HK1098170A - Cell culture system - Google Patents

Description

Cell culture system

Technical Field

The present invention relates to the field of cell culture. The present invention provides novel cell culture systems for growing cells in general, and plant cells in particular. Since such a device can be disposable and effective on a large scale, its use allows to greatly reduce the production costs in different kinds of applications.

Background

Conventional culture systems generally consist of a rigid container (glass or stainless steel) with means for aerating and mixing the culture contents (air distributor, impeller). These systems are complex, and because large scale production is based on stainless steel vessels, sterilized in situ, the usual equipment and support facilities associated with aseptic bioprocessing are extremely expensive. More than 60% of the production cost is due to the fixed cost: high capital cost, depreciation, interest and capital consumption of fermentation plants. The running costs are also high due to the low yield and the need to clean and sterilize the bioreactor after each cultivation cycle.

In the particular industrial application of plant cell culture, different well-known culture systems such as stirred tanks or airlift reactors have been used. Despite much effort to commercialize plant metabolites, little commercial success has been achieved.

One reason is that although it is possible to obtain higher contents of compounds (rosmarinic acid, shikonin, etc.) than required by the whole plant, up to 20% of the dry weight, the productivity is low. The main limitation leading to low productivity is the low growth rate of the plant cells compared to bacteria (less than 0.7 per day, minimum 20h doubling time). Batch cultivation in industrial fermenters means that the plant cell culture is run no more than 10-20 times per year in very high cost facilities. This means that the bottleneck in industrial production is an economic bottleneck rather than a biological bottleneck.

To overcome these problems and reduce production costs, new technologies based on the use of various disposable plastic bags instead of stainless steel fermenters have recently emerged. These new systems with pre-sterilized disposable plastic bags are promising because they reduce capital investment since plastics are low cost materials and they do not require time and cost consuming cleaning, sterilization, validation and maintenance of equipment. Since the bag provided is pre-sterilized, it can be handled by a person not skilled in the art, and therefore it also has greater flexibility in the process.

Different aeration/mixing systems have been proposed in such disposable devices. WaveBiotech (Singh V, U.S. patent No. 6,190,913) developed a system for inflating a bag with a vibrating mechanism placed on the bag that moves the bag to induce wave-like motion to the liquid contained therein. Since such mechanical agitation requires complicated equipment to achieve high culture volumes, the vibrating mechanism limits the size of the tank.

Another proposal is for gas permeable plastic bags to be agitated with a mechanical system or not at all. In U.S. Pat. No. 5,057,429, a gas permeable bag is rotated or vibrated to diffuse oxygen and nutrients to the animal cells. A stationary gas permeable bag is also described in U.S. patent No. 5,225,346. Such culture systems have not been developed industrially to date, on the one hand because it is difficult to enlarge the external stirring device and, on the other hand, because of the problem of insufficient oxygen supply to the cells in a stationary bag containing several liters of culture medium.

The reactor may consist of a plastic bag with a gas spray in the tank and a head plate with the ability to inoculate and remove media samples. Disposable conical plastic bags produced by Osmotec are intended for small-scale use (a few liters) and are aerated with air bubbles through air inlets. U.S. Pat. No. 6,432,698 also describes a disposable bioreactor for culturing microorganisms or cells, which, in addition to being made of plastic material here, comprises a gas diffuser for generating bubbles for mixing and supplying gas, close to the airlift bioreactor.

In these inventions, there are two main limitations: at high densities or large volumes of culture, it is desirable to produce smaller gas bubbles or liquid circulation throughout the reactor to achieve convenient mixing and aeration. This results in a complex bubbling system (gas diffuser, distribution tank … …) that is not compatible with simple disposable technology. In addition, small bubbles are harmful to sensitive cells, increase cell-wall adhesion, and/or deprive the medium of some useful gas (e.g., ethylene for plant cells).

The use of gas bubbles for aeration of bioreactors or fermenters is well known. Currently, diffusers are used to inject microbubbles to improve gas transfer into the media. Bioreactors, in which aeration and agitation is performed by a gas stream without mechanical agitation, are also well known and are currently referred to by experts as airlift bioreactors. For example, U.S. Pat. No. 4,649,117 describes a culture system for an airlift bioreactor for performing cell culture and fermentation. Suitable gas flow rates range from 10 to 300 cc/min, with gentle continuous bubbling of gas, and no mention is made of the size of the bubbles or the periodic generation of single large bubbles in our invention. Two chambers, a growth chamber and a mixing chamber, are used.

Known as "large", but less than 3cm³For mixing and blending a plurality of materials, such as chemicals, beverages or oils. WO-A-8503458 describes A method and apparatus for gas-induced mixing and blending without regard to the growth and cultivation of living cells. The method is based on bubbles of a predetermined variable size and the frequency of injection into the tank through one or several air inlets. The purpose of this is to reduce the total time of blending and mixing, which is not the purpose of our invention. The injection is carried out to obtain a single bubble or a few single bubbles, the size of the bubble and the amount of air being empirically determined, the bubble should not be too large (quoted 1 cubic inch (2.54 cm)³) Not a particular bubble diameter close to the diameter of the cell. This is quite different from our invention where bubble size and amount of air are critical to the growth of living cells. In WO-A-8503458, in the case of some air inlets, individual bubbles are generated having A circular, vertical annular flow pattern. The invention of WO-A-8503458 is for open or open tanks, which are not suitable for culturing living cells under sterile conditions.

In the case of the U.S. patent-a-4,136,970 also describes a method and apparatus for adjusting the size and frequency of bubbles used to mix liquids. It does not consider oxygenation and culture of living cells itself, does not consider maximizing bubble size, and does not consider larger than 1.5cm³Large bubbles of (2). The method described in U.S. Pat. No. 4,136,970 can be used to count platelets but is in no way suitable for or required for culturing and growing living cells.

It is an object of the present invention to provide a low cost cell culture system by means of a disposable device, which is efficient and easy to use on a large scale.

Summary of The Invention

The present invention relates to pre-sterilized flexible or non-flexible plastic bags in which cells are cultured by single large bubble agitation/aeration.

In the present invention, a single large bubble is intermittently generated at the bottom of a column that is partially filled with liquid medium and cells. Since the large bubble almost fills the cross section of the column, it forms a thin gap between the bubble and the side wall of the cylindrical tank, in which the liquid can flow as the bubble rises. This trickling liquid film in contact with the gas bubbles allows convenient mixing and aeration of the contents of the device during operation without damaging the cells.

Such a mixing/aeration system allows for efficient scale-up since oxygen and mass transfer reactions occur at the thin liquid film level. In addition, capital and maintenance costs can be greatly reduced due to the simple design of the system.

Such disposable devices are made of sterilizable and flexible plastic cloth sealed along the edges to form a cylinder. Such a disposable system allows for process flexibility and reduces the time to failure since cleaning, sterilization, maintenance or validation as required in conventional stainless steel equipment is not required.

Since the present invention is disposable and effective on a large scale, it is a good alternative system to reduce production costs in industrial applications.

The culture system can be used for plant, animal, insect or microbial cultures suspended or fixed in different carrier systems. The method allows the production of a variety of molecules, such as metabolites (formed de novo or by biotransformation) or recombinant proteins, or the propagation of embryogenic plant cell lines, by batch, fed-batch or continuous culture, as well as any other use that will be apparent to the skilled person.

Brief Description of Drawings

Fig. 1 is a side view of the device showing the bag and the phenomenon created by rising bubbles.

Fig. 2 is a side view of the plastic bag to tubing joint.

Figure 3 is a schematic diagram of the pneumatic and electrical circuits used to generate and control the frequency and size of bubbles.

Fig. 4 shows the top of the upper part of the groove in the form of an inverted cone.

FIG. 5 shows the growth kinetics of soybean cells in flasks, stirred tank reactor, and cell culture system, expressed as fresh weight per liter of liquid culture.

FIG. 6 shows the growth kinetics of soybean cells in flasks, stirred tank reactor, and cell culture system, expressed as dry weight per liter of liquid culture.

Detailed Description

The present invention relates to the use of very large individual bubbles, periodically produced (whatever the method by which they are obtained), having a diameter as close as possible to that of one of the bioreactors itself, for aeration/agitation (providing efficient oxygenation) of cell cultures. The result is that the culture medium flows as a very thin film between the large gas bubbles and the inner wall of the bioreactor.

In the basic design as shown in fig. l, the bioreactor (or reactors) consists of different parts, containing at least one groove (1) made of plastic cloth, for example sealed along their edges (2), for example to create an interior. The trough is stationary. In a preferred embodiment of the invention, the tank is made of flexible polypropylene having sealable and autoclavable properties, so that it can be sterilized with a small laboratory autoclave or by other methods known in the art. However other types of materials such as Pyrex , stainless steel, semi-flexible, rigid or molded plastic, etc. are suitable and any method known to those skilled in the art such as gamma radiation sterilization may be used.

In a preferred embodiment of the invention, the flexible biocompatible water-proof materials are heat-sealed along their edges (2), for example with a heat-pulse sealer. However, other sealing techniques may be used according to methods known in the art, including but not limited to ultrasonic or radio wave welding. Other types of plastics can be made in different ways, such as injection molding.

In the present invention, the reactor may be cylindrical, as shown in FIG. 1, or may have an oval cross-section, which may be 2m high for a working volume of 20 liters, and may be 12cm in diameter.

Smaller or larger volumes may be used in accordance with the present invention. For example, the diameter of the reactor may be as small as 5cm and may be as large as 40cm or more than 40 cm. The height of the reactor can vary depending on the diameter desired and selected by the user.

The reactor may have different shapes but preferably the height of the shape is at least 5 times the width. It may be, for example, a parallelepiped. The size and shape of the tank (1) can be varied to suit the needs of the user; however, a cylindrical shape is preferred. In suspension culture of cells, it is important to avoid dead space (dead space) where no mixing occurs. Dead spaces occur preferentially in corners, which is why it is preferable for cell culture to make the reactor round-bottomed, where cells tend to form dense aggregates (such as plant cells) that settle more rapidly than individual cells.

If the tank is made of a flexible substance, such as plastic, it is recommended to place the tank in a rigid outer container to support the shape and weight of the tank. Such rigid containers may be made of any material, such as polycarbonate, but the hardness and strength properties of such material (as determined by thickness and/or composition) should be selected primarily. If the plastic bag is also transparent, such outer container may be transparent to facilitate viewing of the culture (3) or to improve light transmission, for example when growing photoautotrophic cells. The size and shape of the outer container is preferably designed according to the size and shape of the tank discussed above.

In the basic design shown in fig. 1, at least four tubes are connected to the tank. The first tube is used at the top for removing excess gas (4). The second tube is at the bottom (5) of the tank for supplying air to the liquid culture via air bubbles (6). In the most preferred embodiment, the tubes are equipped with filters (7), for example 0.22 μm filters, to prevent airborne contamination. The inlet pipe may be equipped with a valve to prevent backflow of liquid in the pipe. Furthermore, one inlet pipe (8) located at the top of the tank allows to fill the bioreactor with sterile culture medium and inoculum and one outlet pipe (9) located near the bottom can be used for harvesting and/or sampling of the culture batch (culturebull).

In the preferred embodiment, the tubing is semi-flexible and made of autoclavable silicone, but other types of tubing such as C-flex or PVC can be used. In a preferred embodiment of the invention, the inner diameter of the tube is 8mm, while the inlet tube is larger: the diameter is 11 mm. In the present invention, the length of the tube is about 1 to 2 meters, but these dimensions can be adjusted by the user as desired.

The tube and the groove may be joined by welding the joint to the plastic cloth according to standard techniques such as heat sealing. In a preferred embodiment of the invention, the pipe and channel are connected through a hole in the plastic cloth to an autoclavable panel attachment connector (panel mount) (10) fitted with bolts (11) and seams (12), as shown in figure 2. Imperviousness may be provided by tightening the bolts to clamp the seam in the plastic cloth. The inner diameter of the panel fixing connector is the same as that of the corresponding pipe in the present invention, but may be adjusted in size as required.

However, it should be understood that any method that allows for the circulation of air or gas may be suitable for the present invention. It is important for the purposes of the present invention that aeration and mixing of the culture medium is achieved by large gas/air bubbles, and preferably by a single large bubble produced every few seconds, the diameter of which is determined by the diameter of the tank. The preferred mixing and aeration method of the present invention therefore consists in bubbles having a length greater than the width. However, this system is also feasible when the bubble length and width are the same.

Preferably, the shape of the large bubble is determined by the shape of the groove; in other words, the gap between the bubble and the groove is limited to a minimum: limited to culture basement membranes containing cells. Preferably, the culture medium flows out as a very thin film between the large gas bubbles and the inner wall of the bioreactor. However, the system also works when the film is relatively thin and the bubbles comprise 50 to 99%, preferably 60 to 99%, more preferably 98.5% of the width of the tank.

For large bubbles, it is understood that each individual and large bubble has a volume of at least 65cm³More preferably at least 500cm³. For example, in a reactor having a diameter of about 20cm, the preferred volume for large bubbles may be 2600 to 4100cm³Or more preferably 3000 to 4100cm³Or even more preferably 3500 or 3700 to 4100cm³And (4) changing.

In order to generate large bubbles, a bubble generator (13) is connected to the air inlet pipe. As shown in fig. 3, the bubble generator is, for example, an electro-gate (17) controlled by a timer (18) and connected to an air pump (19). In this configuration, the electric gate, which is electrically controlled by the timer, is directly connected to the air intake and the air pump. The timer (which is changed by the user) sends an electrical signal to the electric gate regularly in a very short time. During this time, the electric gate is open, allowing the gas supplied by the pump to enter the bioreactor. When a high flow of gas is supplied for a very short period of time in the column, a single large bubble is generated which almost fills the cross section of the column. In the present invention, the cross-section of the electric shutter is 15mm, the air pressure of the air pump is 0.5 bar, and the electric signal is sent every 5 seconds for 0.1 second, thus generating one large bubble every 5 seconds. The user can adjust these parameters as desired.

Such a bubble generator is preferred, but other devices that allow large bubbles to be generated in the column may also be used. In the present invention, the gas used is air, but other gases, alone or in admixture or recovered from the bioreactor, may be used to meet the cell requirements, for example CO for photoautotrophic plant cells₂。

When the bubble reaches the top of the column, which is broken in some way, some of the medium/cells can be lost on the walls of the tank (1). To avoid this drawback, in one embodiment of the invention the upper part of the tank is flared (flare), for example in a preferred embodiment it has the shape of an inverted cone, so that the culture medium/cells can fall back into the tank again (indicated by arrow 20 in fig. 4).

Evaporation occurs during the run, reducing the culture volume and concentrating different compounds in the culture medium, which can be detrimental to the cells. To avoid these problems, equipment may be added, such as a condenser for the exhaust gas or a humidifier for the gas supply. Furthermore, it is possible to connect further inlet and/or outlet pipes to the column, which can be used, for example, for adding acids, bases, antifoams or for removing solutions. Optional equipment for controlling and/or adjusting culture conditions such as, but not limited to, thermometers, pH meters, gas evaluation systems, cell density, pressure control and quality control … … may also be added to this culture system, possibly placing around the bioreactor, for example, a light generator for photoautotrophic plant cells. The regulation of the temperature in the bioreactor can be achieved by different systems such as (but not limited to) placing the bioreactor in a room where the temperature is controlled by suitable air conditioning, using a jacketed outer container, where a circulation of temperature regulated water or air is provided, or any other method known to the person skilled in the art.

The invention is based on the fact that the liquid culture trickles between the rising gas bubbles (6) and the bioreactor side wall (as shown by the arrow (14) in fig. 1). This causes a vortex (15) to mix the culture, avoiding cell deposition, and causes contact with the gas bubbles (6) at the thin liquid membrane (16), where mass transfer is easily achieved for aeration.

This culture system is easy to operate because the user can select the volume and frequency of bubbles by programming the bubble generator described above.

Living cells, such as plant cells, animal cells or microorganisms, e.g., yeast cells, can be grown using the system of the invention. The cells can produce, for example, biomass cells, embryogenic plant cells, metabolites, secondary plant metabolites, and/or recombinant molecules.

Examples

The following examples serve to illustrate some of the products within the scope of the invention and the methods of making the products. This example is not to be construed as limiting the invention in any way. Changes and modifications may be made to the invention. That is, the skilled artisan will recognize many variations in this example to cover a wide range of formulations, ingredients, processing, and mixtures to rationally adjust the naturally occurring levels of the compounds of the invention for a variety of applications.

Example (b): comparison of growth with Soybean cell cultures

The ability of the present invention to grow soybean cells was demonstrated using batch culture. Even at larger scales, this is comparable or better than erlenmeyer flask or stirred tank bioreactor.

Glycine max (L.) Merr. tissue culture strains originally from different varieties cultured in Gamborg et al medium (1968) supplemented with 20 g.L.^-1Sucrose, 7g.L^-1Agar (bacto-agar Difco) and lmg.L^-12, 4-dichlorophenoxyacetic acid. The pH was adjusted to 5.8 and then high pressureSterilized (115 ℃ C., 30 minutes). One strain (13406, cv. maple arrow) was transferred to liquid medium (with no agar and 30 g.L)^-1Sucrose tissue culture same medium), under the same conditions as the tissue culture center, every two weeks in 250mL Erlenmeyer flasks (100mL medium with 3 g.l)^-1Fresh weight) in a culture medium. The erlenmeyer flasks were placed on an orbital shaker at 100 rpm (shaking diameter 20 mm).

A14L stirred tank bioreactor (New Brunswick scientific) with two impellers (six flat blades per impeller) was used with the same media and temperature and pH conditions as mentioned above. The bioreactor containing 9L of fresh medium was autoclaved at 115 ℃ for 40 minutes. Fourteen day old soybean cells were filtered from two 1L Erlenmeyer flasks (500ml of medium). 300g fresh weight of soy cells was placed in 1L fresh medium in a sterile tank with a specific output device aseptically connected to the bioreactor for inoculation. The stirrer speed was adjusted to 100 rpm. Dissolved oxygen was maintained at 30% by increasing or decreasing the air flow rate using a biosensor equipped with a sterilizable oxygen probe (Ingold) and a mass flow rate meter.

A25L cell culture system, called a large bubble column (as described above), was placed in a rigid outer container, filled with 20L of soy cells (30g/L fresh weight) in fresh medium. The room temperature was adjusted to 25 c and a 12cm diameter bubble (approximately 10cm height) was generated every 5 seconds (by programming the bubble generator as noted above).

And (3) growth determination: samples of the culture batch were taken from erlenmeyer flasks, stirred tank bioreactors and large bubble columns at specific growth periods and the sample volumes were determined. The cells were then removed from the liquid culture by filtration. Biomass was weighed (fresh weight). An aliquot of this biomass was weighed accurately (approximately 1g), placed in a drying chamber at 100 ℃ for 24 hours and then weighed accurately (dry weight).

This example shows that a 20L scale column provides a gentle environment for cells, comparable to erlenmeyer flasks and better than a stirred tank reactor. Cell damage is limited and mass and gas transfer is effective under operating conditions.

As already mentioned above, the present invention provides a number of advantages, which in turn are critical for economic benefit:

it provides a mild environment for growing plant cells;

easy to enlarge;

it is disposable;

it is easy to handle.

Claims (9)

1. A bioreactor for culturing living cells in a liquid culture medium, comprising:

at least one stationary groove surrounding the cells and the liquid medium, and

at least one means for introducing a single large bubble at the bottom of the container, wherein the width of the single large bubble is 50 to 99%, preferably 60 to 99%, more preferably 98.5% of the width of the groove.

2. Bioreactor according to claim 1, wherein a single large bubble deviceHas a length of at least 65cm³The volume of (a).

3. Bioreactor according to claims 1 and 2, wherein the bioreactor further comprises at least one means for programming the volume and frequency of the large bubbles.

4. Bioreactor according to claims 1 to 3, wherein the tank is a flexible or inflexible plastic bag.

5. Bioreactor according to claims 1 to 4, wherein the holding trough is surrounded by a rigid outer container.

6. Bioreactor according to claims 1 to 5, wherein the upper part of the tank is flared.

7. Bioreactor according to claims 1 to 6, wherein the tank is cylindrical or has an oval cross section.

8. Use of a bioreactor according to claims 1 to 7 wherein the cells are plants, animal cells or microorganisms.

9. Use of a bioreactor according to claims 1 to 8 wherein the cells are biomass producing cells, embryogenic plant cells, metabolites, secondary plant metabolites and/or recombinant molecules.