially impermeable to high molecular weight proteins, bacteria

and cells to grow.

  Figure 2 schematically represents a

  apparatus for culturing cells according to the invention.

  tion. It comprises a tank 10 of 50 1 capacity, formed of polished 316 L stainless steel

  electrolytic, comprising a plate 12 forming a cover

  key. A motor 14 drives a shaking shaft 16 having vanes 18 and 20 which rotate in the culture 22. The latter is a conventional medium such as modified Eagle medium, with addition of serum. In addition, it may be a hypertonic medium having an osmolarity of about 360 milliosmoles, of the type described in the patent application filed this same

  by the Applicant under the title "Activation Process".

  Protein production by cell cultures. "Many capsules such as that shown in Figure 1 are suspended in the medium, for example, the capsules are spherical or spheroidal and have a diameter of order of less than 2 mm approximately.

  average of about 0.8 mm are satisfactory.

  The quantities of gas required for the cells are obtained by circulating a gas containing oxygen and carbon dioxide in a sterile filter 24, a sparging tube 26 and a sparging head 28. This head 28 may be a microporous porcelain filter of 2.5 microns. When growing animal cells, the gas can contain% air and 5% CO2 (by volume). The bubbles of gas pass from the head to the middle going up among the capsules. The bubbling rate must be sufficient for the oxygen partial pressure in the medium to remain substantially equal to the partial pressure of the oxygen in the gas. The oxygen pressure in the medium can thus be set at a value equal to or slightly greater than that required for growth.

  optimal cells. Given the presence of

  In the middle of the pool, bubbling causes the collection of foam in the upper space 30 of the reservoir 10. However, the capsules protect the

  cells against dehydration if they are temporally

  transported in the foam, and they protect

  also cells against mechanical damage.

  Thus, the bubbling of the gas in an encapsulated culture

  allows the transmission of the amount of oxygen required

  to the increased population of cells, so that the cell densities obtained can be very high. The gas leaving the culture escapes through an outlet channel 32 and a filter 34. For example, the gas flow rate, for a culture of 30 1, is 5.6

  liters per hour, under normal conditions.

  During the implementation of the invention with discontinuous feeding, the medium is simply introduced in a quantity dosed into the reservoir 10 with microcapsules containing the seed culture, and the culture increases to the maximum density in the presence of gas bubbling. . However, the process

  may be the best for the implementation of the invention.

  tion and circulation of the medium in the culture by introducing a continuous or intermittent current of the medium through the inlet channel 38, and evacuation of the medium by the element 40 filtration. The latter comprises a stainless steel cloth which is

  because its pores are smaller than the diameter of

  sules, for example from 50 to 100 microns. The medium can be removed by the valve 42. The middle level

  in culture can be observed by a transparent tube

  The flow of the medium transmitted by the input channel 38 and extracted by the filter 40 is, for example

between 4 and 12 cm3 / min.

  When we want to harvest proteins or

  the substances used by the cells, the method used depends on the relationship between the effective molecular size of the substance of interest and the permeability of the capsule membranes. As described in the aforementioned patents, the permeability of the membranes

  Capsules can be set between certain limits.

  When the proteins of interest are too large to pass through the membrane, the proteins are collected in the microcapsules; when they are weak enough to cross membranes,

  they are collected in the external medium to the capsules.

  During operation, a user of the system determines the amount of oxygen and / or anhydride

  carbonic acid which must be dissolved in the medium 22.

  The concentrations and proportions chosen for the gases depend on the nature of the cells to be cultured, but they do not depend on the actual or predicted density of the cells. In the case of most animal cells, the desired concentration of oxygen (pressure

  partial) is between about 20 and 270 millibars.

  For example, the concentration chosen for oxygen in the case of a cell culture is greater than the amount sufficient for the cells to receive 0.12 millimole per liter per hour for 109 cells, this magnitude being that which is approximately the weakest for which cell growth can be vigorous, as previously indicated. The concentration of the gases in the medium is substantially proportional to the gas concentration in the sparging gas provided that the sparge flow rate is high enough to conceal the variations

  the concentration of O2 / CO2 due to respiratory activity.

  cell. For a threshold value, the splash rate is sufficient to maintain a balance between

  the gaseous and liquid phases in the tank 10.

  The user of the system can thus adjust the sparging rate so that the desired mixture of gases is transmitted to the sparging head 28. This then transmits the gas to the culture 22 with a flow rate that maintains a substantially constant concentration of oxygen in the medium. Such a sparging rate is 5.6 l / h, under normal conditions, in a culture tank of 1. The capsules 13 protect the cells 11 placed in the medium against mechanical deterioration. Capsules also protect cells that can be transported in placed foam

  in the free space in the tank against dehydration

  tation or deterioration by oxygen.

  Thus, it is not necessary for the concentration

  required in the environment is controlled because an equilibrium is established between the concentrations of the gases

  dissolved in medium 22 and the concentrations previously

  selected gases used for bubbling.

  The bubbling continues and the cells 11

  placed in the capsules 13 may undergo mitosis.

  Finally, when the cell culture has reached the desired density for the cells, the substances produced by the cells, for example antibodies, can be harvested either in the capsules or

in the external environment.

  Bubbling in a cell culture system

  Micro-encapsulation membrane as described above allows the maintenance of a cell density of about 5.108 cells / cm3 of deposited capsules or about 1.108 cells / cm3 of the medium, according to the observations made. This is an increase of around 100 compared to conventional crop techniques. Not only is the product yield of the cells increased by increasing density, but also

  the gas bubbling according to the invention allows a change in

  scaling the application of cell culture techniques that can become industrial processes. Thus, bubbling allows efficient gas exchange in the case of microencapsulated cells

of mammals.

  We now consider examples of

implementation of the invention.

Example 1

  Approximately 800 cm3 of deposited capsules containing approximately 105 hybridoma mouse-mouse cells per cubic centimeter are also distributed in sufficient amount of medium to form two volumes of 3 liters of encapsulated seed cultures. Simultaneously, two volumes of 3 liters of medium containing comparable numbers of the same cells are prepared per unit volume of the medium. This was the normal medium of Eagle supplemented with 5% fetal bovine serum. The four seed cultures underwent growth under

  gentle stirring without adding the medium for ten days.

  An encapsulated culture and an unencapsulated culture were bubbled at 8.4 liters per hour under normal conditions for five days and then 28 liters per hour thereafter. The two remaining seed cultures were not bubbled. The splash gas contained 5% CO2 and 95% air (in volume). The density of the cells in the respective cultures was checked and the results were plotted. These results are indicated by the

figure 3.

  As indicated by the results obtained, after six days, the encapsulated culture without bubbling

  had reached a cell density of about 8,105

  lules per cubic centimeter of culture while the conventional cell culture without bubbling had reached a

  density of about 3.106 cells per cubic centimeter.

  The unencapsulated and encapsulated bubbled cultures had a density of approximately

  1.5.106 and 1.3.106 cells per cubic centimeter respec-

  tively. Then, the cell density of the bubbled and bubbled suspension cultures decreased rapidly until the density of both cultures reached less than 2 × 10 5 cells per cubic centimeter on the tenth day. This shows that the bubbling in the unencapsulated cultures has no significant advantage compared to the same culture using dissolved oxygen in the medium from the space delimited at the top. The encapsulated culture

  and without bubbling gave only moderately more satisfactory

  faction. Its cell density was around 7,105

  lules per cubic centimeter, and it decreased on the ninth day. The bubbled encapsulated culture, on the other hand, exhibited a constant increase in cell density from the second to the ninth day, and the density then remained at about 3.106 cells per cubic centimeter. This example shows that

  vialibity and cell growth are favored, in the case of susceptible animal cells, when they undergo

their growth in a culture

  encapsulated in the presence of a gas sparge, the opera-

  encapsulation and bubbling are all

  two necessary to obtain this result.

Example 2

  Two encapsulated hybridoma cell cultures were prepared and suspended in Eagle's medium, supplemented with 5% fetal bovine serum,

  the concentration of ferric ions being a hundred times greater than

  than the normal concentration, the concentration of amino acids being twice the normal concentration. The medium had an osmotic pressure of about 360 milliosmoles. Culture No. 1 had a derisit. initial cell of 5.105 cells per cubic centimeter of culture, and it received, for thirteen days, a circulation of a new medium at 6 cm3 / min, then, for five days, at 11 cm3 / min. Culture No. 2 (having an initial cell density of 106 cells per cubic centimeter) received constant medium for nine days at 4 cc / min, and was renewed by replacing 10 liters of medium per day with each of days 9 to 20. Both crops were air bubbled during their growth cycle at 5.6 liters per hour under normal conditions. The cell density of both cultures was controlled. The results are shown in FIGS. 4 and 5. As shown, the continuously fed culture (FIG. 4), on the seventeenth day and then, reached a cell density of between

  and 5.108 cells per cubic centimeter of capsule

  Sees. Since the volume of the medium was about five times the volume of the capsules, it means that the density of cells per cubic centimeter of cultures was

  between 5.107 and 108 cells per cubic centimeter.

  As shown in Fig. 5, the discontinuously fed culture also reached a density greater than 108 cells per cubic centimeter of capsules deposited.

Claims (11)

  1. A method of culturing cells that do not   no walls, with high density, characteristic ..   in that it comprises: A. the direct bubbling of a gas in a culture medium so that it has a substantially constant concentration of gas sufficient for the reliability of the cells to be maintained and for their mitosis to be maintained , the cells being placed in spheroidal capsules suspended in the medium, the capsules having semi-permeable membranes which   sufficient to protect the cells against   and permeable enough to allow passage through constituents necessary for maintenance of viability and maintenance of cell mitosis, and   B. spontaneous growth of cells.   2. Method according to claim 1, characterized   in addition to the collection of subsistence   which are produced by the cells, either at   the inside of the capsules is in the middle.   3. Process according to claim 1, characterized   in that the cells are animal cells, and the gas contains oxygen.   4. Process according to claim 3, characterized   in that the gas further contains carbon dioxide.   5. Process according to claim 3, characterized   in that the cells are mammalian cells. mals.   6. Process according to claim 3, characterized   in that the oxygen-containing gas contains balanced amounts of oxygen and carbon dioxide, sufficient to produce a concentration of   optimal and substantially constant oxygen and anhy-   carbonic fluid in the middle so that the metabolism   maintained and that the mitosis of the cells is facilitated bedded.   7. Process according to claim 3, characterized   in that the partial pressure of oxygen in said medium is between 20 and 270 millibar5,   8. Process according to claim 3, characterized   in that the animal cells are associated cells.   9. Process according to claim 3, characterized   rised in that the animal cells are cells   obtained by genetic manipulation.   10. Process according to claim 3, characterized   rised in that the animal cells are cells myeloma.   11. The method of claim 3, characterized   in that the animal cells can grow to a density of at least about 5 × 10 6 cells per cubic centimeter of culture.