CN1260343C - Cell culture device - Google Patents

Abstract

A cell culture assembly comprising at least: the first chamber has a first end, a second end, an inner surface and an outer surface, the inner surface having at least one growth substrate to receive at least one cell and to allow the cell to attach and/or adhere; the second chamber is connected with the second end of the first chamber, the second chamber is provided with a first end, a second end, an inner surface and an outer surface, and at least one separation device is arranged between the second end of the first chamber and the first end of the second chamber; the separation device enables the culture medium to flow back and forth between the first chamber and the second chamber; cell proliferation is promoted by periodically and intermittently supplying cell nutrients and oxygen, and the maximum amount of cell products is produced. Has the advantages of low cost, disposable use, reduced sequelae caused by incomplete sterilization, and periodic and intermittent supply of cell nutrients and oxygen by controlling the frequency of contact between the culture medium and the growth substrate. Cells can be efficiently cultured to achieve minimal mortality and maximum amount of cells and cell products produced.

Description

cell culture device

technical field

The present invention is a cell culture device. When cultivating microorganisms, cells and/or tissues, the biggest obstacle is that the equipment and/or devices used are bulky, expensive, unable to be thrown away after use, difficult to sterilize and maintain a sterile environment , thus increasing the chance of contamination; and when culturing microorganisms, cells and/or tissues, it is easy to cause damage to microorganisms and cells and/or even cause cell death. The present invention provides a technique that can basically be used for culturing any microorganisms and cells in vitro and collecting any by-products produced therefrom. More specifically, the present invention protects and discloses a novel device and/or device and method that can effectively increase and collect by-products produced by microorganisms and cells, such as proteins.

Background technique

As we all know, large-scale cell culture procedures have been widely developed in recent years, and are used for culturing cells with strong cell walls and/or relatively elastic extracellular material (extracellular material), such as bacteria, yeasts and molds. The elastic structure of microbial cells is the key factor for the rapid development of such high-efficiency cell culture procedures. For example, bacterial cells can fully contact the air and maintain good Growth situation. In addition, bacteria can also grow on biofilms, but they need a plane for growth.

On the contrary, the technique of culturing eukaryotic cells, animal cells, mammalian cells and/or tissues is much more difficult and complicated because such cells are more fragile than microbial cells and the nutrients and oxygen required for their growth are maintained It is also more difficult and complex. In addition, unlike microbial cells, animal cells and/or mammalian cells cannot tolerate the severe disturbance and/or shear caused by the introduction of air or mixed gases, such as a mixed gas containing oxygen, nitrogen and carbon dioxide. Stress (shearing force). Moreover, animal cells cannot directly contact the air, and most animal cells can only use oxygen dissolved in the medium. Compared with microbial cells, animal cells and mammalian cells are more likely to be damaged by gases or during ventilation, thereby increasing their mortality. Bioreactors used in large-scale culture usually have an internal rotator, such as: impeller, which makes cells withstand very high liquid shear stress, which causes cell damage or death, resulting in low cell survival rate and its The yield of protein and/or by-products is also reduced. Similarly, bioreactors using other mechanical devices, violent gas movement or strong liquid movement in order to make cells evenly suspended and/or properly exposed to air will also cause cell damage and hinder the growth of cells or tissues, further Causes cellular by-products such as decreased protein production.

The main function of the bioreactor is to cooperate with the research to cultivate a large number of cells to extract the trace active substances derived from them, including but not limited to, proteins or antibodies secreted by the cells into the medium; another function of the bioreactor is to large-scale cell Cultivation is used to mass-produce active proteins produced by cells or tissues to meet commercial needs. Due to the need for eukaryotic cells, and/or prokaryotic cells, and/or animal cells, and/or mammalian cells to be cultured in large quantities in the laboratory, bioreactors and culture devices are used to research and produce cells to produce active proteins, and/or or antibodies, and/or any cellular byproducts play an important role.

Many traditional methods can be used to grow cells on a large or small scale. In terms of small-scale cultivation, many cultivation containers have been developed in recent years, for example, a petri dish is an example. A petri dish basically consists of a bottom pan that can be used to contain culture medium, and a top cover that can be lifted. Although the liftable upper cover is indeed convenient to operate, the frequent lifting of the cover during the culture process often causes the cells to be contaminated by microorganisms. In fact, how to avoid contamination is the biggest challenge for a successful cell or tissue culture technology.

In order to avoid the pollution problems that often occur with petri dishes, culture flasks (cultureflasks) were developed. The culture bottle basically comprises a culture chamber, a tubular opening at one end of the bottle and a corresponding cap. This design is to reduce the chances of cells being exposed to dust, bacteria, yeast and other sources of contamination. Patents on culture flasks include, for example, US Patent No. 4,334,028 owned by Carver Company, US Patent No. 4,851,351 owned by Akamine Company, and US Patent No. 5,398,837 owned by Degrassi.

Although the culture bottle is an improvement over the culture dish, it still cannot completely solve the problem of contamination. In addition, neither the culture dish nor the culture bottle can provide sufficient air, and the growth area provided by the culture bottle is not as sufficient as that of the culture dish, thus limiting the scale of cultivation.

Another technology used to culture cells and tissues is roller bottles, which has been widely used in cell culture for several years, although it has advantages that some dishes or bottles do not have, such as : Provides larger growth and attachment area for cells, but still cannot overcome all shortcomings, especially regarding the expansion of the culture scale. Taken together, these disadvantages include, but are not limited to, enormous fluid shear stresses due to gas headspace and large amounts of rotational agitation. Tissue culture of larger three-dimensional structures is nearly impossible due to the high shear stress environment of roller flasks. Only cells that are immune to shear stress and/or remain attached to the inner wall of the roller flask can be cultured for extended periods of time. It can be seen that the difficulty of maintaining a cell line in a rolling flask for a long time lies in its high shear stress environment and the possibility of contamination. Examples of roller flasks include US Patent No. 5,527,705 owned by Mussi Corporation and US Patent No. 4,962,033 owned by Serkes Corporation.

In addition, although the overall area of a rolling flask is larger than that of a dish or flask, the area provided for each cell to attach is not necessarily larger than that provided by the dish or flask, especially in large-scale cultures.

Therefore, an invention aimed at increasing the growth area provided by the rolling flask, for example, US Patent No. 5,010,013 owned by Serkes Company, describes a rolling flask that can increase the surface area for cell attachment. A corrugated channel is added to the inner wall to increase the inner surface area of the roller flask for cell attachment. However, the general rolling culture bottle only provides an area of about 850-1700 square centimeters, so a large number of rolling culture bottles are still needed to achieve mass production. While the automated, mass-rolling flask culture system saves time and manpower, its operation is quite expensive and restrictive.

In addition to fluid shear stress issues and growth area limitations, obtaining and maintaining adequate oxygen supply is also a central issue in cell and tissue culture technology. As is well known to those skilled in the art, the growth of prokaryotic cells, eukaryotic cells, including animal cells, mammalian cells, insect cells, yeasts and molds, has a major rate-limiting step (rate-limiting step), i.e. oxygen mass Conversion (oxygen mass transfer).

Except for some eukaryotic microorganisms that perform fermentation, such as yeast, for most prokaryotic cells and eukaryotic cells, oxygen metabolism is very important for the metabolic function of cells, especially for mammalian cells and animal cells In culture technology, the supply of oxygen is especially important for the early stages of rapid cell division. Oxygen demand is greatest when cells are in suspension and decreases when cells are aggregated or differentiated. Some mammalian cells and animal cells are anchorage-dependent, and some mammalian cells and animal cells can grow in suspension in a liquid environment, that is, non-attachment-dependent (anchorage-independent), but all cells need to grow in In a fully oxygenated environment. In the later stages of cell culture, whether attachment-dependent or attachment-independent cells, the number of cells per unit volume will increase greatly, thus again requiring a large amount of oxygen mass conversion to provide sufficient oxygen.

Basically, at least for attached growth cells, mechanical agitation and gas injection can be used to supply the oxygen they need. However, as mentioned above, the actions of agitation and aeration will damage the cells, thus reducing their survival rate and Efficiency and yield in whole cell and/or tissue culture. In addition, directly passing gas into the cell and tissue culture medium will cause air bubbles, which is also not conducive to cell survival.

Devices invented to solve the problem of oxygen supply during cell culture, for example: US Patent No. 5,153,131 owned by Wolf et al., is about a bioreactor vessel without blades. The bioreactor is to pass the air through the air channel (airinlet passageway), through a support plate member (support plate member) through the screen (screen), and through the sandwich between the two sides of the outer box (housing) of the reaction tank A flat disk permeable membrane uses the oxygen concentration gradient between the two sides of the outer box to allow oxygen to diffuse through the permeable membrane to the culture chamber.

However, Wolf's bioreactor has a number of disadvantages:

1. In particular, the rate of oxygen diffusion through the flat disk-shaped permeable membrane becomes the main limiting factor of the size of the culture chamber.

2. Another disadvantage is that the flat disk-shaped permeable membrane is designed to be stretchable to have a stirring function, but this point may cause cell death. It is very important to use the stirring function to distribute the air evenly in the medium. However, stirring will also increase the shear stress inside the culture chamber, which may cause damage to the cells as mentioned above. Therefore, when designing bioreactors or culture tanks, how to provide sufficient gas exchange to maintain cultured larger cell structures is quite important and is a practical limitation.

In order to solve the disadvantages of the device, the reactor made of gas permeable material is utilized, for example: U.S. Patent No. 5,702,941 owned by Schwarz et al., entitled "Gas permeable bioreactor and method of use thereof ", is about a container that can rotate horizontally, and its wall is partially made of gas-permeable material, in order to achieve the purpose of gas exchange with the culture medium directly through the gas-permeable material.

However, the scale of the device disclosed by Schwarz is still limited, because the gas exchange needs to be determined according to the size of the air-permeable area. Although Schwarz emphasized that when the surface area of the device increases, the volume of the culture medium also increases relatively, and in its specification The scale of the disclosed preferred embodiments is limited to 1 to 6 inches in diameter and 1/4 to 1 inch in width, which is not suitable for growing three-dimensional cell aggregates and tissues and/or any mass mass production.

Likewise, U.S. Patent No. 5,449,617 to Falkenberg et al., entitled "Culture Vessel for Cell Culture," relates to a horizontally rotating device with a dialysis membrane separating the interior for cell culture. Chamber and nutrient solution storage tank (nutrient medium reservoir), the gas-permeable material is used on the wall of this container, so that the cell culture room can perform gas exchange, but the nutrient solution storage tank is not completely filled with nutrient solution, but the culture solution of the two chambers A large amount of gas is maintained above. However, the Falkenberg vessel was not designed to reduce turbulence inside the cell culture chamber; instead, agitation is necessary to keep the dialysis membrane moist; furthermore, Falkenberg does not mention the use of this vessel for culturing cell aggregates or any type of organization.

There are also inventions to solve the problem of oxygen supply, such as: U.S. Patent No. 5,766,949 owned by Liau, entitled "Method and Apparatus for Culturing Attachment-Dependent Monolayer Cells", which describes a medium that shakes up and down to increase Oxygen supplied culture system. Its main disadvantages are:

1. One of them is the complexity of its device. Liau's system consists of two external storage tanks and a separate culture chamber containing a series of vertical substrate plates. In addition, several peristaltic pumps are required to push the culture medium from One storage tank flows through the culture chamber, to another storage tank, and finally back to the first storage tank. Because Liau's device is complex and part of it is the equipment outside the culture room, such as external pipelines, storage tanks and pumps, it is very likely to introduce external pollution sources into the interior. In addition, because the device is huge, sterilization is not easy To carry out, and consume manpower.

2. Another disadvantage of Liau's invention is that when the culture fluid flows through the system, the fluid shear stress caused may disturb and move the cells growing on the substrate plate, thereby reducing the survival rate of the cells.

3. In addition, the design of the vertical substrate plate is not conducive to cell attachment, because some cells that cannot be attached immediately will fall and accumulate at the bottom of the plate, and most of these cells will eventually die, thus reducing the survival rate of cells As well as protein production, the system has to be restarted over and over again, which is quite inefficient and not ideal.

4. Furthermore, due to the complexity of this system, collecting any secreted proteins or cellular products is cumbersome and time-consuming. Finally, when the growth fluid is lower than the substrate plate on which the cells are grown, the cells will be exposed to air, ie, directly in contact with a gaseous environment, thus resulting in cell death.

Contents of the invention

The present invention provides and discloses a method and device for cell and tissue culture, because cell and tissue culture technology is very important for biotechnology research, drug research, patient care and academic research. In order to overcome the defects and limitations of traditional technology, and make up for its inconvenience, the present invention provides more reliable, less complicated, more efficient, less troublesome, less expensive, less manpower and can increase cell survival rate and cell by-products A new method and device for high output to meet long-standing needs. The device of the present invention simultaneously reduces contamination and increases cell lifespan.

The object of the present invention is to provide a bacterial culture method and its device, which are reliable, simple and cheap, disposable and efficient for culturing cells and/or tissues and collecting the products. More specifically, the present invention provides and discloses a novel method and device that can effectively cultivate cells to be cultured, such as prokaryotic cells, eukaryotic cells, animal cells and mammalian cells, and can continuously provide oxygen and nutrients It does not allow any cells to come into contact with the air, thus reducing cell damage and even cell death.

In addition, the method and device of the present invention can reduce pollution and prevent cells from being directly damaged by shear stress when gas is supplied, thus preventing negative effects of air bubbles and gas on cells. The device of the present invention can be operated automatically or manually. Moreover, the device of the present invention provides a simpler and more convenient method for producing and collecting cell products, such as proteins, and/or antibodies, and/or any cells and/or or a product of an organization.

The object of the present invention is achieved in that a kind of cell culture device is characterized in that: it at least includes:

the first chamber has a first end, a second end, an inner surface and an outer surface, the inner surface having at least one growth substrate for receiving at least one cell and allowing the cell to attach and/or attach;

The second chamber is connected to or connected to the second end of the first chamber, the second chamber has a first end, a second end, an inner surface and an outer surface, at least one partition device is placed on the Between the second end of the first chamber and the first end of the second chamber; the partition means allows culture to flow back and forth between the first chamber and the second chamber;

The cell culture device alternately exposes the growth substrate to air or culture medium intermittently, allowing cells to pass through the thin air-medium interface to increase oxygen exchange.

The second chamber is a compressible member having a first end and a second end, and optionally a first cover is provided on an outer surface of the first end of the compressible member, and a first cover is provided on an outer surface of the second end of the compressible member. The surface provides a second cover, and the first cover is combined with the second cover such that the compressible element in the second chamber is compressed between the first cover and the second cover.

It further includes: an air environment is located inside the first chamber; culture medium is located inside the compressible member in the second chamber, the culture medium passes through a partition device and/or membrane, in the freely move between the first chamber and the second chamber; and optionally compress and expand the compressible member in the second chamber using an actuator, so that the culture is based on the first chamber and the second chamber Moving between chambers, at least one cell-attached and/or attached growth substrate can be submerged in the medium when the second chamber is compressed; the second chamber is stretched through a thin layer of air-medium The interface is indirectly exposed to the air environment to provide oxygen.

The first chamber includes at least one opening, which is used to receive or move culture medium and cells, and to allow the cell culture device to exchange air with the outside environment. The opening of said first chamber is optionally provided with a closing device. The closing device is selectively installed with an air filter to prevent pollution.

The driving device is automatic or manual. The partition device placed between the second end of the first chamber and the first end of the second chamber is a permeable membrane or a porous partition, so that the culture is based on the first chamber The flow back and forth between the chamber and the second chamber.

The permeable membrane is optionally porous.

The growth substrate is a loosely assembled matrix. The matrix has a dissolved oxygen generator, a static mixer or a deep bed filter.

The growth substrates are semipermeable capsules, or porous particles, that provide static agitation, increase dissolved oxygen, filter and trap cells.

The growth substrate includes hollow fibers that are at least half permeable.

Said cells are selected from eukaryotic cells or prokaryotic cells.

The amount of the culture medium of the growth substrate is regulated and/or controlled through a floating bag and an air delivery tube.

The second chamber further comprises a volume regulating device to regulate the amount of culture medium entering the first chamber; a control device is to control the volume regulating device to make the culture medium flow between the first and second chambers, so that When the volume of medium in the second chamber is at a minimum, the medium completely covers the growth substrate, and when the volume of medium in the second chamber is at a maximum, the growth substrate is in indirect contact through an air-medium interface to the air environment of the first chamber.

The volume adjusting device is a bellows, through compression and expansion, to control the volume of the culture medium inside the second chamber, so that when the bellows is compressed to the limit, the volume of the culture medium in the second chamber is the minimum, and when the bellows When stretching to the limit, the volume of the medium in the second chamber is the largest; or a balloon, through inflation and deflation, to control the volume of the medium in the second chamber, so that when the balloon is inflated to the limit, the medium in the The volume of the second chamber is the smallest, and when the balloon is deflated to the limit, the volume of the medium in the second chamber is the largest; or a piston, wherein the piston is pushed up or pulled down to control the medium inside the second chamber volume of.

The driving device is an oil pressure cylinder, a pneumatic cylinder, a screw jack or a gas compressor.

The growth substrate is a porous growth substrate, the cells are covered in the porous growth substrate, and when the growth substrate floats out of the medium, the cells on the growth substrate pass through an air-medium The interface is indirect contact with the air environment, so as to obtain enough oxygen.

The material of the porous growth substrate is selected from the following groups: ceramics, woven substrate, non-woven substrate, polyamide, polyester, polyurethane, fluorocarbon polymer, polyethylene, polypropylene or polyvinyl alcohol.

The form of the porous growth substrate is dish, flake, block, plate, sheet, ribbon, granular, semipermeable particle or semipermeable membrane.

The present invention includes but is not limited to providing a novel method and apparatus for preparing, growing and maintaining cells; providing a novel method and apparatus for growing and/or preparing tissue culture;

Provides a novel method and apparatus for preparing and growing organ cultures;

Provide a novel method and device for culturing cells in an environment with sufficient oxygen and nutrient supply and minimal pollution;

To provide a novel method and device for culturing cells on a porous growth substrate to enhance the exchange of oxygen and nutrients of the cells without directly exposing the cells to air bubbles and/or shear due to gas injection stress;

Provide a growth substrate to enhance cell adhesion, increase the contact area between air and culture medium, and serve as a static medium stirrer in a device;

Provide a novel method and device to allow cells to intermittently immerse or emerge from the culture medium;

To provide a method and apparatus which can minimize cell contamination, which can minimize cell death, thereby increasing the yield of cell products;

Provide a method and device that can greatly reduce or even eliminate fluid shear stress during culture, and minimize cell death;

Provide a method and device that do not need to supply medium oxygen through aeration during cultivation;

To provide a method and apparatus to allow movement of medium, to redistribute dropped cells and to help enhance cell attachment;

Provide a method and device for continuously supplying cell culture medium and oxygen to maximize the yield of cell products and provide more area for cell attachment;

Provide a method and device to efficiently prepare and collect proteins and other products secreted by cells and/or tissues; and provide a method and device for culturing cells, so that the survival rate of cells can be improved after a period of culture.

The present invention provides a cell culture device, which at least includes a chamber. The cell culture device may further include:

The first chamber has a first end, a second end, an inner surface, and an outer surface, wherein the inner surface has at least one structure for receiving at least one cell and allowing cell attachment and/or attachment;

The second chamber is connected to or connected to the second end of the first chamber, the second chamber has a first end, a second end, an inner surface and an outer surface, and includes at least one membrane or a connecting The device is disposed between the second end of the first chamber and the first end of the second chamber.

The second chamber comprises a compressible element having a first end and a second end, and optionally a first cap is provided on the outer surface of the first end of the compressible element, and the compressible The outer surface of the second end of the element provides a second cover, wherein when the second chamber is not in use, the first cover is engageable with the second cover such that the compressible element in the second chamber is compressed between the first cover and the second chamber. between the second cover.

The cell culture device may further include: an air environment is located inside the first chamber; the culture medium is located inside the compressible element in the second chamber, and the culture medium can pass through a communication device and and/or the membrane is free to move between the first chamber and the second chamber; and selectively compresses and expands the compressible element in the second chamber using an actuator, so that the culture is based on the first chamber and the second chamber moving between chambers such that at least one cell-attached and/or attached structure is submerged in medium when the second chamber is compressed and passes through a thin air-medium layer when the second chamber is expanded interface, while indirectly exposed to the air environment to provide oxygen.

The first chamber of the cell culture device includes at least one opening, which can be used to receive or move the culture medium and cells, and allow the cell culture device to exchange air with the outside environment. The opening of the first chamber can optionally be equipped with a closing device, and can optionally be equipped with an air filter to prevent contamination. By controlling the closing device, combined with the compression and expansion of the second chamber, the ability to control the pressure environment of the first chamber can be achieved, and the pressure can be directly exerted on the growth substrate to increase the pressure. For example, constricting the closure while compressing the second chamber increases the pressure in the first chamber; constricting the closure while extending the second chamber decreases the pressure in the first chamber. By increasing or decreasing the pressure on the growth substrate, it can be used to cultivate special types of cells or tissues, such as osteoblast or artificial bone tissue.

The cell culture device can be used for three-dimensional tissue culture.

A first type of embodiment of the present invention is a cell culture device, which at least includes: a first chamber in which growth substrate means (growth substrate means) for cell attachment and growth are placed, wherein the growth substrate is placed in the first chamber In the air environment of a chamber, the novel growth substrate of the present invention can be used as a deep bed filter (depth filter) to capture and retain cells when inoculating cells (cell plating), and can also increase air and medium The contact area and as a static mixer (static mixer). The device also includes a second chamber selectively connected to the first chamber by means of a permeable, and preferably porous, membrane, and the first chamber may be located in the second chamber Above, below, and/or beside, and/or in a surrounding manner, it can help the culture medium to freely flow between the first and second chambers.

The device of the present invention includes a driving device, which can drive the culture base to go back and forth between the chambers, so as to gently redistribute the dropped cells back to the growth substrate, thereby enhancing the cell attachment ability. By mixing the culture medium, the Facilitates the contact of gases with the medium and ensures an even distribution of nutrients and gases without harming or killing cells.

The driving device used in the present invention includes: lifter, hydraulic cylinder, pneumatic cylinder, gas compressor, screw jack (screw jack), pulley (pulley) and the like.

Another type of embodiment of the present invention is a cell culture device comprising at least: a first chamber containing and/or supporting a growth substrate that provides cell attachment and growth, wherein the growth substrate is placed in an air In the first chamber of the environment, the device of the present invention may also optionally include a second chamber connected to the first chamber and used to preserve the culture medium, wherein the second chamber includes at least one adjustable and/or regulating A volumetric device that can be used to adjust the amount of medium in the second chamber so that when the medium in the second chamber is at a minimum, most of the medium can flow into the first chamber, whereas when the second chamber When the culture medium is adjusted to the maximum amount, most of the culture medium flows back into the second chamber. Such volume adjustment devices may include, but are not limited to, a compressible chamber such as bellows, pistons, balloons, buoyant bladders with air tubes, air pressure, and/or any other means that can move culture-based back and forth between chambers.

The device of the present invention may also include a drive device and a volume adjustment device for controlling fluid and/or liquid, which can further help the medium to flow intermittently between the first chamber and the second chamber, so that the medium can completely cover the growth Substrate to provide cell nutrients and dissolved oxygen. Thus, when the medium is at a minimum, the cells on the growth substrate will be exposed to a thin air-medium interface for adequate oxygen supply. When the medium in the first chamber is at its maximum, the cells are exposed to the nutrients in the medium. By regulating the amount of medium in the first chamber, when the cells touch the air-medium interface, there is a thin layer of water film between the cells and the air, so that sufficient oxygen can be provided to the cells without allowing The cells are too dry, and the cells will not be directly exposed to shear stress and/or air bubbles due to the entry of air, causing damage or adverse effects to the cells.

Yet another embodiment of the present invention relates to a method for culturing cells, such as prokaryotic cells, eukaryotic cells and/or mammalian cells. The novel method provided by the present invention at least includes the following steps: providing a first chamber; placing a growth substrate for cultivating cells and strengthening cell adhesion; providing a second chamber; connecting the second chamber to the first chamber ; providing culture based on the second chamber; selectively installing a fluid and/or liquid regulating device in the second chamber, so that the medium intermittently flows between the first and second chambers to provide cells Sufficient nutrients and oxygen without harming or killing cells; redistributing dropped cells to increase cell attachment; and providing an air-medium interface and providing a means to replenish fresh media and collect mechanism of cellular production. Therefore, the present invention not only improves cell yield by increasing cell attachment, but also provides a simple collection channel, and the device of the present invention can be sterilized in advance and discarded after use, which minimizes the chance of infection in culture and avoids Problems caused by repeated sterilization. Furthermore, the present invention provides a novel method that intermittently, but indirectly, provides oxygen to cells through a thin air-medium interface without harming and/or killing the cells.

Another embodiment of the present invention is the method according to the previous embodiment, further comprising collecting the desired cell-secreted product from the culture medium. The method at least includes the following steps: collecting the medium from the cell culture device; collecting the protein secreted by the cells in the medium; providing and replenishing fresh medium to the first chamber to an amount sufficient for cell growth; and periodically and intermittently moving the medium back and forth between the first and second chambers so that when the medium in the second chamber is adjusted to a minimum, the medium can flow to the first chamber so that the growth substrate can be immersed in the medium, When the medium in the second chamber is adjusted to the maximum amount, the medium will return to the second chamber, so that the growth substrate can contact the air environment through the air-medium interface to obtain oxygen. This intermittent, but indirect, method of exposing cells on the growth substrate to an oxygen-enriched environment using the air-medium interface and submerging them in the medium provides a balanced supply of oxygen and nutrients. supply without harming and/or killing cells.

Yet another embodiment of the present invention is a cell culture device comprising at least: a chamber having a first end and a second end capable of containing a culture medium and culturing cells at the second end and containing an air environment at the first end ; a growth substrate, placed inside the chamber for cell attachment and growth; a medium storage tank, which is equipped with a medium and connected to the chamber; and a driving device, which can be used intermittently and periodically The culture is based on the flow between the medium storage tank and the chamber, so that the growth substrate is immersed in the medium or indirectly exposed to the air environment through a layer of air-medium film; a scale combination is combined with a driving device that can sense The weight of the chamber to control the opening or closing of the actuator. The cell culture device can further utilize a combination of a rotating shaft and a platform to increase the mixing effect of cell inoculation and culture medium by rotating and agitating.

The following describes in detail in conjunction with preferred embodiments and accompanying drawings.

Description of drawings

Figure 1 is a schematic diagram of embodiment 1 of the cell culture device of the present invention, wherein the second chamber is located below the first chamber, and can optionally have compressibility, so that the culture medium can be in the first and second chambers Between flow, wherein the compressible second chamber is not compressed at this time, so the growth substrate is exposed to an air-medium interface to provide oxygen.

FIG. 2 shows the second chamber of FIG. 1 in a compressed state, and the growth substrate is immersed in the culture medium to allow the cells to obtain nutrients.

3 is a schematic diagram of embodiment 2 of the cell culture device of the present invention, wherein an expandable balloon is placed and/or located in the second chamber, and the balloon is in a deflated state, so that the culture medium is retained in the second chamber , so the growth substrate in the first chamber is exposed to the air environment through an air-medium thin layer interface.

Fig. 4 shows that the balloon of Fig. 3 is inflated, thereby pushing the culture medium from the second chamber to the first chamber, so that the cells on the growth substrate can be submerged in the culture medium.

Fig. 5 is a schematic diagram of embodiment 3 of the cell culture device of the present invention, wherein a piston is placed in the second chamber, and is fully elongated so that the culture medium stays in the second chamber, so the growth medium in the first chamber The material can be indirectly exposed to the air environment through an air-medium thin layer interface, so the cells are never directly exposed to gas or air.

Fig. 6 is that Fig. 5 pushes up the piston of the second chamber to force the culture medium to flow or transport or move into the first chamber from the second chamber so that the growth substrate can be immersed in the culture medium to Provides nutrients for cells.

Fig. 7 is a schematic diagram of embodiment 4 of the cell culture device of the present invention, wherein the growth substrate is a hollow fiber.

Fig. 8 is a schematic diagram of embodiment 5 of the cell culture device of the present invention, wherein the growth substrate is a semi-permeable membrane capsule and/or container and/or carrier.

Fig. 9 is a schematic diagram of embodiment 6 of the cell culture device of the present invention, wherein after the inflation device of the second chamber is inflated, the growth substrate in the first chamber will float on the culture medium, and through an air-culture The base interface is indirectly exposed to the air environment.

Fig. 10 shows that the growth substrate in the first chamber will be immersed in the culture medium after the inflation device of the second chamber in Fig. 9 is deflated.

Fig. 11 is a schematic diagram of embodiment 7 of the cell culture device of the present invention, wherein the upper chamber is compressible, and the flow direction of the culture medium in the lower chamber can be controlled.

Fig. 12 is Fig. 11. By compressing the upper chamber, the growth substrate in the lower chamber can be immersed in the culture medium.

Fig. 13 is a schematic diagram of embodiment 8 of the cell culture device of the present invention, wherein the medium content of the first chamber is controlled by the air pressure of the second chamber surrounding it.

Fig. 14 is the growth substrate of the present invention, which is immersed in the growth medium by compressing the compressible second chamber.

Figure 15 is a growth substrate of the present invention wherein oxygen is obtained by indirect exposure of the growth substrate to the ambient air through an air-medium interface by expanding the compressible second chamber.

16 is a schematic diagram of Embodiment 9 of the cell culture device of the present invention, which is combined with Embodiment 1 of the present invention, wherein the movement of the second chamber is controlled by a piston mechanism, and when the second chamber is not compressed, The culture medium will stay in it.

Fig. 17 is Fig. 16 using the piston mechanism to control the second chamber in the compressed state.

Fig. 18 is a schematic diagram of Embodiment 10 of the cell culture device of the present invention, which is combined with Embodiment 1 of the present invention, wherein a screw jack is combined as a tool for controlling the compression of the second chamber.

Fig. 19 is the screw jack combination of Fig. 18 used to compress the second chamber so that the culture medium can flow from the second chamber to the first chamber.

Figure 20 is a schematic illustration of Embodiment 11 of the cell culture device of the present invention, which includes a drive mechanism for compressing the chamber containing the culture medium.

Fig. 21 is Fig. 20 including a driving device for compressing the second chamber containing the medium so that the medium can flow from the second chamber to the first chamber.

FIG. 22 is a schematic diagram of embodiment 12 of the cell culture device of the present invention, which uses a balloon as a driving tool, as described in FIGS. 3 and 4 .

Fig. 23 is the balloon of Fig. 22 in an inflated state.

Fig. 24 is a schematic diagram of the thirteenth embodiment of the cell culture device of the present invention, in which different driving devices are used as the volume adjustment device of the cell culture device to make the culture medium flow between the chambers.

Fig. 25 is a schematic diagram of embodiment 14 of the cell culture device of the present invention, which combines embodiment 6 of the present invention with another driving device, so that the pressure of the external chamber is higher than that of the internal chamber, so most culture medium can be produced by The outer chamber flows to the inner chamber.

Figure 26 is a schematic diagram of Embodiment 15 of the cell culture device of the present invention, which is combined with Embodiment 1 of the present invention, and additionally includes two storage tanks connected to the cell culture device of the present invention, so that fresh medium is added to the cells , and the cell product dissolved in the culture medium is collected via connectors and/or tubing connected to the cell culture device.

Figure 27 is a schematic diagram of embodiment 16 of the cell culture device of the present invention, which is combined with embodiment 1 of the present invention, and additionally includes two storage tanks connected to the cell culture device of the present invention, so that fresh medium is added to the cells , and the cell product dissolved in the medium is collected via the pump body contained in the cell culture device.

Figure 28 is Figure 27 selectively using the electronic device system or computer to control the pump body so as to collect the cell products dissolved in the culture medium.

Figure 29 is a schematic diagram of embodiment 17 of the cell culture device of the present invention, which is combined with embodiment 1 of the present invention, and additionally includes two storage tanks connected to the cell culture device of the present invention, so that fresh medium is added to the cells , and use the pump body to collect the cell product dissolved in the culture medium.

Figure 30 is the alternative use of electronic and/or computer controlled pumps of Figure 29 to collect cell products dissolved in culture medium.

Figure 31 is a schematic diagram of an embodiment 18 of the cell culture device of the present invention, comprising at least one cell growth chamber, at least one internal open space, and further comprising a movable growth substrate connected to a driving device, the driving device is A lift allows the growth substrate to be submerged in the medium.

Fig. 32 shows that the driving device in Fig. 31 is a lifter, so that the growth substrate can freely move up and down or left and right, so as to float out of the culture medium into the growth chamber.

Fig. 33 is a schematic diagram of embodiment 19 of the cell culture device of the present invention, which includes at least one cell growth chamber, at least one internal open space, and further includes a movable growth substrate connected with a driving device, and the driving device is A screw jack to allow the growth substrate to be submerged in the medium.

Fig. 34 is Fig. 33 and further includes a movable growth substrate connected with a driving tool, and the driving tool is a screw jack, so that the growth substrate can freely move up and down or left and right, so as to float out of the culture medium, and each The amount of oxygen in the growth chamber is increased to the maximum.

35 is a schematic diagram of Embodiment 20 of the present invention, which includes a chamber filled with a growth substrate for cell attachment and growth, and a storage tank for a culture medium. Use a scale to sense the weight of the chamber. When the weight of the chamber is lower than a certain set value, the controller can send a signal to start the air pump body, and open or close the valve to control the flow of culture medium between the storage tank and the chamber. The growth substrate is immersed in the medium, or indirectly exposed to the air environment through an air-medium thin layer interface. In order to improve the stirring efficiency of the reactor, a combination of a rotating shaft and a platform is used to increase the effect of cell inoculation and medium mixing by rotating and stirring.

Fig. 36 is a schematic diagram of embodiment 21 of the cell culture device of the present invention, comprising a first chamber located above a compressible second chamber, and the interior of the first chamber contains a growth substrate for cell growth, and the second chamber The chamber contains the culture medium; through the movement of a lifting platform and the movement of the movable valve, the liquid flow field inside the cell culture device can be made uniform, that is, the liquid flow inside the growth substrate is from top to bottom, which can facilitate cell retention inside the carrier.

Fig. 37 is a schematic diagram of embodiment 22 of the cell culture device of the present invention, which controls the opening and closing of the gas valve to control the air pressure in the chamber and the flow of the culture medium, so as to perform the action of medium replacement; the medium replacement action The process can be controlled by the computer, and the replacement volume and replacement times can be regulated.

Fig. 38 is a schematic diagram of embodiment 23 of the cell culture device of the present invention, which is an extension of embodiment 22. A catheter is used to connect the culture medium replacement device on the right side and the high-efficiency cell culture device on the left side. The operation of this system is Through the compression and expansion of the compressible chamber, the internal pressure is changed, so the medium is moved from the medium storage chamber to the cell culture chamber, so that the growth substrate is immersed in the medium, or through an air- The thin layer interface of the culture medium is indirectly exposed to the air environment; this device can avoid the trouble of frequent replacement of the culture medium.

Detailed ways

The following examples are only for illustrating the present invention, and should not be used to limit the protection scope of the present invention. The detailed description of the present invention may be referred to in conjunction with the accompanying drawings to help gain a more complete understanding. The following description only describes some preferred examples, but it is not appropriate to impose unnecessary limitations on the present invention. Embodiments of the present invention can be used to culture any cell, such as eukaryotic and prokaryotic cells, especially animal cells and/or mammalian cells. The present invention includes providing a device having at least one chamber, at least one opening and at least one closing device for closing the opening. The chamber may include, for example, cells to be cultured, culture medium, and a growth substrate that provides a surface for cell attachment and growth. In a preferred embodiment, the growth substrate is a loose aggregate that can be used as a filter to capture and retain cells when seeding cells to achieve maximum cell attachment. The growth substrate can also be passed through a The thin layer interface of the gas-medium increases the contact area between the air and the liquid, and the growth substrate can also be used as a static mixer (static mixer). The growth substrate is preferably a porous substrate, and can be of any size, shape, and composition of any material, especially the porous growth substrate of the present invention is one that provides the largest area for cell attachment, growth, gentle agitation, and gentle mixing. Evenly and provide oxygen, but never let the cells directly contact with the air.

The chamber system of the present invention can also promote cell growth by periodically and intermittently exposing the cells to nutrients and air.

The system of the present invention also provides a simple method for harvesting culture medium containing cell products and refreshing medium.

The cell culture device of the present invention also protects the cells from direct contact with any air, air bubbles or any shear stress caused by aeration, thereby avoiding any adverse effects on the cells.

Specifically, the present invention relates to a reliable, simple, inexpensive, disposable, sterile and efficient method and device for culturing cells and/or tissues and collecting their products.

More precisely, the present invention relates to a novel method and device that can be used to culture any cell, no matter eukaryotic, prokaryotic, mammalian or animal cells, and can provide sufficient nutrients and oxygen for cell growth without damage the cells.

In addition, the methods and devices of the present invention prevent or substantially reduce any form of contamination, avoidance of shear stress and protection of cells from direct contact with air, air bubbles or gases.

Furthermore, the device of the present invention can be operated automatically or manually, which can save labor and/or management manpower. In addition, the present invention provides a relatively simple and convenient method and device, which can be used to produce and collect cell or tissue products , such as proteins, antibodies, etc.

The first embodiment of the present invention is a cell culture device comprising at least one chamber, more specifically, the first chamber of the cell culture device contains a growth substrate for cell attachment and growth, Growth substrates can be intermittently and periodically, but not directly, exposed to the air environment for oxygen using a thin air-medium interface, or submerged in the medium to aid in cell growth and make cells product. The first chamber comprises at least one membrane, preferably two, the membrane is preferably porous, more preferably permeable, so that the growth substrate can be placed between the two membranes to control the flow of oxygen and nutrients to the cells absorb. The membrane is permeable to the medium and thus acts as a channel for the medium to pass from one chamber to the other chamber at room temperature.

The device of the present invention may optionally include a second chamber as a storage tank for culture medium, and is connected to the first chamber through a membrane, and part of the second chamber is used as an adjustable volume device, through compression and Decompression allows media to flow between chambers. The first chamber can be located above, below, beside, or surrounding the second chamber.

In addition, the present invention may optionally include a driving device to control the volume adjustment device, thus further helping the medium to flow between the chambers periodically and intermittently, so that the medium can completely cover the cells to provide nutrients and grow The substrate can also float out of the medium, allowing the cells to receive sufficient oxygen through an air-medium interface. This indirect exposure to the air environment allows the cells to obtain sufficient and efficient oxygen supply without harming and/or killing the cells.

Another embodiment of the present invention is a growth substrate that provides cell growth and promotes cell attachment, wherein the growth substrate is placed in the air environment of the first chamber, and the first chamber and the second chamber are combined . In addition, there is a thin film placed between the first chamber and the second chamber. The thin film is preferably porous to serve as a channel for the culture-based flow between the chambers, and the thin film can support the growth substrate. The second chamber is a device with a volume adjustment and/or liquid control, so that the medium can flow between the chambers, so that when the gas in the second chamber is adjusted to a minimum, most of the medium remains. However, when the gas in the second chamber is adjusted to the maximum amount, most of the medium flows to the first chamber. The volume adjusting device can be further selectively regulated, adjusted or controlled by the driving device, so that the culture medium flows back and forth between the first chamber and the second chamber.

Therefore, when the medium volume of the second chamber is at a minimum, the growth substrate of the first chamber is periodically and intermittently immersed in the medium, however when the medium volume of the second chamber is at a maximum , the growth substrate in the first chamber will indirectly contact the air environment through an air-medium interface to obtain sufficient oxygen. This method of indirect exposure to the air environment through the air-medium interface to provide sufficient oxygen does not harm and/or kill the cells because the cells are not directly exposed to the air flow.

The first and second chambers of the present invention may be constructed of any material including, but not limited to, polypropylene, plastic or thermoplastic. The volume of each chamber can be changed, and can be the same or different volumes, the volume of each chamber is preferably between about 10 milliliters and about 5000 milliliters, more preferably between about 50 milliliters and about 2500 milliliters , and optimally between about 100 milliliters and about 1000 milliliters.

In another embodiment, the cell culture device of the present invention comprises at least one membrane or partition device capable of separating the first chamber and the second chamber. The membrane or partition device is permeable and preferably porous. material, and can support and/or support a growth substrate, and also act as a platform.

The permeable membrane may be composed of any material, such as, but not limited to, semi-permeable fibers, semi-permeable polymer support, metal or plastic. The permeable membrane can be of any form capable of supporting, supporting or bearing the weight of the growing substrate. In a preferred embodiment, the membrane is porous and permeable so that medium can pass in and out between the chambers without disturbing the cell culture and/or moving any cells, while acting as a static agitator. In particular, another permeable membrane of the same or similar nature as the first membrane can be added, placed on the other side of the growth substrate, so that the growth substrate can maintain a fixed position in the first chamber when immersed in the medium , not to move.

The growth substrate of the present invention can be any material, including but not limited to, for example: ceramics, biodegradable matrix, high molecular polymer, woven substrate (woven substrate), non-woven substrate (non-woven substrate), polyester Amides, polyesters, polyurethanes, fluorocarbon polymers, polyethylene, polypropylene or polyvinyl alcohol, trimethylamine, glass, silicon and diethylaminoethyl (DEAE). The porous growth substrate can be in any form, shape or size including, but not limited to, discs, flakes, blocks, disks, flakes, ribbons, pellets, microcarriers, microparticles, semipermeable particles, microparticles, Semipermeable membrane or semipermeable hollow fiber. The use of the growth substrates of the present invention increases the surface area for cell attachment and the air-medium area.

The form of the growth substrate can also be, but not limited to, hollow fibers, fiber bundles, cell growth cubes and channeled ceramic cores. The advantages of such growth substrates are: a large number of Increases surface area for cell growth and attachment. The growth substrate of the present invention can also use commercially available microcarrier substrate systems, such as hollow fibers, fiber bundles, cell growth cubes and channeled ceramic cores as described above. The exterior of the microcarriers used in the present invention and their associated substrates can be coated with proteins and/or other biological or chemical features to enhance cell adhesion or selectively increase the adhesion of specific types of cells.

The microcarrier system can help cells grow at the speed required for commercial mass production, and does not require a lot of labor. Compared with rolling flasks and other systems, the cell culture device of the present invention can be used for both small-scale and large-scale Cell replication and cell production, in addition, microcarrier bioreactor systems are well suited for automated large-scale cultivation of attachment-dependent cells.

Traditionally, when microcarrier production systems are used to cultivate attachment-dependent cells, a stirring system is required to provide oxygen to the cells, and one component of the system interacts with the other so that a large number of microcarrier particles carrying cells can be suspended in the medium. In contrast, the present invention does not require such an agitation system to provide sufficient nutrients and oxygen, as the medium flows periodically and intermittently between the chambers to aid agitation and allow any dropped cells to reattach Opportunities for growth substrates to improve cell adhesion, thereby improving cell growth and increasing cell product production.

As a microcarrier material, its surface chemical composition must be able to support cell attachment and growth, and it must not be toxic to cells or their derivative products. The ideal microcarrier diameter is about 75-225μm, but there are also larger or smaller ones. has been used, for example: (U.S. Patent No. 5,114,855 (May 1992); J.Varani, S.Josephs and W.Hillegas, "Human Diploid Fibroblast growth in polystyrenemicrocarriers in aggregates", Cytotechnology, 22:111- 117 (1996)).

The present invention provides a novel granular growth substrate, which can exist in various sizes, and its diameter can be between about 1 mm and about 100 mm, however, any diameter is suitable according to individual needs. In addition, in the present invention, the particles can be in any available shape or form. In a preferred embodiment of the present invention, the growth substrate is an irregular, loosely assembled matrix.

The ideal density of the particles of the present invention can be between 1.02-1.05 g/cc, however, according to the needs of different applications, lighter or heavier materials can also be used. In addition to the difference in surface chemical properties, the hardness, porosity and absorption degree of particles of different materials are also different. The operating characteristics, durability, shelf life and ease of disinfection will all affect the cost of the process. The present invention can be appropriately adjusted from the viewpoint of commercial potential.

A preferred embodiment of the present invention is to provide a porous growth substrate and/or particles that can increase cell growth and survival in at least three ways.

First, when the porous particles are submerged in the medium, there will be gentle agitation in the pores of the particles, for example: using semi-permeable particle microcarriers instead of stirring devices, when the porous growth substrate particles When it flows, it creates a gentle stirring environment, so it can be used as a static stirrer. This gentle stirring effect can promote the growth of cells, distribute nutrients evenly, and redistribute the dropped cells to Increases the likelihood of it reattaching and/or reattaching to the growth substrate. Redistribution of dropped cells, especially targeting attachment-dependent cells, can increase overall cell culture survival.

Second, when the porous growth substrate particle is not submerged in the medium, its internal structure can provide cells with sufficient oxygen, yet protect the cells from direct contact with the gas environment. Therefore, the porous growth substrate particle can be used as an efficient solution. Oxygenator. Direct exposure of cells to gaseous environment will be detrimental to the cells, and further damage the cells or cause death, especially for animal cells, when the growth substrate emerges from the medium, a part or thin layer of the medium will remain in the porous particles On the surface of the porous particles, a thin layer of medium is formed to cover the cells growing or soaked on the surface of the porous particles, so that a thin layer of air-medium interface can be formed to allow the efficient diffusion of oxygen, so that the cells can absorb oxygen efficiently. It does not come into direct contact with the air. In addition, the porous growth substrate particles increase the surface area for cell growth and allow cells to access the air-medium interface.

Thirdly, the porous growth substrate particles can be used as a filter to catch cells when plating and/or inoculate cells in a flat plate, so as to increase the number of cell attachments. The porous growth substrate particles are A loosely assembled matrix allows simple and efficient distribution of cells during seeding and ensures maximum cell attachment to the surface of the porous growth substrate.

The gas used in the present invention can be any gas mixture suitable for culturing any cell, including, but not limited to, air. The first chamber of the device of the present invention comprises at least one opening for receiving or moving medium and cells, and for allowing the cell culture device to exchange air with the outside environment. The opening can have a closing device, and an air filter can be optionally installed between the opening and the closing device to receive air and reduce pollution. The opening can be of any shape or diameter.

This closure may be of any shape or form including, but not limited to, screw caps or spring caps. Additionally, the closing device can be made of any material, including but not limited to, plastic. In a preferred embodiment, the closing device may include an air filter to prevent microorganisms, cells or any contaminants from entering or exiting the cell culture device. Sterile air filters are known to those skilled in the art and are commercially available, for example, from Millipore, MA.

The cells of the invention may be eukaryotic cells and/or prokaryotic cells. In a preferred embodiment, the cells are animal cells, mammalian cells, preferably human cells. Cells can be any prokaryotic or eukaryotic cells, recombinant or not, including: insect cells, such as: Sf-9; primate cells, such as: Vero; mouse cells, such as: BHK or C-127; hamsters Cells, such as CHO; yeasts, such as Saccharomyces or Scizosaccharomyces; or human cells, such as tumors, osteoblasts and mesenchymal stem cells.

Prokaryotic cells can be any aerobic or anaerobic, Gram positive or Gram negative bacteria or recombinant or non-recombinant, including, but not limited to, Escherichia coli and Bacillus subtilis. Any cell can be grown in the cell culture device of the present invention, and in addition, attachment-dependent or non-attachment-dependent cells are also suitable for use in the present invention. Attachment-dependent cells require surface attachment to grow, whereas attachment-independent cells grow in liquid suspension, and all cell types require sufficient oxygen, nutrients, and growth factors for their growth.

In another embodiment, the present invention provides a cell culture device capable of cultivating three-dimensional tissue. The tissue cultured for transplantation needs to meet the standards of the US Food and Drug Administration (FDA) to obtain approval. The FDA standards include: But not limited to, disease-modifying functionality, consistency and reproducibility of tissue culture, and certified sterilization process. In order to achieve the effect of in vivo implementation, the cultured tissue must have a three-dimensional structure, and in order to achieve reproducibility, the environment of the cultured cells must conform to the physiological environment of the human body. As for the certification of the sterilization process, it can be detected through the aseptic detection of the cultured tissue data, to demonstrate its sterility, and to establish instructions.

Portable tissues have three key characteristics:

1) It has an extracellular matrix to maintain mechanical stability and scaffolding.

2) It has the contact between cells to maintain its survival and normal function.

3) It has a three-dimensional shape, which can distinguish the growth and reproduction of sub-cell populations (cell subpopulation).

Standard tissue culture methods (e.g., T-flasks, Petri dishes, roller bottles, and stirred roller bottles) are often unable to grow transplantable tissues that directly replace organ function because of the lack of multi-dimensional ) cell-to-cell contacts, and the overgrowth of unwanted daughter cell populations.

The present invention overcomes the limitations and defects of the traditional technology, for example: overcomes the three-dimensional growth inhibition of the rolling culture flask and the stirring reactor of the traditional technology; provides the control of the pressure/vacuum environment to simulate the growth environment of the real tissue. In addition, regarding the production of virus, the virus output of traditional rolling culture bottle is 195pfu/cell, microcarrier/spinner flask (spinner flask) is 109pfu/cell, and the present invention then is 313pfu/cell, is to increase 2 to 3 times output.

Traditional techniques cause damage to fragile cells due to excessive shear stress. The present invention reduces, reduces and controls the applied disturbance and accompanying stress reduction to avoid damage to certain types of cells.

In traditional techniques, in addition to the culture procedure of the standard rolling flask, there are other mechanical operations that can affect the normal growth of cells, especially some mechanical operations that feed cells. When culturing cells in roller flasks and stirred tank bioreactors, the operation of renewing, exchanging or supplementing the medium, the "medium exchange mode", has several deficiencies. Tank bioreactors are difficult to use, such as perfusion or fed-batch "medium renewal mode", because of the need to add other complex devices, such as centrifugal filters (spin filters), inclined separation plate or tangential flow apparatuses. In addition, when using conventional methods and devices to implement the perfusion mode to refresh the medium, it may happen that the attached cells are washed away.

The present invention can remedy the deficiency of the traditional technology by reducing the shear stress and cell drop during perfusion.

The modulating and/or modulating means of the present invention may include, but is not limited to, a collapsible chamber such as a bellows, float bladder, piston combination or balloon. Furthermore, the means of the present invention for actuating the compressible chambers may be automatic or manual.

The device for adjusting and/or adjusting the volume is further described below. The automatic driving device of the present invention can be controlled by an electronic device, such as a computer. The driving device of the present invention can control its automation by operating any conventional automation device, such as a robot.

The volume adjusting device of the present invention is a compressible chamber, such as a bellows, which can adjust the volume of the culture medium in the bellows by compressing and stretching the bellows, for example: when the bellows is compressed to the limit, the cultivation inside the bellows The volume of the base is the smallest, and when the bellows is stretched to the limit, the volume of the culture medium inside it is the largest. In particular, when the bellows are compressed, the opposite medium will flow from the bellows to the first chamber, and the growth substrate in the first chamber is placed in such a way that the cultured cells are not damaged by shear stress. The chamber containing the growth medium can be located above, below, beside, or in a surrounding form the chamber containing the growth substrate. By compressing and extending the bellows, medium can flow between the chambers at regular and/or irregular intervals to provide the cultured cells with an optimal oxygen concentration, which is conventional to those skilled in the art, For example: those generally familiar with the art know that excessively high oxygen concentrations are harmful to cells, especially highly differentiated cells. Likewise, those of ordinary skill in the art understand that too low an oxygen concentration is detrimental to cell growth, especially animal and mammalian cells that require oxygen for metabolism and growth.

The present invention can also achieve the ability to control the pressure environment of the first chamber by controlling the closing device of the first chamber and matching the compression and expansion of the second chamber, and directly exert pressure on the growth substrate to increase the pressure. effect. For example, constricting the closure while compressing the second chamber increases the pressure in the first chamber; constricting the closure while extending the second chamber decreases the pressure in the first chamber. By increasing or decreasing the pressure, it can be used to cultivate special types of cells or tissues, such as osteoblasts.

In the second embodiment of the present invention, the device for adjusting and/or adjusting the volume is an inflatable balloon, and the volume of the medium retained in the second chamber can be adjusted by inflation or deflation. Therefore, when the balloon is inflated to the limit, the volume of the culture medium in the second chamber is the minimum, and when the balloon is deflated to the limit, the volume of the culture medium in the second chamber is the maximum. The balloon of the present invention can be made of any material, for example: rubber, latex or any stretchable plastic. In addition, because the balloon is placed in the chamber containing the medium, when the balloon is inflated, the relative medium will flow from the original chamber to the chamber containing the growth substrate without subjecting the cultured cells to shear stress s damage.

In addition, when the balloon is deflated, the relative culture medium will flow back to the original chamber from the chamber with the growth substrate, so that the growth substrate is exposed to the air environment, and the cells grown on it can pass through an air environment. -Oxygen is obtained at the interface of the thin layer of the medium. Therefore, no matter what volume adjustment and/or volume adjustment device is used in the cell culture device of the present invention, the cultured cells can be intermittently and periodically immersed in the culture medium to obtain nutrients, or indirectly pass through a layer The gas-liquid thin layer interface comes into contact with the air environment. Inflation and deflation of the balloon can be at regular and/or irregular intervals and can be adjusted for a particular cell type to provide the most appropriate oxygen supply.

In the third embodiment of the present invention, the piston is used as the volume adjustment device, and the culture medium is moved between the chambers by moving the piston, for example: the piston is pushed to the limit, and the volume of the culture medium in the second chamber reaches the maximum. A large amount, and when the piston is drawn back to the limit, the volume of the culture medium located in the second chamber reaches the minimum amount.

In another embodiment, the cell culture device of the present invention includes at least one chamber, and the device may optionally include two chambers, namely, a first chamber and a second chamber. The first chamber can be positioned above, below, beside, or around the second chamber, and vice versa.

A growth substrate can be installed in the first chamber to help cells attach and grow, at least one film, preferably two porous, permeable films, to promote the flow of the culture medium, and provide an air environment to Oxygen is provided to grow the substrate. The growth substrate is optionally fixed and/or attached and/or suspended on the inner wall of the first chamber. The second chamber may contain culture medium, with a device for controlling the culture based movement between the chambers, the moving device in the second chamber may be automatic and/or manual, at any time intervals, regular and/or irregular , to provide optimal oxygen and nutrients to the cultured cells. The first chamber is combined with the second chamber through the thin film. In a preferred embodiment, the growth substrate is placed between two porous thin films.

In addition, the present invention provides a first chamber containing a cell growth substrate and a second chamber containing a culture medium, wherein the first and second chambers have a first end (or a distal end) (distal end) and the second end (or proximal) (proximal end) difference. The second chamber contains culture medium at the distal end and an air environment at the proximal end. Cell culture chambers can be made of any material, such as plastic. The first chamber and the second chamber can be bonded together, or can be selectively integrated.

A fourth embodiment of the present invention utilizes an airtight buoyant capsule and/or a container combination as a device for adjusting volume or adjusting liquid, which can be fixed on a surface or selectively porous membrane, so that the growth substrate is located in the Above it, the growth substrate can be moved vertically up and down by using the air inside the floating bag, and at the same time promote the flow of the medium. The growth substrate can provide oxygen indirectly through an air-liquid thin layer interface; and when the floating bladder is deflated, the growth substrate will be pushed down and immersed in the medium to nourish the cells. The floating bag combination device can be made of any suitable material.

The present invention can optionally be equipped with a driving device, such as an air compressor, to automatically or manually inflate or deflate the floating bag assembly to help provide oxygen and nutrients to the cells attached to the growth substrate.

A sixth embodiment of the present invention provides a cell culture device comprising a first chamber with a growth substrate placed and suspended therein. The first chamber contains a first end (or proximal end) and a second end (or distal end), wherein the distal end of the first chamber has an opening facing the second chamber, and the second chamber also contains the first end (or proximal end) and a second end (or distal end), wherein the proximal end is substantially closed. When the gas is injected from the proximal end of the second chamber, the growth substrate will be immersed in the medium; when the gas is drawn out from the second chamber, the growth substrate will indirectly pass through a thin layer of gas-medium interface And come into contact with the air environment. The pumping or filling of gas into the second chamber can be driven by any driving device, such as an air compressor, and the driving device can be operated automatically or manually.

The present invention provides a novel method for culturing cells, which can be used for culturing eukaryotic cells and/or prokaryotic cells, especially animal cells and mammalian cells. The operating steps of the method of the present invention at least include:

adding culture medium to the first chamber of the cell culture device of the present invention, and suspending the cells on the growth substrate;

providing at least one surface and/or a membrane to support the growth substrate, wherein the surface and/or the membrane are preferably porous;

At least one opening is provided at the first end of the first chamber for adding and/or removing medium and cells, and at least one closing device is used for closing the opening. The closure device is optionally provided with an air filter for exchanging air and reducing contaminants entering the chamber.

The device also provides a second chamber to receive the culture medium, a volume adjustment means to allow the culture medium to move between the chambers, and a method of collecting cell products in the culture medium.

The cell culture device of the present invention may further comprise at least one membrane or a compartment. The membrane or compartment may be permeable and porous, and may be used to separate the first and second chambers.

Furthermore, in a preferred embodiment, both sides of the growth substrate are provided with a thin film or a partitioning device as a compartment for confining the cell-containing particles. The second chamber can contain culture medium and can be located above, below, and/or next to the first chamber, and/or in a surrounding manner, so that the culture medium can be gently mixed between the first chamber and the second chamber. Flow between chambers. The material of the membrane or the partition device can be, for example, semi-permeable fiber, semi-permeable polymer support, metal or plastic. Additionally, the membrane or spacer may support or hold and/or constrain the growing substrate in a fixed position.

The present invention may further comprise the step of seeding and/or plating the cells onto the growth substrate.

In addition, the present invention also provides a novel method that can periodically and/or intermittently move the growth substrate or medium in the cell culture device, so that the cells on the growth substrate can be immersed in the medium to obtain nutrients, Or allow the cells on the growth substrate to float out of the medium, indirectly allowing the cells to pass through a thin layer of gas-medium interface and contact the air environment to obtain oxygen, but without harming and/or kill cells. The present invention also provides a novel method that can directly exert pressure on the growth substrate by controlling the closing device of the first chamber, in conjunction with the compression and expansion of the second chamber, to achieve the ability to simulate the tissue growth environment. For example, constricting the closure while compressing the second chamber increases the pressure in the first chamber; constricting the closure while extending the second chamber decreases the pressure in the first chamber. By increasing or decreasing the pressure, it can be used to cultivate special types of cells or tissues, such as osteoblasts.

In yet another embodiment of the present invention, the method of said embodiment may further comprise collecting the cell product secreted in the culture medium. Secreted cellular products can include, but are not limited to, proteins, DNA, RNA, plastids, antibodies, and viruses. The step of collecting the culture medium, in addition to the steps described, additionally includes:

The culture medium is collected from the opening of the first chamber of the present cell culture device, and the proteins secreted by the cells contained in the culture medium are collected by draining and/or any general method.

Any conventional method of collecting culture medium can be applied to the present invention, for example, draining and/or aspirating the culture medium from the culture chamber. The method of the present invention also provides a method for reinjecting an appropriate amount of fresh medium into the device after removing the spent medium to provide nutrients for the cells. The growth substrate will again be immersed in the medium periodically and intermittently, and as the volume of the second chamber increases from the smallest to the largest, the cells will pass indirectly through a thin layer of gas-medium interface , while exposed to the air environment. Repeat the steps above until the cells can no longer produce the desired cell product. Since the cell device of the present invention is a simple device, it is not expensive, and it is a disposable device, so as to avoid the problems related to device sterilization.

Embodiments of the present invention are further described below in conjunction with the accompanying drawings,

Example 1

Referring to Figures 1-2, Embodiment 1 of the device of the present invention includes that the first chamber 110 contains a growth substrate 120, and the growth substrate 120 can be made of any material, for example: ceramics, biodegradable matrix, polymer Polymer, woven substrate, non-woven substrate, polyamide, polyester, polyurethane, fluorocarbon polymer, polyethylene, polypropylene or polyvinyl alcohol, trimethylamine (tri-methyl amine), glass, silicon and diethylaminoethyl (DEAE). The growth substrate 120 can optionally be porous, and the growth substrate 120 of the present invention can be used for cell attachment, and the attached cells can obtain oxygen without being directly exposed to the air environment. Direct exposure to air can cause injury and death to cells, especially animal and mammalian cells, and loss of culture viability.

The growth substrate 120 can be in any form, shape or size, including, but not limited to, discs, flakes, blocks, disks, flakes, ribbons, granules, microcarriers, semipermeable particles, microparticles, semipermeable Permeable membrane or semi-permeable hollow fiber.

The first chamber 110 is connected with the second chamber 130, and the first chamber 110 and the second chamber 130 can be bonded, or integrated with the second chamber 130, wherein the first chamber 110 and/or The second chamber 130 can contain culture medium, and both chambers have at least one side of an opening that can communicate with each other, allowing liquid and gas to flow and move between the chambers. In Embodiment 1, the second chamber 130 is equipped with a compressible element, which may be in the form of a retractable bellows. The second chamber 130 may contain growth fluid or culture medium.

Figure 1 shows that the compressible element of the second chamber 130 is in a non-compressed state, so that the medium is full of the second chamber, so that the growth substrate 120 will pass indirectly through a thin layer of gas-medium. The layer interface is exposed to the air environment to obtain oxygen.

FIG. 2 shows that the compressible element of the second chamber 130 is in a compressed state. When the second chamber 130 is in a compressed state, the culture medium is moved to the first chamber to immerse the growth substrate 120 in the culture medium. However, when the second chamber 130 is in a non-compressed state, the medium stays in the second chamber 130 and the growth substrate 120 is not immersed in the medium. The first chamber 110 can be provided with at least one opening 140 to load or discharge cells and culture medium to the first chamber 110. The opening 140 can be equipped with a closing device 150, and the closing device 150 includes at least one air filter 160. Used to filter the air entering and leaving the cell culture device to reduce the occurrence of contamination.

Example 2

Referring to Fig. 3-shown in Fig. 4, the embodiment 2 of the device of the present invention comprises that the first chamber 210 contains a growth substrate 220, the growth substrate 220 can be composed of a loosely assembled matrix, and its material includes but not limited to: ceramics , biodegradable matrix, polymer, woven substrate, non-woven substrate, polyamide, polyester, polyurethane, fluorocarbon polymer, polyethylene, polypropylene or polyvinyl alcohol, trimethylamine, glass, silicon and Diethylaminoethyl.

The growth substrate 220 in embodiment 2 can optionally be porous, and the cells attached to the growth substrate 220 can obtain oxygen, but will not be directly exposed to the air environment. Direct exposure to air can cause injury and death to cells, especially animal cells and/or mammalian cells, and loss of viability in culture.

Growth substrate 220 can be any particle in any form, shape, or size, including, but not limited to, discs, flakes, blocks, disks, flakes, ribbons, granules, microcarriers, semipermeable particles, micro Granules, semipermeable membranes, or semipermeable hollow fibers.

The first chamber 210 is combined with the second chamber 230, wherein the first and/or second chambers may include growth fluid and/or culture medium, and have openings at the interface of the two chambers to facilitate liquid Exchange and movement with gas. In the second embodiment, the second chamber 230 is equipped with a compressible element 280, which can be a balloon 280 as a volume adjustment device. The second chamber 230 may optionally contain culture medium.

FIG. 3 shows the balloon 280 in the second chamber 230 in a deflated state, and FIG. 4 shows the balloon 280 in the second chamber 230 in an inflated state. The material of the balloon 280 can be any material, such as rubber, latex or any stretchable plastic.

As shown in FIG. 4 , when the balloon 280 is inflated, the medium in the second chamber 230 is the least amount, and most of the medium is forced to flow into the first chamber 210 so that the growth substrate 220 can be submerged. in the culture medium.

As shown in Figure 3, when the balloon 280 is in the deflated state, the medium in the second chamber 230 is the largest amount, therefore, the growth substrate 220 in the first chamber 210 will pass through a layer of gas-culture medium indirectly. The thin layer interface of the base is exposed to the air environment to obtain oxygen. The first chamber 210 may be provided with at least one opening 240 to load, plate, inoculate, or expel cells onto the growth substrate, to allow culture medium to enter and exit the first chamber 210, and to provide communication between the first chamber 210 and the external environment. gas exchange between them. Opening 240 may be provided with a closure device 250 comprising at least one air filter 260 for filtering air entering and exiting the cell culture device.

Example 3

Referring to Figures 5-6, Embodiment 3 of the device of the present invention includes a growth substrate 320 in the first chamber 310, the growth substrate 320 can be composed of a loosely assembled matrix, and its materials include but are not limited to: ceramics , biodegradable matrix, polymer, woven substrate, non-woven substrate, polyamide, polyester, polyurethane, fluorocarbon polymer, polyethylene, polypropylene or polyvinyl alcohol, trimethylamine, glass, silicon and Diethylaminoethyl. The growth substrate 320 in embodiment 3 can optionally be porous or a loosely assembled matrix that can be composed of particles. The porous growth substrate 320 can make the cells attached to it obtain oxygen, but it will not be directly exposed. in the air environment. Because direct exposure to air may cause injury and death to cells, especially animal cells and/or mammalian cells, and loss of viability in culture.

Growth substrate 320 may be in any form, shape or size, including, but not limited to, discs, flakes, blocks, disks, flakes, ribbons, granular, microcarriers, semipermeable particles, microparticles, semipermeable Permeable membrane or semi-permeable hollow fiber.

The first chamber 310 is combined with the second chamber 330, wherein the first and/or the second chamber may contain culture medium, and both chambers have an opening at the interface of the two chambers. In Embodiment 3, a piston 380 is installed in the second chamber 330 as a volume adjusting device. The piston 380 can be pushed up or pulled down to control the medium flow between the first chamber 310 and the second chamber 330 .

Figure 5 shows the situation where the plunger 380 is pulled down, where the medium volume of the second chamber 330 is at its maximum.

Figure 6 shows the situation where the piston 380 is pushed up, where the medium volume of the second chamber 330 is at a minimum. The piston can be made of any material such as rubber, plastic, metal, synthetic material or polypropylene and can be operated by any conventional method.

When the piston 380 is in the pull-down state, as shown in FIG. 5 , the culture medium stays in the second chamber 330, and the growth substrate 320 in the first chamber 310 can obtain oxygen supply. When the piston 380 is in the push-up state , as shown in FIG. 6 , most of the medium is pushed toward the first chamber 310 so that the growth substrate 320 can be immersed in the medium. The first chamber 310 can optionally be provided with at least one opening 340 to load or discharge cells and medium to enter and exit the first chamber 310, and to provide gas exchange between the first chamber 310 and the external environment. The opening 340 can be provided with a closing device 350, and the closing device 350 can optionally be provided with at least one air filter 360 for filtering the air entering and exiting the cell culture device.

Example 4

Referring to Fig. 7, embodiment 3 of the device of the present invention includes, in the first chamber 410, at least the growth substrate 420 comprising semi-hollow fibers, the growth substrate 420 of the semi-hollow fibers can be made of any material, for example: braided substrate materials, nonwoven substrates, biodegradable matrices, polyamides, polyesters, polyurethanes, fluorocarbon polymers, polyethylene, polypropylene or polyvinyl alcohol, trimethylamine, glass, silicon, and diethylaminoethyl. The semi-hollow fiber growth substrate 420 allows cells attached thereto to obtain oxygen without being directly exposed to the air environment. Because direct exposure to air may cause injury and death to cells, especially animal cells and/or mammalian cells, and loss of viability in culture.

The first chamber 410 is attached or integrated with the second chamber 430, wherein the first and/or the second chamber may contain culture medium, and both chambers have an opening at the interface of the two chambers. In this preferred embodiment 4, the second chamber 430 can be in the form of a bellows, which adjusts the volume of the culture medium inside the second chamber 430 through compression or decompression, as shown in FIGS. 1 and 2 .

When the second chamber 430 is in a compressed state (not shown in the figure), the culture medium flows into the first chamber 410 so that the semi-hollow fiber growth substrate 420 is immersed in the culture medium so that the cells can obtain nutrients. When the second chamber 430 is in the uncompressed state, the culture medium is returned to and/or retained in the second chamber 430 to provide oxygen to the cells growing on or in the semi-hollow fiber growth substrate 420 . The first chamber 410 can optionally be provided with at least one opening 441 for loading cells into the semi-hollow fiber growth substrate 420 , and can be provided with a closing device 451 to repeatedly open and close the opening 441 . The first chamber 410 may also be provided with a second opening 440 to fill or drain culture medium, as well as to provide gas exchange with the external environment. The opening 440 can also be equipped with a closing device 450, and the closing device 450 is equipped with at least one air filter 460 for filtering the air entering and leaving the cell culture device.

Example 5

Referring to Fig. 8, Embodiment 5 of the device of the present invention includes, in the first chamber 510, at least a growth substrate 520 comprising a semi-permeable capsule, the growth substrate 520 of the semi-permeable capsule can be made of any material, for example: braided substrate materials, non-woven substrates, polyamide, polyester, polyurethane, fluorocarbon polymers, polyethylene, polypropylene or polyvinyl alcohol. The growth substrate 520 of the semi-permeable capsule allows attached cells to pass through a thin air-medium interface, obtaining oxygen without direct exposure to the air environment. The growth substrate 520 of the semi-permeable capsule or container is optionally permeable to gases but not to cells.

The first chamber 510 is combined with the second chamber 530, wherein the first and/or the second chamber may contain culture medium, and there is an opening at the interface of the two chambers for communication. In Embodiment 5, the second chamber 530 can be a compressible chamber, such as a bellows, through which the culture can flow between the first chamber 510 and the second chamber 530 through compression or expansion.

When the second chamber 530 is in a compressed state, the culture medium is flowed or forced into the first chamber 510, so that the growth substrate 520 of the semi-permeable capsule is immersed in the culture medium to allow the cells to obtain nutrients. When the second chamber 530 is in an uncompressed state, the culture medium is returned to and/or retained in the second chamber 530 to oxygenate the growth substrate 520 of the semi-permeable capsule in the first chamber 510 . The first chamber 510 may be further provided with at least one opening 541 for loading cells into the growth substrate 520 of the semi-permeable capsule, and may be provided with a suitable closing device 551 for repeatedly opening and closing the opening 541 . The first chamber 510 may also be further provided with a second opening 540 for loading or discharging culture medium, as well as providing gas exchange with the external environment. The second opening 540 may also be provided with a second closing device 550 which is optionally provided with at least one suitable air filter 560 for filtering air entering and exiting the cell culture device.

Example 6

Referring to Figures 9-10, Embodiment 6 of the device of the present invention includes that the growth substrate 620 is included in the first chamber 610, and the growth substrate 620 is floated on or Immerse in medium. As shown in FIG. 9 , when the floating bag assembly 628 is fully inflated, the growth substrate 620 will indirectly contact the air environment of the culture chamber 610 , and the cells can obtain oxygen through an air-medium interface. As shown in FIG. 10 , when the floating cell assembly 628 is largely deflated, the growth substrate 620 will be immersed in the culture medium, allowing the cells on it to obtain nutrients.

The culture chamber 610 can optionally be provided with at least one opening 640 to fill or drain culture medium, and to provide gas exchange with the external environment. The opening 640 can be equipped with a closing device 650 so that the opening 640 can be opened and closed repeatedly. The closing device 650 may further be provided with at least one air filter 660 for filtering the air entering and exiting the cell culture device.

Example 7

Referring to Figures 11-12, Embodiment 7 of the device of the present invention includes that the first chamber 710 contains a culture medium, and the second chamber 730 is combined with the first chamber 710 and placed in the first chamber 710 above. The second chamber 730 is connected to the growth substrate 720 through the cable 729. The growth substrate 720 can be composed of any material such as: ceramics, biodegradable matrix, high molecular polymer, woven substrate, non-woven substrate, polyester Amides, polyesters, polyurethanes, fluorocarbon polymers, polyethylene, polypropylene or polyvinyl alcohol, trimethylamine, glass, silicon and diethylaminoethyl. The growth substrate 720 in embodiment 7 can optionally be porous, a porous growth substrate that allows the cells attached to it to obtain oxygen, but will not be directly exposed to the air environment, because air flow or air bubbles produce Direct exposure to air may cause injury and death to cells, especially animal cells and/or mammalian cells, and loss of viability in culture.

Growth substrate 720 may be in any form, shape or size, including, but not limited to, discs, flakes, blocks, discs, flakes, ribbons, granules, microcarriers, semipermeable particles, microparticles, semipermeable Permeable membrane or semi-permeable hollow fiber.

The first chamber 710 is combined with the second chamber 730, wherein the first and/or the second chamber may contain culture medium, and the two chambers may communicate directly through an interface. In Embodiment 7, the second chamber 730 can be a compressible chamber, such as a compressible or expandable bellows. FIG. 11 shows the second chamber 730 in an expanded state, and FIG. 12 shows the second chamber 730 in a compressed state.

As shown in Figure 12, when the second chamber 730 is compressed, the growth substrate 720 is lowered and immersed in the medium in the first chamber 710, and when the second chamber 730 is not compressed, the growth substrate 720 in the first chamber The growth substrate 720 of 710 can float out of the culture medium and contact the air environment of the second chamber 730 indirectly through a thin layer of gas-medium interface to obtain oxygen. The second chamber 730 may further be provided with at least one opening 740 . The opening 740 can be provided with a closing device 750, and the closing device 750 can be further provided with at least one air filter 760 for filtering the air entering and exiting the cell culture device.

Example 8

Referring to Figure 13, Embodiment 8 of the device of the present invention includes two culture chambers 810 and 810', wherein the chamber 810 is installed in the chamber 810', and the chamber 810' surrounds the chamber 810 . Chamber 810 includes a growth substrate for receiving cells and has an opening at the bottom so that medium can freely flow between chambers 810 and 810' through the opening. The bottom surface of the chamber 810' can optionally be designed with a raised surface or a curved contour (contour) 821' (such as a cone), so that the water flow is streamlined and can avoid accumulation of unattached growth substrate 820. cell.

The chamber 810 may further be provided with at least one opening 840 to load or drain cells and/or allow medium to enter and exit the culture chamber 810 . The opening 840 can be provided with a closing device 850 to repeatedly open and close the opening 840 . The closing device 850 may further be provided with at least one air filter 860 for filtering the air entering and exiting the cell culture device. The chamber 810' may further be provided with at least one opening 840' and may be provided with a suitable closing device 850'. The closing device 850' can further be equipped with at least one air filter 860' for filtering the air entering and exiting the cell culture device.

The control of the culture medium content in the chamber 810 is to change the gas space of the chamber 810' by blowing air into the chamber 810' through the air filter 860', and this pressure will drive the culture medium to flow into the chamber from the chamber 810' 810. When the gas chamber 810' becomes smaller, the growth substrate 820 is immersed in the medium; when the gas chamber 810' becomes larger, the vacuum of the gas chamber 810' causes the medium to flow back into the chamber 810', thus allowing The growth substrate 820 of the chamber 810 may be exposed to the air environment indirectly through a thin gas-medium interface.

The growth substrate 820 can be placed in the chamber 810 alone, in the chamber 810' alone, or in the chamber 810 and the chamber 810' at the same time.

In addition, as shown in Figure 14, the growth substrate 120 of the device of the present invention is immersed in the culture medium, and can obtain a considerable amount of nutrients. When the selective porous growth substrate is immersed in the culture medium, a kind of The phenomenon, called static agitation, gently agitates the medium and nutrients and redistributes dropped cells evenly back into the growth substrate 120 .

Referring to shown in Figure 15, it is that when the medium height is lower than the growth substrate 120 of the present invention, the growth substrate 120 can obtain a considerable amount of oxygen. At the same time, there will be a thin layer of culture medium remaining on the porous growth substrate. This thin layer can protect the cells on and/or in the growth substrate 120 from being directly exposed to the air environment due to air flow. the resulting shear stress.

Example 9

Referring to Fig. 16-shown in Fig. 17, embodiment 9 of the device of the present invention is that embodiment 1 of Fig. 1 and Fig. 2 is combined with a driving device 1012, for example: an oil pressure cylinder (oil pressure cylinder) or a pneumatic cylinder ( air pres sure cylinder) as a combination, installed in the bottom of the compressible second chamber 1030. As shown in FIG. 17 , when the pressure cylinder 1012 is raised; or as shown in FIG. 16 , when the pressure cylinder 1012 is lowered, the second chamber 1030 is periodically and intermittently compressed and expanded. The compression and stretching of the second chamber 1030 causes the growth substrate 1020 to move periodically and intermittently between the chamber 1010 and the chamber 1030, thereby immersing the growth substrate 1020 in the culture medium to obtain nutrients, or The surfacing medium allows the cells on the growth substrate 1020 to receive oxygen.

Example 10

Referring to Fig. 18-Fig. 19, the embodiment 10 of the cell culture device of the present invention is to combine the embodiment 1 of Fig. 1 and Fig. 2 with a volume adjustment device 1117, for example: a screw jack is combined, and the installation At the bottom of the second chamber 1130 with a compression element. As shown in FIG. 19 , when the screw jack 1117 is raised; or as shown in FIG. 18 , when the pressure cylinder 1117 is lowered, the second chamber 1130 is compressed and expanded periodically and intermittently. The compression and expansion of the second chamber 1130 causes the growth substrate 1120 to be periodically and intermittently immersed in the culture medium, and indirectly exposed to the air environment through a thin layer of gas-medium interface to obtain oxygen.

Example 11

Referring to Fig. 20-shown in Fig. 21, the embodiment 11 of the device of the present invention is to combine the embodiment 1 of Fig. 1 and Fig. 2 with volume adjustment devices 1219 and 1220, for example: drive shaft rod (shaft driver) and drive device 1218, for example : A motor or a step motor (step motor), installed at the bottom of the compressible second chamber 1230. As shown in FIG. 21 , when the driving shaft is raised; or as shown in FIG. 20 , when the driving shaft is falling, the second chamber 1230 is periodically and intermittently compressed and stretched. The compression and expansion of the second chamber 1230 enables the growth substrate 1220 of the first chamber 1210 to immerse or float in the culture medium periodically and intermittently.

Example 12

Referring to Fig. 22-Fig. 23, embodiment 12 of the device of the present invention is the combination of embodiment 2 in Fig. 3 and Fig. 4 and a gas compression driving device. Valve port (valve port) 1322 is in open state, and when valve hole 1324 is opened, and when valve hole 1323 is closed, air just is driven into balloon 1380 by air pump body 1325 and makes it inflate. Therefore, as shown in FIG. 23 , the volume in the second chamber 1330 will decrease, and the medium will be pushed from the second chamber 1330 to the first chamber 1310 , so that the growth substrate 1320 is immersed in the medium. The switching of the helical valve holes 1323 and 1324 can be controlled by the following, for example: a timer 1326 to control, the timer 1326 can change the opening/closing state of the helical valve holes 1323 and 1324 . When the steam valve hole 1324 is closed and the steam valve hole 1323 is opened, the gas in the balloon will be squeezed out due to the pressure caused by the weight of the medium, so that the medium flows from the first chamber 1310 to the second chamber 1330, The growth substrate 1320 is exposed to air. Switching the inflated and deflated states of the balloon 1380 allows the growth substrate 1320 to immerse and float in the culture medium periodically and intermittently, and indirectly contact the air environment through a thin layer of gas-medium interface.

Example 13

Referring to Figure 24, Embodiment 13 of the device of the present invention is the combination of Embodiment 6 of Figures 9-10 and a gas compression driving device. The growth substrate 1420 can be submerged and floated in the culture medium by adjusting the gas content in the floating bag 1428 and the gas delivery tube 1427 . The floating bladder 1428 is attached to the bottom of the growth substrate 1420, and the floating bladder 1428 can be inflated or deflated by a driving device connected to at least one solenoid valve hole, such as an air pump body 1430 . During operation, the valve holes 1433 and 1436 are always open, and when the valve holes 1432 and 1437 are opened, the valve holes 1434 and 1435 are closed, and the air is driven into the floating bag 1428 by the air pump body 1430 and makes the floating bag 1428 Inflated, the growth substrate 1420 is then pushed up and exposes the cells to the air environment of the first chamber 1410 indirectly through a thin layer of gas-medium interface. The switch of the solenoid valve hole can be controlled as follows, for example: a timer 1431 is controlled. The timer 1431 can change the opening/closing state of the solenoid valve holes. When the valve holes 1432 and 1437 are closed, and the valve holes 1434 and 1435 are opened, the gas in the floating bag 1428 will be emptied by the air pump body 1430. The growth substrate 1420 is allowed to submerge in the medium.

Example 14

Referring to Fig. 25, embodiment 14 of the device of the present invention combines embodiment 8 of Fig. 13 with a gas compression driving device. The valve holes 1533 and 1536 are open, and when the solenoid valve holes 1534 and 1535 are opened, the other valve holes 1532 and 1537 are closed, and the gas compressor 1530 will push air into the inner surface of the first chamber 1510, so , the volume of the culture medium in the first chamber decreases due to the air pressure, because the lower side of the chamber 1510 and the chamber 1510' can communicate, so when the volume of the culture medium in the chamber 1510 decreases, the volume of the culture medium in the chamber 1510' is just will rise, and vice versa. Secondly, when the valve holes 1532 and 1537 are open, and when the valve holes 1534 and 1535 are closed, the valve holes 1533 and 1536 are still open and will not change. After that, the air will be pushed into the external culture chamber 1510', so that The medium volume in chamber 1510' decreases while at the same time the medium volume in chamber 1510 increases. By repeating this operation, the culture medium in the chamber 1510 and the chamber 1510' is periodically and indirectly moved between the two chambers, so that the growth substrate 1520 is immersed in the culture medium to obtain nutrients, or to float for culture. The substrate obtains oxygen through a thin gas-medium interface.

Example 15

Referring to Fig. 25, embodiment 15 of the device of the present invention is the combination of embodiment 1 in Fig. 1 and Fig. 2 with a culture medium exchange system. The media exchange system can be operated in two different modes: media exchange mode and growth mode. In the growth mode, the valves 1643 and 1644 are closed to close the couplers 1645 and 1646. Similarly, the valve and the air inlet 1647 are opened to allow the embodiment 1 of FIGS. 1 and 2 to pass through the air filter 160 ( The figure is not) for gas exchange, in this state, the culture medium cannot enter and exit the cell culture device. Once the medium is exhausted and/or the cells have produced a sufficient amount of protein, the medium is harvested for protein and replaced with fresh medium.

In medium exchange mode, valve and air inlet 1647 are closed, and valve 1643 is closed. Using the residual gas pressure of the first chamber 1610 plus the compression of the second chamber 1630 (eg, a bellows), the valve 1644 is opened to allow the spent culture medium to flow out through the coupler 1646 to the storage tank 1642 . After the old medium flows out, the valve 1644 will be closed, and then the valve 1643 will be opened, and the fresh medium located in the storage tank 1641 will be sucked into the second chamber 1630 through the coupler 1645 . The expansion of the second chamber 1630 attracts fresh medium from the storage tank 1641 through the coupler 1645 to the cell culture device. Valve 1643 is closed once a sufficient amount of medium has been reached. To return to the normal growth mode again, the valve and the air inlet 1647 are opened, and all valves can be controlled by an electronic device, such as a computer 1648, and the operation mode can be automatic or manual.

Example 16

Referring to Fig. 27-Fig. 28, embodiment 16 of the device of the present invention is the combination of embodiment 1 in Fig. 1 and Fig. 2 and a culture medium exchange system. Wherein Fig. 27 is manual operation, Fig. 28 is automatic operation.

The cell culture device can be operated in two different modes: medium exchange mode and growth mode.

In the growth mode, the pump bodies 1743 and 1744 are in a closed state. Similarly, the air inlet 1760 is opened to allow the embodiment 1 of FIGS. 1 and 2 to exchange gas through the air filter 160 (not shown). , in this state, fresh medium cannot enter or exit the first and/or second chamber of the cell culture device, once the medium is used up and/or the protein produced by the cells has reached a sufficient amount, the medium will be collected , to obtain protein, and replace with fresh medium.

In the medium exchange mode, the valve and the air inlet 1760 are closed, and the pump body 1744 is activated to push out the used medium in the second chamber 1730 , through the coupler 1750 , and then transported to the storage tank 1742 . After the old medium flows out, the pump body 1744 is closed. To return to normal growth mode again, the air inlet 1760 is open and all pump controls can be driven by a computer and/or electronic device 1748, either automatically or manually.

Example 17

Referring to Figures 29-30, Embodiment 17 of the device of the present invention is a cell culture device as shown in Figures 1 and 2 combined with a medium exchange system. Figure 29 is manual operation, Figure 30 is automatic operation.

The medium exchange device can be operated in two different modes: medium exchange mode and growth mode.

In the growth mode, the pump bodies 1843 and 1844 are in a closed state. Similarly, the air inlet 1860 is opened to allow the cell culture device as shown in FIGS. 1 and 2 to pass through the air filter 160 (not shown) for gas Exchange, in this state, fresh medium cannot enter and exit the first and/or second chamber of the cell culture device, once the medium is used up and/or the protein produced by the cells has reached a sufficient amount, the medium will be exchanged Harvest for protein and replace with fresh medium.

In medium exchange mode, air inlet 1860 is closed and pump 1844 is activated to push spent medium out of the cell culture device and into storage tank 1842 . After the old medium flows out, the pump body 1844 is closed. The pump body 1843 is activated to replenish the fresh medium in the storage tank 1841 to the cell culture device. When enough fresh medium is reached, the pump 1843 is turned off. To return to normal growth mode again, the air inlet 1860 is open and all pump controls can be driven by a computer and/or electronic device 1848, either automatically or manually.

Example 18

Referring to Figures 31-32, Embodiment 18 of the device of the present invention includes at least one cell growth chamber, which contains at least one chamber inner wall 1992, wherein each chamber has a first end, a second end, a The inner surface and an outer surface are used to define an opening. The cell culture device of the present invention may have a second chamber, which may be located above, below, and/or beside the first chamber, and/or in an inner/outer surrounding manner. At least one of the growth chambers is provided with wheels or supports 1990, so that the cell culture device of this embodiment can be supported by a flat surface, such as a floor, a desktop or a laboratory operating table. Each growth chamber has at least one growth substrate 1991 to grow at least one cell and to allow cell attachment and/or attachment.

At least one of the growth chambers contains a culture medium and a gas environment such that the culture medium is located at the second end of the culture chamber and the air environment is located and/or contained at the first end of the culture chamber. Embodiments of the present invention also include a driving device 1995 for moving the growth substrate up and down or left and right between the first end and the second end of the growth chamber, so that the growth substrate can be immersed periodically and intermittently, and / Or float out of the culture medium to provide cells with proper nutrients and oxygen. The driving device 1995 may optionally include a first connecting device 1994, which is used to connect the driving device 1995 and the second connecting device 1993. The driving device 1995 is connected with the growth substrate 1991, and its function is to make the growth substrate repeatedly and periodically Intermittently move from the first end of the growth chamber to the second end, that is, move the growth substrate in the growth chamber so that it can repeatedly immerse and float out of the medium to provide suitable nutrients and oxygen. The driving device of the present invention can be a lifter, and the lifter system can help the growth substrate to move up and down or left and right.

The growth substrate 1991 in this embodiment 18 can optionally be porous, and the porous growth substrate can allow the cells attached to it to obtain oxygen, but will not be directly exposed to the shear caused by air flow or air bubbles in the air environment. stress. Because direct exposure to air may cause injury and death to cells, especially animal cells and/or mammalian cells, and loss of culture viability.

The growth substrate 1991 can be in any form, shape or size, including, but not limited to, discs, flakes, blocks, disks, flakes, ribbons, granular, microcarriers, semipermeable particles, microparticles, semipermeable Permeable membranes or semi-permeable hollow fibers, optionally utilizing a porous membrane housing to accommodate growth substrates such as porous particles or microcarriers, allowing media to flow. Alternatively, a fixed support structure may be used to support the growth substrate so that it can be moved into and out of the medium.

Example 19

Referring to Fig. 33-shown in Fig. 34, embodiment 19 of the device of the present invention comprises that the first growth chamber contains at least one chamber inner wall 2092, wherein each chamber has a first end, a second end, an inner surface and An outer surface is used to define an opening. The cell culture device of the present invention may have a second chamber, which may be located above, below, and/or beside the first chamber, and/or in an inner/outer surrounding manner. At least one of the growth chambers is provided with wheels or supports 2090, so that the cell culture device of this embodiment 19 can be supported by a flat surface, such as a floor, a desktop or a laboratory operating table. Each growth chamber has at least one growth substrate 2091 to grow at least one cell and to allow cell attachment and/or attachment.

At least one growth chamber contains a culture medium and a gaseous environment, such that the culture medium is placed at the second end of the culture chamber, and the air environment is disposed and/or contained at the first end of the culture chamber. Embodiment 19 of the present invention also includes a driving device 2095, which is used to move the growth substrate up and down or left and right between the first end and the second end of the growth chamber, so that the growth substrate can be repeatedly, periodically and intermittently Permanently immerse and/or float out of the medium to provide the cells with the proper nutrients and oxygen. The driving device 2095 may optionally include a first connecting device 2094, which is used to connect the driving device 2095 and the second connecting device 2093. The driving device 2095 is connected to the growth substrate 2091, and its function is to make the growth substrate repeatedly and periodically Intermittently move from the first end of the growth chamber to the second end, that is, move the growth substrate in the growth chamber so that it can repeatedly immerse and float out of the medium to provide suitable nutrients and oxygen. The driving device of the present invention can be a screw jack, and the screw jack system can help the growth substrate to move up and down or left and right.

The growth substrate 2091 in this embodiment 19 can optionally be porous, and the porous growth substrate can allow the cells attached to it to obtain oxygen, but will not be directly exposed to the shear caused by air flow or air bubbles in the air environment. stress. Cells, especially animal cells and/or mammalian cells, may be injured and killed and lose culture viability due to direct exposure to air.

The growth substrate 2091 can be in any form, shape or size, including, but not limited to, discs, flakes, blocks, discs, flakes, ribbons, granular, microcarriers, semipermeable particles, microparticles, semipermeable Permeable membranes or semi-permeable hollow fibers, optionally utilizing a porous membrane housing to accommodate growth substrates such as porous particles or microcarriers, allowing media to flow. Alternatively, a fixed support structure may be used to support the growth substrate so that it can be moved into and out of the medium.

Example 20

As shown in FIG. 35 , the embodiment 20 of the device of the present invention includes a chamber 2110 filled with a growth substrate 2120 for cell attachment and growth, and a storage tank 2130 for a culture medium. Use a scale 2180 to sense the weight of the chamber 2110. When the weight of the chamber 2110 is lower than a certain set value, a signal can be sent through the controller 2181 to start the air pump body 2125 and close the valve 2144, and open the valve 2143 and close the valve. Air pressure pump body 2126, so that the gas released by air pressure pump body 2125 will pass through air filter 2160, so that the culture medium is pushed to the chamber 2110 from the storage tank 2130, and the growth substrate 2120 is soaked in the culture medium at this time. When the weight of the sensing chamber 2110 of the scale 2180 exceeds a certain set value, a signal can be sent by the controller 2181 to start the air pump body 2126 and close the valve 2143, and open the valve 2144 and close the air pump body 2125, so that the culture medium From the chamber 2110 to the storage tank 2130, the growth substrate 2120 will indirectly contact the air environment through an air-medium thin layer interface.

In order to improve the stirring efficiency of the reactor, a combination of a rotating shaft and a support platform 2170 is used to increase the effect of cell seeding and medium mixing by rotating and stirring.

Example 21

Referring to FIG. 36, the twenty-first embodiment of the device of the present invention at least includes a first chamber 2210 and a compressible second chamber 2230, wherein the first chamber 2210 is located above the second chamber 2230, and The first chamber 2210 contains a growth substrate 2220 for cell growth, and the second chamber 2230 contains a culture medium. In order to make the liquid flow field inside the cell culture device consistent, that is, to make the liquid inside the growth substrate 2220 flow from top to bottom, it may be beneficial for the cells to be trapped in the carrier. When an elevating platform 2250 moves upward, the movable valve 2240 located inside the second chamber 2230 moves upward to close the hole, so the medium flows upward through the middle hollow cylinder 2270 to overflow, and then flows downward to cover the growth substrate 2220 . When the lifting platform 2250 moves downward, the movable valve 2240 moves downward to open the hole, so the culture medium in the first chamber 2210 flows down into the second chamber 2230 through the outer ring hole, so that the growth substrate 2220 has indirect contact with the air environment through a thin air-medium interface.

Example 22

Referring to Figure 37, the embodiment 22 of the device of the present invention, when the culture medium is to be replaced, the gas valve 2345 is closed to prevent the air pressure in the chamber from leaking out, and at the same time, the valve 2343 is closed to prevent fresh culture medium from being sucked in. Opening the valve 2344 and compressing the bottom, the compressible device 2330 thus increases the air pressure in the chamber, so as to squeeze out the culture medium in the chamber and discharge it into the first storage tank 2341 through the connector 2350 . After this action is completed, the gas valve 2345 is still in the closed state to prevent the air pressure in the chamber from leaking. The negative pressure generated sucks the fresh culture medium from the second storage tank 2342 through the connector 2351 . The medium replacement action can be controlled by a computer to regulate the opening and closing state of the valve and the compression times of the compressible device 2330 to regulate the replacement volume.

During normal cultivation, the valves 2343 and 2344 are closed. Therefore, the connectors 2351 and 2350 connected to both sides of the chamber are closed, and the gas valve 2345 is opened for gas exchange.

Example 23

Referring to Figure 38, Embodiment 23 of the device of the present invention is an extension of Embodiment 22. As shown in Figure 38, the right side is the culture medium replacement device, which at least includes the upper medium storage chamber 2410 and the lower compressible chamber The chamber 2430, on the left is a high-efficiency cell culture device, at least including an upper cell culture chamber 2411 and a lower compressible chamber 2431, and a conduit 2460 is used to connect these two devices. The operation of this system is through the compression and expansion of the compressible chamber 2430, causing a change in the internal pressure, so that the medium is squeezed from the medium storage chamber 2410 through the connector 2450, and through the conduit 2460 and the connector 2451. Pressed to the cell culture chamber 2411 of the cell culture device, no matter whether the compressible chamber 2431 below it is compressed or not, the growth substrate 2421 can be immersed in the culture medium, and when the compressible chamber 2430 is stretched, the culture The substrate will be sucked back from the cell culture device through the same channel. This device avoids the trouble of frequent medium replacement by increasing the volume of the total medium.

The present invention is illustrated by preferred embodiments, and it should be understood that the present invention should not be limited to the provided implementations, but should include various implementations of the invention (as conventionally known by those skilled in the art) ), therefore, the protection scope of the present invention should be interpreted in the broadest sense to include all modifications and similar combinations.

The present invention is further illustrated by a non-limiting embodiment. This embodiment is used to describe the content of the present invention, and should not be used to limit the present invention and many obvious and possible possibilities that do not depart from the spirit and scope of the present invention. difference.

Example 24

Application examples of cell culture and collection of protein secreted by cells during cell growth of the present invention.

Obtain a Chinese hamster ovary cell line (CHO cell line) that contains a stably inserted and expressed DNA through genetic engineering to encode the foreign protein to be expressed, and inoculate this cell line into the cell culture device of the present invention. Semipermeable microcarriers grown on substrates. The cell culture device of the present invention contains two chambers: a first chamber and a second chamber, wherein the first chamber is located above the second chamber and contains the growth substrate of the present invention, and the second chamber is Contains a compressible bellows and contains culture medium.

Once the medium and cells are added, the cell culture device of the present invention can be installed on the driving device to periodically and intermittently compress and expand the bellows. The chamber is pushed towards the first chamber, so that the microcarrier growth substrate is immersed in the medium, and when the bellows is extended by the driving device, the medium will flow from the first chamber to the second chamber, so that the microcarrier growth substrate The cells on and above are indirectly exposed to the air environment of the first chamber, which contains the desired oxygen concentration, through an air-medium interface maintained on the surface of the growth substrate.

In addition, when the culture medium returns to the second chamber, a small amount of culture medium will be maintained on the surface of the microcarrier substrate to protect the cells from being damaged by contact with air. The compression and expansion of the bellows can make the medium move periodically and intermittently to contact the growth substrate and the cells on it, so as to provide the most nutrients to the cells. Furthermore, the periodic and intermittent movement of cells between chambers can help redistribute and reattach any dropped cells, thereby increasing overall culture viability and productivity

Using the cell culture device described above, cultivate a Chinese hamster ovary cell line (CHO cell line) containing DNA that stably inserts and encodes the desired foreign protein. The movement between chambers and the interactive provision of oxygen and nutrients do not directly expose the cells to the air environment to achieve ideal cell growth conditions and foreign protein production and secretion. The desired foreign protein will be secreted into the medium, and its Concentrations will accumulate over time.

Once the cell growth reaches the ideal state, for example: all the nutrients in the medium are used up, the old medium in the cell culture device must be removed and fresh medium is added. The old medium contains the desired recombinant protein. It can be purified according to generally conventional methods.

The above descriptions are preferred embodiments of the present invention, and the described embodiments should not be used to limit the present invention.

Claims (27)

1, a kind of cell culture apparatus is characterized in that: which comprises at least:

First chamber has first end, second end, an internal surface and an outside surface, and this internal surface has at least one growth base material, to accept at least one cell and to allow this cell to attach and/or adhere to;

Second chamber is with the handing-over of second end of described first chamber or links to each other that this second chamber has first end, second end, an internal surface and an outside surface, at least one separating between first end that device is positioned over second end of this first chamber and second chamber; This separates device makes cultivation flow based on coming and going between this first chamber and second chamber;

This cell culture apparatus is exposed in air or the substratum this growth base material off and in turn, and the thin layer interface that makes cell pass through air-substratum increases the exchange of oxygen.

2, cell culture apparatus according to claim 1, it is characterized in that: described second chamber is the compressible element with first end and second end, and optionally the outside surface in first end of compressible element provides first lid, and provide second lid in the outside surface of second end of compressible element, this first lid combines with second lid, makes that the compressible element in second chamber is to be compressed between first lid and second lid.

3, cell culture apparatus according to claim 1, it is characterized in that: it has further comprised:

One air ambient is the inside that is positioned at this first chamber; Substratum is the compressible element inside that is arranged in this second chamber, and this substratum separates device and/or film via one, moves freely between this first chamber and second chamber; And optionally use a drive unit to compress and the compressible element that stretches in this second chamber, so that should cultivate, have at least a cell to attach and/or the growth base material that adheres to can be immersed in this substratum when this second chamber compresses based on moving between this first chamber and second chamber; When this second chamber stretches, be exposed to air ambient indirectly by one air-substratum thin layer interface, so that oxygen to be provided.

4, cell culture apparatus according to claim 1 is characterized in that: described first chamber comprises at least one opening, is to be used for accepting or mobile substratum and cell, and allows cell culture apparatus and extraneous environment exchange of air.

5, cell culture apparatus according to claim 4 is characterized in that: the opening of described first chamber optionally is equipped with a stopping device.

6, cell culture apparatus according to claim 5 is characterized in that: described stopping device is optionally to install an air filter screen, to prevent pollution.

7, cell culture apparatus according to claim 3 is characterized in that: described drive unit is an automatic or manual.

8, cell culture apparatus according to claim 1, it is characterized in that: separate device between first end of described second end that places first chamber and second chamber, be one to have infiltrative film or porousness dividing plate, this cultivation is flowed based on coming and going between first chamber and second chamber.

9, cell culture apparatus according to claim 8 is characterized in that: described have infiltrative film for optionally having porous.

10, cell culture apparatus according to claim 1 is characterized in that: described growth base material is the matrix of a loose assembly.

11, cell culture apparatus according to claim 10 is characterized in that: described matrix also includes dissolved oxygen hyperplasia device, static mixer or deep-bed filter.

12, cell culture apparatus according to claim 1 is characterized in that: described growth base material is half permeable sac.

13, cell culture apparatus according to claim 1 is characterized in that: described growth base material is a porous particle, static agitation, increase dissolved oxygen, filtration is provided and intercepts cell.

14, cell culture apparatus according to claim 1 is characterized in that: described growth base material comprises at least one semi-permeable tubular fibre.

15, cell culture apparatus according to claim 1 is characterized in that: described cell is to be selected from eukaryotic cell or prokaryotic cell prokaryocyte.

16, cell culture apparatus according to claim 1 is characterized in that: the amount of the substratum of described growth base material is to regulate and/or control by a floatoblast and a pneumatic tube.

17, cell culture apparatus according to claim 1 is characterized in that: this second chamber further includes a volume setting device, enters the substratum deal of first chamber with regulation and control;

One control device is the described volume adjustment means of control, substratum is flowed between first and second chamber, make when substratum at the volume of second chamber to hour, substratum covers the growth base material fully, and when substratum at the volume of second chamber when being maximum, the interface indirect contact of growth base material by one air-substratum is to the air ambient of first chamber.

18, cell culture apparatus according to claim 17, it is characterized in that: described volume adjustment means is bellows, by compression and stretching, extension, to control the volume of the second chamber interior substratum, make when bellows are compressed to the limit, substratum is minimum at the volume of second chamber, and when bellows were stretched over the limit, substratum was maximum at the volume of second chamber.

19, cell culture apparatus according to claim 17, it is characterized in that: described volume adjustment means is a balloon, by inflation and disappointing, to control the volume of the second chamber interior substratum, make when balloon inflation arrives the limit, substratum is minimum at the volume of second chamber, and when balloon lost heart to the limit, substratum was maximum at the volume of second chamber.

20, cell culture apparatus according to claim 17 is characterized in that: described volume adjustment means is a piston, wherein pushes away on the piston or drop-down, to control the volume of the second chamber interior substratum.

21, cell culture apparatus according to claim 3 is characterized in that: described drive unit is an oil cylinder.

22, cell culture apparatus according to claim 3 is characterized in that: described drive unit is a pneumatic cylinder.

23, cell culture apparatus according to claim 3 is characterized in that: described drive unit is a screw jack.

24, cell culture apparatus according to claim 3 is characterized in that: described drive unit is a gas compression device.

25, cell culture apparatus according to claim 1, it is characterized in that: described growth base material is a porous growth base material, cell is coated within the porous growth base material, and when growth base material emersion during in substratum, cell on the growth base material arrives air ambient by the interface indirect contact of one air-substratum, and obtains enough oxygen.

26, cell culture apparatus according to claim 25 is characterized in that: the material of described porous growth base material is selected from following group: pottery, braiding base material, non-braiding base material, polymeric amide, polyester, urethane, fluorocarbon polymer, polyethylene, polypropylene or polyvinyl alcohol.

27, cell culture apparatus according to claim 25 is characterized in that: the form of described porous growth base material is dish shape, laminar, block, plate-like, sheet, band shape, granular, semipermeability particle or semipolar linkage.

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