GB2154246A - Improved method of culturing cells - Google Patents

Abstract

A method of growing fragile cells, i.e., cells having no cell membrane such as animal cells or genetically modified animal cells, to high density in culture comprises the steps of suspending permeable microcapsules containing a seed culture of such cells in a medium, directly sparging a gas through the medium along the capsules, and allowing the cells to grow. The gas comprises a gas or a selected mixture of gases and is sparged at a rate high enough to attain a substantial partial pressure equilibrium between the gas and liquid phase. The partial pressure of gases essential for cell growth and normal rates of cell mitosis can be maintained in the medium by controlling the composition of the gas and maintaining a threshold sparging level. By sparging, oxygen availability need no longer be a growth-rate limiting factor, and cell densities on the order of 10<8> cells/ml may be achieved.

Description

SPECIFICATION Improved method of culturing cells The present invention relates to an improved method of culturing cells.

More particularly, this invention relates to a method of culturing fragile cells capable of proliferation and, still more specifically, to a method of optimizing gaseous exchange in cultures of such cells. Cells benefitting from the method of the present invention include animal cell lines such as various myelomas cells, cell lines naturally or artificially transformed from primary cultures, fused cells such as hybridoma, malignant transformants, and other cells having no cell walls, e.g., spheroplasts.

It is well known that oxygen is essential in animal cell culturing, and that the amount of oxygen available for diffusion into a cell is important to the cell's viability and growth. Generally, too little oxygen can kill a cell or, at best, limit its growth. As an example, growing hybridoma cultures require at least about 0.12 millimoles of oxygen/liter/hour/l 09 cells. If less oxygen is available, mitosis and protein production are limited. Also, too much oxygen can lead to oxygen toxicity that can kill a cell by oxidizing essential proteins. A cell culture's rate of oxygen uptake increases with the growth of the cell culture, and as the density of the cell culture increases, it is more difficult to transfer adequate amounts of oxygen from the culture surface to the inner volume of the culture.

Carbon dioxide, which influences pH and bicarbonate concentration, is also required for sustained animal cell growth; its control is essential if one seeks to grow large quantities of cells.

Conventional, laboratory scale mammalian cell culturing techniques involve suspending the cells in a medium disposed in a Petri dish. Oxygen and carbon dioxide diffuse through the surface of the medium and into the cells. The depth of the culture medium influences the rate of oxygen and carbon dioxide diffusion to cells. As the depth of a medium increases, the ratio of the surface area of the medium to its volume decreases. At some point, the concentration of oxygen and carbon dioxide in the air above the dish and/or the rate of dissolution into the medium is insufficient to provide throughout the volume of the medium sufficient gas concentration to satisfy the requirements of the growing cells.At that point oxygen availability becomes a growth limiting factor. Accordingiy, the prior art teaches that one should keep the depth of the medium for such static cultures within the millimeter range. Generally, the upper limit of cell density for such cultures, using air in the headspace for its oxygen supply, is about 105 cells/milliliter of culture.

When cell culturing techniques are scaled up to grow mammalian cells in production amounts, difficulties are encountered in maintaining the availability of sufficient amounts of oxygen to satisfy the requirements of the cells. See Glacken et al., Mammalian Cell Culture: Engineering Principles and Scale-up Trends in Biotechnology, Elsevier Science Publications, 0166-9430/83, p. 102 (1983). One solution to this problem involves providing increased concentrations of oxygen and carbon dioxide in the headspace above the medium surface as cell density increases. However, with this approach, too much oxygen may be present near the surface of the medium, causing oxygen toxicity, and expensive metering devices and oxygen sensors must continually test and adjust the concentration of oxygen in the medium in order to maintain optimal proportions of the gases throughout the medium.

In growing cells having cell walls, e.g. prokaryotic cells, gases are often sparged directly through the medium in order to supply the cells with requisite gas concentrations. However, direct sparging is unsuitable in culturing cells which have no cell wall, are quite fragile, and are physically damaged when the gas is sparged at high enough rates to provide adequate gas concentrations in the medium. Also, protein components in the medium, which currently are required in all animal cell cultures, produce a foam on the medium surface that can trap the cells.

Cells trapped in the foam rapidly expire. Antifoaming agents, which may be added to the medium to prevent foaming, are potentially toxic to the cells in the culture.

As described in the Glacken article, recent developments in the cell culturing field have included enclosing cells in capsules comprising calcium alginate in order to produce high cell densities. U.S.

Patent No. 4,352,883 entitled, "Encapsulation of Biological Material," teaches encapsulating cells to protect them from harmful agents such as bacteria.

U.S. 4,409,331 discloses a method of producing animal and other cell products by harvesting such products from encapsulated cell cultures wherein the cells are disposed within semipermeable capsule membranes.

As is apparent from the above, it would be desirable to achieve improved gaseous exchange in the culturing of mammalian and other fragile cells.

Accordingly, it is an object of the invention to optimize gas concentration in a cell culture medium without damaging the cells or resorting to expensive monitoring techniques. Desirably, the process may be used for growing large cell cultures, e.g. 20-30 liter cultures, and/or to densities comparable to cell densities in many normal tissues or to high cell densities, e.g. 1 x 108 cells/ml. The process to be disclosed in detail can culture cells having no cell walls, such as animal cells or spheroplasts, employing gas sparging to satisfy the cells' gas requirements without damaging the cells.

According to the present invention, there is provided a method of culturing cells having no cell walls to a high density comprising the steps of: A. directly sparging a gas, through a growth medium, to supply said medium with a substantially constant gas concentration sufficient to maintain viability and support mitosis of the cells which are disposed within spheroidal capsules suspended in the said medium, the capsules having semipermeable membranes sufficient to protect the cells from physical damage and sufficiently permeable to permit traverse of components needed to maintain viability and to support mitosis of said cells; and B. allowing the cells to grow.

The present method is capable of efficiently culturing fragile cells to high density from an encapsulated seed culture of the cells. The capsules are semipermeable membrances which protect the cells from physical damage and are sufficiently permeable to permit traverse of essential nutrients, vitamins, ions, gases etc., needed to support growth of the cells. The gas containing components needed by the cells is sparged directly through the capsulecontaining medium either continuously or intermittently, during cell growth. A preferred composition of the sparged gas is sufficient to maintain a substantially constant oxygen, and/or carbon dioxide concentration in the medium, the concentration being sufficient to supply the cells with oxygen and/or carbon dioxides in amounts well suited to support cell mitosis and metabolism.Using conventional media and this gas sparging technique, it is possible to grow cells within the capsules at densities on the order of 5 x 108 cells/ml of settled capsules. If desired, substances that are produced by the cells may be harvested either from within the capsules or from the medium.

In preferred embodiments, when growing animal cells, the sparging gas has carbon dioxide and oxygen in balanced amounts sufficient to produce a desired 02/CO2 content in the medium. Preferably the 02/CO2 content of the medium is sufficient to maximize cell density within the capsules by promoting optimal cell growth. This means that the oxygen is present in the sparging gas at a partial pressure level of about 0.15 mm Hg, to 450 mm Hg, preferably at least 0.15 and up to 200 mm Hg, and optimally for mammalian cells, 150-170 mm Hg.

Advantageous, mammalian cells that may be grown in the process include genetically engineered cells, myeloma cells, and hybridoma and otherfused cells.

A principle advantage of the present method for culturing mammalian cells using sparging for gaseous exchange is that the cells are provided with access to abundant gas supplies. Thus, even dense and voluminous cultures may be supplied with oxygen and/or carbon dioxde at levels where gaseous exchange does not become a growth limiting factor. This is possible because the cells are encapsulated within membranes, and thus are protected from the physical damage normally caused by the sparging. The sparging is an efficient method of providing the medium with optimal concentrations of oxygen and carbon dioxide because an equilib rium can be established at a threshold sparging rate between the concentration of gases in the sparged gas and the concentration of gases in the medium.

Oxygen and carbon dioxide concentrations within the medium can thus be set at optimum levels by controlling their concentrations in the gas being sparged. The need for expensive monitoring and gas metering equipment is thereby obviated.

The invention will now be described in more detail by way of example only with reference to the accompanying drawings, in which: Figure 1 schematically illustrates mammalian cells enclosed within a microcapsule suitable for use in the present invention; Figure2 schematically illustrates a cell culture apparatus suitable for use in the practice of the invention; Figure 3 is a graph of total cells per milliliter of medium versus time for various batch-fed cultures with and without gas sparging; Figure 4 is a graph of cells/ml of settled capsules versus time for a continuously fed, gas-sparged encapsulated culture illustrating the cell densities achievable using the process of the invention; and Figure 5 is a graph similar to Figure 4 showing the growth of a batch-fed culture.

The present invention is based on the observation that cells without cell walls, e.g., mammalian cells, encapsulated within semipermeable membranes are substantially immune from physical damage and oxygen toxicity problems caused by gas sparging, and that gas sparging may be employed efficiently to supply oxygen and/or carbon dioxide to such cells. Using the method of this invention, the concentration of oxygen and carbon dioxide may be balanced to provide optimal levels in a medium, and increased cell yields can be achieved in large scale cell culture vessels. Because of 2 and CO2 concentrations in the medium will be proportional to their concentration in the sparged gas, concentration monitoring and its attendant expense is eliminated.

Turning to Figure 1, mammalian cells 11 may be encapsulated within spheroidal, typically spherical capsules 13 by means of the procedure set forth in the United States Patent No. 4,352,883, the disclosure of which is incorporated herein by reference.

The currently preferred encapsulation techniques is disclosed in our copending GB patent application (corresp. U.S.S.N. 579,494), filed on even date herewith (Agent's ref: 6278). The disclosure of this application is also incorporated by reference herein.

A typical encapsulation procedure involves forming a suspension containing about 106 cells/ml in 1% (w/v) sodium alginate (NaG-Kelco LV). The density of the cells within this suspension will determine the number of cells per capsule in the finally produced seed culture. The suspension is transferred to a jet-head apparatus consisting of a housing having an upper air in take nozzle and an enlongate hollow body friction fitted into a stopper. A syringe, e.g., a 10 cc syringe, equipped with a stepping pump is mounted atop the housing with a needle, e.g., a 0.01 inch (0.25 mm) I.D. Teflon-coated needle, passing through the length of the housing. The interior of the housing is designed such that the top of the needle is subjected to a constant laminar airflow which acts as an air knife. In use, the syringe full of the solution containing the material to be encapsulated is mounted atop the housing, and the stepping pump is activated to incrementally force drops of the solution to the tip of the needle. The air stream "cuts off" each drop, which then falls approximately 2.5-3.5 cm into a 1.2% (w/v) calcium chloride solution, forming gelled masses which are collected by aspiration. The gelled masses are incubated in three replenished volumes of isotonic saline for gel expansion for a total of approximately 11 minutes. Next, a membrane is formed about the gelled masses by contact with a 750 mg/l poly-L-lysine (Sigma Chemical Company, 65,000 dalton molecular weight) in isotonic saline solution.After 12 minutes of reaction, the resulting capsules are washed for 10 minutes with 1.4 g/l solution of CHES (2-cyclohexylamino ethane sulfanic acid) buffer (Sigma) containing 0.2% (w/v) calcium chloride in saline. The capsules are washed for approximately 8 minutes with 0.3% (w/v) calcium chloride in saline, and a second membrane is formed about the capsules by a 10 minute reaction with 105 mg/l polyvinyl amine (Polyscience, 50,000150,000 dalton molecular weight) in saline. The capsules are washed again with two volumes of isotonic saline over 7 minutes and post-coated with a 7 minute immersion in 5 x 10-2% (w/v) NaG in saline solution.The capsules are washed for an additional 4 minutes in saline, then the intracapsular volumes are reliquified by two immersions in 55mM sodium citrate in saline solution, the first for 20 minutes and the second for 6 minutes. The capsules are washed twice in saline and washed once for 4 minutes in medium. The capsules 13 are then ready for incubation in a growth medium 16.

Capsules such as are illustrated in Figure 1 prepared according to this encapsulation procedure have membranes which are substantially impermeable to high molecular weight proteins, bacteria, and the cells to be cultured.

Figure 2 schematically illustrates apparatus for culturing cells in accordance with the invention. It comprises a 316L stainless steel, electropolished, 50 liter capacity vessel 10 fitted with a headplate 12. A motor 14 drives paddle shaft 16 to rotate paddles 18 and 20 disposed within the culture 22. Culture 22 comprises a conventional medium such as Eagle's modified medium with added serum. Alternatively, it may comprise a hypertonic medium having an osmolarity of about 360 milliosmoles of the type described hereinafter and described in greater detail in our copending GB patent application (corresp.

U.S.S.N. 579,492) (Agent's ref: 6280), filed on even date herewith. The disclosure of this application is incorporated herein by reference. A multiplicity of capsules such as the capsule depicted in Figure 1 are suspended in the medium. Typically, the capsules are spherical or spheroidal and have a diameter of the order of less than about 2 mm. Capsules having an average diameter of the order of 0.8 mm work well.

The gas requirements of the cells are met by passing an oxygen and carbon dioxide-containing gas through sterile filter 24, air tube 26, and sparging head 28. Sparging head 28 may comprise a 2.5 micron microporous porcelin filter. For growing animal cells, the gas may comprise 95% air and 5% CO2 (by volume). Gas bubbles pass from the sparging head up through the medium among the capsules. The rate of sparging should be sufficient to maintain the partial pressure of oxygen in the medium substantially equal to the partial pressure of oxygen in the gas. The pressure of oxygen in the medium may thereby be set to a level at or slightly above that which the cells require for optimal growth. Because of the serum components in the medium, sparging causes foam to collect in the headspace 30 of vessel 10.However, the capsules protect the cells from dehydration should they temporarily be transported into the foam, and also protect the cells from mechanical damage. Thus, gas sparging through an encapsulated culture can satisfy the need of the increasing cell population for oxygen, thereby enabling extremely high cell densities to be achieved. Gas exiting the culture passes through exit port 32 and filter 34. A typical gas flow rate for a 30 liter culture is 0.2. standard cubic feed per hour (5.7 l/hr).

When practicing the invention in the batch-feeding mode, the medium is simply metered into vessel 10 together with microcapsules containing the seed culture, and the culture is grown to maximum density with gas sparging. However, perhaps the best way of practicing the invention is to pass the medium through the culture by introducing a continuous or intermittent flow of the medium into entry port 38, and draining off the medium through filter element 40. Filter element 40 may comprise a stainless, microporous mesh having pores smaller than the diameter of the capsules, e.g. 50 to 100 microns. Medium may be withdrawn through valve 42. The level of medium in the culture may be observed in transparent sight tube 44. A typical rate of medium flow into entry port 38 and out through filter 40 is 4 ml to 12 ml per minute.

If one seeks to harvest protein or other substances produced by the cells, the method employed will depend upon the relationship of the effective molecular dimensions of the substance of interest and the permeability of the capsule membranes. As disclosed in the above-reference patents, the permeability of the capsule membranes can be controlled within limits. If the protein of interest is too large to traverse the membrane, the protein collects within the microcapsules; if it is small enough to traverse the membranes, it will collect in the extracapsular medium.

In use, the user determines a desired amount of oxygen and/or carbon dioxide to be dissolved within the medium 22. The selected gas concentrations and proportions depend upon the type of cells being cultured, but is independent of current or projected cell density. For most animal cells, the desired concentration (partial pressure) of oxygen will be between about 15 and 200 mm Hg. For example, the selected oxygen concentration level for a culture of cells will be greater than that sufficient to provide the cells with 0.12 millimoles/literihour/106 cells, the earlier stated approximate minimum requirement for vigorous cells growth.

The concentration of gases in the medium is substantially proportional to the concentration of the gases in the sparging gas provided that the sparging rate is sufficiently high to mask variations in the O2/CO2 concentration caused by cell respiration activity. At a threshold level, the sparging rate is sufficient to sustain an equilibrium between the gas and liquid phases in vessel 10. The system user can accordingly set the sparging rate to supply the desired blend of gases to sparging head 28. The sparging head 28 then supplies the gas to the culture 22 at a rate that maintains a substantially constant concentration of oxygen in the medium. One such sparging rate is 0.2 standard cubic foot/hour (5.71/her) in a 30 liter culture vessel. The capsules 13 protect the cells 11 within the medium from mechanical damage.The capsules also protect cells which may be transported into the foam in the head space in the vessel from oxygen damage or dehydration.

Thus, oxygen concentration in the medium need not be monitored, because an equilibrium becomes established between the concentrations of the gases dissolved in the medium 22 and the preselected concentrations of the gases sparged therethrough.

Sparging continues, and the cells 11 within capsules 13 are allowed to undergo mitosis. Eventually, when the cell culture has reached the desired cell density, substances produced by the cells such as antibodies, may be harvested either from within the capsules or from the extracapsular medium.

Sparging in a microencapsulated cell culture system such as described above, has been observed to support a cell density of about 5 x 108 cells/milliliter of settled capsules or about 1 x 106 cells/ml of medium. This is an improvement of approximately two orders of magnitude over conventional culturing techniques. Besides improving cell product yield by increasing cell density, gas sparging in accordance with the invention allows cell culturing techniques to be scaled up to commercial production levels.

Thus, it can be seen that sparging provides improved, efficient gaseous exchanges for microencapsulated mammalian cells.

The invention will be further understood from the following, non-limiting examples.

Example 1 Approximately 800 ml of settled capsules containing approximately 105 mouse-mouse hybridoma cells per milliliter were distributed equally in sufficient medium to make two, three liter volumes of encapsulated seed cultures. Simultaneously, two, three liter volumes of medium containing comparable numbers of the same cells per unit volume of medium were prepared. The medium was standard Eagle's medium supplemented with 5% fetal bovine serum. The four seed cultures were grown with gentle stirring and without supplementing the medium for 10 days. One encapsulated culture and are unencapsulated culture were sparged at a rate of 0.3 standard cubic feet per hour (8.5 l/hr) for five days, and 1.0 standard cubic feet per hour (28.31/her) thereafter. The remaining two seed cultures were not sparged.The sparged gas consisted of 5% CO2 and 95% air (by volume). Cell density in the respective cultures was monitored and the data was plotted. The results are shown in Figure 3.

As is evident from the data, after six days the unsparged encapsulated culture had grown to a cell density of about 8 x 105 cells/ml of culture and the unsparged conventional cell culture had grown to a density of about 3 x 106 cells/mi. The sparged unencapsulated and encapsulated cultures had grown to a density of about 1.5 x 106 cells/ml and 1.3 x lO6ceIls/ml, respectively. Thereafter, the cell density of both the unsparged and sparged suspension cultures fell rapidly, until, at day 10, the density of both cultures was less than 2 x 105 cellsiml. This shows that sparging unencapsulated cultures has no significant beneficial effect as compared with the same culture utilizing oxygen dissolved in the medium from the headspace. The unsparged, encapsulated culture was only moderately more successful.Its cell density was at about 7 x 105 cells/ml and declining on day 9. The encapsulated, sparged culture, in contrast, showed a steady increase in cell density from day 2 through day 9, and cell density was maintained thereafter at about 3 x 106 cells/ml.

This example demonstrates that cell viability and cell growth is promoted in sensitive animal cells if they are grown in a gas sparged, encapsulated culture, and that both the encapsulation and sparging steps are required to achieve this result.

Example 2 Two encapsulated hybridoma cell cultures were prepared and suspended in Eagle's medium supplemented with 5% fetal bovine serum, 100 times the normal ferric ion concentration, and twice the normal amino acid concentration. The medium had an osmotic pressure of about 360 milliosmoles. Culture number 1 had an initial cell density of 5 x 105 cells/ml of culture, was fed for 13 days by a circulation of fresh medium at 6 ml/minute, and thereafter for 5 days at 11 ml/minute. Culture number 2 (with an initial cell density of 1 x 106 cells/ml was continuously fed at 4 ml/minute for nine days, and batch fed by replacement of 10 liters of medium per day on each of days 9-20. Both cultures were sparged with air throughout their growth cycle at 0.2 standard cubic feet per hour (5.71/her). The cell density of each culture was monitored. The results are displayed in Figures 4 and 5. As illustrated, the continuously fed culture (Figure 4) at and after day 17, attained a cell density of between 1 x 108 cells/ml of settled capsules and 5 x 106 cells/ml settled capsules. Since the volume of the medium was about 5 times the volume of the capsules, this means that the cell density per ml of culture was between about 5 x 107 and 1 x 108 cellsiml.

As shown in Figure 5, the batch fed culture also attained a density of greater than 1 x 108 cells/ml of settled capsules.

Those skilled in the art may determine other modifications or variations of the procedures and products described herein. Such other modifications and variations are included within the following

Claims (10)

claims. CLAIMS

1. A method of culturing cells having no cell walls to a high density comprising the steps of: A. directly sparging a gas, through a growth medium, to supply said medium with a substantially constant gas concentration sufficient to maintain viability and support mitosis of the cells which are disposed within spheroidal capsules suspended in the said medium, the capsules having semipermeable membranes sufficient to protect the cells from physical damage and sufficiently permeable to permit traverse of components needed to maintain viability and to support mitosis of said cells; and B. allowing the cells to grow.

2. The method according to claim 1, comprising the additional step of harvesting substances that are produced by the cells either from within the capsules or from the medium.

3. The method according to claim 1 or claim 2, wherein the cells are animal cells and the gas is an oxygen-containing gas.

4. The method according to claim 3, wherein the gas further contains carbon dioxide.

5. The method according to claim 3 or claim 4, wherein the cells comprises mammalian cells.

6. The method according to any of claims 1 to 5, wherein the gas contains balanced amounts of oxygen and carbon dioxide sufficient to produce an optimal, substantially constant oxygen and carbon dioxide concentration in the said medium to support metabolism and to promote mitosis of the cells.

7. The method according to any of claims 1 to 6, wherein the medium contains oxygen at a partial pressure of oxygen in said medium within the range of 15-200 mmHg.

8. The method according to any of claims 1 to 7, wherein the animal cells comprise fused cells, genetically engineered cells or myeloma cells.

9. The method according to any of claims 1 to 8, wherein the cells are animal cells and are allowed to grow to a density of at least about 5 x 106 cells/ml of culture.

10. A method of growing cells according to claim 1 and substantially as hereinbefore described.