TW200424311A - Cell-cultivating device and method - Google Patents

Abstract

The present invention discloses a novel cell-cultivating device, which at least includes more than one chamber, a loading construction for loading a growth substrate for receiving at least more than one cell and allowing adherence and/or attachment of cells, and a bubble exclusion structure for preventing interference on cells and/or growth substrate caused by bubbles generated during the cultivation process. The present invention also provides a cell-cultivating method, which mainly comprises: intermittently and/or periodically contacting a growth substrate attached and/or adhered with cells with an air-cultivation medium interface to obtain oxygen, which is characterized in: using a bubble exclusion construction to avoid interference on the cells and the growth substrate caused by bubbles during the cultivation process, and using a pressure regulation device to develop a constant or gradient variation on the internal pressure of the chamber. Thus, cells can be effectively cultivated to a maximum amount with minimal mortality. Meanwhile, special cells can be cultivated in an environment with a specific pressure variation.

Description

200424311 V. Description of the invention (1) [Technical field to which the invention belongs] The present invention relates to a cell culture device and method, which can be used to cultivate any microorganisms and cells, and to collect any by-products produced by them. [Previous technology] Large-scale cell culture programs have been widely developed in recent years and have been used to culture bacteria, yeasts, and molds with strong cell walls and / or extracellular matrix (e X trace 1 1 u 1 armateria 1). More elastic cells. The elastic structure of microbial cells is a key factor in the rapid development of such efficient cell culture programs. For example, bacterial cells can be exposed to the air in a large amount of medium using vigorous vibration, shaking, and aeration techniques, and maintain good Growth situation. In addition, bacteria can also grow on the biofilm, but a plane is needed to provide growth. On the contrary, the technology of culturing eukaryotic cells, animal cells, mammalian cells and / or tissues is 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 complicated. In addition, unlike microbial cells, animal cells and / or mammalian cells cannot tolerate the introduction of air or mixed gases, such as: a mixed gas containing oxygen, nitrogen, and carbon dioxide, resulting in severe preferential action and / or Shearing force. Also, animal cells cannot directly contact air, and most animal cells can only use oxygen dissolved in the culture medium. Compared with microbial cells, animal cells and mammalian mammalian cells are more likely to be injured by gas or during ventilation, thus increasing their mortality. Bioreactors used in large-scale culture usually have an internal runner, such as: agitating wings (i mpe 1 1 er), to make cells

Page 5 200424311 V. Description of the invention (2) Suffering from very high liquid shear stress and causing cell injury or death, resulting in a low survival rate of cells and a decrease in the production of proteins and / or by-products. Similarly, in order to make cells uniformly suspended and / or properly exposed to air, bioreactors using other mechanical devices, intense gas motion or strong liquid motion will also cause cell damage and hinder cell or tissue growth, further Causes by-products of cells, such as protein, yield reduction. The main function of the bioreactor is to cooperate with research that requires the cultivation of a large number of cells to extract trace active substances derived from them, including but not limited to proteins or antibodies secreted by the cells into the culture medium; Another function of the bioreactor is large-scale cell culture to mass-produce active proteins produced by cells or tissues to meet commercial needs. Due to the need for large-scale cultivation of eukaryotic cells and / or prokaryotic cells and / or animal cells and / or mammalian cells in the laboratory, bioreactors and culture devices are researching and producing cells to produce active proteins and / or antibodies and / or Any aspect of cellular by-products plays an important role. Many conventional methods are available for culturing cells on a large or small scale. In terms of small-scale cultivation, many culture vessels have been developed in recent years, for example: cultivation of jiel is an example. The culture m basically includes a bottom plate for holding the culture medium, and a top cover which can be opened. Although the upper cover can be lifted to facilitate operation, the frequent lifting of the cover during the culture process often causes the cells to be contaminated with 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 in the cultivation of cultivation,

Page 6 200424311

Culture flasks. The culture bottle basically contains a # culture chamber, a tubular opening at one end of the bottle, and a corresponding one.丄 Shu Ding. This design is designed to reduce the chance of cells coming into contact with dust, bacteria, yeast, and other sources of contamination. The patents of the teaching flasks are, for example: US Patent No. 4,334,028 owned by Carver Company, US Patent No. 4,8 5 1, 3 51, owned by Akamine Company, and Degrassi U.S. Patent No. 5,3 98,8 37. Although culture flasks are an improvement over petri dishes, they still cannot completely solve the problem of contamination. In addition, neither the culture dish nor the flask can provide sufficient air, and the growth area provided by the culture flask is not as sufficient as that of the culture dish, thus limiting the scale of the culture. Another technique for culturing cells and tissues is roller bottles. Roller culture bottle technology has been widely used in cell culture for several years, although it has some advantages not found in some culture dishes or culture bottles. For example: providing a large area for cell growth and attachment, but still cannot overcome all the disadvantages, especially in terms of expanding the culture scale. In summary, these disadvantages include, but are not limited to, the huge fluid shear stress caused by the gas headspace and the large amount of rotary agitation. Due to the high shear stress environment of rolling culture flasks, tissue culture of larger three-dimensional structures is almost impossible. Only cells that are not damaged by shear stress and / or can maintain the attachment to the inner wall of the rolling culture flask can be continuously cultured for a period of time. It can be seen that the difficulty in maintaining a cell line in a rolling culture flask for a long time lies in its high shear stress environment and the possibility of contamination. Examples of rolling culture flasks include: US Patent No. 5,527,705 owned by Mussi and US Patent 200424311 owned by Serkes. V. Description of Invention (4) No. 4, 9 6 2, 0 3 3 number. In addition, although the overall area of a rolling culture flask is larger than that of a petri dish or culture bottle, the area provided for each cell attachment is not necessarily larger than that provided by a petri dish or culture bottle, especially in large-scale culture. Therefore, an invention aimed at increasing the growth area provided by a rolling culture flask, for example, Se]: US Patent No. 5,0 1 0,0 1 3 owned by kes Company describes a rolling type that can improve the cell attachment surface area. A culture flask is attached to the inner wall of the rolling culture flask and a crepe-shaped channel is added to increase the inner surface area of the rolling culture flask for cell attachment. However, the general rolling culture flask only provides an area of about 850-1700 square centimeters, so a large number of rolling culture flasks are still needed to achieve mass production. The automated, large-volume rolling culture flask cultivation system can save time and manpower, but its operation is quite responsible and restrictive. In addition to the problems of fluid shear stress and the limitation of growth area, obtaining and maintaining an adequate oxygen supply are also central issues 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 main rate-limiting step, which is the quality of oxygen. Conversion (oxygen mass transfer). Except for some eukaryotic microorganisms that undergo fermentation, such as yeast, for most prokaryotic and eukaryotic cells, the metabolism of oxygen φ is very important for the metabolic function of cells, especially for mammalian cells and animals Cell culture technology and oxygen supply are particularly important for the dry phase of rapid cell division. When cells are suspended, the need for oxygen

Page 8 200424311 V. Description of the invention (5) The maximum amount is required, and when the cells aggregate or differentiate, the oxygen demand will decrease. Some mammalian cells and animal cells are anchorage-dependent, while other mammalian cells and animal cells can grow in suspension in a liquid environment, that is, anchorage independent. However, all cells require Grow in a fully soluble environment. In the later stage of cell culture, whether it is attachment-dependent or non-attach-dependent cells, the number of cells per unit volume will increase significantly, and therefore a large amount of oxygen mass conversion is required again to provide sufficient oxygen. Basically, at least for attached and growing cells, a machine can be used; falling and aeration can be used to supply the oxygen they need '. However, as mentioned earlier, the act of mixing and aerating will damage the cells and reduce their survival. Rate and overall cell and / or tissue culture efficiency and yield. In addition, directly injecting gas into the cells and tissue culture medium will cause air bubbles, which is also not conducive to cell survival. Device invented to solve the problem of oxygen supply during cell culture, for example: W ο 1 feta 1 · Owned US Patent No. 5,1 5 3,1 3 1 is related to a creature without blades Reaction tank (bioreactor · vessel). The biological reaction tank passes air through an air inlet passageway, passes through a screen through a support plate member, and is sandwiched between two boxes of the reaction tank (h 〇using). A flat disk-shaped permeable membrane (f 1 atdisk permeable membrane) between the sides uses the oxygen concentration gradient between the two sides of the outer box to allow oxygen to diffuse through the permeable membrane and reach the cu 11 ure chamber.

Page 9 200424311 V. Description of the invention (6) However, the Wo 1 f bioreactor has many disadvantages. In particular, the rate of oxygen diffusion through the disc-shaped permeable membrane has become the main limiting factor for the size of the culture chamber. Another disadvantage is that The flat disk-shaped permeable membrane is designed to be retractable to have a stirring function, but this may cause cell death. It is very important to use the stirring function to uniformly distribute the air in the culture medium. However, the stirring at the same time will increase the shear stress inside the culture chamber, which may cause damage to the cells as described above. Therefore, when designing a bioreactor or culture tank, how to provide sufficient gas exchange to maintain a large cell structure in culture is very important and a practical limitation. In order to solve the aforementioned shortcomings, a reactor made of a gas permeable material such as Schwarz e 1 a 1. U.S. Patent No. 5,7 0 2,9 4 1 owned under the name π 可The breathable bioreactor and its use method π are about a horizontally rotatable container, and the container wall part adopts a breathable material, so as to achieve the purpose of directly exchanging gas with the culture medium through the breathable material. However, the scale of the device disclosed by S chwar ζ is still limited, because the gas exchange needs to be determined according to the size of the ventilation area. Although S chwarz emphasizes that when the surface area of the device increases, the volume of the culture medium also increases relatively. The size of the preferred embodiment disclosed in this article is limited to 1 to 6 inches in diameter and 1/4 to 1 inch in width. This size is not suitable for growing cell aggregates and tissues with three-dimensional space. And / or any mass production. Similarly, F a 1 k e n b e r g e t a 1. U.S. Patent No. 5,4 4 9,6 1 7 owned by him, entitled “Culture vessel n for cell culture n”, related

Page 10 200424311 V. Description of the invention (7) In a horizontally rotating device, this culture container is divided into a cell culture chamber and a nutrient liquid storage tank (nutr 1 entmed 1 um reservoir) by a dialysis membrane, and a breathable material It is used on the wall of the container to allow gas exchange in the cell culture chamber, but the nutrient solution storage tank is not completely filled with nutrient solution, but a large amount of gas is maintained above the culture solution in the two chambers. However, the Fa 1 kenberg container is not designed to reduce the disturbance inside the cell culture chamber. On the contrary, in order to keep the dialysis membrane wet, stirring is a necessary step. In addition, the Fa 1 kenberg container is not used and used in the culture. Cell aggregates or any type of tissue. In order to solve the problem of 氡 IL supply, there are also inventions such as US Patent No. 5,7 6 6, 9 4 9 owned by Liau, entitled π Method and Apparatus for Cultivating Dependent Monolayer Cells " It describes a culture system that shakes the medium up and down to increase the supply of oxygen. However, Liau ’s invention has many shortcomings, one of which is the complexity of its device. Liau ’s system includes two external storage tanks and one containing a series of Separate culture chambers with vertical substrate plates. In addition, several peristaltic pumps (pe 1: 1 sta 11 icpumps) are required to push the culture medium from one storage tank through the culture chamber and then to another. Storage tank, and finally flow back to the first storage tank. Because L.iau's device is complex and part of the equipment is external to the culture chamber, 5 t, 2 pipelines, storage tanks and pumps, so it is very likely The external > dyeable source is introduced into the interior. In addition, because the equipment of the device is huge, the sterilization work is not easy and labor-intensive. Another disadvantage of Lia U's invention is that the culture fluid flows through the system. Caused by

ill

Page 11 200424311 V. Description of the invention (8) The fluid shear stress can be reduced, which is not good for the cells and accumulates on the plate. Low cell survival. New start, quite complicated, and time-consuming to collect. Finally, they will be exposed to empty cell death. Detailed research, defects in patient care technology, and disclosure of a cell that is not complicated, but also increases the cell survival rate to meet the long-term requirements of the present invention. The present invention relates to the reliability and efficiency of its products, and to reveal a new cell, such as: Cells and human cells will not allow any fine energy to attach to the cell, so there is no interference between the cell and the reason and the efficiency of the cell and the fineness of the cell when it is angry, and the secretion of the long fluid, ie, the tissue Academic and weaving, less demanding and viability of some of these white matter and eggs are lower, direct culture research to make up the nourishing product of the mobile production. Others are unable to produce the most cells. Not ideal white matter or cell contact technology are quite inconvenient. The less output is longer than that, which is large immediately, and this is the important point of the slender gas growth. One of the main shellfishes is a substrate with a multi-system attachment system. The fine death of the new flat disc material must be flat due to the fact that the technology research in order to invent a disc saver for a more labor-saving method, because when the system is very numb, it will be investigated and overcome. The teaching is reliable, and the design of the system is very difficult to drop. The cells cause drug habituation and can be augmented. It provides a method for culturing cells and / or tissues and collecting, simple, cheap, and easy to use. Recyclable and / or disposable methods and devices. More specifically, the present invention teaches that the method and device of Ying can effectively culture the nucleus cells, eukaryotic cells, animal cells, mammalian cells, bacteria or molds that are to be cultured, and can continuously provide oxygen and nutrients in direct contact with the cells. Air, so it can reduce cell damage

Page 12 200424311 V. Description of the invention (9) Even cell death rate. In addition, the method and device of the present invention can reduce pollution and prevent cells from being directly damaged by shear stress when supplying gas, so that the negative effects of bubbles and gas on the cells can be prevented. Furthermore, the device of the present invention provides a simpler and more convenient method for the production and collection of cellular products, such as proteins, and / or antibodies, and / or viruses, and / or products of any cells and / or tissues. At the same time, the gas outlet pressure sensing and regulating equipment is used to cause a fixed or gradient change in the pressure in the culture chamber. Using this change will facilitate the growth and differentiation of specific cells. The objects of the present invention include, but are not limited to: providing a novel method and device for preparing, growing and maintaining cells; providing a novel method and device for growing and / or preparing tissue culture; providing a novel method and device To prepare and grow organ cultures; provide a novel method and device for culturing cells in an environment with sufficient oxygen and nutrient supply and minimal pollution; provide a novel method and device on a porous growth substrate (porous growth substrate) to enhance the exchange of oxygen and nutrients in the cell, but will not directly contact the cells with air bubbles and / or shear stress due to the injection of gas; provide a growth substrate to strengthen cell adhesion and increase air The contact area with the culture solution, and as a static medium stirrer in the device; providing a method and device to minimize cell contamination, and providing a method and device to minimize cell death, thereby increasing the cell product Yield, providing a method and device which can greatly reduce or even complete the fluid shear stress during cultivation No, to minimize the cell death rate; providing a method and apparatus need not be supplied by the bubble venting radon in the culture medium; providing a method and apparatus so that the medium can move

Page 13 200424311 V. Description of the Invention (ίο) The ability to move, let fall; provide cell products; provide a method and device for tissue secretion, good. The characteristics of providing a cell plus the present invention include: one to receive at least; and a bubble to interfere with the aforementioned cell of the cell; a filled cell and structure to prevent the substrate; wherein the aforementioned is in the outer chamber, and is suitable for the culture The aforesaid cells with similar structure and exhaust gas can be redistributed and a method and device can continuously supply the maximum output; provide methods and devices to efficiently produce protein and other products; and make cells after a period of culture The method and device can adjust the culture cavity to promote the growth of cells. The main purpose is to provide more than one thin cavity; a structure to fill more than one cell and a bubble-removal structure to prevent and / or grow the substrate. The cell culture device includes at least: a structure for filling a growth substrate to allow cells to attach and / or stop air bubbles generated in the culture process, and the position of the filling structure between the inner chamber and the outer chamber is not set and its filling The type of growth substrate is a substrate for culturing cells or tissues. The bubble exclusion structure includes: stepped channels, pressure-sensing valves used alone or in combination, which can help increase the area of fine cell culture media and collection. Provide a culture survival rate. Can be used for pressure or / and differentiation in the room. < Cell culture device, which is used to fill cell attachment and oxygen during cell attachment and culture, so that cell attachment and / or cell access can be changed with fine D, which at least grows the substrate / or attached gas An inner cavity; an outer cavity to receive at least one and more than one air bubble to exclude nodes interfering with cells and / or growth lines can communicate with each other. Restricted, the preferred system is not restricted, can be freely selected inclined plate structure, fin type and any bubble generation can be excluded

Page 14 200424311 V. Description of the invention (11) Room or outer chamber. The cell culture device of the present invention may further include units such as an air pump, a feed pump, a discharge pump, a culture medium container, a product collection container, a pressure sensing valve, and a timer. With the cell culture device of the present invention, the medium can be moved back and forth between the chambers to gently redistribute the dropped cells back to the growth substrate, thereby enhancing the cell attachment ability and helping the gas by mixing the medium _ Contact with the culture medium and ensure that nutrients and gases are evenly distributed without harming or killing the cells. The first embodiment of the present invention is a cell culture device, which includes at least: an inner chamber; an outer chamber; a growth substrate filling structure; and is arranged in the aforementioned outer chamber; a step The inclined plate structure is arranged in the outer chamber to block air bubbles generated during the cultivation process; the inner chamber and the outer chamber can communicate with each other. A second embodiment of the present invention is a cell culture device, which includes at least: an inner chamber; an outer chamber; a growth substrate filling structure, which is arranged in the aforementioned chamber; and a fin The structure is arranged on the wall edge of the filling structure of the growth substrate to block air bubbles generated during the cultivation process; the inner chamber and the outer chamber can communicate with each other. A third embodiment of the present invention is a cell culture device, which includes at least: an inner chamber; an outer chamber; a growth substrate filling structure; φ is arranged in the aforementioned outer chamber; an exhaust The channel is arranged on the top of the inner chamber to eliminate air bubbles generated during the cultivation process; the inner chamber and the outer chamber can communicate with each other.

Page 15 200424311 V. Description of the invention (12) The aforementioned cell culture device further includes a floating plate membrane structure arranged below the aforementioned exhaust channel, which can block the radon channel to prevent the medium from flowing out of the inner chamber and contaminating the air. Strainer. A fourth embodiment of the present invention is a cell culture device, which includes at least: an inner chamber; an outer chamber; a growth substrate filling structure, arranged in the aforementioned outer chamber; a pressure-sensing type The valve is arranged on the top of the inner chamber to block air bubbles generated during the cultivation process; the inner chamber. And the outer chamber can communicate with each other. The aforementioned pressure-sensing valve can be further equipped with a timer, so that the pressure-sensing valve can be opened and closed by pulse. In the foregoing embodiments, the air pump can be optionally installed during the introduction of the air pump into the chamber: an air filter to prevent pollution. The cell culture device of the present invention can be used for two-dimensional or three-dimensional tissue culture. The cavity system of the present invention may be composed of any material, including but not limited to glass, metal or polymer materials. The growth substrate used in the present invention may be of any material, including, but not limited to, for example, ceramics, biodegradable matrix, polymer, woven substrate, non-woven substrate Polyamine, polyester, polyurethane, fluorocarbon polymer, polyethylene, polypropylene or polyvinyl alcohol, trimethylamine, glass, silicon and diethylaminoethyl φ group (DEAE). The porous growth substrate can be of any type, shape or size, including, but not limited to, dish, sheet, block, disc, sheet, ribbon, granular, semi-permeable, semi-permeable membrane, or semi-permeable Pervious hollow fiber.

Page 16 200424311 V. Description of the invention (13) Another object of the present invention is to provide a cell culture method, which mainly uses a growth substrate attached and / or attached to cells, and intermittently and / or regularly contacts the air. -The interface of the culture medium to obtain oxygen, which is characterized by a bubble elimination structure to prevent bubbles generated during the culture process from interfering with cells and growth substrates. The invention provides a method and a device which can adjust the pressure in the culture chamber according to the characteristics of the cultured cells to promote cell growth or / and differentiation. Any cell can be grown in the cell culture device of the present invention, such as: prokaryotic cells, eukaryotic cells, and / or mammalian cells. In addition, cells that are dependent or non-attached are also suitable for use in the present invention. Attachment-dependent cells require surface attachment growth, while non-adherence-dependent cells grow in liquid suspensions. All types of cells require sufficient oxygen, nutrients, and growth factors for their growth. The present invention provides a novel method that is intermittent, but not direct, and provides cellular oxygen through a thin layer of air-culture medium interface without harming and / or killing the cells. [Embodiment] The following embodiments are for illustration only and should not be used to limit the scope of the present invention. The detailed description of the invention can be combined with the accompanying drawings to help obtain a better understanding. Embodiment 1 Figures A and B show the first embodiment of the present invention, a cell culture device 100, which mainly includes: an inner chamber 1005;-an outer chamber

Page 17 200424311 V. Description of the invention (14) Chamber 1 0 0 4; —Filling structure 1 0 7 is provided in the aforementioned outer chamber 1 0 4 to fill the growth substrate 1 0 1 0; — A stepped inclined plate structure 1 0 6 is arranged in the outer chamber 1 0 4 to block air bubbles generated during the cultivation process; between the inner chamber 1 0 5 and the outer chamber 1 0 0 4 Departments can communicate with each other. The culture device 1 0 0 further includes: an induction valve 1 0 0 1, an air pump 1 0 1 1, a fresh culture medium container 1 0 0 8, a product collection container 1 0 0 3, and a feed pump 1 0 0 9 、 Discharge pump 1 0 2. The cell culture device 100 can be further equipped with an air filter 1012 to prevent contamination during the introduction of the air pump 1011 into the chamber. The operation method of the cell culture device 100 is as follows: As shown in the first diagram A, before the air pump 10 1 1 is moved, most of the culture medium is in the lower layer, so the growth substrate 1010 is exposed to the air. ; As shown in Figure 1B, when the air pump 1011 is activated, the culture medium in the inner chamber 10 05 will be subjected to air pressure and compressed from the inner chamber 1 0 0 5 to the outer chamber 1 0 4 to promote The culture medium rises in the outer chamber 1 0 0 and covers the growth substrate of the attached cells 1 0 1 0; when the pressure value in the outer chamber 1 0 4 rises to a specific value, the air pump is closed 1 0 1 1 and open the pressure sensing valve 1 0 0 1 to release the pressure in the outer chamber 1 0 4 and cause the cultured genes in the outer chamber 1 0 4 to flow back to the inner chamber 1 0 0 5 At this time, the growth substrate 1 0 1 0 is again exposed to the air, as shown in FIG. 1A. Repeating the procedures of the first A picture and the first B picture in this way is intermittent, but not direct, and provides cell oxygen through a thin layer of air-medium interface without harming and / or killing the cells. At the same time, in order to avoid excessive air blowing, the culture medium is completely squeezed into the outer chamber 104, and excess air is pushed from the inner chamber 1 0 5 into the outer chamber 1 0 0, causing bubbles to flush directly.

Page 18 200424311 V. Description of the invention (15) Hitting the growth substrate 1 0 1 10, so a stepped inclined plate structure 1 06 is used to introduce excess air bubbles into the outer chamber 1 04 wall edge to block the air bubbles directly Shock cells, causing cell damage. ^ In addition, the feed pump 1 009 and the discharge pump 1 00 were used for continuous culture. The pressure sensing valve 1 0 0 1 can also be opened forever. ° ▲ 2 Timer (not shown) is used as the basis for the air pump 1 〇 11 to act. The bite is to use a timer and pressure sensing valve 1 0 0 1 together, that is, the pressure sensing valve 1 0 0 1 adopts pulse opening and closing, so that it is easier to control the pressure of the outer chamber 1 0 0 4 ′ and The air pump can also be turned off when the timer expires. The cell culture device 100 of the present invention can be selectively operated automatically or manually. Example Two cell culture devices 2000, including at least __chambers 20003;-a filling structure 2005, is provided in the outer chamber 2003 to fill the growth substrate 2 〇〇8;-The second A and B diagrams of the upwardly inclined fin structure show the second embodiment of the present invention, _ ^ ^ ^ ^ 〇π π ^ ^ 1 ^ 内 内 室 2 0 0 4; -Xi Buzhong "^ ^ ^ yj υ υ, an inner 丄; < 7/7 eight, cultivation 2 0 0 9, is set on the wall edge of the aforementioned growth substrate filling structure 2000, to Block the air bubbles generated during the cultivation process; the aforementioned inner chamber 2004 and the outer chamber 2003 = can be communicated with each other. This cultivation device 2000 further includes: a sensing element 2 01 and an air pump 2 0 〇 7. Fresh medium container 2 〇 丨 〇, product container 2 0 1 1. Feed pump 2 006, discharge pump 2 002. This cell culture set 200 can be further used in air pump 2 0 0 7 An air filter 20 1 2 can be installed during the introduction into the chamber to prevent: loss. The operation of the cell culture device 2 0 0 is as follows: as shown in Figure 2A

Page 19, 200424311 V. Description of the invention (16) shows that before the air pump 2 0 7 was moved, most of the culture medium was located in the lower layer of the chamber, so the growth substrate 2 008 was exposed to the air; As shown in Figure B, when the air pump 2 0 7 is activated, the medium is compressed from the inner chamber 2 0 4 to the outer chamber 2 0 3, and the medium rises to the outer chamber 2 0 3 to cover the cells. The growing substrate filling structure 2 0 5 is grown. When the pressure value of the outer chamber 2 0 3 rises to a certain value, the air pump 2 0 7 is closed and the pressure-sensitive valve 2 0 0 1 is opened. The pressure in the outer chamber 2003 was released, and the outer chamber 2000 was released. The cultured gene gravity flowed back to the inner chamber 2 0 4, at which time the growth substrate 2 0 8 was exposed again. In the air, as shown in Figure A. Repeating the procedures of Figures 2A and 2B in this manner is intermittent, but not direct. Cellular oxygen is provided through a thin layer of air-medium interface without harming and / or killing dead cells. . At the same time, in order to avoid excessive air blowing, the culture medium is completely introduced into the outer chamber 2003, and excess air is pushed into the outer chamber 2 by the inner chamber 2004, causing bubbles to directly impact the growth substrate 2 0 8 Partly, therefore, an upwardly inclined fin structure 2 0009 provided at the wall edge of the filling structure 2 0 9 is used to introduce bubbles into the wall edge of the outer chamber 2 0 3 to block the direct impact of the bubbles on the cells. In addition, a continuous perfusion culture was performed using the feed pump 2006 and the feed pump 2000. In addition, the pressure sensing valve 2 0 1 can always be opened, and the timer (not shown) is used as the basis for the air pump 2 0 7 to operate, or the timer and the pressure sensing valve 2 0 0 1 is used together, that is, the pressure sensing valve 2 0 0 1 adopts pulse opening and closing, so it is easier to control the pressure in the outer chamber φ 2 0 0 3, and the air can be closed when the timer expires. Pump 2 0 0 7. The cell culture device 200 of the present invention can be selectively operated automatically or manually.

Page 20 200424311 V. Description of the invention (17) Example 3

The second diagram A and the second diagram B show a third embodiment of the present invention, a cell culture device 300 'includes at least: an inner chamber 3003; a chamber 3004;- The filling structure 3 0 05 is set in the aforementioned outer chamber to fill the growth substrate 3008;-the exhaust channel 3009 is set at the top of the aforementioned inner chamber 3 0 3 to block the bubbles generated during the cultivation process_ ; The inner chamber 3 0 3 and the outer chamber 3 0 4 can communicate with each other. The above-mentioned exhaust channel 3009 further includes a floating plate membrane structure 3〇 丨 2, which can block the exhaust channel 3009 to prevent the culture medium from flowing out of the inner cavity of the chamber 3 and polluting the air. 3013. The culture device 300 further includes: an inductive valve 3007, an air pump 3 0 01, a fresh culture medium container 3 〇 丨 〇, a product collection container 3 〇 丨 i, a feed pump 3 0 6 , Discharge pump 3002. The cell culture device 300 can be further equipped with an air filter 3 01 3 during the introduction of the air pump 3001 into the chamber to prevent contamination.

The operation method of this cell culture device 300 is as follows: As shown in the third A figure, before the air pump 3001 is not moved, most of the culture medium is located in the lower layer of the chamber at this time, and the growth substrate 3 〇08 is immersed in the culture medium; as shown in Figure 3B, when the air pump 3000 is activated, the culture medium is compressed from the outer chamber 3004 to the inner chamber 3003 and the gas is compressed from the inner chamber The radon gas channel 3 0 9 at the top of the chamber 3 0 3 is discharged and the culture medium continues to rise until the liquid surface pushes the floating plate meningeal structure 3012 to block the exhaust channel 3009, the outer chamber 3 0. 〇4 After the pressure value rises to a specific value, the pressure sensing valve 3 0 07 will feedback control the air pump 3 0 1 'to close it and make the culture gene in the inner chamber 3 2003 flow back to the night. The chamber 3 0 04, at this time the growth substrate 3 0 08 is again exposed to air_ '如

Page 21 200424311 V. Description of the invention (18) Figure 3A. Repeating the procedures of the third diagram A and the third diagram B in this manner is intermittent, but indirect, and the cell radon is provided through a thin layer interface of an air-culture medium without harming and / or killing the cells. In addition, a continuous perfusion culture was performed by using the feed pump 3 0 6 and the discharge pump 3 0 2. The cell culture device 300 of the present invention can be selectively operated automatically or manually. Fourth Embodiment Figures A and B show a fourth embodiment of the present invention, a cell culture device 400, which at least includes: an inner chamber 4004;-an outer chamber 4003; —Filling structure 4005, which is set in the outer chamber 4003 to fill the growth substrate 4 0 8; —Pressure sensing valve 4 0 7, which is set on the top of the inner chamber 4 0 4 In order to block the air bubbles generated during the cultivation process, the inner chamber 4 0 4 and the outer chamber 4 0 3 can communicate with each other. The cultivation device 400 further includes: an air pump 4001, a fresh culture medium container 4 0 10, a product collection container 4 0 1 1, a feed pump 4 0 6 and a discharge pump 4002. The cell culture device 400 may further be provided with an air filter 4 0 9 to prevent contamination during the introduction of the air pump 4001 into the chamber. The operation method of the cell culture device 400 is as follows: As shown in the fourth figure A, before the air pump 400 is not moved, most of the culture medium is located in the lower layer of the chamber, and the growth substrate 4008 is immersed in the culture medium. As shown in Figure 4B, when the air pump 4001 is activated, the culture medium is compressed from the outer chamber 4003 to the inner chamber 4004 and the gas in the inner chamber 4004 is compressed. When the inner chamber 4003 is compressed, After the pressure value rises to a specific value, the pressure sensing valve 4 0 7 will feedback control

Page 22 200424311 V. Description of the invention (19) The air pump 4 0 0 1 is closed, and the culture gene of the inner chamber 4 0 4 is gravity flowed back to the outer chamber 4 0 3, at this time the growth base The material 4 0 8 is again exposed to the air, as shown in the fourth A diagram. Repeating the procedures of Figures 4A and 4B in this manner is intermittent, but indirect, and provides cell oxygen through a thin layer of air-medium interface without harming and / or killing the cells. In addition, a continuous infusion culture was performed using the feed pump 4 0 0 and the discharge pump 4 0 2. In addition, the pressure sensing valve 4 0 7 can always be opened, and a timer (not shown) is used as the basis for the air pump 4 0 0 1 to operate, or the timer and the pressure sensing valve The 4 0 0 7 is used together, that is, the pressure sensing valve 4 0 7 is pulsed on and off. This makes it easier to control the pressure in the outer chamber, and the air pump can be closed when the timer expires. 4 0 0 1 . In particular, the present invention relates to a reliable, simple, inexpensive, reusable or disposable, sterile and efficient method for culturing cells and / or tissue and collecting their products. More specifically, the present invention relates to a novel method and device that can be used to culture any cell, whether it is a eukaryotic, prokaryotic, mammalian or animal cell, and can provide sufficient nutrients and oxygen for cell growth without harming At the same time, the pressure in the culture chamber can be adjusted according to the characteristics of the cells through the outlet pressure sensing and regulating equipment to promote cell growth or / and differentiation. In addition, the method and device of the present invention can prevent or substantially reduce any form of pollution, avoid shear stress and protect cells from direct contact with air, air bubbles, or gases. Moreover, 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 simpler and more convenient method and device for producing and collecting cells

Page 23 200424311 V. Description of the invention (20) or products of tissues, such as proteins, antibodies, etc. The present invention can be illustrated by a preferred embodiment. It should be understood that the present invention should not be limited to the implementation modes provided, but should include various implementation modes of the invention (as those skilled in the art know) (Known), therefore, the scope of patent application of the present invention should be interpreted in the broadest sense to include all modifications and similar combinations. The present invention is further described using a non-limiting embodiment, which is used to describe the present invention The content is not suitable for limiting the present invention and many obvious and possible differences without departing from the spirit and scope of the present invention. _

Η

Page 24 200424311 Brief description of the drawings The first A diagram shows the first embodiment of the cell culture apparatus of the present invention, in which the growth substrate is exposed to the air before the air pump is activated. The first diagram B shows the first implementation of the cell culture device of the present invention, in which the growth substrate is immersed in the culture medium after the air pump is operated. Example, state Example, state. Example, state Example, state. Example, state example, state 0 The second A picture shows the second implementation of the cell culture device of the present invention, in which the growth substrate is exposed to the air before the air pump is activated. The second B picture shows this The second implementation of the invented cell culture device wherein the air pump is actuated, the growth substrate is immersed in the culture medium. The third figure A shows the third implementation of the cell culture device of the present invention, in which the air pump Before the operation, the growth substrate is immersed in the culture medium. Figure 3B shows the third implementation of the cell culture device of the present invention. After the air pump is operated, the growth substrate is exposed to the air. Figure A shows the fourth implementation of the cell culture device of the present invention in which the growth substrate is immersed in the culture medium before the air pump is moved. Figure 4B shows the fourth implementation of the cell culture device of the present invention. After the air pump is activated, the growth substrate is exposed to the air.

Page 25 200424311 Brief description of the drawings [Comparison of the symbols of the main originals] 1 00-cell culture device 10 0 1--pressure sensing valve 1 0 0 2-discharge pump 1003-product collection container 1 0 0 4 --- outer chamber 1 0 0 5-inner chamber 1 0 0 6 --- stepped inclined plate structure 1 0 0 7-filled structure 1 0 0 8-fresh medium container 1 0 0 9 ——Feed pump 10 10——Growth substrate 10 11——Air pump 10 12——Air filter 2 0 0 --- Cell culture device 2 0 0 1——Pressure sensing valve 2 0 0 2——Discharge pump 2 0 0 3 --- Xibu chamber 2 0 0 4 --- Inner moon empty chamber 2 0 0 5 --- Filled structure 2 0 0 6--Feed pump 2 0 0 7——air pump 2 0 0 8——growth substrate 2 0 0 9 --- II sheet structure

Page 26 200424311 Brief description of the diagram 2010——Fresh medium container 2 0 1 1 ——— Product collection container 2 0 12——Air filter 3 0 0——Cell culture device 3 0 0 1 --- Air pump 3 0 0 2-discharge pump 3 0 0 3 --- inner chamber 3 0 0 4 --- outer chamber 3 0 0 5-filling structure 3 0 0 6-feed pump 3 0 0 7 --- Pressure sensing valve 3 0 0 8——Growth substrate 3 0 0 9—Exhaust channel 30 10—Fresh culture medium container 30 11—Product collection container 30 12—Floating plate diaphragm 3 0 13 --- air gauze net 4 0 0 --- cell culture device 4 0 0 1-air pump 4 0 0 2-discharge pump 4 0 0 3 --- Xibu moon empty room 4 0 0 4 --- Inner chamber 4 0 0 5--Filling structure 4 0 0 6--Feed pump

Page 27 200424311 Brief description of the diagram 4 0 0 7——Pressure sensing valve 4 0 0 8 --- Growth substrate 4 0 0 9 --- Air filter 40 10-Fresh medium container 40 11- Product collection container

Page 28

Claims (1)

200424311 VI. Application Patent Scope 1. A cell culture device comprising at least: one or more chambers; a filling structure for filling a growth substrate to receive at least one or more cells and allowing cells to attach and / or attach And a bubble elimination structure for preventing bubbles generated in the culture process from interfering with the cells and / or the growth substrate. 2. The cell culture device according to item 1 of the scope of the patent application, wherein the aforementioned bubble elimination structure includes: a stepped inclined plate structure, a fin structure, an exhaust channel, a pressure-sensing valve, and any bubbles that can be eliminated. Similar structures are used alone or in combination. 3. The cell culture device according to item 1 of the scope of patent application, further comprising: an air pump, a feed pump, a discharge pump, a culture medium container, a product collection container, a pressure sensing valve, and a timer And other units. 4. A cell culture device comprising at least: an inner chamber; an outer chamber; a filling structure for filling a growth substrate to receive at least one or more cells and allowing the cells to attach and / or attach; and a bubble The exclusion structure is used to prevent air bubbles generated during the culture process from interfering with the cells and / or the growth substrate; wherein the inner chamber and the outer chamber can communicate with each other. 5. The cell culture device according to item 4 of the patent application, wherein the aforementioned bubble exclusion structure can be selectively disposed in the inner chamber or the outer chamber. 6. The cell culture device according to item 4 of the patent application, wherein Page 29 200424311 VI. Scope of patent application Bubble exclusion structure includes: stepped inclined plate structure, fin structure, exhaust passage, pressure sensing valve and any similar structure that can eliminate the generation of bubbles. It can be used alone or in combination. 7. The cell culture device according to item 4 of the scope of patent application, which may further include: an empty pump, a feed pump, a discharge pump, a culture medium container, a product collection container, a pressure sensing valve, and a timer. Device and other units. 8. A cell culture device comprising at least: an inner cavity: r; an outer cavity; a growth substrate filling structure provided in the aforementioned outer chamber; and a stepped inclined plate structure provided in the aforementioned In the outer chamber, air bubbles generated during the cultivation process are blocked; the inner chamber and the outer chamber can communicate with each other. 9. The cell culture device according to item 8 of the scope of patent application, wherein the stepped inclined plate structure is a bubble discharge structure. 10. The cell culture device according to item 8 of the scope of the patent application, further comprising: an air pump, a feed pump, a discharge pump, a culture medium container, a product collection container, a pressure sensing valve, a timer Device and other units. 11. A cell culture device, comprising at least: an inner chamber; an outer chamber; a growth substrate filling structure arranged in the aforementioned outer chamber; and an inverse slit-type structure 'arranged in the aforementioned growth Substrate-filled structural wall Page 30 200424311 VI. Scope of patent application In order to block the air bubbles generated during the tower cultivation process; the aforementioned inner chamber and outer chamber can communicate with each other. 12. The cell culture device according to item 11 of the scope of patent application, wherein the aforementioned fin structure is a bubble discharge structure. 13. The cell culture device according to item 11 of the scope of the patent application, which may further include: air: air pump, feed pump, discharge pump, culture medium container, product collection container, pressure sensing valve , Timer and other units. ^ 14. A cell culture device comprising at least: an inner chamber; an outer chamber; a growth substrate filling structure arranged in the aforementioned outer chamber; and ® and an exhaust channel arranged in the aforementioned The top of the inner chamber is to exclude air bubbles produced during the cultivation; the inner chamber and the outer chamber can communicate with each other. 1 5 · The cell culture device according to item 14 of the scope of the patent application, further comprising a floating plate membrane structure arranged below the aforementioned exhaust passage, which can block the exhaust passage to prevent the culture medium from flowing out of the inner chamber. Contaminated air filters. 16. The cell culture device according to item 14 of the scope of the patent application, further comprising: an air pump, a feed pump, a discharge pump, a culture vessel, a product collection container, and a pressure sensing valve Units, timers, etc. 17. A cell culture device comprising at least: an inner chamber; Page 31 200424311 VI. Application scope: an outer chamber; a growth substrate filling structure is arranged in the aforementioned outer chamber; and a pressure-sensitive valve is arranged at the opening of the aforementioned inner chamber to The air bubbles generated during the cultivation process are eliminated; the inner chamber and the outer chamber can communicate with each other. 18. The cell culture device according to item 17 of the scope of the patent application, wherein the pressure-sensing valve is a bubble discharge structure. 19. The cell culture device according to item 17 of the scope of patent application, wherein the former The pressure-sensing system described above can be further equipped with a timer, so that the pressure-sensing valve can be opened and closed by pulse. 20. The cell culture device described in item 17 of the scope of patent application, which may further include units such as an air pump, a feed pump, a discharge pump, a culture medium container, a product collection container, and a timer. 2 1 · The sprint culture device as described in item 3, item 7, item 10, item 13 or item 16 of the scope of patent application, wherein the aforementioned air pump can be selectively introduced into the chamber An air filter is installed to prevent pollution. 2 2 · If the cell culture device described in item 1, 4, 8, 8, 11 or Π of the scope of patent application, it can be used for two-dimensional or three-dimensional tissue culture . 2 3 · The cell culture device according to item 1, 4, 8, 8, 11 or 17 of the scope of patent application, wherein the aforementioned chamber is made of glass, metal or Polymer material. 2 4 · The cell culture device according to item 1, item 4, item 8, item 11, item 14, or item 17 of the scope of patent application, wherein Page 32 200424311 VI. The growth substrates in the scope of patent application can be selected from the following groups: ceramics, biodegradable substrates, high molecular polymers, woven substrates, non-woven substrates, polyamide, polyester, polyurethane, Fluorocarbon polymers, polyethylene, polypropylene or polyvinyl alcohol, trimethylamine, glass, silicon and diethylaminoethyl. 2 5 · The cell culture device according to item 1, item 4, item 8, item 11, item 14, or item 17 of the scope of application for a patent, wherein the aforementioned growth substrate may be dish-shaped, Sheet, block, disc, sheet, ribbon, granular, semi-permeable particles, semi-permeable membrane or semi-permeable hollow fiber. 2 6 · The cell culture device according to item 1, 4, 8, 8, 11 or 17 in the scope of patent application, wherein the aforementioned cells can be selected from the following groups: Prokaryotic cells, eukaryotic cells, animal cells, mammalian cells, humans, cells, bacteria and molds. 27. A cell culture method, which mainly uses a growth substrate to which cells are attached and / or attached, and intermittently and / or periodically contacts the air-culture medium interface to obtain oxygen, which is characterized by an air bubble elimination structure To avoid air bubbles generated during the culture process from interfering with cells and growth substrates. 2 8. The cell culture method according to item 27 of the scope of the patent application, wherein the aforementioned bubble elimination structure includes: a stepped inclined plate structure, a fin structure, an exhaust passage, a pressure-sensing valve, and any removable Similar structures generated by bubbles are used alone or in combination. 29. The cell culture method according to item 27 of the scope of the patent application, wherein the above-mentioned method for eliminating bubble interference is to direct excess air or bubbles generated in the chamber out of the chamber through a stepped inclined plate structure. 30. The cell culture method according to item 27 of the scope of patent application, wherein Page 33 200424311 VI. Scope of patent application The best way to eliminate air bubbles is to direct the excess air or air bubbles generated in the chamber out of the chamber through a fin structure. 31. The cell culture method according to item 27 of the scope of the patent application, wherein the above-mentioned method for eliminating air bubbles is to direct excess air or air bubbles generated in the chamber out of the chamber through an exhaust channel. 3 2. The cell culture method according to item 31 of the scope of the patent application, wherein the aforementioned exhaust channel further includes a floating plate diaphragm, and when the medium level continues to rise to push the aforementioned floating plate diaphragm, the foregoing row can be discharged. The air channel is blocked to prevent the medium from flowing out of the chamber and contaminating the air filter. 33. The cell culture method according to item 27 of the scope of the patent application, wherein the above-mentioned method for eliminating bubble interference is to direct excess air or bubbles generated in the chamber out of the chamber through a pressure-sensing valve. 34. The cell culture method according to item 33 of the scope of the patent application, wherein the pressure-sensing valve can be further equipped with a timer to open and close the pressure-sensing valve in pulses. 35. The cell culture method according to item 27 of the scope of the patent application, wherein the aforementioned cells can be selected from the following groups: prokaryotic cells, eukaryotic cells, animal cells, mammalian cells, human cells, bacteria, and molds. 36. The cell culture method described in item 27 of the scope of patent application can be used for two-dimensional or three-dimensional tissue culture. 37. A cell culture method using the cell culture apparatus described in the aforementioned patent application scopes 1, 4, 8, 8, 11, 14 or 17 to culture cells. . 38. — A method of culturing cells to produce proteins, using the aforementioned application Page 34, 200424311 VI. Patent application scope 'The patent scope includes the cell culture device described in items 1, 4, 8, 8, 11, 14 or 17 to produce cells protein. 3 9. —A method for cultivating 纟 cells to produce a virus, using the methods described in items 1, 4, 4, 8, 11 or 17 of the aforementioned patent application. New Zealand cell culture device to culture cells to produce virus. 40. If the cell culture device described in the scope of the patent application is No. 1, No. 4, No. 8, No. 11, No. 14, or No. 17, it is optional to be automated or to Operate manually. 4 1. A cell culture method, which can use the cell culture device described in the scope of patent application for items 1, 4, 8, 8, 11, 14 or 17 to cause Constant or gradient changes in pressure in the culture chamber to facilitate the growth and / or differentiation of specific Putian cells. Page 35