NO850534L - PROCEDURE FOR CULTING CELLS - Google Patents

Description

This invention relates to a method for cultivating fragile cells that are capable of reproduction and more particularly to a method for optimizing gas exchange in cultures of such cells. Cells that benefit from the method of the present invention include animal cell lines, such as various myeloma cells, cell lines naturally or artificially transformed from primary cultures, fused cells such as hybridomas, malignant transformants, and other cells that do not have cell walls, e.g. e.g. spheroblasts.

It is well known that oxygen is necessary in the cultivation of animal cells, and that the amount of oxygen available for diffusion into a cell is important for the cell's viability and growth. In general, too little oxygen can kill a cell or at best limit its growth. As an example, adult hybridoma cultures require at least about 0.12 millimoles of oxygen/liter/hour/10 9 cells. If less oxygen is available,

mitosis and protein production are limited. Also, too much^ oxygen can lead to oxygen toxicity which can kill a cell by oxidizing essential proteins. A cell culture's rate of oxygen uptake increases with the weight of the cell culture, and as the density of the cell culture increases, it becomes more difficult to transfer sufficient amounts of oxygen from the surface of the culture to the inner volume of the culture.

Carbon dioxide, which influences the pH and the bicarbonate concentration, is also needed to maintain the weight of animal cells; it is necessary to control it if one seeks to grow large quantities of cells.

Conventional techniques for culturing mammalian cells

on a laboratory scale involves suspending the cells in a medium 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 affects the rate of oxygen and carbon dioxide diffusion into cells. As the depth of the medium increases, the ratio between the average surface area and the volume of the medium decreases.

At the same time, the concentration of oxygen and carbon dioxide

in the air above the dish and/or the dissolution rate in the medium insufficient to provide sufficient gas concentration throughout the volume of the medium to satisfy the requirements of the adult cells. At that point, the oxygen supply becomes a weight limit-

send factor. Consequently, the previous methods teach that one should keep the depth of the medium for such static cultures within the millimeter range. In general, the upper limit of cell density for such cultures, using air in the supervolume as oxygen supply, is approximately 10^ cells per milliliters of culture.

When cell culture techniques are scaled up to cultivation

of mammalian cells in industrial quantities, difficulties are encountered in maintaining the supply of sufficient amounts of oxygen to satisfy the cells' demands. See Glacken et al., Mammalian Cell Culture: Engineering Principles and Scale-up Trends in Biotechnology, Elsevier Science Publications, 0166-9430/83, page 102 (1983). A solution to this problem involves ensuring increased concentrations of oxygen and carbon dioxide in the space above the medium surface while the cell density increases. However, with this approach, too much oxygen may be present near the surface of the medium, causing oxygen toxicity,

and expensive measuring devices and oxygen sensors must continuously test and adjust the oxygen concentration in the medium to maintain optimal proportions of the gases through the medium.

In growing cells that have cell walls, e.g. prokaryotic cells, gases are often injected directly through the medium to supply the cells with the necessary gas concentrations. However, direct spraying is unsuitable in growing cells that do not have a cell wall, are rather fragile, and are physically damaged when the gas is sprayed at high enough velocities to provide sufficient gas concentrations in the medium. It is also the case that protein components in the medium, which are usually needed in all animal cell cultures, produce a foam on the surface of the medium, which can trap the cells. Cells trapped in the foam die quickly. Antifoaming substances that are 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 field of cell culture have included encapsulating cells in capsules consisting of calcium alginate to produce high cell densities. US Patent No. 4,352,883 entitled "Encapsulation of Biological Material" describes the encapsulation of cells to protect them from harmful factors such as bacteria. US 4,409,331 describes a method for producing products from animal and other cells by harvesting such products from encapsulated cell cultures where the cells are placed in semipermeable capsule membranes.

As can be seen from the above, it would be desirable to provide improved gas exchange in the culture of mammalian cells and other fragile cells. Accordingly, it is an object of the invention to optimize the gas concentration in a medium without damaging the cells or resorting to expensive control techniques. Another aim is to provide a method for growing large cell cultures, e.g. 20 - 30 liter cultures, for high cell densities, e.g. 1x10 Q cells/ml. Another goal is to grow cells that do not have cell walls, such as animal cells or spheroblasts, using gas permeation to satisfy the cells' gas requirements without destroying the cells. Another goal is to grow animal cells in culture to densities comparable to cell densities in many normal tissues.

The present invention relates to a method for the efficient cultivation of fragile cells, i.e. cells which have no cell wall, to a high density. The method includes the step of suspending spheroidal capsules in a medium containing a seed culture of the cells to be cultured. The capsules include semipermeable membranes sufficient to protect the cells from physical damage and sufficiently permeable to allow the passage of necessary nutrients, vitamins, ions, gases, etc., needed to support the weight of the cells. A gas containing components needed by the cells is injected directly through the medium among the suspended capsules, either continuously or intermittently, during cell growth. The composition of the injected gas is sufficient to maintain a constant oxygen and/or carbon dioxide concentration in the medium, a concentration which is sufficient to supply the cells with oxygen and/or carbon dioxide in amounts well adapted to support cell mitosis and metabolism . Using conventional media and this gas permeation technique, it is possible to grow cells inside the capsules at densities on the order of 5 x 10 g cells/ml of bottomed capsules.

If desired, substances produced by the cells can be harvested either from the inside of the capsules or from the medium.

In preferred embodiments, when culturing the animal cells, the permeated gas carbon dioxide and oxygen are harvested in balanced amounts sufficient to produce a desired C₂/CC₂ content in the medium. Preferably, the C^/CG^ content in the medium is sufficient to maximize the cell density inside the capsules by promoting optimal cell growth. This means that the oxygen is present in the permeated gas at levels between about 0.15 mm Hg, and about 450 mm Hg, preferably at least between 0.15 and 200 mm Hg, and optimally for mammalian cells, 150 - 170 mm Hg.

Advantageously, mammalian cells that can be cultured according to

to the method, include genetically engineered cells, myeloma cells, and hybridomas and other fused cells.

A principle advantage of the present technique for growing mammalian cells that uses perfusion for gas exchange is that the cells are supplied with an abundant supply of gas. Thus, even dense and voluminous cultures can be supplied with oxygen and/or carbon dioxide at levels where gas exchange does not become a weight-limiting factor. This is possible because the cells are enclosed in membranes and are thus protected from the physical damage normally caused by the flow. The flow-through is an efficient method of supplying the medium with optimal concentrations of oxygen and carbon dioxide because an equilibrium can be established at a threshold velocity for permeation between the concentration of the gases in the permeating gas and the concentration of the gases in the medium. Oxygen and carbon dioxide concentrations in the medium can thus be set at optimum levels by controlling their concentrations in the gas being injected. The need for expensive control and gas measurement equipment is therefore removed. Other points, objects and advantages of the invention will become apparent to those skilled in the art by reading the following claims and detailed specifications together with the drawings.

FIG. 1 schematically illustrates mammalian cells encapsulated

in a microcapsule suitable for use in the present invention;

FIG. 2 schematically illustrates a suitable cell culture apparatus

for use in the practice of the invention;

FIG. 3 is a graphical representation of the total number of cells

per milliliters of medium in relation to the time of difference

cultures added in equal portions with and without gas permeation; FIG. 4 is a graphic representation of cells per milliliters of bottomed capsules versus time for a continuously fed gas-permeated encapsulated culture illustrating the cell densities achievable using the method of the invention; and FIG. 5 is a graphical representation similar to fig. 4 showing the growth of an aliquoted culture.

The present invention is based on the observation

that cells without cell walls, e.g. mammalian cells, enclosed in semipermeable membranes, are immune to physical damage and oxygen toxicity problems caused by permeation,

and that gas injection can be used effectively to supply oxygen and/or carbon dioxide to such cells. When using the method of this invention, the concentration of oxygen and carbon dioxide can be balanced to provide optimal levels in a medium, and increased cell yields can be achieved in large cell culture vessels. Because the G^ and CG^ concentrations in the medium will be proportional to their concentration in the recirculated gas, concentration cost control and its associated costs are eliminated.

Turning to fig. 1, mammalian cells 11 can be encapsulated in spheroidal, typical spherical capsules 13 by means of the method set forth in US Patent No. 4,352,883, the content of which is referred to here. The commonly preferred encapsulation techniques are explained in co-filed application No. 85,0535. For the content, we refer to this application.

A typical encapsulation method involves the formation of

a suspension containing approximately 10 cells/ml in 1% (vol/wt) sodium alginate (NaG-Kelco LV). The density of the cells in the suspension will determine the number of cells per capsule in the final produced seed culture. The suspension is transferred to a jet head apparatus which consists of a container having an upper air intake nozzle and an elongated hollow part which is friction-fitted into a stopper. A syringe, e.g. a 10 cc syringe, fitted with a step-up pump, is fitted to the top of the container with a needle, e.g. a 0.01 inch I.D. Teflon cover

the needle, which passes through the length of the container. The interior of the container is designed so that the top of the needle is exposed to a constant laminar air flow that acts like an air knife.

In use, the syringe full of the solution containing the material to be encapsulated is mounted on the top of the container, and the stage pump is actuated to incrementally force droplets of the solution to the top 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 gel masses that are collected by aspiration. The gel masses are incubated in three completed volumes of isotonic saline for gel expansion for a total of approximately 11 minutes. A membrane is then formed around the gel masses by contact with a 750 mg/litre 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 a 1.4 g/l solution of CHES (2-cyclohexylaminoethanesulfonic acid) buffer (Sigma) containing 0.2% (w/v) calcium chloride in saline. The capsules are washed for approx. 8 minutes with 0.3%

(w/v) calcium chloride in salt water and a No. 2 membrane is formed around the capsules by a 10 minute reaction with 105

mg/l polyvinylamine (Polyscience, 50,000 - 150,000 dalton molecular weight) in salt water. The capsules are washed again with two volumes of isotonic saline for 7 minutes and covered with a 7-minute

_2

immersion in 5 x 10% (w/v) NaG in saline. The capsules are washed for another 4 minutes in saline, then the intracapsular volumes are rehydrated by 2 immersions in 55 millimolar sodium citrate in saline, 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.

The capsules as illustrated in fig. 1, prepared according to this encapsulation method have membranes impermeable to high molecular weight proteins, bacteria and the cells to be cultured.

Fig. 2 schematically illustrates an apparatus for cell cultivation

according to the invention. It consists of a 316L stainless steel electropolished, 50 liter vessel 10 fitted with a lid

12. A motor 14 drives the rotary shaft 16 to rotate the propellers 18 and 20 which are located in the culture 22. The culture 22 includes a conventional medium such as Eagle's modified medium with added serum. Alternatively, it can pass

of a hypertonic medium having an osmolarity of approximately 360 milliosmoles of the type described below and described in greater detail in the related application No. 85.0533,

entered on the same date as this, the contents of which there

is referred to here. A quantity of capsules such as the capsule pictured in fig. 1 is suspended in the medium. Typically, the capsules are spherical or spheroidal and have a diameter on the order of less than about 2 mm. The capsules, which have an average diameter of around 0.8 mm, work well.

The gas requirements of the cells are met by allowing an oxygen

and carbon dioxide-containing gas pass through sterile filter 24, air tube 26, and spray-through head 28. The spray-through head 28 may contain a 2.5 µm microporous porcelain filter. For growing animal cells, the gas can consist of 95% air,

5% CO? (in volume). Gas bubbles pass from the spray head up through the medium between the capsules. The spray-through rate should be sufficient to maintain the partial pressure of oxygen in the medium equal to the partial pressure of oxygen in the gas. The pressure of oxygen in the medium can thereby be set to a level at or just above what the cells need for optimal growth.

Due to the serum component of the medium, permeation causes foam to accumulate in the supervolume 30 of vessel 10. However, the capsules protect the cells from dehydration should they be temporarily introduced into the foam, and also protect the cells from mechanical damage. Thus, gas spraying through an encapsulated culture can satisfy the growing cell population's need for oxygen, thereby making it possible to achieve extremely high cell densities. The gas exiting the culture passes through outlet opening 32 and filter 34. A typical gas flow rate for a 30 liter culture is 0.2 standard cubic feet per hour.

When the invention is practiced in the portion feeding manner, the medium is simply metered into vessel 10 together with microcapsules containing the seed culture, and the culture is grown to maximum density by gas sparging. However, perhaps the best way to practice the invention is to allow medium to pass through the culture by introducing a continuous or periodic flow of medium into the inlet opening 38, and draining the medium through filter element 40. Filter element 40 may contain a stainless, microporous mesh. which have pores smaller than the diameter of the capsules, e.g. 50 - 100 µm. Medium can be entrained through valve 42. The level of medium

in the culture can be observed in the transparent tube 44. A typical rate of medium flow in inlet opening 38 and out through filter 40 is 4 ml to 12 ml per minute.

If one seeks to harvest proteins or other substances produced by the cells, the method used will depend on the ratio between the effective molecular sizes of the substance of interest and the permeability of the capsule membranes. As described in the above-referenced patents, the permeability of the capsule membranes can be controlled within limits. If the protein of interest is too large to pass through the membrane, the proteins accumulate inside the microcapsules; if there is little enough to pass through the membranes, they will accumulate in the extracapsular medium.

In application, a system user determines a desired amount of oxygen and/or carbon dioxide to be dissolved in medium 22.

The chosen gas concentrations and proportions depend on the type of cells being cultured, but are independent of flow or planned cell density. For most animal cells, the desired concentration (partial pressure) of oxygen will be between approximately 15

and 200 mm Hg. For example, the selected oxygen concentration level for a cell culture will be greater than that which is sufficient to supply the cells with 0.12 mM/l/hour/10 9 cells, the previously determined approximate minimum requirement for vigorous cell growth.

The concentration of gases in the medium is proportional to the concentration of the gases in the permeated gas, provided that the permeation rate is sufficiently high to cover variations in G^/CC^ concentration caused by cell respiration activity. At a threshold level, the permeation rate is sufficient to maintain an equilibrium between gas and liquid pockets in vessel 10. Accordingly, the system user can set the permeation rate to supply the desired mixture of gases to the permeation head 28. The permeation head 28 then supplies the gas to culture 22 at a rate that maintains a constant concentration of oxygen in the medium. Such a spray-through rate is 0.2 standard cubic feet/hour in a 30 liter grow tank. The capsules 13 protect the cells 11 in the medium against mechanical damage. The capsules also protect the cells that can be transported into the foam in the excess volume in the tub, from oxygen damage or dehydration.

Thus, the oxygen concentration in the medium does not need to be controlled, because an equilibrium is established between the concentration of the gases dissolved in medium 22 and the preselected concentrations of the gases that are sprayed through.

The perfusion continues and the cells 11 in the capsules

13 is allowed to undergo mitosis. Finally, when the cell culture has reached the desired cell density, the substances produced by the cells, such as antibodies, can be harvested either from inside the capsules or from the extracapsular medium.

Inoculation in a microencapsulated cell culture system as described above has been observed to support a cell density of approximately 5x10 g cells per ml of bottomed capsules, or approximately 1x10 g of cells per ml medium. This is an improvement of approximately 2 orders of magnitude over conventional cultivation techniques. Apart from improving the cell product yield by increasing the cell density, gas permeation according to the invention allows the cultivation techniques to be scaled up to commercial production levels.

Thus, it can be seen that permeation provides improved, efficient gas exchanges to microencapsulated mammalian cells.

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

EXAMPLE 1

Approximately 800 ml bottomed capsules containing approx. 10<5>mouse-mouse hybridoma cells per milliliters were equally distributed in sufficient medium to make up 2.3 liter volumes of encapsulated seed cultures. At the same time, 2.3 liter volumes of medium containing comparable numbers of the same cells per volume unit of medium produced. The medium was standard Eagle's medium supplemented with 5% fetal bovine serum. The four seed cultures were grown with gentle stirring and without additions to the medium for 10 days. One encapsulated culture and one unencapsulated culture were sprayed at a rate of 0.3 standard cubic feet per second. hour for 4 days, and 1.0 standard cubic feet per hour thereafter. The two remaining seed cultures were not inoculated.

The injected gas consisted of 5% CG^ and 95% air (by volume). The cell density in the respective cultures was checked, and data was plotted. The results are shown in fig. 3.

As shown by the data, after 6 days the uninoculated unencapsulated culture had grown to a cell density of approximately 8x10 5 cells/ml culture, and the uninoculated conventional cell culture had grown to a density of approximately 3x10 5 cells/ml. The inoculated unencapsulated and encapsulated cultures had grown to a density of approximately

6 6

1.5 x 10 cells/ml and 1.3 x 10 cells/ml, respectively. Thereafter, the cell density of both the uninoculated and inoculated suspension cultures fell rapidly, until on day 10, the density of both cultures was less than 2x10 5 cells per cell. milliliters. This shows that permeation of unencapsulated cultures has no significant beneficial effect compared to the same culture that uses oxygen dissolved in the medium from the excess volume. The uninoculated encapsulated culture was only moderately more successful. Its cell density was approximately 7 x 10~* cells/ml and decreased on day 9. The encapsulated, flow-through culture, in contrast, showed a steady increase in cell density from day 2 to day 9, and the cell density was then maintained at approximately 3x10 cells/ml. This example shows that cell viability and cell growth are promoted in susceptible animal cells if grown in a gas-permeated, encapsulated culture, and that both the encapsulation and flow-through steps are necessary to achieve this result.

EXAMPLE 2

Two encapsulated hybridoma cell cultures were prepared and suspended in Eagle<1>'s medium supplemented with 5% fetal bovine serum, 100 times the normal iron ion concentration and 2 times the normal amino acid concentration. The medium had an osmotic pressure of approximately 360 milliosmoles. Culture No. 1 had an initial cell density of 5 x 10 3 cells/ml culture, was fed for 13 days by circulating fresh medium at 6 ml/minute, and then for 5 days at 11 ml/minute. Culture no. 2 (with an initial cell density of 1 x 10 cells/ml) received a continuous nutrient supply of 4 ml/minute for 9 days, and was supplied in portions by replacing 10 liters of medium per day on each of days 9 through 20. Both cultures were aerated during their growth cycle at 0.2 standard cubic feet per hour. The cell density of each culture was checked. The results are presented in fig. 4 and 5. As illustrated, the continuously fed culture (Fig. 4) at and after day 17 achieved a cell density of between 1x10 8 cells/ml of bottomed capsules and 5 x 10<8> cells/ml of bottomed capsules. Since the volume of the medium was approximately 5 times the volume of the capsules, this means that the cell density per milliliters of culture was between approximately 5x10 7 and 1 x 10 cells/ml.

As shown in fig. 5, the aliquoted culture also achieved a density greater than 1x10 cells/ml of bottomed capsules.

The person skilled in the art can determine other modifications or variations of the methods and products described herein. Such other modifications and variations are included in the following claims.

Claims (11)

1. A method for growing cells that do not have a cell wall to high density, characterized by the following steps: A. direct injection of a gas through a growth medium, to supply said medium with a constant gas concentration sufficient to maintain viability and support mitosis of said cells, said cells being placed in spheroidal capsules suspended in said medium, said capsules having semipermeable membranes sufficient to protect said cells from physical damage and sufficiently permeable to allow passage of components needed to maintain viability and support mitosis of said cells; and B. to allow said cells to grow. 2. Method according to claim 1, characterized by an additional step which consists in harvesting substances that are produced by said cells either from the inside of the capsules or from said medium. 3. Method according to claim 1, characterized in that said cells are animal cells and said gas is an oxygen-containing gas. 4. Method according to claim 3, characterized in that said gas further contains carbon dioxide. 5. Method according to claim 3, characterized in that said cells include mammalian cells. 6. Method according to claim 3, characterized in that said oxygen-containing gas contains balanced amounts of oxygen and carbon dioxide sufficient to produce an optimal, constant oxygen and carbon dioxide concentration in said medium to support metabolism and promote mitosis in said cells. 7. Method according to claim 3, characterized in that the partial pressure of oxygen in said medium is within the range 15-200 mm Hg. 8. Method according to claim 3, characterized in that said animal cells include fused cells. 9. Method according to claim 3, characterized in that said animal cells include genetically engineered cells. 10. Method according to claim 3, characterized in that said animal cells include myeloma cells. 11. Method according to claim 3, characterized in that said animal cells are allowed to grow to a density of at least approximately 5 x 10 cells/ml culture.