GB2206892A

GB2206892A - Stationary cell culture system

Info

Publication number: GB2206892A
Application number: GB08816173A
Authority: GB
Inventors: Aftab Alam
Original assignee: Individual
Current assignee: Individual
Priority date: 1987-07-07
Filing date: 1988-07-07
Publication date: 1989-01-18
Also published as: GB8816173D0

Abstract

A cell culture suspension is introduced into at least one environmentally controlled narrow culture channel (7) arranged in a reactor (3) and a supply of nutrient and a gaseous phase containing oxygen and carbon dioxide are brought into contact with the cell culture suspension so as to form desired cell cultures. The surrounding environment (11) within the reactor is controlled to suit the required cell culture in the channel, and the nutrient is brought into direct contact by hollow fibre capillaries (28) with the cell culture suspension in the channel, whereas the wall of the channel is gas-permeable so as to allow the gaseous phase to diffuse through the wall and into contact with the cell culture suspension. The oxygen content of the gaseous phase diffuses into the cell culture and maintains oxygen supply for the cellular respiration and the carbon dioxide content diffuses into the cell culture to maintain a desired pH value for the cell culture. <IMAGE>

Description

This invention relates to a cell culture system.

BACKGROUND OF THE INVENTION An increasing number of useful biomolecules and therapeutic substances are produced using engineered mammalian cells. Monoclonal antibodies, human growth factors, interleukins, interferon, tissue plasminogin activator and insulin are all useful therapeutic substances that are produced from mammalian cells. Therefore, there is rapidly expanding need for developing technology for the production of large quantities of mammalian cell cultures for the extraction of medically important biomolecules.

Mammalian cells are generally more complex and difficult to grow than procaryotic microorganisms because they are larger and lack rigid cell walls. The growth characteristics of mammalian cells varies considerably.

Mammalian cells are divided into two groups. Suspension cells are able to grow when freely suspended in the medium, while anchorage-depended cells must be attached to a surface in order to grow. There are a number of technologies currently available for both types of cell cultures. The most widely used method is the suspension culture technique where cells are suspended in culture medium and the suspension is constantly agitated to prevent the cells from sedimenting to the bottom and facilitate oxygen transfer. Suspension cultures are described in US patent Nos 4059858, 4166768, 4178209 and 4184916.

For anchorage depended cells microcarriers have been employed in suspension cultures as support means. Such microcarries are illustrated in US patent Nos 3717551, 4036693, 4189534, 4203801, 4237033, 4237218, 4266032, 4289854, 4293654 and 4335215.

More complex techniques have utilised capillary hollow fibre membranes as the support and the means of supplying nutrient culture media to the cells. Medium is pumped through the lumen of hollow fibres arranged in an elongated bundle as described in US patent No 3821087, 4220725, 4184922 and 4647539.

A closer examination of the technology currently available for mammalian cell cultures reveals that as the cell culture density and volume increases it requires greater amount of the dissolved oxygen for cellular respiration. In most of the aforesaid methods the oxygen transfer is brought about either by agitation or rapid transfer of oxygen rich media, which in turn creates liquid shear. Mammalian cells do not have a protective cell wall and consequently they are sensitive to liquid shear force.

Because of liquid shear most of the aforesaid methods offer limited scalibility. Attempts have been made to design shear free stirring devices as described in US patent Nos 3572651, 3622129 and 3647632. These devices are useful only for small scale cell cultures.

The present invention relates to a method and a device for the production of shear free stationary cell cultures.

It can be used for the production of batch or continuous cell cultures. It can be scaled up to produce large quantities of cell cultures.

SUMMARY OF THE INVENTION According to the invention there is provided a method of cell culture which comprises: introducing a cell culture suspension into at least one environmentally controlled narrow culture channel which is arranged in a reactor; supplying a gaseous phase containing oxygen and carbon dioxide to the reactor so as to come into contact with the cell culture suspension; and supplying liquid nutrient to the cell culture suspension in the channel and removing metabolically produced substances from the cell culture suspension.

Preferably, the wall of the cell culture channel is gas-permeable so as to allow the gaseous phase to diffuse inwardly through the wall and into contact with the cell culture suspension.

The cell culture suspension may be introduced intermittently into the culture channel, to be succeeded by a gaseous phase.

Preferably, the cell culture suspension is introduced into a plurality of environmentally controlled narrow culture channels.

The term gaseous phase is intended to mean a composition of gas or gaseous required to support and promote healthy cell growth for animal cell cultures, the gaseous phase is generally air or oxygen containing 0-56 carbon dioxide.

The term cell culture suspension is intended to include reference to the suspension of cells in nutrient media, suspension of cells and cell-microcarriers and other culture supplements in nutrient media or nutrient media alone.

The internal diameter and the geometry of the culture channels may be such as to prevent mixing of the culture segments in stationary or agitated state, and preferably the gaseous phase is such as to support and promote healthy cell growth.

The cell culture suspension may be provided with the means to supply nutrient media and with the means to withdraw metabolically produced substances.

The invention is also concerned with a cell culture system which comprises: at least one environmentally controlled narrow culture channel arranged in a reactor and having a gas-permeable wall; means for introducing a cell culture suspension into said channel; means for supplying liquid nutrients to the channel to contact with the cell culture suspension; and, means for supplying a gaseous phase to the reactor so as to diffuse inwardly through the wall of the channel and into contact with the cell culture suspension.

The cell culture suspension may be introduced into the culture channel continuously or intermittently succeeded by a gaseous phase.

Preferably cell culture suspension is introduced intermittently and succeeded by a gaseous phase.

The cell culture suspension may be introduced into the culture channels either under the force of gravity, or against the force of gravity or at an inclination to the direction of gravity.

Preferably the culture channels are bundles of elongated or twisted tubular or composite honeycomb structures, and advantageously the interchannel spaces are available for gaseous and thermal exchanges.

The culture channels are preferably made of materials permeable to gases such as silicone rubber, low density polyethylene and fluorinated ethylene propylene (FEP Teflon).

The culture channels are provided with the means to maintain desired environment and temperature around the culture channels.

The gaseous components of the environment along the culture channels may be maintained either in a static and uniform mode or by dynamic mode changing during the progression of cell growth and metabolism or as a gradient along the length of the cell culture channels.

The device according to the present invention may be constructed either as elongated or twisted columns of culture channels or in the configuration of container vessels such as bottles, jars, barrels, tanks and so forth, filled with culture channels.

The device may be run either in a continuous cyclic mode where the cell culture suspension alternated by gaseous phase continuously migrate along the culture channels or in a batch mode where the culture channels are filled with the cell culture suspension alternated by gaseous phase and incubated to grow until the exhaustion of the nutrient medium.

The culture channels are provided with the means to generate segments of cell culture suspension alternated by gaseous phase in the culture channels.

The culture channels may be provided with the means to harvest the culture.

The culture channels are provided with the means to mix seed culture with fresh nutrient media and deliver it to the culture channels.

Preferably the culture channels are provided with the means to regulate the rate of flow of the culture and the gaseous phase through the culture channels and preferably with the means to regulate the volume of the culture segments and the gaseous phase.

The culture channels could be positioned horizontally, vertically, or diagonally.

Preferably several individual units of the cell culture system could be coupled together for large scale production of cell cultures.

Preferably the culture channels are provided with the means of sterilisation and maintaining the sterile environment inside the culture channels.

The device is provided with the means to measure the density of the ingoing culture suspension and the means to alter the suspension density.

In order to culture photosynthetic plant and alga cell cultures the device may also be provided with the means to illuminate the culture channels to facilitate photosynthesis.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a schematic side view of a culture channel reactor chamber according to the invention for continuous production of cell cultures; Figure 2 is a cross-sectional view of the reactor chamber; Figure 3 is a diagrammatic side view of the reactor chamber; Figure 4 is a diagrammatic illustration of a number of channel reactor chambers coupled together; Figure 5 is an enlarged view of a single channel implanted with a capillary hollow fibre running along the length of the culture channel; and, Figure 6 is a schematic illustration of culture channel encased in a jar configuration for batch cell cultures and without implanted hollow fibre capillaries.

DESCRIPTION OF PREFERRED EMBODIMENTS Referring to the accompanying drawings, the cell culture device in accordance with the present invention is constructed of a plurality of hollow tubings of narrow diameter 7 as shown in Figure 1, Figure 2 and Figure 6.

The plurality of culture channels are encased in a reactor chamber 3, and are made of thin wall gas-permeable fluoroethylene propylene polymer (FEP-Teflon) of 2.5 millimetre internal diameter. The walls of the culture channels are permeable to oxygen as well as carbon dioxide.

The culture channels have free interchannel space 11 as shown in Figure 2.

Figure 2 and Figure 5 show culture channels implanted with hollow fibre capillaries 28. The hollow fibre capillaries are permeable to low molecular weight substances.

Referring again to Figures 1 and 3, a bundle of hollow fibres 31 enters the culture channel reactor chamber, and are individually implanted in the individual culture channels, as shown in Figure 5. The hollow fibres emerge out of the reactor chamber as a bundle 32. The reactor chamber consists of a main cylindrical body 3 and two detachable members 1, 2 which are mounted on the main body 3 using ring mounts 24 and sealing O-rings 4. The reactor chamber consists of plurality of inlets 6 and outlets 5.

Thermostatically controlled liquid or gas mixture is introduced through the inlets (not shown in the drawing) which maintains desired temperature and gaseous environment around the culture channels.

The reactor vessel 3 contains inlets 8, 9 and an outlet 10. The inlet 8 is used for introducing cell culture suspension from a fixed volume delivery container 15 (see Figure 3) which contains an inverted U-tube 16.

The fixed volume delivery container 15 is connected to a cell culture suspension supply line 17, and gas phase supply lines 12, 14 and 25 include gas filters. The inlet 9 is used for supplying a gaseous phase, and 21 and 26 are line connectors. The outlet 10 is connected to supply line 18.

Figure 6 shows cell culture channels encased in a jar configuration. The outlet supply line 10 is positioned on the same side as the inlet supply lines 8, 9. This arrangement is achieved by positioning supply line 10 along the length of the culture channels from one end of the culture channels to the other. The supply lines 8, 9 and 10 are mounted on the jar using a screw mount cap 27, as shown in Figure 6.

The entire device could be sterilised by flushing the channels and the supply lines with sterilising gas or liquid medium. The device could also be autoclaved.

USE OF THIS INVENTION FOR THE PRODUCTION OF CONTINUOUS CELL CULTURES To make continuous cell cultures in accordance with the present invention, the culture channels are first filled with sterile buffer in order to remove air from the channels. A fresh cell culture suspension is supplied by the line 17 into the fixed volume reservoir 15, using a peristaltic pump 19, and cell culture suspension 29 is collected in the reservoir 15. As soon as the reservoir 15 is filled up to the top of the U-tube 16, it discharges a fixed volume of the cell culture suspension into the culture channels 7. The outlet supply line 18 co-operates with a peristaltic pump 20 which assists controlled outflow of cell culture.

The inlet 9 is attached to a gaseous phase reservoir (not shown in the drawing). For animal cell culture the gaseous phase is normally a mixture of 0-5% carbon dioxide in air. The peristaltic 20 pump is run at such a speed that it draws into the culture channels the cell culture suspension delivered from the fixed volume reservoir 15 and a small volume of overhead gaseous phase. The peristaltic pump 20 runs fast enough to draw into the channels the cell culture suspension and a small volume of overhead gaseous phase before the next charge of the cell culture suspension 29 is discharged from the reservoir 15. By controlling the flow speed of the output peristaltic pump 20 and the supply peristaltic pump 19 and the height of the short arm of the U-tube 16, the operator has the option to select the volume of cell culture suspension and gaseous phase 30 entering the culture channels. Figure 3 shows cell culture suspension 29 and gaseous phase 30 alternatingly entering the channels and migrating along the plurality of the culture channels.

The culture suspension in the culture channels is continuously supplied with fresh nutrient medium. Fresh nutrient medium is pumped through the hollow fibre capillaries 31, as shown in Figures 1, 2 and the represented view in Figure 5. Nutrient medium diffuses across into the culture and waste products diffuse back across into the capillaries and are removed by the medium flow.

The functions of the device may be automated and controlled by an electronic means.

The migration of cell culture along the culture channels creates a gentle mixing in culture segments which keeps the cells homogeneously suspended in the liquid medium.

The temperature around the culture channels will be maintained by supplying thermostatically controlled gases or liquid through the inlet 6. For animal cell cultures the optimum temperature is around 370C.

The gaseous components of the environment around the culture channels will be selected depending on the particular need of the culture. Generally, for animal cell cultures, a mixture of 0-5% carbon dioxide in air would be appropriate. Because of the gas permeability of FEP Teflon cell culture channels, oxygen will readily diffuse across the channels, into the culture and maintain sufficient supply of oxygen for the growing and dividing cells.

The dividing and metabolically active cells produce carbon dioxide. The carbon dioxide will diffuse out of the gas permeable culture channels into the interchannel space and into equilibrium with the surrounding environment and prevent excessive build-up of carbon dioxide in the culture segments.

The cell culture system (when used for the production of continuous cell cultures) produces a gentle mixing of the cells within the confines of a single culture segment.

The cells cultured in this system experience no liquid shear.

In the culture channels each culture segment is surrounded, from both sides by an oxygen rich gaseous phase, (air containing 0-5% carbon dioxide), and this arrangement provides sufficient oxygen transfer to the culture bubbles and helps to maintain the pH and oxygen tension of the cell culture bubbles.

The oxygen transfer in the culture segments can be manipulated by varying the size of the culture segments and the oxygen containing gaseous phase sandwiching the culture segments. The smaller the size of the culture segments, the more rapidly will oxygen diffuse along the culture segments.

The culture channels are made of gas permeable materials (FEP Teflon) which provides diffusion of oxygen into the culture, and consequently the cell cultures in the channels are surrounded from all sides with an oxygen rich gaseous environment. The gas permeable culture channels, by allowing the diffusion of metabolically produced carbon dioxide, prevent harmful build-up of carbon dioxide in the cell culture. The hollow fibre capillaries continuously supply nutrient medium and remove waste products. These factors and the absence of agitation create an environment which behaves in a manner more like in situ.

The segments of the culture suspension alternated by the gaseous phase will gradually migrate towards the outlet. The rate of flow of the culture along the channels may be regulated. The appropriate rate of flow of the culture can be estimated by taking into consideration the length of the culture channels and the rate with which the culture will reach growth limiting condition. the flow will be set such that as soon as the growth limiting condition is reached, the culture emerges out of the culture channels through the outlet 10. However, there are situations where cells are cultured beyond log phase, and for such a situations the rate of flow of culture in the culture channels may be slowed down to achieve desired metabolic results.

The cell culture emerges out of the culture channels through the outlet 10. The spent nutrient medium may be separated from the cells by using a filtrative device. The culture emerging out of the culture channels flows through a vessel comprising a filtrative device. The filtrative device withdraws the spent medium from the culture (not shown in the drawing). The harvested cells are resuspended in a fresh nutrient medium, cell density is readjusted, and the cell culture suspension reintroduced into the culture channels through the line 17, thereby creating a continuous cell culture process.

Several units of this device could be coupled together, as shown in Figure 4, to produce large quantity of cell cultures. In Figure 4, 22 are cell culture reactor chambers coupled in series. The device can be run indefinitely to supply a large quantity of cell cultures for research and industrial uses.

The device described here has a configuration of elongated column. Other configuration of the device will be equally efficient such as the configuration of narrow neck bottles, wide neck jars, barrels, drums and so forth.

USE OF THIS INVENTION FOR BATCH CELL CULTURES This invention can be used either for continuous cell cultures or batch cell cultures, where cell culture suspension is incubated until exhaustion of the nutrient media. The production and technical requirements of batch cell cultures are generally less stringent and limited than the continuous cell cultures. Therefore, in batch cell cultures it may not be necessary to continuously feed the cell cultures. A simpler version of the invention can be used, as shown in Figure 6, in which the cell culture channels are encased in a jar configuration. The culture channels are not implanted with the hollow fibre capillaries.

The cell culture channels are filled with the cell culture suspension alternated with gaseous phase and the jar is incubated.

For batch cell cultures the composition of the gases around the channels may be adjusted during the progression of the cell growth. As cells divide and grow they produce lactate and carbon dioxide, the culture acidity begins to rise and consequently requires less environmental carbon dioxide to maintain the pH. The gaseous composition of the environment may be adjusted to counter the effects of lactate, for example, by reducing the carbon dioxide content.

In this cell culture system, (when used for the production of batch cell cultures) there is absolutely no motion in the cell culture segments. Batch culture segment comprises motionless units of cell cultures and there is no liquid shear to damage the cell growth.

This system can be used for the production of large quantities of shear free stationary cell cultures.

USE OF THE PRESENT INVENTION FOR THE CULTIVATION OF ANCHORAGE DEPENDENT CELLS The cells that adhere to surface for growth are cultured either on microbeads or roller bottles. Such microbead cell cultures can be grown in this cell culture device by introducing the cell-leaden microbeads suspension into the culture channels alternated by gaseous phase.

The adhering cells can also be cultured on the inside walls of the culture channels. The inside walls on the culture channels may be treated to improve the cell attachment, either by chemically etching or creating hairy filaments, crevasses, lumens etc. The cell suspension will be introduced into the channels alternated by the gaseous phase. After the channels are filled it will be incubated to allow the cells to attach to the inside walls of the culture channels. After the cells have attached to the channels walls, the cells are fed either by introducing into the culture channels nutrient medium alternated by gaseous phase or nutrient medium along. When cell growth reaches confluency, it can be elevated from the channels by washing the culture channels with trypsin containing saline buffer solution.

USE OF THE PRESENT INVENTION FOR THE CULTIVATION OF OTHER TYPES OF CULTURES Although the device refers to the animal cell cultures it can also be used for the production of bacterial, alga, fungal, plant or other types of cultures by selecting an appropriate culture suspension and a gaseous phase compatible with the culture, and a compatible extrachannel environment. In the animal cell cultures the gaseous phase is generally air containing 0-5% carbon dioxide. Where the function of carbon dioxide is to maintain pH of culture media, anaerobic bacteria can be grown in the absence of oxygen.

The plant and alga cell cultures require light energy to perform photosynthesis. For growing plant and alga cell cultures the culture channels may be assembled in a formation of a single layer of culture channels and each layer will be illuminated with a compatible light source.

Claims

1. A method of cell culture which comprises: introducing a cell culture suspension into at least one environmentally controlled narrow culture channel which is arranged in a reactor; supplying a gaseous phase containing oxygen and carbon dioxide to the reactor so as to come into contact with the cell culture suspension; and supplying liquid nutrient to the cell culture suspension in the channel and removing metabolically produced substances from the cell culture suspension.

2. A method of cell culture which comprises: introducing a cell culture suspension into at least one environmentally controlled narrow culture channel which has a gas-permeable wall and which is arranged in a reactor; supplying liquid nutrient to the cell culture suspension in the channel; supplying a gaseous phase containing oxygen and carbon dioxide to the reactor so as to diffuse across the channel wall and into the cell culture suspension, whereby oxygen diffuses into the cell culture and maintains oxygen supply for the cellular respiration and carbon dioxide diffuses into the culture to maintain a desired pH value in the cell culture, the carbon dioxide produced by the propagating cell culture diffusing outwardly into equillibrium with the surrounding environment so as to prevent excessive build-up of carbon dioxide and to maintain desired pH in the cell culture; and, maintaining the cell culture suspension in the culture channel so that it is allowed to propagate without agitation and without liquid shear to the cell culture.

3. A method according to Claim 2, in which the cell culture suspension is introduced intermittently into the culture channel.

4. A method according to Claim 3, in which a gaseous phase is introduced directly into the channel successively to the introduction of the cell culture suspension whereby the gaseous phase diffuse into the cell culture suspension.

5. A method according to Claim 2, in which the liquid nutrient is introduced into the cell culture suspension in the channel via a hollow fibre capillary.

6. A method according to Claim 2, in which metabolically produced substances in the cell culture are removed via a hollow fibre capillary.

7. A method according to Claim 2, in which cell culturesw are formed on a continuous basis.

8. A method according to Claim 2, in which cell cultures are formed on a batch-line basis.

9. A method according to Claim 2, in which adherent cell cultures are formed.

10. A method according to Claim 1 and comprising introducing cell culture suspension into a plurality of environmentally controlled narrow culture channels arranged in the reactor.

11. A cell culture system which comprises: at least one environmentally controlled narrow culture channel having a gas-permeable wall and arranged in a reactor; means for introducing a cell culture suspension into said channel; means for supplying liquid nutrients to the channel to contact with the cell culture suspension and means for removing metabolically produced substances; means for supplying gaseous phase to the reactor so as to diffuse inwardly through the wall of the channel and into contact with the cell culture suspension and so that the gas generated in the cell culture diffuses outwardly into equilibrium with the gaseous phase surrounding the culture channel and maintaining desired pH in the culture: and, means for regulating the composition of the gas phase around the culture channel to meet the changing requirement of the cell culturs.