Cell sorting and culturing method and apparatus, computer device, and storage medium

Abstract

The present application relates to the technical field of biomedicine. Provided are a cell sorting and culturing method and apparatus, a computer device, and a storage medium. The cell sorting and culturing method comprises separating a sample solution by means of a density gradient centrifugation method to obtain a cell solution to be cultured; conveying said cell solution to a culturing device by means of a first pipeline switching operation, and culturing said cell solution to obtain a cultured cell solution; and acquiring the cultured cell solution by means of a second pipeline switching operation, and concentrating and washing the cultured cell solution to obtain a target cell solution. The cell sorting and culturing method can ensure a fully enclosed one-stop sterile operating environment, avoid the risk of contamination during cell operation site changing, and improve the cell treatment stability. In addition, continuous operation between cell solution sorting and culturing treatments can be realized by means of the two pipeline switching operations, thereby improving the degree of automation and continuity of cell treatment, saving operation time, and improving cell treatment efficiency.

Description

Cell sorting and culture method, device, computer device and storage medium

[0001] The present application claims priority to the Chinese patent application No. 202411353279.1 filed on September 26, 2024, and entitled "Cell sorting and culture method, device, computer device and storage medium", the whole content of which is incorporated herein by reference. TECHNICAL FIELD

[0002] The present application relates to the field of biological medical technology, in particular to a cell sorting and culture method, device, computer device and storage medium. BACKGROUND

[0003] Peripheral blood mononuclear cells (PBMCs) are single-nucleated cells isolated from peripheral blood and widely used in antibody research. The isolated PBMC cell solution usually needs to be transduced with antibodies and cultured and coated before it can become a cell solution with antibodies. The inventors realized that in the prior art, the environment required for different steps in the process of cell separation, antibody addition and culture is different (such as different temperatures, different humidities, etc.), so different cell operation sites need to be changed, which increases the time and labor consumption. At the same time, the different cell operation sites cannot be fully closed, and the sterile operation environment can be easily damaged during the change of sites, affecting the continuity and stability of cell processing.

[0004] Therefore, there is an urgent need for a fully closed cell processing scheme that can automatically sort and culture. SUMMARY

[0005] Therefore, it is necessary to provide a cell sorting and culture method, device, computer device and storage medium to solve the problem of insufficient continuity and stability in the existing cell sorting and culture process.

[0006] A cell sorting and culture method, comprising:

[0007] Separating and processing a sample solution by density gradient centrifugation to obtain a cell solution to be cultured;

[0008] Transferring the cell solution to be cultured into a culture device by a first switching pipeline operation, and culturing the cell solution to be cultured to obtain a cultured cell solution;

[0009] Obtaining the cultured cell solution by a second switching pipeline operation, and concentrating and cleaning the cultured cell solution to obtain a target cell solution.

[0010] A cell sorting and culturing device comprises:

[0011] A separation processing module is configured to separate and process a sample liquid by a density gradient centrifugation method to obtain a cell liquid to be cultured.

[0012] A culturing processing module is configured to deliver the cell liquid to be cultured into a culturing device by a first switching pipeline operation and to process the cell liquid to be cultured to obtain a cultured cell liquid.

[0013] A concentration and cleaning processing module is configured to obtain the cultured cell liquid by a second switching pipeline operation and to process the cultured cell liquid by concentration and cleaning to obtain a target cell liquid.

[0014] A computer device comprises a memory, a processor, and computer readable instructions stored in the memory and executable on the processor, and the processor executes the computer readable instructions to implement the above cell sorting and culturing method.

[0015] A computer readable storage medium stores computer readable instructions, and the computer readable instructions are executed by one or more processors to implement the above cell sorting and culturing method.

[0016] In the above cell sorting and culturing method, device, computer device, and storage medium, the sample liquid is first separated and processed by a density gradient centrifugation method to obtain a cell liquid to be cultured; then the cell liquid to be cultured is delivered into a culturing device by a first switching pipeline operation and processed to obtain a cultured cell liquid; finally, the cultured cell liquid is obtained by a second switching pipeline operation and processed by concentration and cleaning to obtain a target cell liquid. The separation processing is performed in a centrifugation device, and the centrifugation device and the culturing device are connected by a pipeline to realize integrated integration of the cell processing device, which can ensure a full-closed one-stop sterile operation environment, avoid the pollution risk when changing the cell operation site, and improve the stability of cell processing. At the same time, the two switching pipeline operations can realize continuous operation between the sorting and culturing of the cell liquid, improve the automation degree and continuity of cell processing, save operation time, and improve processing efficiency.

[0017] The details of one or more embodiments of the present application are presented in the following drawings and description, and other features and advantages of the present application will become apparent from the description, drawings, and claims. BRIEF DESCRIPTION OF DRAWINGS

[0018] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings needed to be used in the description of the embodiments of the present application will be briefly introduced. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor.

[0019] Fig. 1 is a flowchart of a cell sorting and culturing method according to an embodiment of the present application;

[0020] Fig. 2 is a schematic diagram of the distribution of devices in a cell sorting and culturing method according to an embodiment of the present application;

[0021] Fig. 3 is another schematic diagram of the distribution of devices in a cell sorting and culturing method according to an embodiment of the present application;

[0022] Fig. 4 is a schematic diagram of the structure of a cell sorting and culturing device according to an embodiment of the present application;

[0023] Fig. 5 is a schematic diagram of a computer device according to an embodiment of the present application. DETAILED DESCRIPTION

[0024] The technical solutions of the embodiments of the present application will be described clearly and completely below with reference to the drawings of the embodiments of the present application. Obviously, the described embodiments are only some embodiments of the present application, rather than all the embodiments. Based on the embodiments of the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of the present application.

[0025] The cell sorting and culturing method provided in the embodiments can be applied in application scenarios of automatic sorting and culturing of cell liquid, especially in scenarios of automatic sorting of peripheral blood mononuclear cells (PBMC) from blood samples and culturing. The whole process of sorting and culturing needs to be completed by cooperation of hardware devices and software servers. The hardware devices and the software servers are connected in communication so as to exchange data and transmit instructions between the hardware devices and the software servers. The hardware devices include but are not limited to various containers, pipelines and instrument devices. The software server can be implemented by an independent server or a server cluster composed of multiple servers.

[0026] In an embodiment, as shown in Fig. 1, a cell sorting and culturing method is provided, including the following steps S10-S30:

[0027] S10, separating and processing the sample liquid by using a density gradient centrifugation method to obtain a cell liquid to be cultured.

[0028] It is understandable that the sample liquid refers to a cell liquid sample that needs to be centrifuged, and the cell liquid to be cultured refers to a liquid separated from the sample liquid. When the cell sorting and culturing method is applied to the scenario of separating PBMCs from peripheral blood, the sample liquid is a peripheral blood sample, and the cell liquid to be cultured refers to the PBMC cell liquid separated from the peripheral blood. The density gradient centrifugation method is a method of separating cells by forming a continuous or discontinuous density gradient in a centrifuge container with a specific medium and allowing the cells to stratify and separate under the action of gravity or centrifugal force.

[0029] The peripheral blood sample includes red blood cells, granulocytes, lymphocytes, and monocytes. Due to the differences in physical properties such as size, shape, and density, various cells can be effectively separated by density gradient centrifugation. Specifically, red blood cells and granulocytes have a higher density and will settle at the bottom of the gradient medium, while lymphocytes and monocytes (which are the main components of PBMCs) have a lower density and will stay at a specific layer of the gradient medium, forming a clear cell band. By collecting the cells in this layer, the PBMC cell liquid, i.e., the cell liquid to be cultured, can be obtained.

[0030] S20, delivering the cell liquid to be cultured to the culture device through a first switching pipeline operation, and culturing the cell liquid to be cultured to obtain a cultured cell liquid.

[0031] It is understandable that the separation process of the sample liquid needs to be performed in a centrifuge device, and the subsequent culturing process needs to be performed in a culture device. The centrifuge device and the culture device are connected by a pipeline to form an integrated cell processing device system. The centrifuge device is an operating instrument for centrifuging the cell liquid, such as a centrifuge. The culture device is an operating instrument for culturing the cell liquid, such as a culture box. After obtaining the cell liquid to be cultured, the first switching pipeline operation can deliver the cell liquid to be cultured from the centrifuge device to the culture device. The centrifuge device and the culture device are connected by a pipeline, and different positions in the pipeline are provided with pipeline valves. The pipeline valves are in a default closed state, and at this time the cell liquid cannot be delivered through the pipeline. The switching pipeline operation is an operation of controlling the pipeline to be on or off by opening and closing the pipeline valves. The first switching pipeline operation includes opening the pipeline valves and closing the pipeline valves again after the cell liquid to be cultured is delivered from the centrifuge device to the culture device. The cell liquid to be cultured is mixed with the culture medium in the culture device, and the cell liquid to be cultured is cultured under specific temperature, humidity, and other conditions to obtain a cultured cell liquid. The cultured cell liquid refers to the product of culturing the cell liquid to be cultured. When the cell sorting and culturing method is applied to the scenario of separating PBMCs from peripheral blood, the cultured cell liquid refers to a cell liquid containing PBMCs with antibodies.

[0032] S30, obtaining the target cell liquid by operating the second switching pipeline to obtain the culture cell liquid, and performing concentration and cleaning treatment on the culture cell liquid.

[0033] It is understandable that the culture cell liquid contains not only PBMC with antibodies, but also by-products and impurities in the culture process. Therefore, the culture cell liquid needs to be concentrated and cleaned to obtain the target cell liquid. The target cell liquid refers to the PBMC cell liquid combined with antibodies with required purity. The concentration and cleaning treatment process of the culture cell liquid needs to be performed in a centrifugal device. Therefore, the culture cell liquid needs to be transported from the culture device to the centrifugal device by operating the second switching pipeline. The second switching pipeline operation includes opening the pipeline valve and closing the pipeline valve after the culture cell liquid is transported from the culture device to the centrifugal device.

[0034] The centrifugal device for concentration and cleaning treatment of the culture cell liquid can be the same centrifugal device as the centrifugal device for separation treatment of the sample liquid, or can be a different centrifugal device. When the same centrifugal device is used, the culture device only needs to be connected to one centrifugal device, and the pipeline valve between the first switching pipeline operation and the second switching pipeline operation is the same, except that the direction of cell liquid transportation is different. Specifically, the multifunctional cell sorting device is used as the centrifugal device. The culture cell liquid in the culture device is drawn back into the centrifugal container of the same multifunctional cell sorting device. The centrifugal container is controlled to rotate to concentrate and clean the culture cell liquid. The rotation speed of the centrifugal container is 500-2500 rpm. Continuous flow concentration and cleaning liquid replacement operation is performed by adding cleaning liquid during centrifugal rotation. The target cell liquid is obtained after concentration and cleaning.

[0035] When a different centrifugal device is used, the culture device needs to be connected to two centrifugal devices at the same time, as shown in FIG. 2. The two centrifugal devices are a first centrifugal device and a second centrifugal device. The first centrifugal device is a centrifugal device for separation treatment of the sample liquid, which realizes the sorting and cleaning process of PBMC before the culture process. The second centrifugal device is a centrifugal device for concentration and cleaning treatment of the culture cell liquid, which realizes the concentration and cleaning process of the culture cell liquid after the culture process. At this time, the first switching pipeline operation controls the pipeline connection and disconnection between the first centrifugal device and the culture device, and the second switching pipeline operation controls the pipeline connection and disconnection between the second centrifugal device and the culture device, thereby realizing the transportation process of the cell liquid from the first centrifugal device to the culture device and then to the second centrifugal device.

[0036] The embodiment first separates the sample liquid by using the density gradient centrifugation method to obtain the cell liquid to be cultured; then the cell liquid to be cultured is transported into the culture device through the first switching pipeline operation, and the cell liquid to be cultured is cultured to obtain the cultured cell liquid; finally, the cultured cell liquid is obtained through the second switching pipeline operation, and the cultured cell liquid is concentrated and cleaned to obtain the target cell liquid. The separation process is carried out in the centrifugal device, and the centrifugal device and the culture device are connected by the pipeline to realize the integration of the cell processing equipment, which can ensure a closed one-stop sterile operation environment, avoid the pollution risk when changing the cell operation site, and improve the stability of the cell processing. At the same time, the embodiment can realize the continuous operation between the sorting and culture of the cell liquid through two switching pipeline operations, improve the automation and continuity of the cell processing, save the operation time, and improve the processing efficiency.

[0037] In an embodiment, in step S10, the sample liquid is separated by using the density gradient centrifugation method to obtain the cell liquid to be cultured, comprising:

[0038] S101, based on the density gradient centrifugation method, the sample liquid and the separation liquid are added into the centrifugal container, and the separation mixed liquid after mixing is subjected to centrifugal separation treatment to obtain the separation cell liquid;

[0039] S102, the washing liquid is added into the centrifugal container, and the washing mixed liquid after mixing is subjected to centrifugal washing treatment to obtain the washing cell liquid;

[0040] S103, the liquid culture medium is added into the centrifugal container, and the resuspension mixed liquid after mixing is subjected to resuspension treatment to obtain the cell liquid to be cultured.

[0041] As shown in FIG. 3, the centrifugal device includes a centrifugal container (such as a centrifugal cup) and a waste liquid container (such as a waste liquid bag), and the centrifugal container and the waste liquid container are connected by a pipeline. In the scenario of separating PBMC from peripheral blood, the sample liquid is a peripheral blood sample, the separation liquid is a Ficoll solution, and the separation cell liquid refers to the PBMC cell liquid preliminarily separated from the peripheral blood. Specifically, a multifunctional cell sorting device is used as the centrifugal device, and the Ficoll solution is first pumped into the centrifugal cup through the hook on the multifunctional cell sorting device, and then the peripheral blood sample with a preset sample volume (which can be set as needed, such as less than 150 mL) is pumped into the centrifugal cup to obtain the mixed separation mixed liquid. The centrifugal cup is controlled to rotate to centrifugally separate the separation mixed liquid, extract the white membrane layer cells formed in the centrifugal cup to obtain the separation cell liquid, and output the cells outside the white membrane layer to the waste liquid container.

[0042] The separated cell solution can be mixed with impurities, and needs to be washed and resuspended to obtain the cell solution to be cultured. When washing, the washing solution is added to the centrifugal cup containing the separated cell solution to obtain a mixed washing mixture, the centrifugal cup is controlled to rotate to centrifugally wash the mixed washing mixture, the supernatant outside the white membrane layer is output to a waste liquid container, and the white membrane layer is retained to obtain the washed cell solution. When resuspending, the liquid medium is added to the centrifugal container to obtain a mixed resuspension mixture, the centrifugal cup is controlled to rotate to resuspend the mixed resuspension mixture, so that the cells are uniformly dispersed in the cell solution, and the cell solution to be cultured is obtained. The liquid medium is a medium for providing nutrients required for cell growth for the cell solution to be cultured, such as RPMI-1640 medium containing 10% fetal bovine serum.

[0043] In specific embodiments, after obtaining the cell solution to be cultured, all the cell solution to be cultured can be transported from the centrifugal device to the culture device, or part of the cell solution to be cultured can be transported from the centrifugal device to the culture device as needed. The centrifugal device further comprises a cryopreservation container (such as a cryopreservation bag). When only part of the cell solution to be cultured needs to be transported from the centrifugal device to the culture device, the centrifugal device has remaining cell solution to be cultured, that is, the resuspended PBMCs remain in the centrifugal cup. At this time, the resuspended PBMCs remaining in the centrifugal cup are subjected to a freezing solution centrifugal replacement, and then are extracted into the cryopreservation bag for cryopreservation.

[0044] The embodiment is based on the centrifugal device, which first performs centrifugal separation on the sample solution, and then performs centrifugal washing and resuspension to finally obtain the cell solution to be cultured, thereby realizing automatic and continuous operation of separation, washing and resuspension, and improving the efficiency of cell sorting.

[0045] In an embodiment, in step S102, the mixed washing mixture is centrifugally washed in the centrifugal container to obtain the washed cell solution, including:

[0046] S1021, transporting the separated cell solution from the centrifugal container to an intermediate product container, and determining the centrifugal container as a centrifugal container to be washed;

[0047] S1022, adding a washing solution to the centrifugal container to be washed to perform container washing, so that the centrifugal container to be washed is changed into a centrifugal container that has been washed;

[0048] S1023, transporting the separated cell solution from the intermediate product container to the centrifugal container that has been washed, and adding a washing solution to the centrifugal container that has been washed to centrifugally wash the mixed washing mixture to obtain the washed cell solution.

[0049] As shown in Fig. 3, the centrifugal device can further include an intermediate product container (e.g., an intermediate product bag) in addition to the centrifugal container and the waste liquid container, and the intermediate product container and the centrifugal container are connected by a pipeline. In the process of centrifugal washing, the inner wall of the centrifugal container can be contaminated by residual sample liquid, which can affect the washing effect. Therefore, the centrifugal container needs to be washed separately. The separated cell liquid is transferred from the centrifugal container to the intermediate product container, and the centrifugal container is empty at this time. The empty centrifugal container is determined as a centrifugal container to be washed. The washing liquid is added to the centrifugal container to be washed to perform container washing treatment, so that the centrifugal container to be washed is changed to a centrifugal container that has been washed. The container washing treatment process is a process of replacing liquid by adding washing liquid and pumping the washing liquid to the waste liquid container, and the process is continued for a certain period of time (e.g., 1 minute). After the centrifugal container is washed, the separated cell liquid is transferred from the intermediate product container to the centrifugal container that has been washed, and the washing liquid is added to the centrifugal container that has been washed to wash the mixed washing mixture, and the washed cell liquid is obtained.

[0050] The embodiment utilizes the intermediate product container to empty the centrifugal container, realizes the separate washing of the centrifugal container, and realizes the washing process of the separated cell liquid by the washed centrifugal container, thereby ensuring the washing effect.

[0051] In an embodiment, the culture device includes a first temperature-controlled culture device and a second temperature-controlled culture device. In step S20, the cell liquid to be cultured is transferred to the culture device by the first switching pipeline operation, and the cell liquid to be cultured is cultured to obtain the cultured cell liquid, including:

[0052] S201, the cell liquid to be cultured is transferred to the culture container in the first temperature-controlled culture device by the first switching pipeline operation, and the antibody stock solution and the liquid culture medium are added to the culture container to activate the mixed antibody mixture, and the activated cell liquid is obtained;

[0053] S202, the culture container containing the activated cell liquid is transferred to the second temperature-controlled culture device, and the coating liquid is added to the culture container to perform static coating treatment on the mixed coating mixture, and the coated cell liquid is obtained;

[0054] S203, the culture container containing the coated cell liquid is transferred to the first temperature-controlled culture device, and the virus cell liquid is added to the culture container to perform static culture treatment on the mixed virus mixture, and the cultured cell liquid is obtained.

[0055] Understandably, the process of culturing the cell liquid to be cultured includes three stages of activation, coating and virus transduction, and a specific carbon dioxide gas environment is required in the three stages of culturing process, so a carbon dioxide incubator is used as the culturing equipment. The culturing conditions are different between different stages, especially the culturing temperature, and the culturing temperature in the coating stage is lower than that in the activation and virus transduction stages. In order to ensure the continuity of the culturing process, the culturing equipment includes a first temperature-controlled culturing equipment and a second temperature-controlled culturing equipment, the temperature set in the first temperature-controlled culturing equipment is a first culturing temperature, the temperature set in the second temperature-controlled culturing equipment is a second culturing temperature, and the first culturing temperature is higher than the second culturing temperature. For example, the first culturing temperature is set to 35-37℃, and the second culturing temperature is set to 2-8℃. The culturing container refers to a container used to hold the cell liquid in the process of culturing, and the liquid in the culturing container is the cell liquid to be cultured at the beginning of the culturing process, and is the cultured cell liquid at the end of the culturing process.

[0056] In the activation stage, the cell liquid to be cultured is transported from the centrifugal equipment to the empty culturing container in the first temperature-controlled culturing equipment through the operation of the first switching pipeline, and the antibody stock solution and the liquid culture medium are added to the culturing container to obtain the mixed antibody mixture. The antibody mixture is subjected to activation culturing treatment for a preset activation time length to obtain the activated cell liquid. The antibody stock solution is a solution containing specific antibodies, which can bind to receptors or other molecules on the surface of cells, thereby activating the cells. The preset activation time length is a fixed time length for culturing the mixed solution composed of the cell liquid to be cultured, the antibody stock solution and the liquid culture medium. The preset activation time length is set to 48 hours by default. After the activation stage is completed, the liquid contained in the culturing container is the activated cell liquid, and the culturing container is also called the activation container.

[0057] In the coating stage, the activation container (the culturing container containing the activated cell liquid) is transferred to the second temperature-controlled culturing equipment, and the coating liquid is added to the activation container to obtain the mixed coating mixture. The coating mixture is subjected to static coating treatment to obtain the coated cell liquid. During the transfer of the culturing container, manual transfer can be used, or mechanical automatic transfer can be used, but a sterile operating environment needs to be ensured. Coating refers to fixing a substance (such as an antibody, a protein, an enzyme, etc.) on a carrier for subsequent cell culturing, antigen-antibody reaction or biological molecule detection. The coating liquid is a solution containing the substance that needs to be fixed on the carrier. The coating mixture in the static state can enable the substance in the coating liquid to fully contact the surface of the carrier and react, such as adsorption and combination. After the coating stage is completed, the liquid contained in the culturing container is the coated cell liquid, and the culturing container is also called the coating container.

[0058] In the virus transduction stage, first, the coated container (a culture container containing a coating cell solution) is transferred to the first temperature-controlled culture device, the temperature in the first temperature-controlled culture device is 35-37°C, and the virus cell solution composed of virus and virus activation solution is added to the coated container to obtain the mixed virus mixture. Then, the virus mixture is subjected to static culture treatment to obtain the cultured cell solution. During the static culture treatment, the virus mixture is first allowed to stand for 24 hours to complete virus transduction, and then the cell solution after virus transduction is subjected to subsequent static culture. During the subsequent static culture, the liquid medium needs to be supplemented at regular intervals, and the waste liquid generated during the culture process is extracted, and the carbon dioxide concentration is maintained at 5%, and the subsequent static culture lasts for 7 days. During the static culture treatment of the virus mixture, the cells will try to resist the infection of the virus and may produce an immune response against the virus. Therefore, when the cell solution to be cultured contains PBMC, the cultured cell solution contains PBMC with antibodies.

[0059] The present embodiment completes the culture of the activation and virus stages based on the first temperature-controlled culture device, completes the culture of the coating stage based on the second temperature-controlled culture device, and ensures that the temperature conditions of the activation and virus stages are consistent under the condition of meeting the appropriate coating temperature, thereby realizing the continuity of the culture process. In addition, when the cells are transferred between the first temperature-controlled culture device and the second temperature-controlled culture device in the present embodiment, the culture container and the cell solution in the container are directly transferred together, thereby avoiding the waste of the cell solution and improving the cell culture efficiency.

[0060] In an embodiment, after the antibody stock solution and the liquid culture medium are added to the culture container in step S201, the following steps are included:

[0061] S2011, the cell solution to be cultured, the antibody stock solution and the liquid culture medium in the culture container are subjected to a mixing and exhaust operation for a first preset mixing time to obtain a mixed antibody mixture.

[0062] As shown in FIG. 3, the first temperature-controlled culture device and the second temperature-controlled culture device are both provided with a mixing assembly, which is an instrument assembly for carrying the culture container and mixing the cell solution in the culture container. In the present embodiment, the mixing assembly is a mixing shaker, which realizes the mixing of the cell solution in the culture container by driving the culture container to swing at a certain angle and direction. Specifically, the inlet / outlet port of the culture container is disposed above the inclined mixing shaker, so that the bubbles can gather above the culture container, and then the mixing process can be subjected to exhaust treatment, thereby realizing the mixing and exhaust operation.

[0063] In the activation stage, the temperature of the first temperature-controlled culture device is 35-37℃, and the carbon dioxide concentration is 5%. The cell liquid to be cultured, the antibody stock solution, and the liquid culture medium in the culture container are subjected to a first preset mixing time length of the mixing and exhaust operation to obtain a mixed antibody mixture. The first preset mixing time length is a time period preset for limiting the mixing and exhaust operation of the cell liquid to be cultured, the antibody stock solution, and the liquid culture medium. The first preset mixing time length can be the time point of starting to add the antibody stock solution, or the time point of completing the addition of the antibody stock solution and the liquid culture medium. Specifically, the culture container containing the cell liquid to be cultured is added with a preset antibody volume of the antibody stock solution, and after the cell liquid to be cultured and the antibody stock solution are mixed, the liquid culture medium is continuously added to the culture container to reach a preset mixing volume (such as 1 liter) and continue to mix. The culture container has a container port (such as a bag port of a culture bag), and when the mixing is performed by using a mixing shaker, the culture container is tilted with the container port upward, so that the gas bubbles move to the container port and gather in the container port, and then the gas bubbles are exhausted. After the gas bubbles are exhausted, the activation culture process is performed.

[0064] In this embodiment, when the cell liquid to be cultured, the antibody stock solution, and the liquid culture medium in the culture container are mixed, the operation of mixing and exhausting at the same time is performed to ensure that the cell liquid to be cultured, the antibody stock solution, and the liquid culture medium are fully mixed and contacted, which helps to improve the efficiency of the activation culture.

[0065] In an embodiment, in step S202, the culture container containing the activated cell liquid is transferred to the second temperature-controlled culture device, including:

[0066] S2021, the activated cell liquid is subjected to a sampling treatment by a third switching pipeline operation, and the activated cell liquid sample obtained by sampling is subjected to a counting treatment to obtain an activation counting result;

[0067] S2022, when the activation counting result meets the preset antibody binding condition, the culture container containing the activated cell liquid is transferred to the second temperature-controlled culture device;

[0068] S2023, when the activation counting result does not meet the preset antibody binding condition, the activated cell liquid is subjected to a continuous activation culture treatment.

[0069] Understandably, after the mixed antibody mixture is subjected to the activation culture treatment to obtain the activated cell solution, and before the culture container containing the activated cell solution is transferred to the second temperature-controlled culture device, the activated cell solution needs to be sampled and judged. Specifically, when the fixed time length of the activation culture process is 48 hours, the connection pipeline between the centrifugal device and the culture device is cleaned with the liquid culture medium after the activation culture reaches 48 hours, so as to avoid the influence of impurity cells on the sampling result. The centrifugal device includes a sampling container (such as a sampling tube), and the capacity of the sampling container is the preset sampling volume. The activated cell solution is sampled and treated through the third switching pipeline operation, so that the activated cell solution with the preset sampling volume is transported from the culture device to the sampling container, and the activated cell solution sample obtained by sampling is counted to obtain an activation count result. The activation count result refers to the proportion of the number of cells that have completed antibody binding in the activated cell solution sample to the total number of all cells in the sample. The preset antibody binding condition includes a preset activation threshold, which is a minimum critical value preset for determining whether the activation count result meets the preset antibody binding condition. The activation count result is compared with the preset activation threshold. If the activation count result is greater than or equal to the preset activation threshold, it is determined that the activation count result meets the preset antibody binding condition, and the culture container containing the activated cell solution can be transferred to the second temperature-controlled culture device. If the activation count result is less than the preset activation threshold, it is determined that the activation count result does not meet the preset antibody binding condition, and the activated cell solution needs to be subjected to the activation culture treatment.

[0070] During the continued activation culture treatment, the activated cell solution is subjected to the activation culture treatment for a preset extension time, and the sampling and counting treatment is performed after the preset extension time ends to obtain a continued activation count result. The preset extension time is a time period preset for limiting each time the continued activation culture treatment is performed, and can be set to a default value or adjusted as needed. The preset extension time is set to 2 hours by default. The continued activation count result refers to the count result after the activated cell solution is subjected to the continued activation culture treatment. When the continued activation count result meets the preset antibody binding condition, the culture container containing the activated cell solution is transferred to the second temperature-controlled culture device.

[0071] Before the culture container containing the activated cell solution is transferred to the second temperature-controlled culture device, the embodiment judges whether the activated cell solution has completed antibody binding through sampling and counting, and performs continued activation culture treatment when antibody binding has not been completed, which can ensure the effectiveness of the activation culture and improve the yield of subsequent culture.

[0072] In an embodiment, in step S202, the coated mixture after mixing is subjected to the static coating treatment by adding the coating liquid into the culture container to obtain the coated cell solution, including:

[0073] S2024, mixing the activated cell solution and the coating solution in the culture container for a second preset mixing time to obtain a mixed coating solution;

[0074] S2025, performing a preset coating time of the mixed coating solution, and performing a third preset mixing time of the mixed coating solution after the preset coating time to obtain a coated cell solution.

[0075] It can be understood that, in the coating stage, the temperature of the second temperature-controlled culture device is 2-8℃, and the carbon dioxide concentration is 5%. Specifically, a preset coating volume of coating solution is added to the culture container containing the activated cell solution, and a second preset mixing time of the activated cell solution and the coating solution is performed to obtain a mixed coating solution. The second preset mixing time is a preset time period for limiting the mixing time of the activated cell solution and the coating solution. In order to ensure the mixing effect, the coating solution is added while mixing, and the mixing is continued for the second preset mixing time after the addition is completed. The starting time point of the second preset mixing time is the time point when the addition of the coating solution is completed. After the mixing of the activated cell solution and the coating solution, the mixed coating solution is obtained. The mixed coating solution is subjected to a preset coating time of the mixed coating solution, and a third preset mixing time of the mixed coating solution is performed after the preset coating time to obtain a coated cell solution. The preset coating time is a preset time period for completing the coating culture, and the preset coating time is 16-24 hours. After the preset coating time, the third preset mixing time of the mixed coating solution is performed to facilitate the removal of air bubbles in the static process, and the waste liquid is discharged to the waste liquid container of the centrifugal device to obtain the coated cell solution. The third preset mixing time is a preset time period for limiting the mixing of the activated cell solution and the coating solution after the static treatment. After the third preset mixing time of the mixed coating solution is completed, the cell state of the coated cell solution can also be determined through the sampling container in the centrifugal device to determine whether the coated cell solution needs to be subjected to a static treatment.

[0076] In this embodiment, the activated cell solution and the coating solution in the culture container are mixed, which ensures that the activated cell solution and the coating solution are fully mixed and contacted. At the same time, by performing the mixing and exhaust operation again after the static treatment, the influence of air bubbles is avoided, which helps to ensure that the cell coating culture is fully carried out.

[0077] It should be understood that the size of the serial number of each step in the above embodiment does not mean the order of execution, and the execution order of each process should be determined according to its function and inherent logic, and should not constitute any limitation on the implementation process of the embodiment of the present application.

[0078] In an embodiment, a cell sorting and culturing device is provided, which corresponds to the cell sorting and culturing method in the above embodiment. As shown in FIG. 4, the cell sorting and culturing device includes a separation processing module 10, a culturing processing module 20, and a concentration and cleaning processing module 30. Each functional module is described in detail as follows:

[0079] The separation processing module 10 is configured to separate the sample liquid by using the density gradient centrifugation method to obtain a cell liquid to be cultured;

[0080] The culturing processing module 20 is configured to deliver the cell liquid to be cultured into a culturing device by a first switching pipeline operation, and to perform culturing processing on the cell liquid to be cultured to obtain a cultured cell liquid;

[0081] The concentration and cleaning processing module 30 is configured to obtain the cultured cell liquid by a second switching pipeline operation, and to perform concentration and cleaning processing on the cultured cell liquid to obtain a target cell liquid.

[0082] In an embodiment, the separation processing module 10 includes:

[0083] A cell liquid separation processing unit is configured to add the sample liquid and a separation liquid into a centrifuge container based on the density gradient centrifugation method, and to perform centrifugal separation processing on a mixed separation mixture to obtain a separation cell liquid;

[0084] A cleaning processing unit is configured to add a cleaning liquid into the centrifuge container, and to perform centrifugal cleaning processing on a mixed cleaning mixture to obtain a cleaning cell liquid;

[0085] A resuspension processing unit is configured to add a liquid culture medium into the centrifuge container, and to perform resuspension processing on a mixed resuspension mixture to obtain the cell liquid to be cultured.

[0086] In an embodiment, the separation processing module 10 further includes:

[0087] A separation cell liquid delivery unit is configured to deliver the separation cell liquid from the centrifuge container into an intermediate product container, and to determine the centrifuge container as a centrifuge container to be cleaned;

[0088] A container cleaning processing unit is configured to add a cleaning liquid into the centrifuge container to be cleaned to perform container cleaning processing, so that the centrifuge container to be cleaned is changed into a cleaned centrifuge container;

[0089] A cell liquid cleaning processing unit is configured to deliver the separation cell liquid from the intermediate product container into the cleaned centrifuge container, and to add a cleaning liquid into the cleaned centrifuge container to perform centrifugal cleaning processing on a mixed cleaning mixture to obtain the cleaning cell liquid.

[0090] In an embodiment, the culturing processing module 20 includes:

[0091] The activation culture processing unit is configured to deliver the cell liquid to be cultured into a culture container in the first temperature-controlled culture device through the first switching pipeline operation, add the antibody stock solution and the liquid culture medium into the culture container, and perform activation culture processing on the mixed antibody mixture to obtain an activated cell liquid;

[0092] The static coating processing unit is configured to transfer the culture container containing the activated cell liquid to the second temperature-controlled culture device, add the coating solution into the culture container, and perform static coating processing on the mixed coating mixture to obtain a coated cell liquid.

[0093] The static culture processing unit is configured to transfer the culture container containing the coated cell liquid to the first temperature-controlled culture device, add the virus cell liquid into the culture container, and perform static culture processing on the mixed virus mixture to obtain a cultured cell liquid.

[0094] In an embodiment, the culture processing module 20 further includes:

[0095] The first mixing processing unit is configured to perform mixing and exhaust operation on the cell liquid to be cultured, the antibody stock solution and the liquid culture medium in the culture container for a first preset mixing time to obtain the mixed antibody mixture.

[0096] In an embodiment, the culture processing module 20 further includes:

[0097] The sampling and counting processing unit is configured to perform sampling processing on the activated cell liquid through the third switching pipeline operation, and perform counting processing on the activated cell liquid sample obtained by sampling to obtain an activation counting result.

[0098] The temperature-controlled culture transfer unit is configured to transfer the culture container containing the activated cell liquid to the second temperature-controlled culture device when the activation counting result meets a preset antibody binding condition.

[0099] The continued activation culture processing unit is configured to continue activation culture processing on the activated cell liquid when the activation counting result does not meet the preset antibody binding condition.

[0100] In an embodiment, the culture processing module 20 further includes:

[0101] The second mixing processing unit is configured to perform mixing operation on the activated cell liquid and the coating solution in the culture container for a second preset mixing time to obtain the mixed coating mixture.

[0102] The third mixing processing unit is configured to perform static processing on the coating mixture for a preset coating time, and perform mixing and exhaust operation on the coating mixture for a third preset mixing time after the static processing is completed to obtain the coated cell liquid.

[0103] The specific limitations of the cell sorting and culture device can refer to the limitations of the cell sorting and culture method described above, which will not be repeated here. Each module of the above cell sorting and culture device can be realized by software, hardware and their combination. The above modules can be embedded in or independent of the processor in the computer device in hardware form, or stored in the memory of the computer device in software form, so that the processor calls and executes the operations corresponding to the above modules.

[0104] In one embodiment, a computer device is provided, which can be a server, and its internal structure diagram can be as shown in FIG. 5. The computer device includes a processor, a memory, a network interface and a database connected by a system bus. The processor of the computer device is used to provide computing and control capabilities. The memory of the computer device includes a readable storage medium and an internal memory. The readable storage medium stores an operating system, computer readable instructions and a database. The internal memory provides an environment for the operation of the operating system and computer readable instructions in the readable storage medium. The database of the computer device is used to store the data involved in the cell sorting and culture method. The network interface of the computer device is used to communicate with the external terminal through the network connection. The computer readable instructions are executed by the processor to implement a cell sorting and culture method. The readable storage medium provided in this embodiment includes a non-volatile readable storage medium and a volatile readable storage medium.

[0105] In one embodiment, a computer device is provided, which includes a memory, a processor and computer readable instructions stored on the memory and executable on the processor, and the processor executes the computer readable instructions to implement the following steps:

[0106] The sample liquid is separated and treated by the density gradient centrifugation method to obtain the cell liquid to be cultured;

[0107] The cell liquid to be cultured is delivered to the culture device by the first switching pipeline operation, and the cell liquid to be cultured is cultured to obtain the cultured cell liquid;

[0108] The cultured cell liquid is obtained by the second switching pipeline operation, and the cultured cell liquid is concentrated and cleaned to obtain the target cell liquid.

[0109] In one embodiment, one or more computer readable storage media storing computer readable instructions are provided. The readable storage medium provided in this embodiment includes a non-volatile readable storage medium and a volatile readable storage medium. The computer readable instructions are stored on the readable storage medium, and the computer readable instructions are executed by one or more processors to implement the following steps:

[0110] The sample liquid is separated and treated by the density gradient centrifugation method to obtain the cell liquid to be cultured;

[0111] The to-be-cultured cell liquid is transported into the culture device by the first switching pipeline operation, and the to-be-cultured cell liquid is cultured to obtain a cultured cell liquid;

[0112] The cultured cell liquid is obtained by the second switching pipeline operation, and the cultured cell liquid is concentrated and cleaned to obtain a target cell liquid.

[0113] A person of ordinary skill in the art can understand that all or part of the processes in the above-mentioned embodiment methods can be completed by computer readable instructions instructing related hardware, and the computer readable instructions can be stored in a non-volatile readable storage medium or a volatile readable storage medium, and when the computer readable instructions are executed, the processes of the above-mentioned embodiments can be included. Wherein, any reference to memory, storage, database or other medium used in each embodiment provided by the present application can include non-volatile and / or volatile memory. Non-volatile memory can include read-only memory (ROM), programmable ROM (PROM), electrically programmable ROM (EPROM), electrically erasable programmable ROM (EEPROM) or flash memory. Volatile memory can include random access memory (RAM) or external cache memory. As an illustration but not limitation, RAM is available in various forms, such as static RAM (SRAM), dynamic RAM (DRAM), synchronous DRAM (SDRAM), double data rate SDRAM (DDR SDRAM), enhanced SDRAM (ESDRAM), synchronous link (Synchlink) DRAM (SLDRAM), memory bus (Rambus) direct RAM (RDRAM), direct memory bus dynamic RAM (DRDRAM), and memory bus dynamic RAM (RDRAM) and the like.

[0114] Those skilled in the art can clearly understand that, for the convenience and brevity of description, only the division of the above-mentioned functional units and modules is exemplified, and in actual application, the above-mentioned functions can be completed by different functional units and modules according to needs, that is, the internal structure of the device is divided into different functional units or modules to complete all or part of the functions described above.

[0115] The above embodiments are only used to illustrate the technical solutions of the present application, but not to limit them; although the present application has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions recorded in the foregoing embodiments, or make equivalent replacements for part of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present application, and should be included in the protection scope of the present application.

Claims

1. A method of cell sorting and culturing, wherein, The method comprises the following steps: separating the sample liquid by using a density gradient centrifugation method to obtain a cell liquid to be cultured; delivering the cell liquid to be cultured into a culture device by a first switching pipeline operation, and culturing the cell liquid to be cultured to obtain a cultured cell liquid; obtaining the cultured cell liquid by a second switching pipeline operation, and concentrating and cleaning the cultured cell liquid to obtain a target cell liquid.

2. The cell sorting and culturing method of claim 1, wherein, The method for separating the sample liquid by using the density gradient centrifugation method to obtain the cell liquid to be cultured comprises the following steps: based on the density gradient centrifugation method, the sample liquid and a separation liquid are added into a centrifugal container, and the mixed separation mixture is subjected to centrifugal separation treatment to obtain a separated cell liquid; the washing liquid is added into the centrifugal container, and the mixed washing mixture is subjected to centrifugal cleaning treatment to obtain a washed cell liquid; the liquid culture medium is added into the centrifugal container, and the mixed resuspension mixture is subjected to resuspension treatment to obtain the cell liquid to be cultured.

3. The cell sorting and culturing method of claim 2, wherein, The method for adding the washing liquid into the centrifugal container, and subjecting the mixed washing mixture to centrifugal cleaning treatment to obtain the washed cell liquid comprises the following steps: the separated cell liquid is delivered from the centrifugal container into an intermediate product container, and the centrifugal container is determined as a centrifugal container to be cleaned; the washing liquid is added into the centrifugal container to be cleaned to perform container cleaning treatment, so that the centrifugal container to be cleaned is changed into a cleaned centrifugal container; the separated cell liquid is delivered from the intermediate product container into the cleaned centrifugal container, and the washing liquid is added into the cleaned centrifugal container to perform centrifugal cleaning treatment on the mixed washing mixture, so as to obtain the washed cell liquid.

4. The cell sorting and culturing method of claim 1, wherein, The culture device comprises a first temperature-controlled culture device and a second temperature-controlled culture device. The method for delivering the cell liquid to be cultured into the culture device by the first switching pipeline operation, and culturing the cell liquid to be cultured to obtain the cultured cell liquid comprises the following steps: the cell liquid to be cultured is delivered into a culture container in the first temperature-controlled culture device by the first switching pipeline operation, and the antibody stock solution and the liquid culture medium are added into the culture container to perform activation culture treatment on the mixed antibody mixture, so as to obtain an activated cell liquid; the culture container containing the activated cell liquid is transferred into the second temperature-controlled culture device, and the coating liquid is added into the culture container to perform standing coating treatment on the mixed coating mixture, so as to obtain a coated cell liquid; the culture container containing the coated cell liquid is transferred into the first temperature-controlled culture device, and the virus cell liquid is added into the culture container to perform standing culture treatment on the mixed virus mixture, so as to obtain the cultured cell liquid.

5. The cell sorting and culturing method of claim 4, wherein, After the antibody stock solution and the liquid culture medium are added into the culture container, the following step is performed: the cell liquid to be cultured, the antibody stock solution and the liquid culture medium in the culture container are subjected to mixing and exhaust operation for a first preset mixing time, so as to obtain the mixed antibody mixture.

6. The cell sorting and culturing method of claim 4, wherein, The method for transferring the culture container containing the activated cell liquid into the second temperature-controlled culture device comprises the following steps: The activated cell solution is sampled by a third switching pipeline operation, and the sampled activated cell solution sample is counted to obtain an activation count result; When the activation count result meets a preset antibody binding condition, the culture container containing the activated cell solution is transferred to the second temperature-controlled culture device; When the activation count result does not meet the preset antibody binding condition, the activated cell solution is continuously activated and cultured.

7. The cell sorting and culturing method according to claim 4, wherein The coated cell solution is obtained by adding a coating solution into the culture container, and performing standing coating on the mixed coating mixed solution. The activated cell solution and the coating solution in the culture container are mixed for a second preset mixing time to obtain a mixed coating mixed solution; The coating mixed solution is statically treated for a preset coating time, and after the static treatment is completed, a third preset mixing time is mixed and exhausted to obtain a coated cell solution.

8. A cell sorting and culturing device, wherein, It comprises: The separation processing module is used for separating the sample liquid by density gradient centrifugation method to obtain a cell liquid to be cultured; The culture processing module is used for transporting the cell liquid to be cultured into a culture device by a first switching pipeline operation, and culturing the cell liquid to be cultured to obtain a cultured cell liquid; The concentration and cleaning processing module is used for obtaining the cultured cell liquid by a second switching pipeline operation, and performing concentration and cleaning processing on the cultured cell liquid to obtain a target cell liquid.

9. A computer device comprising a memory, a processor, and computer readable instructions stored in the memory and executable on the processor, wherein, The processor executes the computer readable instructions to realize the cell sorting and culture method in any one of claims 1-7.

10. A computer-readable storage medium storing computer-readable instructions, wherein, The computer readable instructions are executed by one or more processors to cause the one or more processors to perform the cell sorting and culture method in any one of claims 1-7.

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