A cell culture method and device

Abstract

The present application discloses a cell culture method and device, the method comprising the following steps: constructing a cell culture environment; delivering target cells and culture medium to a culture container through a pipeline system, the culture container being located in the cell culture environment; shaking the culture container through a shaking device to disperse the target cells and culture medium in the culture container; culturing target cells and vibrating the culture container based on the cell culture environment and the culture medium to obtain a cell product; and outputting the cell product from the culture container through the pipeline system. Compared with the prior art, the cell culture method and device provided by the present application can achieve large-scale mass production and avoid contamination of cells by the external environment during the culture process.

Description

Technical Field

[0001] The present application relates to the technical field of cell culture and cell engineering, and more specifically, to a cell culture method and device. Background Art

[0002] Cell culture is a method for studying the behavior of animal cells that was created in the early 20th century. It refers to taking cells from body tissues, simulating the in vivo growth environment, and allowing cells to grow and reproduce under sterile, appropriate temperature, pH, and nutritional conditions, while maintaining their structure and function. Cell preparation refers to the process in which researchers use relatively mature cell culture protocols to cultivate high-purity target cell products, which can be directly used in scientific research and clinical fields after passing functional and quality tests.

[0003] The current method of cell culture is manual culture by researchers in clean laboratories using culture bottles or culture dishes. The culture efficiency is low, large-scale mass production cannot be achieved, and it is difficult to meet the growing demand for cell culture. In addition, cells are easily contaminated by the external environment during the culture process.

[0004] Therefore, there is an urgent need for a cell culture method and device that can achieve large-scale mass production and prevent cells from being contaminated by the external environment during the culture process. Summary of the invention

[0005] In order to solve the above technical problems, the present application provides a cell culture method and device, which can achieve large-scale mass production and prevent cells from being contaminated by the external environment during the culture process.

[0006] The technical solutions provided by this application are as follows:

[0007] A cell culture method comprises the following steps:

[0008] Constructing cell culture environment;

[0009] transporting target cells and culture medium into a culture container through a pipeline system, wherein the culture container is located in the cell culture environment;

[0010] Shaking the culture container by a shaking device to disperse the target cells and the culture medium in the culture container;

[0011] Based on the cell culture environment and the culture medium, culturing target cells and vibrating the culture container to obtain a cell product;

[0012] exporting the cell product from the culture container via the piping system;

[0013] in,

[0014] Shaking the culture container by a shaking device comprises the following steps:

[0015] Flipping the culture container around a first axis to a first position so that a first preset angle exists between the bottom surface of the culture container and a horizontal plane;

[0016] shaking the culture container around a second axis to mix the target cells and the culture medium;

[0017] Continue shaking the culture container around the second axis to a second position;

[0018] Flipping the culture container in the opposite direction around the first axis so that the bottom surface of the culture container is parallel to the horizontal plane;

[0019] Shaking the culture container in the opposite direction around the second axis to reset the culture container;

[0020] There is a third preset angle between the first axis and the second axis.

[0021] Preferably, the culturing of target cells comprises the following steps:

[0022] Get the current cell state of the target cell;

[0023] Comparing the current cell state with a preset cell state;

[0024] If the current cell state does not reach the preset cell state, the target cell continues to grow;

[0025] If the current cell state reaches the preset cell state, the target cells are harvested.

[0026] Preferably, obtaining the current cell state of the target cell specifically comprises the following steps:

[0027] Obtaining a morphological image of target cells through an image acquisition device;

[0028] Comparing the target cell morphology image with a preset cell morphology image;

[0029] If they are inconsistent, the focal length of the image acquisition device is adjusted until the acquired target cell morphology image is consistent with the preset cell morphology. Figure 1 To;

[0030] Analyze the target cell morphology image according to an image analysis device to obtain cell uniformity, total cell count, and active cell count;

[0031] Comparing the cell uniformity with a preset cell uniformity;

[0032] If the cell uniformity is greater than the preset cell uniformity, vibrating the culture container until the obtained cell uniformity is no greater than the preset cell uniformity;

[0033] The current cell status includes the target cell morphology diagram, the total cell count and the active cell count.

[0034] Preferably, before the target product and the culture medium are transported to the culture container through the pipeline system, the following steps are also included:

[0035] The culture container is first cleaned and first sterilized.

[0036] Preferably, the outputting of the cell product from the culture container through the pipeline system specifically comprises the following steps:

[0037] Extract cell products and output them through the pipeline system;

[0038] Outputting the culture waste liquid in the culture container;

[0039] The culture container is subjected to a second cleaning and a second sterilization.

[0040] Preferably, the method further comprises the following steps:

[0041] constructing cold storage space;

[0042] The target cells are directly transported to the culture container through the pipeline system, or the target cells are transported to the culture container through the pipeline system after being transported to a cold storage space for storage.

[0043] Preferably, before the target cells and the culture medium are transported into the culture container through the pipeline system, the following steps are also included:

[0044] Thaw the frozen cells and obtain the target cells.

[0045] Preferably, after the cell product is output from the culture container through the pipeline system, the method further comprises the following steps:

[0046] Centrifuging the cell product to obtain centrifuged cells;

[0047] Outputting the centrifuged cells through a tubing system;

[0048] The centrifuged cells are packaged.

[0049] Preferably, the constructing of the cell culture environment comprises the following steps:

[0050] Setting environmental parameters, including CO concentration, O concentration, N concentration, temperature and humidity;

[0051] The culture container is arranged in the cell culture environment. A diaphragm is arranged on the culture container. The diaphragm is configured to provide a gas exchange channel for the gas medium and isolate the liquid medium.

[0052] A cell culture device, used in any of the above cell culture methods, comprising:

[0053] Environment construction unit, constructing cell culture environment;

[0054] An input unit, used for delivering target cells and culture medium into a culture container in the cell culture environment through a pipeline system;

[0055] A shaking device, the shaking device is used to shake the culture container so that the target cells and the culture medium are dispersed in the culture container;

[0056] A culture unit, the culture unit is used to culture target cells based on the cell culture environment and the culture medium and vibrate the culture container to obtain a cell product;

[0057] an output unit, used for outputting the cell product from the culture container through the pipeline system;

[0058] in,

[0059] The shaking device includes: a placement table for placing the culture container; a rotating component for driving the culture container to rotate on the placement table; a flipping component connected to the rotating component, the flipping component is used to drive the placement table and the culture container to flip around the first side of the placement table; a vibration component is arranged on a side of the culture container away from the placement table, the vibration component is used to drive the culture container to vibrate along the height direction of the placement table.

[0060] Preferably, the pipeline system comprises:

[0061] An input tube, at least two of which are provided, and are used to transport target cells and culture medium respectively;

[0062] An output tube, used for outputting the cell product in the culture container;

[0063] A driving member, the driving member is used to drive the medium to flow in the pipe;

[0064] The switch valves are arranged on the input pipe and the output pipe.

[0065] Preferably, the pipeline system further includes:

[0066] A waste liquid pipe, used for outputting the culture waste liquid in the culture container;

[0067] A one-way valve is arranged in the waste liquid pipe.

[0068] Preferably, the pipeline system further includes:

[0069] Connectors provided at the ends of the waste liquid pipe, the input pipe, and the output pipe;

[0070] A protective cover is sleeved on the outer side of the connector, and the protective cover and the connector are specifically detachably connected.

[0071] Preferably, the pipeline system further includes:

[0072] A manifold, wherein a first end of the manifold is connected to the culture container, and a second end of the manifold is connected to the input pipe and the output pipe.

[0073] Preferably, the pipeline system further includes:

[0074] An exhaust pipe is connected to the input pipe, and a gas sterilizing filter is provided at the end of the exhaust pipe away from the input pipe.

[0075] Preferably, the culture container comprises:

[0076] A shell, wherein a culture chamber is provided in the shell;

[0077] A diaphragm disposed on the housing, the diaphragm being configured to: provide a gas exchange channel for the gas medium and isolate the liquid medium;

[0078] The filling port is used to be connected to the pipeline system to input target cells and culture medium into the culture chamber.

[0079] Preferably, the culture container further comprises:

[0080] a partition, disposed in the culture chamber, for dividing the culture chamber into at least two culture layers, wherein the target cells can move in the culture layers;

[0081] The channel is used to connect two adjacent culture layers.

[0082] Preferably, at least two partitions are provided, and the partitions are arranged at intervals along the height direction of the culture container.

[0083] Preferably, the rotating assembly comprises:

[0084] A rotary drive member, wherein an output shaft of the rotary drive member is connected to the culture container;

[0085] A drive member seat is used to install the rotating drive member.

[0086] Preferably, the flip assembly comprises:

[0087] A turning table, the turning table is fixedly connected to the driving member seat;

[0088] A fixed seat, the first side of the turning table is rotatably connected to the fixed seat;

[0089] A turning driving member is connected to the turning platform and drives the turning platform to rotate around the fixing seat.

[0090] Preferably, the maximum turning angle between the turning platform and the horizontal plane is not less than 90°.

[0091] Preferably, the filling port is arranged on a first side of the top of the culture container.

[0092] Preferably, the vibration component comprises:

[0093] A buffer member disposed between the culture container and the placement table, wherein at least two buffer members are provided;

[0094] A mounting plate is arranged above the culture container, the mounting plate is in contact with the top of the culture container away from the buffer, and two sides of the mounting plate are respectively fixedly connected to the turning table;

[0095] A vibrator is used to drive the mounting plate and the culture container to vibrate in a vertical direction.

[0096] Preferably, the shaking device further comprises:

[0097] A rotation angle detector, used for detecting the rotation angle of the culture container on a horizontal plane;

[0098] Horizontal angle detection is used to detect the angle between the flip table and the horizontal plane.

[0099] Preferably, the culture unit further comprises:

[0100] An image acquisition device, the image acquisition device is used to obtain real-time morphology;

[0101] A comparison mechanism, the comparison mechanism is used to compare the real-time form with a preset form;

[0102] A focusing device is used to automatically focus the image acquisition device according to the comparison result until the real-time form meets the preset form.

[0103] Preferably, the image acquisition device is arranged on the outside of the culture container, and the culture container is made of a transparent material.

[0104] Preferably, the image acquisition device comprises:

[0105] A microscope camera assembly is arranged below the placement table, and the microscope camera assembly is used to obtain the real-time status of cells;

[0106] The placement table is provided with a collection window used in conjunction with the microscope camera assembly;

[0107] An illumination assembly is arranged above the culture container and used in conjunction with the microscope camera assembly.

[0108] Preferably, the focusing device comprises:

[0109] A mounting base, used for mounting the microscope camera assembly;

[0110] A camera bracket is arranged on the fixing seat, and the camera bracket is slidably connected to the mounting seat;

[0111] A sliding drive member, used for driving the mounting seat to move along the height direction of the camera bracket;

[0112] The focus controller controls the sliding drive member to drive the mounting seat to move according to the comparison result until the real-time shape meets the preset shape.

[0113] Preferably, it also includes:

[0114] A refrigeration unit is used to construct a refrigerated space.

[0115] The cell culture method provided by the present invention comprises the following steps: constructing a cell culture environment; transporting target cells and culture medium to a culture container located in the cell culture environment through a pipeline system; culturing the target cells to obtain cell products; and outputting the cell products from the culture container through the pipeline system.

[0116] Compared with the existing technology, the target cells and culture medium are transported to the culture container through the pipeline system, and the cell products are output from the culture container through the pipeline system, which effectively avoids contamination from the external environment during the input and output process. The target cells do not need to rely too much on manual labor during the culture process, the efficiency of cell culture is increased, and large-scale mass production can be achieved to meet the growing demand for cell culture.

[0117] The present invention also provides a cell culture device, which adopts the above-mentioned cell culture method. Since it belongs to the same technical concept as this method, solves the same technical problem, and should have the same beneficial effects, it will not be described in detail here. BRIEF DESCRIPTION OF THE DRAWINGS

[0118] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0119] Figure 1 A flow chart of a cell culture method provided by an embodiment of the present invention;

[0120] Figure 2 A schematic diagram of a shaking culture container provided in an embodiment of the present invention: wherein, Figure 2 (a) is a schematic diagram of a structure in which the culture container is placed horizontally in the initial state. Figure 2 (b) is a schematic diagram of a structure in which the culture container is flipped around the first axis to a first position, Figure 2 (c) is a schematic diagram of a structure in which the culture container continues to rotate around the second axis to a second position, Figure 2 (d) is a schematic diagram of a structure in which the culture container is reversely flipped around the first axis until the bottom surface of the culture container is parallel to the horizontal plane. Figure 2 (e) is a schematic diagram of a structure in which the culture container rotates in the opposite direction around the second axis to an initial state;

[0121] Figure 3 A structural block diagram of a cell culture device provided by an embodiment of the present invention;

[0122] Figure 4 A schematic diagram of the structure of a pipeline system provided by an embodiment of the present invention;

[0123] Figure 5 A schematic diagram of the structure of a cell culture device provided in an embodiment of the present invention;

[0124] Figure 6 A schematic diagram of the structure of a shaking device provided in an embodiment of the present invention;

[0125] Figure 7 A schematic diagram of the internal structure of a shaking device provided in an embodiment of the present invention (with a placement table);

[0126] Figure 8 A schematic diagram of the internal structure of a shaking device provided in an embodiment of the present invention (without a placement table);

[0127] Fig. 9 An embodiment of the present invention provides a structural schematic diagram of an image acquisition device.

[0128] 1. Inlet pipe; 120. Outlet pipe; 130. Drive member; 140. Switch valve; 150. Connector; 160. Protective cover; 170. Manifold; 180. Drain pipe; 190. Gas sterilizing filter; 121. Waste liquid pipe; 122. One-way valve; 200. Culture container; 300. Environment construction unit; 400. Refrigeration unit; 500. Waste liquid storage area; 210. Filling port; 220. Diaphragm; 230. Channel; 61. Placement table; 62. Rotation assembly; 63. Flip assembly; 64. Vibration assembly; 611. Collection window; 631. Flip table; 632. Fixing seat; 633. Flip drive member; 641. Mounting plate; 642. Vibrator; 7. Image acquisition device; 71. Microscope camera assembly; 72. Illumination assembly; 73. Illumination bracket. DETAILED DESCRIPTION

[0129] In order to enable those skilled in the art to better understand the technical solutions in this application, the technical solutions in the embodiments of this application will be clearly and completely described below. Obviously, the described embodiments are only part of the embodiments of this application, not all of them. Based on the embodiments in this application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of this application.

[0130] It should be noted that when an element is referred to as being "fixed on" or "set on" another element, it can be directly on the other element or indirectly set on the other element; when an element is referred to as being "connected to" another element, it can be directly connected to the other element or indirectly connected to the other element.

[0131] It should be understood that the terms "length", "width", "up", "down", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", "inside", "outside", etc., indicating the orientation or position relationship, are based on the orientation or position relationship shown in the drawings, and are only for the convenience of describing the present application and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore should not be understood as a limitation on the present application.

[0132] In addition, the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of the features. In the description of this application, "multiple" and "several" mean two or more, unless otherwise clearly and specifically defined.

[0133] It should be noted that the structures, proportions, sizes, etc. illustrated in the drawings of this specification are only used to match the contents disclosed in the specification for people familiar with this technology to understand and read, and are not used to limit the conditions under which this application can be implemented. Therefore, they have no substantive technical significance. Any structural modification, change in proportional relationship or adjustment of size should still fall within the scope of the technical content disclosed in this application without affecting the effects and purposes that can be achieved by this application.

[0134] The embodiments of the present invention are written in a progressive manner.

[0135] like Figure 1 As shown, an embodiment of the present invention provides a cell culture method, comprising the following steps:

[0136] Constructing cell culture environment;

[0137] The cell culture environment in this embodiment specifically refers to providing environmental conditions suitable for cell culture.

[0138] The target cells and culture medium are transported to a culture container 200 located in a cell culture environment through a pipeline system;

[0139] It should be noted that the target cells in the present technical solution refer to cells to be cultured, and the cell product specifically refers to a mixture of target cells and nutrient medium after the target cells are cultured.

[0140] In this embodiment, a pipeline system is used to transport the target cells and the culture medium to the culture container 200. The culture container 200 is located in a cell culture environment, and the target cells in the culture container 200 can obtain the gas required for cell growth from the cell culture environment. Among them, the target cells and the culture medium can be directly transported from the preparation equipment to the culture container 200 through the pipeline system, and the target cells and the culture medium can be indirectly transported to the culture container 200 through the pipeline system.

[0141] In this embodiment, a pipeline system is used to transport the target cells and the culture medium to the culture container 200, thereby preventing the target cells and the culture medium from being contaminated by the external environment from the source.

[0142] Shaking the culture container 200 by a shaking device so that the target cells and the culture medium are dispersed in the culture container 200;

[0143] After the target cells and the culture medium are transported into the culture container 200 through the pipeline system, in order to avoid the target cells and the culture medium from being locally concentrated in the culture container 200, which affects the efficiency of the target cell culture, the method provided by the present invention includes shaking the culture container 200 so that the target cells and the culture medium can be dispersed in the culture container 200. In particular, for the target cells that are adherent cells, the contact area between the inner wall of the culture container 200 and the target cells is increased so that the target cells can adhere to the wall.

[0144] Based on the cell culture environment and the culture medium, culturing target cells and vibrating the culture container to obtain a cell product;

[0145] During the culture process of the target cells, the culture medium provides the target cells with the nutrients they need for growth, and the cell culture environment provides the target cells with the gases they need for growth, so that the target cells grow under suitable conditions, thereby obtaining cell products.

[0146] It should be noted that, with respect to both culturing the target cells and vibrating the culture container, the culture container may be vibrated while the target cells are being cultured, or after the cells are cultured, or the culture container may be vibrated while the cells are being cultured but not after the cells are cultured. The selection may be made according to the state of the target cells during culture:

[0147] For cells that grow adherently, the vibrating culture container can be vibrated during the culture of the target cells and / or after the target cells are cultured. On the one hand, during the culture process, the culture container is vibrated slightly to allow the target cells to adhere to the wall. On the other hand, when harvesting, the culture container 200 is vibrated greatly to allow the target cells to detach from the wall and disperse in the culture medium to obtain the target cells.

[0148] For cells growing in suspension, there is no need to vibrate the culture container to remove the target cells;

[0149] For cells that tend to clump during growth, the culture container may be vibrated during the culture process to disperse the target cells so that the target cells can be better cultured.

[0150] The cell product is transported out of the culture container 200 via a pipeline system.

[0151] Specifically, in this embodiment, after the cell product is obtained, the cell product is output from the culture container 200 through the pipeline system. The cell product is output through the pipeline system to prevent the cell product from being contaminated during the output process. The output cell product can be transported to a storage device for storage through the pipeline system, and can also be transported to the next process through the pipeline system.

[0152] The shaking of the culture container 200 provided in the embodiment of the present invention comprises the following steps:

[0153] Flipping the culture container 200 around a first axis to a first position so that a first preset angle exists between the bottom surface of the culture container 200 and a horizontal plane;

[0154] shaking the culture container 200 around a second axis to mix the target cells and the culture medium;

[0155] Continue to rotate the culture container 200 around the second axis to a second position;

[0156] Flipping the culture container 200 around the first axis in the opposite direction so that the bottom surface of the culture container 200 is parallel to the horizontal plane;

[0157] Rotate the culture container 200 in the opposite direction around the second axis to reset the culture container 200;

[0158] There is a third preset angle between the first axis and the second axis.

[0159] like Figure 2 As shown, during the shaking process, the target cells and the culture medium can be better mixed by combining flipping and rotating the culture container 200 on a horizontal plane.

[0160] Please Figure 2 As shown in (a), in the initial state, the culture container 200 is placed horizontally, and the bottom surface of the culture container 200 is parallel to the horizontal plane, driving the culture container 200 to flip around the first axis to the first position, see Figure 2 As shown in (b), at this time, there is a first preset angle between the bottom surface of the culture container 200 and the horizontal plane. At this time, the target cells and the culture medium are located in the culture layer below the culture container 200. The culture container 200 is shaken left and right at the first position to mix the target cells and the culture medium at the channel 230; the culture container 200 is continuously rotated around the second axis to the second position. At this time, the channel 230 is located at the bottom of the culture container. Figure 2 (c); Flipping the culture container 200 around the first axis so that the bottom surface of the culture container 200 is parallel to the horizontal plane, and the target cells and the culture medium are redistributed to each culture layer through the channel 230, see Figure 2 (d), rotating the culture container 200 in the opposite direction around the second axis, so that the target cells and the culture in each culture layer are further mixed and dispersed, so that the culture container 200 is reset, see Figure 2 (e) The mixed target cells and culture medium are dispersed in each culture layer more evenly.

[0161] There is a third preset angle between the first axis and the second axis.

[0162] In the above method, the first preset angle is specifically 90° to 110°, the third preset angle is preferably 90°, and the angle of rotating the culture container 200 around the second axis is specifically 0° to 30°.

[0163] Furthermore, the first preset angle is specifically 90° to 110°, so that when the culture container 200 is located Figure 2 In state (c), the filling port 210 is located at the lowest point of the culture container. When it is necessary to harvest the target product in the culture container or output the waste liquid, the liquid in the culture container can be output more completely.

[0164] In summary, the target cells and culture medium in the embodiment of the present invention are transported to the culture container 200 through the pipeline system, and the cell product is output through the pipeline system. The target cells are completely closed during the culture process, thereby preventing the cell product from being contaminated by the outside; secondly, since the process of culturing the target cells does not need to rely too much on artificial culture, the efficiency of cell culture can be further improved, and large-scale mass production can be achieved to meet the growing demand for cell culture.

[0165] In addition, by shaking the culture container with a shaking device, the target cells and the culture medium can be mixed more evenly in the culture container. By rotating the culture container to a preset position, the target cells in the culture container can be harvested or waste liquid can be output through the pipeline system, so that the liquid in the culture container can be output more thoroughly.

[0166] It can be seen that, compared with the prior art, the cell culture method in the embodiment of the present invention can achieve large-scale mass production and avoid the contamination of cells by the external environment during the culture process.

[0167] In the above method, the nutrient medium and target cells in the embodiment of the present invention are transported in the form of liquid medium in the pipeline system.

[0168] In the above method, as one embodiment, the method provided in the embodiment of the present invention for culturing target cells comprises the following steps:

[0169] Get the current cell state;

[0170] Compare the current cell state with the preset cell state;

[0171] If the current cell state does not reach the preset cell state, the target cell continues to grow;

[0172] If the current cell state reaches the preset cell state, the target cells are harvested.

[0173] Specifically, current cell culture often involves placing the target cells and culture medium in a culture container 200 for a certain period of time to culture the target cells. However, the choice of culture time often depends mainly on the experience of the technician, and it is impossible to determine whether the cells meet the requirements after a certain period of culture. In the method provided in an embodiment of the present invention, the current cell state is compared with the preset cell state. When the current cell state does not reach the preset cell state, the target cells continue to be cultured. When the current cell state reaches the preset cell state, the target cells are harvested. In this way, the target cells are prevented from being harvested when they are not well cultured.

[0174] Furthermore, as one implementation mode, the method provided in the embodiment of the present invention, after the current cell state reaches a preset cell state and before the target cells are harvested, further includes the following steps: issuing an alarm signal.

[0175] Specifically, when the current cell state obtained reaches a preset cell state, an alarm signal can be issued to remind the user to harvest.

[0176] In the above structure, as one implementation mode, the method provided in the embodiment of the present invention obtains the current state of the target cell, specifically comprising the following steps:

[0177] Obtaining a target cell morphology image through an image acquisition device 7;

[0178] Compare the target cell map with the preset cell morphology map;

[0179] If they are inconsistent, the focal length of the image acquisition device is adjusted until the acquired target cell morphology image is consistent with the preset cell morphology. Figure 1 To;

[0180] Analyze the target cell morphology image according to an image analysis device to obtain cell uniformity, total cell count, and active cell count;

[0181] Comparing the cell uniformity with a preset cell uniformity;

[0182] If the cell uniformity is greater than the preset cell uniformity, vibrating the culture container until the obtained cell uniformity is no greater than the preset cell uniformity;

[0183] The current cell status includes the target cell morphology diagram, the total cell count and the active cell count.

[0184] Specifically, when the target cell morphology image is obtained by the image acquisition device 7, the clarity of the target cell morphology image may be insufficient, and it is difficult to determine whether the target cell morphology image is consistent with the preset cell morphology image. Figure 1At this time, it is necessary to adjust the focal length of the image acquisition device in time until the target cell morphology image obtained by the image acquisition device is consistent with the preset cell morphology. Figure 1 Then the target cell morphology image is analyzed by an image analysis device to obtain the total cell count and active cell count.

[0185] Furthermore, the image acquisition device 7 in the embodiment of the present invention can acquire the target cell morphology image in the acquisition area in real time, and the image acquisition device 7 in the embodiment of the present invention can also acquire the target cell morphology image in the acquisition area at a fixed time.

[0186] Preferably, the image acquisition device 7 in the embodiment of the present invention acquires the target cell morphology in the acquisition area in real time, analyzes the acquired cell morphology according to the image analysis device, determines whether the cell is an active cell, counts the active cells, and obtains the total cell count and the active cell count in the acquisition area.

[0187] In order to avoid the phenomenon of agglomeration during cell culture, the current cell state of the target cells in the embodiment of the present invention is obtained, and further includes the following steps: comparing the cell uniformity with the preset cell uniformity; if the cell uniformity is greater than the preset cell uniformity, vibrating the culture container until the obtained cell uniformity is no greater than the preset cell uniformity. The cell uniformity can be obtained by analyzing the target cell morphology image according to the image analysis device. The cell uniformity is used to characterize the uniformity of cell dispersion. If the detected cell uniformity is higher than the preset cell uniformity, it indicates that agglomeration may have occurred during the cell culture process. At this time, the target cells are dispersed by vibrating the culture container until the obtained cell uniformity is no greater than the preset cell uniformity.

[0188] In the above method, as one implementation mode, the cell status in the embodiment of the present invention includes but is not limited to the target cell morphology diagram, the total cell count and the active cell count.

[0189] Furthermore, the image acquisition device 7 in the embodiment of the present invention may acquire the cell status of target cells in only one region, or may acquire the cell status of target cells in multiple regions.

[0190] Preferably, in the method provided in the embodiment of the present invention, after shaking the culture container 200, the target cells and the culture medium are dispersed throughout the culture container 200. The image acquisition device 7 can only collect the cell state of the target cells in one area and compare it with the preset cell state, so as to determine whether the target cells in the entire culture container 200 have reached the preset cell state.

[0191] Furthermore, the growth stopping medium in the embodiment of the present invention flows in the tube in a liquid state.

[0192] In order to better distribute the target cells and culture medium in the culture container 200, specifically, the culture container 200 has at least two culture layers and a channel 230, wherein the channel 230 is arranged on the first side of the shell, and the channel 230 is connected to the culture layer.

[0193] In the above method, the target cell harvesting provided by the embodiment of the present invention includes the following two situations: For target cells that need to grow by adhering to the wall, the target cells grow by adhering to the inner surface of the culture container 200, and the method for harvesting the target cells is specifically as follows: a stopping medium is transported into the culture container 200 through a pipeline system, the target cells are detached from the wall, and the culture waste liquid is output through the pipeline system, and a filter is provided in the pipeline system, and only liquid and fragments smaller than the cell diameter are allowed to pass through, and the rest is the cell product. For target cells in suspension culture, the target cells grow in suspension in the culture medium, and the method for harvesting the target cells is specifically as follows: a stopping medium is transported into the culture container 200 through a pipeline system, and the culture waste liquid is output through the pipeline system, and a filter is provided in the pipeline system, and only liquid and fragments smaller than the cell diameter are allowed to pass through, and the rest is the cell product.

[0194] Furthermore, in the method provided in the embodiment of the present invention, before the target product and the culture medium are transported into the culture container 200 through the pipeline system, the following steps are also included:

[0195] Performing a first cleaning on the pipeline system and the culture container 200;

[0196] The culture container 200 is first sterilized.

[0197] In order to avoid contamination by residues in the pipeline system and the culture container 200, as one embodiment, before the target product and the culture medium are transported to the culture container 200 through the pipeline system, the pipeline system and the culture container 200 need to be first cleaned.

[0198] During the first cleaning process, if the culture container 200 is a culture container with at least two culture layers, in order to better clean the culture container, the culture container will also be shaken during the first cleaning. The process of shaking the culture container is similar to the above case and will not be repeated.

[0199] Specifically, in this embodiment, the pipeline system is emptied by physiological saline to discharge the residue in the pipeline system, and then the culture container 200 is first cleaned by the pipeline system. The first cleaning method is specifically: the physiological saline is delivered to the culture container 200 by the pipeline system, the culture container 200 is cleaned by the physiological saline, and finally the physiological saline is output by the pipeline system to prevent the residue in the culture container 200 from contaminating the cell culture process.

[0200] After the cleaning is completed, the culture container 200 is first disinfected. The specific steps of disinfecting the culture container 200 are: adjusting the cell culture environment temperature to 120° C. and disinfecting the culture container 200 by high temperature.

[0201] Furthermore, the number of cleaning times of the first cleaning is preferably 2 to 3 times.

[0202] In the above method, as one embodiment, the method provided in the embodiment of the present invention outputs the cell product from the culture container 200 through the pipeline system, which specifically includes the following steps:

[0203] Extract cell products and output them through the pipeline system;

[0204] Outputting the culture waste liquid in the culture container 200;

[0205] The culture container 200 is subjected to second cleaning and second sterilization.

[0206] Specifically, a preset volume of solution is extracted from the culture container 200 and outputted through the pipeline system, and the remaining culture waste liquid in the culture container 200 is outputted. The culture waste liquid can be outputted through other channels, or the culture waste liquid can also be outputted through the pipeline system to perform a second cleaning and a second disinfection on the culture container 200. Preferably, the culture waste liquid in the embodiment of the present invention is outputted through the pipeline system.

[0207] Furthermore, as one of the embodiments, the method in the embodiment of the present invention also includes the following steps: constructing a cold storage space; directly transporting the target cells to the culture container 200 through the pipeline system, or, after transporting the target cells to the cold storage space for storage, transporting them to the culture container 200 through the pipeline system.

[0208] The refrigerated space in the embodiment of the present invention uses a refrigerator to cool the refrigerated space. In order to further prevent the target cells from being contaminated by the external environment, after the target cells are prepared in the embodiment of the present invention, they can be directly transported to the culture container 200 through the pipeline system for culture. If the prepared target cells cannot be directly transported to the culture container 200 through the pipeline system, the target cells can also be transported to the refrigerated space for storage and then transported to the culture container 200 through the pipeline system.

[0209] Furthermore, the target cells in the embodiment of the present invention can be transported to a refrigerated space for direct storage. Alternatively, after the target cells in the embodiment of the present invention are prepared, they can also be transported to a storage device for storage, and then the storage device can be placed in a refrigerated space for refrigerated storage.

[0210] Preferably, the storage device in the embodiment of the present invention is a storage bag. A connector 150 is provided on the storage bag to facilitate the use with the pipeline system, which is more convenient and safe.

[0211] As one embodiment, the method in the embodiment of the present invention further includes the following steps: thawing the frozen cells to obtain target cells.

[0212] Specifically, the frozen cells are revived by placing the frozen cells in warm water at 37° C. to resuscitate the target cells. The frozen cells are heated more evenly by resuscitating the frozen cells in warm water.

[0213] Furthermore, after the cell product is output from the culture container 200 through the pipeline system, the method in the embodiment of the present invention also includes the following steps: centrifuging the cell product to obtain centrifuged cells; outputting the centrifuged cells through the pipeline system; and packaging the centrifuged cells.

[0214] Specifically, the cell product is output from the culture container 200 to the centrifugal device through the piping system, the cell product is centrifuged by the centrifugal device to obtain centrifuged cells, the centrifuged cells are then output through the piping system, the centrifuged cells are output to the packaging device, and the centrifuged cells are packaged by the packaging device.

[0215] Furthermore, the method in the embodiment of the present invention constructs a cell culture environment, comprising the following steps:

[0216] Environmental parameters are set, including CO2 concentration, O2 concentration, N2 concentration, temperature and humidity; a culture container 200 is set in a cell culture environment, and a diaphragm 220 is provided on the culture container 200, and the diaphragm 220 is configured to provide a gas exchange channel for the gas medium and isolate the liquid medium.

[0217] Furthermore, the cell culture environment in the embodiment of the present invention is specifically an incubator, in which a chamber for placing a culture container is formed. The environmental parameters in this embodiment include CO2 concentration, O2 concentration, N2 concentration, temperature and humidity. The environmental parameters can be set according to the culture requirements.

[0218] Specifically, the culture container 200 is located in a cell culture environment, and the culture container 200 may be provided with an opening at the top to facilitate the culture environment to provide an environmental medium for the growth of the target cells in the culture container 200. However, this way of setting the opening may cause the target cells and culture medium in the culture container 200 to be contaminated.

[0219] As a preferred embodiment, the culture container 200 is provided with a diaphragm 220, through which a gas exchange channel can be provided, so that the inside of the culture container 200 can exchange gas with the cell culture environment, providing the required gas environment for the culture of cells in the culture container 200. The diaphragm 220 isolates the liquid medium to prevent the target cells and the culture medium in the culture container 200 from being contaminated. The gas-permeable and liquid-impermeable film is an existing material, which will not be described in detail here.

[0220] Please Figure 3 As shown, the present invention also provides a cell culture device, which adopts any of the above-mentioned cell culture methods, and the device includes: an environment construction unit 300, which constructs a cell culture environment; an input unit, which is used to transport target cells and culture media to a culture container 200 located in the cell culture environment through a pipeline system; a culture unit, which is used to culture target cells to obtain cell products; and an output unit, which is used to output cell products from the culture container 200 through a pipeline system.

[0221] The cell culture device provided in the embodiment of the present invention adopts the above-mentioned cell culture method and can also achieve large-scale mass production to prevent cells from being contaminated by the external environment during the culture process.

[0222] In the above structure, in order to disperse the target cells and the culture medium in the culture container 200, as one embodiment, the culture unit provided in this embodiment includes: a shaking device, which is used to shake the culture container 200 so that the target cells and the culture medium are dispersed in the culture container 200. By setting the shaking device to shake the culture container 200, the target cells and the culture medium can be dispersed in the culture container 200, the contact area between the target cells and the culture container 200 is increased, and the target cells are easy to grow by adhering to the wall.

[0223] Furthermore, the shaking device in the embodiment of the present invention is preferably arranged in a cell culture environment.

[0224] Please Figures 5 to 9As shown, the shaking device provided in this embodiment includes: a placement table 61, used to place the culture container 200; a rotating component 62, used to drive the culture container 200 to rotate around the height direction of the placement table 61; a flipping component 63, connected to the rotating component 62, and the flipping component 63 is used to drive the culture container 200 to flip around one side of the placement table 61; a vibration component 64, arranged on one side of the culture container 200 raw material placement table 61, and the vibration component 64 is used to drive the culture container 200 to vibrate along the height direction of the placement table 61.

[0225] Specifically, the rotating assembly 62 can drive the culture container 200 to rotate around the height direction of the placement table 61, so that the target cells and culture medium of each layer are dispersed in the culture layer. The flip assembly 63 can drive the culture container 200 to flip around one side of the placement table 61, so that the target cells and culture medium can enter each culture layer. The vibration assembly 64 is used to drive the culture container 200 to vibrate along the height direction of the placement table 61. On the one hand, during the culture process, the vibration assembly 64 drives the culture container 200 to vibrate slightly so that the target cells adhere to the wall. On the other hand, when harvesting, the vibration assembly 64 drives the culture container 200 to vibrate greatly so that the target cells detach from the wall and disperse in the culture medium to obtain the target cells.

[0226] Furthermore, please Figure 4 As shown, Figure 4 A specific structural schematic diagram of a pipeline system provided in an embodiment of the present invention, the pipeline system in the method provided in an embodiment of the present invention includes: an input tube 110, at least two input tubes 110 are provided, respectively used to transport target cells and culture medium; an output tube 120, used to output the cell product in the culture container 200; a driving member 130, the driving member 130 is used to drive the medium to flow in the tube; and a switch valve 140 arranged on the input tube 110 and the output tube 120.

[0227] Specifically, the pipeline system in the embodiment of the present invention includes an input tube 110, an output tube 120, a driving member 130 and a switch valve 140, wherein at least two input tubes 110 are provided, which are respectively used to transport culture medium and target cells into the culture container 200, the output tube 120 is used to output cell products, and the driving member 130 is used to drive the medium to flow in the tube. The medium here refers to the culture medium and the target cells. The switch valve 140 is used to open and close the input tube 110 and the input valve, so that the medium is transported in the tube along a preset path.

[0228] Furthermore, the driving member 130 in the embodiment of the present invention is specifically a peristaltic pump, which can drive the medium to flow in the tube in a forward or reverse direction.

[0229] Furthermore, the switch valve 140 in the embodiment of the present invention is specifically a pipe pressure valve.

[0230] Furthermore, in the embodiment of the present invention, at least four input tubes 110 are provided, which are used to transport target cells, culture medium, growth stopping medium and physiological saline respectively.

[0231] Furthermore, as one of the implementation modes, the pipeline system in the embodiment of the present invention also includes a controller, wherein the controller is connected to the pipe pressure valve and the peristaltic pump, and the opening and closing of the pipe pressure valve and the peristaltic pump are controlled by the controller so that the medium can flow along a preset path.

[0232] Furthermore, the piping system in the method provided by the embodiment of the present invention also includes: a waste liquid pipe 121 for outputting the culture waste liquid in the culture container 200, and a pipe pressure valve is provided on the waste liquid pipe 121; and a one-way valve 122 arranged in the waste liquid pipe 121.

[0233] Specifically, the waste liquid in the culture container 200 is output through the pipeline system, and the pipeline system also includes a waste liquid pipe 121. The waste liquid in the culture container 200 is discharged through the waste liquid pipe 121. A pipe pressure valve is also provided on the waste liquid pipe 121 for opening and closing the waste liquid pipe 121. A one-way valve 122 is provided in the waste liquid pipe 121. The medium in the waste liquid pipe 121 can only be output from the culture container 200, and the external medium cannot enter the culture container 200 through the waste liquid pipe 121, thereby avoiding external contamination.

[0234] Furthermore, the piping system in the method provided in an embodiment of the present invention also includes: a connector 150 arranged at the ends of the waste liquid pipe 121, the input pipe 110, and the output pipe 120; a protective cover 160 sleeved on the outside of the connector 150, and the protective cover 160 and the connector 150 are specifically detachably connected.

[0235] Specifically, the end in this embodiment refers to the end of the pipeline away from the culture container 200. In order to prevent the end from being contaminated by the external environment, as one of the implementation methods, the pipeline system in the embodiment of the present invention also includes a connector 150 and a protective cover 160, wherein the connector 150 is arranged at the end of the waste liquid pipe 121, the input pipe 110 and the output pipe 120, and the connector 150 is used in conjunction with the quick connector on the storage bag. A protective cover 160 is arranged on the outside of the connector 150, and the protective cover 160 is detachably connected to the connector 150. When the connector 150 is in use, the protective cover 160 protects the connector 150 to prevent external harmful substances from entering the culture container 200 through the pipeline system.

[0236] In the above method, as one implementation mode, the first ends of the input tube 110 and the output tube 120 in the embodiment of the present invention can be directly connected to the culture container 200 .

[0237] As another embodiment, the piping system in the method provided in the embodiment of the present invention further includes: a manifold 170, wherein the first end of the manifold 170 is connected to the culture container 200, and the second end of the manifold 170 is connected to the input pipe 110 and the output pipe 120.

[0238] Specifically, in the embodiment of the present invention, since a manifold 170 is provided, the first ends of the input tube 110 and the output tube 120 are connected to the manifold 170, and are transported to the culture container 200 through the manifold 170. There is only one connection port between the pipeline system and the culture container 200, which is safer.

[0239] Furthermore, the manifold 170 and the culture container 200 are detachably connected, so that the culture container 200 can be easily replaced.

[0240] Furthermore, the pipeline system in the method provided by the embodiment of the present invention further includes: an exhaust pipe 180 connected to the input pipe 110 , and a gas sterilization filter 190 is provided at the end of the exhaust pipe 180 away from the input pipe 110 .

[0241] Specifically, the driving member 130 needs to discharge the gas in the pipe during the process of driving the medium to flow in the pipe to avoid excessive air pressure in the pipe. In the embodiment of the present invention, an exhaust pipe 180 is provided, and the exhaust pipe 180 is connected to the input pipe 110. The gas in the input pipe 110 and the output pipe 120 is discharged through the exhaust pipe 180. In order to prevent external harmful substances from entering the pipeline system through the exhaust pipe 180, a gas sterilization filter 190 is provided at the end of the exhaust pipe 180 away from the input pipe 110. The air entering the exhaust pipe 180 is filtered by the gas sterilization filter 190, which is safer.

[0242] In the above structure, as one of the implementation modes, the culture container 200 provided in this embodiment includes: a shell, in which a culture chamber is provided; a diaphragm 220 arranged on the shell, and the diaphragm 220 is configured to: provide a gas exchange channel for the gas medium and isolate the liquid medium; a filling port 210, which is used to be connected to the pipeline system to input target cells and culture medium into the culture chamber.

[0243] Specifically, the pipeline system is connected to the filling port 210, and the target cells and culture medium are transported into the culture chamber through the filling port 210. The culture medium provides the target cells with the nutrients needed for growth. The diaphragm 220 is set on the shell, and the gas in the cell culture environment can exchange gas with the culture chamber through the diaphragm 220, which is used to update the gas medium in the culture chamber and provide a gas growth environment for the target cells. Furthermore, the diaphragm 220 is configured to provide a gas exchange channel for the gas medium and isolate the liquid medium, so as to prevent the liquid medium in the cell culture environment from entering the culture chamber through the diaphragm 220, which is safer.

[0244] In the above structure, in order to further increase the contact area between the target cells and the inner wall of the culture container 200, it is convenient for the target cells to grow by adhering to the wall during the culture process. As one embodiment, the culture container 200 provided in this embodiment also includes: a partition, which is arranged in the culture chamber, and is used to separate the culture chamber into at least two culture layers, and the target cells can move in the culture layer; a channel 230, which is arranged on the first side of the shell, and the channel is connected with the culture layer, and the filling port is connected with the channel, and the culture medium and the target cells can be transported into the channel through the filling port, and the culture medium and the target cells enter each culture layer through the channel.

[0245] Furthermore, by setting a partition in the culture chamber, the culture chamber is divided into at least two culture layers, and the culture layer is configured so that the target cells can move in the culture layer. When the culture cells are shaken, the target cells and the culture medium can be dispersed in the culture container 200. The various culture layers are connected by channels 230 to facilitate the flow of target cells and culture medium.

[0246] More specifically, the filling port 210 is connected to at least one of the culture layers, and the target cells and the culture medium are input into the culture layer through the filling port 210, and the culture container 200 is shaken to disperse the target cells and the culture medium.

[0247] At least one filling port 210 is provided, and multiple filling ports 210 may also be provided. In order to further prevent the cell culture environment from being polluted by the outside world, only one filling port 210 is provided in the embodiment of the present invention.

[0248] In the above structure, as one implementation mode, at least two partitions are provided in this embodiment, and the partitions are arranged at intervals along the height direction of the culture container 200 .

[0249] In the above structure, as one implementation mode, the rotating assembly 62 provided in this embodiment includes: a rotating driving member, the output shaft of which is connected to the culture container 200; and a driving member seat for mounting the rotating driving member.

[0250] Specifically, the rotating component 62 provided in the embodiment of the present invention includes a rotating driving member and a driving member seat, wherein the output shaft of the rotating driving member is connected to the culture container 200, and is used to drive the culture container 200 to rotate around the output shaft, and the driving member seat is used to install the driving member, and the driving member seat is connected to the flipping component 63. Under the action of the flipping component 63, the rotating driving member and the culture container 200 are driven to flip around one side of the placement table 61.

[0251] Furthermore, the rotary driving member in the embodiment of the present invention is specifically a rotary motor.

[0252] Furthermore, the rotating assembly 62 in the embodiment of the present invention can drive the culture container 200 to rotate forward and reverse around the output shaft, and the rotation angle of the culture container 200 specifically ranges from -15° to 15°.

[0253] In the above structure, as one of the implementation modes, the flip assembly 63 provided in this embodiment includes: a flip table 631, which is fixedly connected to the driving member seat; a fixed seat 632, and the first side of the flip table 631 is rotatably connected to the fixed seat 632; and a flip driving member 633 connected to the flip table 631 to drive the flip table 631 to flip around the fixed seat 632.

[0254] More specifically, the flip table 631 is connected to the driving member seat, and the driving member seat is installed through the flip table 631. The first side of the flip table 631 is rotatably connected to the fixed seat 632. The flip driving member 633 is connected to the flip table 631. The flip driving member 633 drives the flip table 631 to flip around the fixed seat 632, which makes it easier for target cells and culture media to enter the culture layers of each layer.

[0255] Furthermore, as one implementation mode, the flip driving component 633 in the embodiment of the present invention is specifically any one of a cylinder, a hydraulic cylinder and a linear motor.

[0256] The telescopic end of the flip driving member 633 is connected to the bottom surface of the flip platform 631 away from the driving member seat. The telescopic end of the flip driving member 633 is telescoped to drive the flip platform 631 to flip.

[0257] Furthermore, in order to better harvest the target cells, as one implementation mode, the maximum flip angle between the flip table 631 in the embodiment of the present invention and the horizontal plane is not less than 90°.

[0258] As one specific implementation manner, the angle range between the turning platform 631 and the horizontal plane in the embodiment of the present invention is specifically -5° to 95°.

[0259] As one implementation manner, the filling port 210 in the cell culture device in the embodiment of the present invention is arranged on the first side of the top of the culture container 200 , which makes it easier to clean the culture container 200 .

[0260] In the above structure, as one of the implementation modes, the vibration component 64 provided in this embodiment includes: a buffer member arranged between the culture container 200 and the placement table 61, at least two buffer members are arranged; a mounting plate 641 arranged above the culture container 200, the mounting plate 641 is in contact with the top of the culture container 200 away from the buffer member, and the two sides of the mounting plate 641 are respectively fixedly connected to the turning table 631; a vibrator 642, the vibrator 642 is used to drive the mounting plate 641 and the culture container 200 to vibrate in the vertical direction.

[0261] Specifically, the vibration component 64 is used to drive the culture container 200 to vibrate along the height direction of the placement table 61. By setting a buffer between the culture container 200 and the placement table 61, uneven vibration of the culture container 200 during the vibration process is avoided. The vibrator 642 is fixed to the top of the culture container 200 through the mounting plate 641, and the two sides of the mounting plate 641 are respectively fixedly connected to the turning table 631.

[0262] Furthermore, the vibrator 642 is specifically a vibration motor.

[0263] In the above structure, as one of the implementation modes, the shaking device provided in this embodiment further includes: a rotation angle detector for detecting the rotation angle of the culture container 200 on the horizontal plane; and a horizontal angle detection for detecting the angle between the turning table 631 and the horizontal plane.

[0264] In the above structure, the cells need to be placed in the horizontal direction during the culture process. The shaking device in the embodiment of the present invention also includes a rotation angle detector and a horizontal angle detector. The rotation angle detector is used to detect the rotation angle of the culture container 200 on the placement table 61. Before the culture container 200 is turned over, according to the angle detected by the rotation detector, the rotary drive member drives the culture container 200 to rotate until the rotation angle detected by the rotation detector is 0°, so that it can be ensured that the filling port 210 is located at the lowest point of the culture container 200 when turning over. The horizontal angle detector is used to detect the angle between the turning table 631 and the horizontal plane. Before the culture is carried out, based on the angle detected by the horizontal angle detector, the turning drive member 633 drives the turning table 631 to rotate so that the angle detected by the horizontal angle detector is 0°.

[0265] In the above structure, as one of the implementation modes, the culture unit provided in this embodiment also includes: an image acquisition device 7, which is used to obtain the real-time morphology of the target cells; a comparison mechanism, which is used to compare the real-time morphology with the preset morphology; and a focusing device, which is used to adjust the focal length of the image acquisition device 7 according to the comparison result until the real-time morphology meets the preset morphology.

[0266] Specifically, in the process of collecting the real-time morphology of the target cells, the image acquisition device 7 often needs to be focused. As one implementation method, the culture unit provided in the embodiment of the present invention also includes an image acquisition device 7, which is used to obtain the real-time morphology of the target cells. The real-time morphology collected is compared with the preset morphology through a comparison unit. If the real-time morphology meets the preset morphology, the current cell state is output; if the real-time morphology does not meet the preset morphology, the focal length of the image acquisition device 7 is adjusted until the real-time morphology meets the preset morphology, and then the current cell state is output.

[0267] Furthermore, the image acquisition device 7 in the embodiment of the present invention is arranged on the outside of the culture container 200, and the culture container 200 is made of a transparent material.

[0268] Furthermore, the image acquisition device 7 can be set at any position outside the culture container 200, and multiple microscope camera components 71 can also be used to collect cell morphology at multiple positions.

[0269] Please Fig. 9 As shown, an embodiment of the present invention provides a specific image acquisition device. The image acquisition device 7 provided in this embodiment includes: a microscopic camera assembly 71 disposed below a placement table 61, and the microscopic camera assembly 71 is used to obtain the real-time state of cells; a collection window used in conjunction with the microscopic camera assembly 71 is disposed on the placement table 61; and an illumination assembly 72 disposed above the culture container 200 and used in conjunction with the microscopic camera assembly 71.

[0270] Specifically, the image acquisition device 7 includes a microscope camera assembly 71, which is used to obtain the real-time status of the cells. The microscope camera assembly 71 is arranged below the placement table 61, and the real-time status of the cells is obtained through the microscope camera assembly 71. A collection window 611 is arranged on the placement table 61, and the collection window 611 is used in conjunction with the microscope camera assembly 71. The cell morphology is collected through the collection window by the microscope camera assembly 71. The lighting assembly 72 is arranged above the culture container 200, and the lighting assembly 72 is used in conjunction with the microscope camera assembly 71. The lighting assembly 72 is used to supplement the light for the microscope camera assembly 71.

[0271] Furthermore, as one of the implementation modes, the image acquisition device 7 in the embodiment of the present invention also includes a lighting bracket 73, and the lighting bracket 73 is used to install the lighting component 72. The lighting bracket 73 is slidably connected to the lighting component 72, and the position of the lighting component 72 on the lighting bracket 73 can be adjusted.

[0272] Furthermore, the collection window 611 in the embodiment of the present invention is specifically a hollow window, or the collection window 611 in the embodiment of the present invention is made of a transparent material.

[0273] In the above structure, as one of the implementation modes, the focusing device provided in this embodiment includes: a mounting seat for mounting the microscope camera assembly 71; a camera bracket arranged on the fixed seat 632, and the camera bracket is slidably connected to the mounting seat; a sliding drive member for driving the mounting seat to move along the height direction of the camera bracket; and a focusing controller, which controls the sliding drive member to drive the mounting seat to move according to the comparison result until the real-time shape meets the preset shape.

[0274] When the real-time form captured by the microscope camera assembly 71 does not conform to the preset form, the focus device needs to adjust the focal length, and the focus device adjusts the focal length mainly by adjusting the distance between the microscope camera assembly 71 and the culture container 200. Specifically, the focus device includes a mounting seat, the mounting seat is used to mount the microscope camera assembly 71, and a camera bracket is provided on the fixed seat 632. The camera bracket and the mounting seat are connected by sliding, and the sliding drive is used to drive the mounting seat to move along the height direction of the camera bracket to adjust the focal length. In order to achieve automatic focus, the focus device provided by the present invention also includes a focus controller. The focus controller controls the sliding drive to drive the mounting seat to move according to the comparison result between the real-time form and the preset form until the real-time form meets the preset form.

[0275] In the above structure, as one implementation mode, the cell culture device provided in this embodiment further includes: a refrigeration unit 400, the refrigeration unit 400 is used to construct a refrigeration space, and the refrigeration space can be used to store any one of the culture medium or the target cells.

[0276] In the above structure, as one implementation mode, the cell culture device provided in the embodiment of the present invention further includes a waste liquid storage area 500 for storing waste liquid output by the pipeline system.

[0277] In the above structure, as one implementation mode, the cell culture device provided in the embodiment of the present invention further includes a display, which can be used to display the real-time morphology and alarm signals of the cells in the culture unit during the culture process.

[0278] In the above structure, as one of the implementation modes, the cell culture device in the embodiment of the present invention also includes an Internet of Things platform, through which remote monitoring and management of the cell preparation process can be achieved, and the culture process can be controlled in real time through mobile platforms such as mobile phones, thereby realizing unattended fully automatic cell preparation.

[0279] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A cell culture method, characterized in that: The following steps are involved: Constructing cell culture environment; transporting target cells and culture medium into a culture container through a pipeline system, wherein the culture container is located in the cell culture environment; Shaking the culture container by a shaking device to disperse the target cells and the culture medium in the culture container; Based on the cell culture environment and the culture medium, culturing target cells and vibrating the culture container to obtain a cell product; exporting the cell product from the culture container via the piping system; in, Shaking the culture container by a shaking device comprises the following steps: Flipping the culture container around a first axis to a first position so that a first preset angle exists between the bottom surface of the culture container and a horizontal plane; shaking the culture container around a second axis to mix the target cells and the culture medium; Continue shaking the culture container around the second axis to a second position; Flipping the culture container in the opposite direction around the first axis so that the bottom surface of the culture container is parallel to the horizontal plane; Shaking the culture container in the opposite direction around the second axis to reset the culture container; There is a third preset angle between the first axis and the second axis; The shaking device comprises: a placement table (61) for placing the culture container; a rotating component (62) for driving the culture container to rotate on the placement table (61); a flipping component (63) connected to the rotating component (62), the flipping component (63) being used to drive the placement table (61) and the culture container to flip around a first side of the placement table (61); and a vibration component (64) being arranged on a side of the culture container away from the placement table (61), the vibration component (64) being used to drive the culture container to vibrate along the height direction of the placement table (61); The culture container comprises: A shell, wherein a culture chamber is provided in the shell; A diaphragm disposed on the housing, the diaphragm being configured to: provide a gas exchange channel for the gas medium and isolate the liquid medium; A filling port, used to be connected to the pipeline system to input target cells and culture medium into the culture chamber; a partition, disposed in the culture chamber, for dividing the culture chamber into at least two culture layers, wherein the target cells can move in the culture layers; A channel, used to connect two adjacent culture layers; The filling port is disposed on a first side of the top of the culture container.

2. The cell culture method according to claim 1, characterized in that The culturing of target cells comprises the following steps: Get the current cell state of the target cell; Comparing the current cell state with a preset cell state; If the current cell state does not reach the preset cell state, the target cell continues to grow; If the current cell state reaches the preset cell state, the target cells are harvested.

3. The cell culture method according to claim 2, characterized in that The obtaining of the current cell state of the target cell specifically comprises the following steps: Obtaining a morphological image of target cells through an image acquisition device; Comparing the target cell morphology image with a preset cell morphology image; If not, adjusting the focal length of the image acquisition device until the acquired target cell morphology image is consistent with the preset cell morphology image; Analyze the target cell morphology image according to an image analysis device to obtain cell uniformity, total cell count, and active cell count; Comparing the cell uniformity with a preset cell uniformity; If the cell uniformity is greater than the preset cell uniformity, vibrating the culture container until the obtained cell uniformity is no greater than the preset cell uniformity; The current cell status includes the target cell morphology diagram, the total cell count and the active cell count.

4. The cell culture method according to any one of claims 1 to 3, characterized in that: Before the target product and the culture medium are transported to the culture container through the pipeline system, the following steps are also included: The culture container is first cleaned and first sterilized.

5. The cell culture method according to any one of claims 1 to 3, characterized in that: The method of outputting the cell product from the culture container through the pipeline system specifically includes the following steps: Extract cell products and output them through the pipeline system; Outputting the culture waste liquid in the culture container; The culture container is subjected to a second cleaning and a second sterilization.

6. The cell culture method according to any one of claims 1 to 3, characterized in that: The following steps are also included: constructing cold storage space; The target cells are directly transported to the culture container through the pipeline system, or the target cells are transported to the culture container through the pipeline system after being transported to a cold storage space for storage.

7. The cell culture method according to claim 6, characterized in that: Before the target cells and the culture medium are transported into the culture container through the pipeline system, the following steps are also included: Thaw the frozen cells and obtain the target cells.

8. The cell culture method according to any one of claims 1 to 3 and 7, characterized in that: After the cell product is output from the culture container through the pipeline system, the following steps are also included: Centrifuging the cell product to obtain centrifuged cells; Outputting the centrifuged cells through a tubing system; The centrifuged cells are packaged.

9. The cell culture method according to any one of claims 1 to 3 and 7, characterized in that: The cell culture environment is constructed, comprising the following steps: Setting environmental parameters, including CO2 concentration, O2 concentration, N2 concentration, temperature and humidity; The culture container is arranged in the cell culture environment. A diaphragm is arranged on the culture container. The diaphragm is configured to provide a gas exchange channel for the gas medium and isolate the liquid medium.

10. A cell culture device, characterized in that: The cell culture method according to any one of claims 1 to 9 comprises: An environment construction unit (300) constructs a cell culture environment; An input unit, used for transporting target cells and culture medium into a culture container (200) in the cell culture environment through a pipeline system; A shaking device, the shaking device is used to shake the culture container (200) so that the target cells and the culture medium are dispersed in the culture container (200); shaking the culture container by the shaking device comprises the following steps: flipping the culture container around a first axis to a first position so that a first preset angle exists between the bottom surface of the culture container and a horizontal plane; shaking the culture container around a second axis so that the target cells and the culture medium are mixed; continuing to shake the culture container around the second axis to a second position; flipping the culture container in the opposite direction around the first axis so that the bottom surface of the culture container is parallel to the horizontal plane; shaking the culture container in the opposite direction around the second axis so that the culture container is reset; a third preset angle exists between the first axis and the second axis; A culture unit, the culture unit is used to culture target cells based on the cell culture environment and the culture medium and vibrate the culture container to obtain a cell product; an output unit, used for outputting the cell product from the culture container (200) through the pipeline system; in, The shaking device comprises: a placement table (61) for placing the culture container; a rotating component (62) for driving the culture container to rotate on the placement table (61); a flipping component (63) connected to the rotating component (62), the flipping component (63) being used to drive the placement table (61) and the culture container to flip around a first side of the placement table (61); and a vibration component (64) being arranged on a side of the culture container away from the placement table (61), the vibration component (64) being used to drive the culture container to vibrate along the height direction of the placement table (61); The culture container comprises: A shell, wherein a culture chamber is provided in the shell; A diaphragm disposed on the housing, the diaphragm being configured to: provide a gas exchange channel for the gas medium and isolate the liquid medium; A filling port, used to be connected to the pipeline system to input target cells and culture medium into the culture chamber; a partition, disposed in the culture chamber, for dividing the culture chamber into at least two culture layers, wherein the target cells can move in the culture layers; A channel, used to connect two adjacent culture layers; The filling port is disposed on a first side of the top of the culture container.

11. The cell culture device according to claim 10, characterized in that: The pipeline system comprises: An input tube (110), wherein at least two input tubes (110) are provided, and are used to transport target cells and culture medium respectively; An output tube (120), used for outputting the cell product in the culture container; A driving member (130), the driving member (130) being used to drive the medium to flow in the pipe; A switch valve (140) is arranged on the input pipe (110) and the output pipe (120).

12. The cell culture device according to claim 11, characterized in that: The pipeline system further comprises: A waste liquid pipe (121), used for outputting the culture waste liquid in the culture container; A one-way valve (122) is arranged in the waste liquid pipe (121).

13. The cell culture device according to claim 12, characterized in that: The pipeline system further comprises: Connectors (150) disposed at the ends of the waste liquid pipe (121), the input pipe (110), and the output pipe (120); A protective cover (160) is sleeved on the outside of the connecting head (150); the protective cover (160) and the connecting head (150) are specifically detachably connected.

14. The cell culture device according to any one of claims 11 to 13, characterized in that: The pipeline system further comprises: A manifold (170), wherein a first end of the manifold (170) is connected to the culture container, and a second end of the manifold (170) is connected to the input tube (110) and the output tube (120).

15. The cell culture device according to any one of claims 11 to 13, characterized in that: The pipeline system further comprises: An exhaust pipe (180) is connected to the input pipe (110), and a gas sterilization filter (190) is provided at the end of the exhaust pipe (180) away from the input pipe (110).

16. The cell culture device according to claim 15, characterized in that: At least two partitions are provided, and the partitions are arranged at intervals along the height direction of the culture container (200).

17. The cell culture device according to claim 16, characterized in that: The rotating assembly (62) comprises: A rotary drive member, wherein an output shaft of the rotary drive member is connected to the culture container (200); A drive member seat is used to install the rotating drive member.

18. The cell culture device according to claim 17, characterized in that: The flip assembly (63) comprises: A turning table (631), the turning table (631) being fixedly connected to the driving member seat; A fixed seat (632), the first side of the turning platform (631) being rotatably connected to the fixed seat (632); A turning driving member (633) connected to the turning platform (631) and driving the turning platform (631) to rotate around the fixing seat (632).

19. The cell culture device according to claim 18, characterized in that: The maximum turning angle between the turning platform (631) and the horizontal plane is not less than 90°.

20. The cell culture device according to any one of claims 18 to 19, characterized in that: The vibration component (64) comprises: A buffer member disposed between the culture container (200) and the placement table (61), wherein at least two buffer members are provided; A mounting plate (641) is arranged above the culture container (200), the mounting plate (641) is in contact with the top of the culture container (200) away from the buffer, and two sides of the mounting plate (641) are respectively fixedly connected to the turning platform (631); A vibrator (642), the vibrator (642) being used to drive the mounting plate (641) and the culture container (200) to vibrate in a vertical direction.

21. The cell culture device according to any one of claims 18 to 19, characterized in that: The shaking device further comprises: A rotation angle detector, used for detecting the rotation angle of the culture container (200) on a horizontal plane; Horizontal angle detection is used to detect the angle between the flip table (631) and the horizontal plane.

22. The cell culture device according to any one of claims 18 to 19, characterized in that: The culture unit further comprises: An image acquisition device (7), the image acquisition device (7) being used to obtain real-time morphology; A comparison mechanism, the comparison mechanism is used to compare the real-time form with a preset form; A focusing device is used to automatically focus the image acquisition device according to the comparison result until the real-time form meets the preset form.

23. The cell culture device according to claim 22, characterized in that: The image acquisition device (7) is arranged outside the culture container (200), and the culture container (200) is made of a transparent material.

24. The cell culture device according to claim 23, characterized in that: The image acquisition device (7) comprises: A microscope camera assembly (71) disposed below the placement table (61), the microscope camera assembly (71) being used to obtain the real-time status of cells; The placement table (61) is provided with a collection window for use with the microscope camera assembly (71); An illumination assembly (72) is disposed above the culture container (200) and is used in conjunction with the microscope camera assembly (71).

25. The cell culture device according to claim 24, characterized in that: The focusing device comprises: A mounting base, used for mounting the microscope camera assembly (71); A camera bracket arranged on the fixing seat (632), the camera bracket being slidably connected to the mounting seat; A sliding drive member, used for driving the mounting seat to move along the height direction of the camera bracket; The focus controller controls the sliding drive member to drive the mounting seat to move according to the comparison result until the real-time shape meets the preset shape.

26. The cell culture device according to claim 10, characterized in that: Also includes: A refrigeration unit (400), wherein the refrigeration unit (400) is used to construct a refrigeration space.

Images