Methods, apparatus, systems, and media for processing cell preparations

Abstract

The present invention provides a method, apparatus, system, and medium for processing cell preparations. The method involves performing a cell proliferation treatment on the target cell solution in a cell culture vessel to obtain a post-culture cell solution, then performing a washing and concentration treatment on the post-culture cell solution in the cell culture vessel to obtain a concentrated cell solution, and finally dispensing the concentrated cell solution from the cell culture vessel into multiple preparation bags. By performing cell culture, cell washing and concentration, and preparation dispensing all within the cell culture vessel, dispersed placement between devices is avoided, reducing occupied space. Executing the above steps sequentially within the cell culture vessel ensures that the entire process from cell culture to cell preparation is carried out in a single, sealed, sterile environment, eliminating the need for cell solution transfer and improving the quality of cell culture. By integrally executing the above steps sequentially within the cell culture vessel, manual intervention is reduced, the entire process from cell culture to cell preparation is intelligently executed, and the efficiency of cell culture is improved.

Description

Technical Field

[0001] This application claims the priority of a Chinese patent application filed with the Chinese Patent Office on August 22, 2023, with the application number 202311064991.5 and the invention title "Method, Apparatus, System, and Medium for Processing Cell Preparations", and the entire content thereof is incorporated herein by reference. This application relates to the technical field of biological processing, and particularly to a method, apparatus, system, and medium for processing cell preparations.

Background Art

[0002] Cell therapy is one of the key development fields at the forefront of international medicine. In the process of cell therapy, multiple important steps such as cell culture, cell processing, and cell preparations are involved. In the current process of cell therapy, cell culture, cell processing, and cell preparations are usually carried out separately by area. That is, they are realized by manual operations within the areas of these three important steps. The inventors have recognized that although in some steps it may be necessary to use equipment in different areas, the decentralized arrangement of equipment occupies a large amount of space and requires a high degree of manual involvement. In fact, due to differences in the experience, operation methods, and habits of operators, operation errors are likely to occur in any step, which may affect the quality and culture efficiency of cell culture. Therefore, how to realize an integrated process of cell culture, cell processing, and cell preparations has become an urgent problem to be solved by those skilled in the art.

Summary of the Invention

[0003] Embodiments of this application provide a method, apparatus, system, and medium for processing cell preparations to solve the problem of the decline in the quality and culture efficiency of cell culture caused by the high manual involvement and low automation level of equipment in the prior art.

[0004] A method for processing cell preparations, the method comprising: Performing cell proliferation treatment on the culture target cell liquid in the cell culture container to obtain the cell liquid after culture; The post-culture cell solution in the aforementioned cell culture vessel is subjected to a washing and concentration process to obtain a concentrated cell solution. This includes dispensing the concentrated cell solution in a cell culture vessel into multiple formulation bags.

[0005] A device for processing cell preparations, the device is A cell proliferation module for performing cell proliferation treatment on the target cell medium in a cell culture vessel to obtain a post-culture cell medium, A washing and concentrating module for performing a washing and concentrating process on the post-culture cell solution in the cell culture vessel to obtain a concentrated cell solution, The system comprises a first dispensing formulation module for dispensing the concentrated cell solution in a cell culture vessel into multiple formulation bags.

[0006] A cell preparation processing system comprising a cell culture vessel and a controller connected to the cell culture vessel, wherein the controller is used to perform the following cell preparation processing method, the cell preparation processing method is: The process involves performing a cell proliferation treatment on the target cell medium in a cell culture vessel to obtain a post-culture cell medium, and The post-culture cell solution in the aforementioned cell culture vessel is subjected to a washing and concentration process to obtain a concentrated cell solution. This includes dispensing the concentrated cell solution in a cell culture vessel into multiple formulation bags.

[0007] One or more readable storage media storing computer-readable commands, wherein when the computer-readable commands are executed by one or more processors, the one or more processors are instructed to perform the following steps: The process involves performing a cell proliferation treatment on the target cell medium in a cell culture vessel to obtain a post-culture cell medium, and The post-culture cell solution in the aforementioned cell culture vessel is subjected to a washing and concentration process to obtain a concentrated cell solution. This includes dispensing the concentrated cell solution in a cell culture vessel into multiple formulation bags.

[0008] According to the above-described method, apparatus, system, and medium for processing cell preparations, cell culture, cell washing and concentration, and preparation dispensing can all be performed in a single cell culture vessel, thereby avoiding the need for dispersed placement of equipment and reducing the occupied space. By sequentially executing the above steps in a cell culture vessel, it is possible to ensure that the entire process from cell culture to cell preparation is carried out in a single sealed, sterile environment, eliminating the need for cell saturation transfer and improving the quality of cell culture. By integrally executing the above steps sequentially in a cell culture vessel, manual intervention is reduced, the entire process from cell culture to cell preparation is intelligently executed, and the efficiency of cell culture is improved.

[0009] Details of one or more embodiments of the present application are described in the following drawings and description. Other features and advantages of the present application will become apparent from the specification, drawings and claims. [Brief explanation of the drawing]

[0010] To more clearly illustrate the technical solutions of the embodiments of the present application, the drawings necessary for describing the embodiments are briefly described below. Clearly, the drawings described below are only a few embodiments of the present application, and those skilled in the art can obtain other drawings based on these without expending any creative effort. [Figure 1] This is a flowchart of the processing method for a cell preparation in one embodiment of the present invention. [Figure 2] A schematic diagram of a cell preparation processing device according to one embodiment of the present invention. [Figure 3] A schematic diagram of a cell preparation processing system in one embodiment of the present invention. [Modes for carrying out the invention]

[0011] The technical solutions in the embodiments of this application will be described clearly and completely below with reference to the drawings of the embodiments. Clearly, the embodiments described are only some, and not all, embodiments of this application. All other embodiments that can be obtained by a person skilled in the art without creative work based on the embodiments of this application are all within the scope of protection of this application.

[0012] In one embodiment, a method for processing cell preparations is provided, as shown in Figures 1 and 2, which includes the following steps.

[0013] S10: Cell proliferation treatment is performed on the cell culture medium in the cell culture vessel to obtain the post-culture cell medium.

[0014] To make it clear, a cell culture medium bag (which may be sample bag 1 in Figure 2) containing the cell culture medium can be connected to one of the inlets / outlets of a cell culture vessel, and the cell culture medium can be introduced into the cell culture vessel along the connected piping using a fluid delivery device (e.g., a peristaltic pump). The cell culture medium can consist of T cells and T cell subsets. So-called T cells refer to T lymphocytes, which are a type of pluripotent stem cell derived from bone marrow. T cell subsets are obtained by classifying T cells using different classification methods. That is, T cell subsets are a subcategory of T cells. Cell proliferation treatment is used to induce proliferation of the cell culture medium, thereby enabling the mass proliferation of T cells and T cell subsets to meet the number of cells required for cell therapy. The cell medium after culture contains T cells, T cell subsets, and non-cellular impurities (i.e., impurities generated during the cell proliferation treatment). Note that T cells in the cell culture medium can be obtained by differentiating T cell progenitor cells. During the cell proliferation process, a culture nutrient solution can be added to the cell culture vessel, and this culture nutrient solution can be used as the culture medium. The culture medium is an artificial nutrient solution used for the culture and proliferation of microorganisms, cells, and tissues, and consists of multiple nutrients such as carbohydrates, nitrogen sources, minerals, and vitamins.

[0015] Furthermore, the cell preparation processing method provided in this embodiment is implemented by an integrated cell preparation processing apparatus, the entire apparatus being installed in a sterile environment. A cell culture vessel is attached to the apparatus, the cell culture vessel is in a sealed sterile environment, and an input pipe is connected to the cell culture vessel, the other end of which may be connected to a collection bag (the collection bag is used to store solutions such as cell solution or other reagents). In this way, it can be ensured that when the cell solution to be cultured is added to the cell culture vessel, and when activation reagents, etc., are added to the cell culture vessel in subsequent steps, it is possible to ensure that these processes are carried out in a sealed sterile environment, preventing the quality of the cell solution or the reagents themselves from being compromised by environmental influences, thereby improving the quality of cell culture.

[0016] The cell culture vessel may be a rotatable centrifuge, bottle, tank, or piston-type centrifuge. Preferably, during the cell proliferation process, the cell culture vessel can be oscillated, rotated, or vibrated to improve the culture efficiency of the target cell solution and accelerate the incubation of the target cell solution. For example, it may be rotated intermittently from side to side at a predetermined rotation speed within one culture cycle. The predetermined rotation speed may be 10 to 400 rpm (Revolutions Per Minute), and the culture cycle may be 24 hours. The intermittent setting may involve performing left-right rotations for a predetermined time every hour within the culture cycle. The predetermined rotation speed can be adjusted according to the cell density of the target cell solution.

[0017] S20: The cultured cell solution in the cell culture vessel is subjected to a washing and concentration process to obtain a concentrated cell solution.

[0018] As can be understood, the washing liquid bag 2 is connected to the cell culture container. The washing liquid bag contains a washing liquid, which may be a mixture of physiological saline and HSA (human serum albumin) at a concentration of 0.5%. The washing liquid can be allowed to flow into the cell culture container, and the cell culture container can be provided with two inlets / outlets (I / O ports). The washing and concentration process is a process for washing impurities in the cell liquid after culture and extracting a purer cell liquid at a higher concentration. During the process of cell growth treatment for the target cell liquid to be cultured, when T cells or T cell subsets divide, there may be impurities other than some cells generated. The concentrated cell liquid obtained after the washing and concentration process contains only T cells and / or T cell subsets. During the washing and concentration process, the intermediate product bag 5 can be used as a temporary storage bag during the process.

[0019] S30: Dispense the concentrated cell liquid in the cell culture container into a plurality of formulation bags.

[0020] Specifically, after performing a washing and concentration process on the cell liquid after culture in the cell culture container to obtain a concentrated cell liquid, the concentrated cell liquid obtained at this time contains only T cells and / or T cell subsets. Then, the concentrated cell liquid in the cell culture container can be dispensed into a plurality of formulation bags.

[0021] Furthermore, if the concentrated cell liquid is directly dispensed into the formulation bag, the activity of the cells in the concentrated cell liquid may decrease over time. Therefore, before dispensing the concentrated cell liquid into a plurality of formulation bags, a cryopreservation solution is introduced into the culture container containing the concentrated cell liquid, and the concentrated cell liquid is cryopreserved with the cryopreservation solution. After mixing the cryopreservation solution and the concentrated cell liquid and then dispensing it into the formulation bag, the activity of the cells can be maintained.

[0022] Furthermore, if the cryopreservation solution and concentrated cell solution are mixed and then directly dispensed into formulation bags as described above, the amount dispensed into each formulation bag may differ. In cases where there are specific dispensing requirements (for example, the need to dispense into formulation bags of different volumes), a quantitative dispensing tube is used to accurately dispense the mixture of cryopreservation solution and concentrated cell solution into the corresponding formulation bags. That is, the volume of the mixture of cryopreservation solution and concentrated cell solution is measured using a quantitative dispensing tube and dispensed into the corresponding formulation bags.

[0023] In one embodiment, during the process of dispensing the concentrated cell solution into each of the formulation bags 4, a replacement solution bag 3 may be connected to the cell culture vessel to adapt it to the cell storage environment. The replacement solution bag contains a replacement solution, such as a cryopreservation solution or a resuspension, which is necessary to replace the components in the concentrated cell solution. The replacement solution is delivered into the cell culture vessel by a liquid delivery device (e.g., a peristaltic pump 10), and the cells are resuspended with the replacement solution and the concentrated cell solution. After rotating the cell culture vessel from side to side to uniformly mix the replacement solution and the concentrated cell solution, the mixture is dispensed into each formulation bag. The dispensing process involves dispensing the liquid in the cell culture vessel into each formulation bag in the corresponding volume according to the formulation requirements of the formulation bag.

[0024] Here, sensors may be installed in all piping routes in this invention as needed to monitor the piping. For example, a bubble sensor may be used to monitor the direction of liquid flow and the flow rate in the piping, a pressure sensor 9 may be used to monitor the pressure conditions in the piping, a flow sensor may be used to monitor the transmitted flow rate, and a photoelectric sensor may be used to detect the volume of liquid in the cell culture vessel. On / off valves 7 may be installed in all piping routes in this invention as needed to open and close the piping. For example, solenoid valves may be used to open and close the input and output of each liquid bag.

[0025] In one embodiment, the quantitative formulation of the cryopreservation solution or resuspension and concentrated cell solution in the cell culture vessel 12 is performed using the quantitative formulation tube 11, and the quantitatively formulated cryopreservation solution and concentrated cell solution are dispensed into each formulation bag. During the quantitative formulation process, a bubble sensor 8 and a liquid sensor are installed in the inlet and outlet pipes of the quantitative formulation tube, respectively. The bubble sensor and liquid sensor monitor the time points at which the liquid flows into the quantitative formulation tube and the time points at which it flows out of the quantitative formulation tube, and these are defined as the initial time point and the end time point, respectively. Based on the fixed volume of the quantitative formulation tube and the time difference between the two time points, the steady flow rate of the liquid flowing through the peristaltic pump at a constant rotation speed during this period can be determined, and this steady flow rate allows for highly accurate dispensing into each formulation bag. This is because the value obtained by dividing the volume of the formulation bag by the steady flow rate is the operating time for dispensing into each formulation bag, and by detecting the time point at which the liquid flows into the formulation bag using a sensor provided in each formulation bag, the accuracy of the amount dispensed into each formulation bag can be improved. Before monitoring the time points at which the liquid enters and exits the metering tube using the bubble sensor / liquid sensor, a bubble removal process may be performed on the liquid entering the metering tube. Initially, the inlet of the metering tube is positioned below the outlet, and bubbles inside the metering tube are discharged using the principle of bubble rise. After filling the metering tube, the metering tube is then positioned so that its inlet is above its outlet, and the metering tube is kept filled with liquid.

[0026] In this embodiment, by performing cell culture, cell washing and concentration, and formulation dispensing all within the cell culture vessel, dispersed placement of equipment can be avoided, and occupied space can be reduced. By sequentially executing the above steps in the cell culture vessel, it is possible to ensure that the entire process from cell culture to cell formulation is carried out in a sealed, sterile environment, eliminating the need to transfer cell solution and improving the quality of cell culture. By sequentially executing the above steps integrally in the cell culture vessel, manual intervention is reduced, the entire process from cell culture to cell formulation is intelligently executed, and the efficiency of cell culture is improved.

[0027] In one embodiment, step S10, that is, performing a cell proliferation treatment on the cell culture medium in the cell culture vessel to obtain a post-culture cell medium, includes adding an activation reagent to the cell culture vessel. The activation reagent is one or more of the following: stimulating antibody CD3, stimulating antibody CD28, cytokine recombinant protein, costimulatory molecule, lectin, ionophore, synthetic molecule, antigen-presenting cell, artificial antigen-presenting cell, and feeder cell.

[0028] To ensure clarity, a portion of the activation reagent may be pre-coated onto the cell culture vessel. A reagent bag containing the activation reagent is connected to one of the inlets / outlets of the cell culture vessel, and the activation reagent is introduced into the cell culture vessel via a fluid delivery device (e.g., a peristaltic pump) along the connected piping. Here, the piping route connecting the cell culture medium to the cell culture vessel and the piping route connecting the activation reagent to the cell culture vessel can largely overlap (when using a single peristaltic pump). A three-way valve is used to switch the flow direction of the cell culture medium and the activation reagent. The piping route connecting the cell culture medium to the cell culture vessel and the piping route connecting the activation reagent to the cell culture vessel may be provided separately and delivered by different peristaltic pumps.

[0029] As described above, cell proliferation treatment is used to induce proliferation of the target cell medium, and the reagent used to induce T cell proliferation is the activation reagent. In this example, cell proliferation can be achieved by performing an activation reaction between a single activation reagent and the target cell medium, or by performing an activation reaction with multiple activation reagents simultaneously with the target cell medium. Here, the total amount of activation reagent to be added can be determined based on the total amount of target cell medium in the cell culture vessel. For example, if the target cell medium is 4 x 10 6 If the concentration is individual cells / mL, the total amount of activation reagent added can be 8 μg / mL. (3 x 10) 6When the cell density is individual cells / mL, the total amount of activating reagent added can be 5 μg / mL. While the addition of the activating reagent can induce cell proliferation more rapidly, excessive activating reagent can cause cell overgrowth in the cell culture vessel, leading to decreased cell viability and reduced cell activity. Therefore, in this example, the culture medium of the target cells is 3 x 10⁶. 6 When the concentration is individual cells / mL, the evaluation criterion is that the total amount of activating reagent added should be 5 μg / mL.

[0030] In this way, before adding the activation reagent to the cell culture vessel, the total amount of activation reagent required can be calculated based on the total volume of the target cell medium in the cell culture vessel. For example, the ratio between the target cell medium and the total amount of activation reagent in the above-established evaluation criteria can be defined as the set ratio, thereby allowing the total amount of activation reagent to be determined based on the total volume of the target cell medium in the cell culture vessel and the set ratio. Next, the type of activation reagent to be used in this cell proliferation treatment is determined. For example, one or more of the types exemplified above. If only one type of activation reagent is included, the total amount of that activation reagent determined as described above is directly added to the cell culture vessel. If multiple types of activation reagents are included, the amount of each activation reagent to be added can be determined based on the total volume and type determined as described above.

[0031] Furthermore, when performing cell proliferation treatment on the target cell medium in a cell culture vessel for the first time, it is necessary to add glucose in addition to the activation reagent, and a culture medium 6 containing the activation reagent and glucose may be connected to the cell culture vessel. The glucose supplies nutrients to the target cell medium. Subsequently, when performing cell proliferation treatment on the target cell medium in a cell culture vessel again, it is sufficient to add a culture medium containing only glucose, and it is not necessary to add the activation reagent. This is because, once the cells are activated, they maintain their activated state and proliferate continuously.

[0032] The cell culture medium containing the target cell solution and the activation reagent is mixed by shaking to obtain the post-culture cell solution.

[0033] Specifically, after adding the activation reagent to the cell culture vessel, a rocking and mixing operation is performed on the cell culture vessel containing the target cell solution and the activation reagent. At this time, a mechanism for gripping the cell culture vessel (e.g., a manipulator) can be attached to the device in advance, and a mechanical rocking mechanism with a rocking function can also be provided in the gripping mechanism that fixes the cell culture vessel. This enables rocking motion of the cell culture vessel, thoroughly fusing the activation reagent and the target cell solution, inducing cell proliferation in the target cell solution, and obtaining post-culture cell solution. It should be noted that the rocking and mixing operation may be started simultaneously with the start of adding the activation reagent to the cell culture vessel, or after the addition of the activation reagent is complete, and is not particularly limited.

[0034] In one embodiment, the oscillating mixing operation may be performed by an oscillating method that rotates from side to side.

[0035] In one embodiment, the step of performing a shaking and mixing operation on the cell culture vessel containing the cell medium to be cultured and the activation reagent to obtain the post-culture cell medium is as follows: The process includes measuring the density of cells in the cell culture vessel using a density acquisition device during the agitation mixing operation, and determining the density of cells in the cell culture vessel.

[0036] As can be understood, after adding the activation reagent to the cell culture vessel, a shaking and mixing operation is performed to thoroughly fuse the activation reagent with the target cell medium and induce the proliferation of the target cell medium. However, it is necessary to determine when to stop the shaking and mixing operation. In this embodiment, a density acquisition device is used to measure the cell density in the cell culture vessel and determine whether the cell density in the cell culture vessel meets the corresponding requirements. The detection of cell density by the density acquisition device may be started immediately after the start of the shaking and mixing operation, or it may be started after a certain period of time (for example, 5 seconds, 10 seconds, or 15 seconds) has elapsed since the start of the shaking and mixing operation. Here, the cell density reflects the number of cells in the cell culture vessel.

[0037] In one embodiment, a sampling port is provided near the entrance and exit of the cell culture vessel. A small amount of liquid from inside the cell culture vessel is sampled through this sampling port and flowed into a density acquisition device, which measures the cell density of the sampled liquid.

[0038] As mentioned above, cell proliferation treatment involves mass-proliferating T cells and T cell subsets to meet the cell count required for cell therapy. Therefore, cell density can be used to determine whether the current number of proliferated cells meets the required number for cell therapy.

[0039] If it is detected that the cell density is above a predetermined density threshold, the shaking and mixing operation is stopped to obtain the post-culture cell solution.

[0040] To ensure clarity, the pre-set density threshold can be determined based on factors such as the volume of the cell culture vessel and the number of cells required for cell therapy. Specifically, the cell density of the cells in the cell culture vessel is measured using a density acquisition device, and after determining the cell density, it is compared with the pre-set density threshold. When it is detected that the cell density is equal to or greater than the pre-set density threshold, the agitation and mixing operation is stopped, and the solution currently in the cell culture vessel is considered the post-culture cell solution. If the cell density is less than the pre-set density threshold, the agitation and mixing operation is continued, indicating that the number of cells in the cell culture vessel has not yet reached the number required for cell therapy. When it is detected that the cell density is equal to or greater than the pre-set density threshold, the agitation and mixing operation is stopped, and the solution currently in the cell culture vessel is considered the post-culture cell solution.

[0041] In one embodiment, the density acquisition device comprises an image acquisition device and a cell identification device. The step of measuring the cell density of the cells in the cell culture vessel using the density acquisition device and determining the cell density of the cells in the cell culture vessel is as follows: This includes obtaining cell images by acquiring images of the cells in the cell culture vessel using the image acquisition device.

[0042] To ensure clarity, the image acquisition device may be a camera, video camera, or micro-surveillance camera mounted on the device. The image acquisition device can be installed above the cell culture vessel. A cell image is an image containing cells obtained by photographing the cell culture vessel with the image acquisition device.

[0043] The cell identification device performs cell counting and identification on the cell image and determines the number of cells in the cell image.

[0044] To understand this, cell counting and identification refers to the process of identifying cells within a cell image and then calculating the number of cells contained in that image. Specifically, an image acquisition device is used to acquire images of cells in a cell culture vessel. After acquiring the cell images, a cell identification device is used to perform cell identification on the cell images and determine the cells contained in the image. At this time, all cells in the cell image can be marked, and the number of marked cells can be determined as the number of cells in the cell image.

[0045] In one embodiment, the step of performing cell count identification on the cell image using the cell identification device and determining the number of cells in the cell image is as follows: The process includes inputting the cell image into a cell recognition model and extracting cell shape features from the cell image using the cell recognition model. The cell recognition model is a neural network model trained to identify cell regions in the input image, and is obtained by convergence through deep learning of cell shape features, where the cell shape features are the features exhibited by the edges of the cell and the shape enclosed by those edges. When extracting the cell shape features, image preprocessing, such as grayscale processing and edge enhancement, can be performed on the cell image.

[0046] Using the cell recognition model described above, cell recognition is performed on the cell image based on the extracted cell shape features to obtain a cell region having the cell shape features. The cell region may be the region of a single cell, or it may be a region surrounded by overlapping or adjacent cells.

[0047] Count statistics are performed on the identified cell regions to obtain the number of cells in the cell image. In the count statistics process, the actual number of cells is obtained by outputting a number corresponding to the number of cells in the overlapping or adjacent regions. For example, if the overlapping region is within a region size range of one to two cells, the region is counted as containing two cells. In this way, it becomes possible to identify the number of cells more accurately, and the reliability of cell count identification is improved.

[0048] The network structure of this neural network model can be YOLO, VGG, or ResNets, and the cell shape features include cell size, external shape, aggregation, and superposition features.

[0049] The YOLO (You Only Look Once) network structure is a network structure for algorithms that enable real-time object detection. The YOLO algorithm uses a convolutional neural network to detect objects in a single forward pass, allowing it to detect and identify various objects in an image in real time.

[0050] In the Visual Geometry Group (VGG) network structure, three 3x3 convolutional kernels are used to replace a 7x7 convolutional kernel, and two 3x3 convolutional kernels are used to replace a 5x5 convolutional kernel. The main purpose of this configuration is to achieve network deepening under the condition that the same receptive field is maintained, thereby enhancing the effectiveness of the neural network to a certain extent.

[0051] The Residual Networks (ResNets) structure weakens strong inter-layer connections by having certain layers of the neural network skip neuronal connections in the next layer, instead creating inter-layer connections. This helps solve the problems of vanishing gradients and gradient explosions, allowing for the learning of deeper networks while simultaneously ensuring good network performance.

[0052] Image parameters corresponding to the cell image are acquired, and the cell density is determined based on the number of cells and the image parameters.

[0053] To understand this, image parameters are parameters necessary to convert cell count to density, such as the resolution of the cell image. Specifically, a cell identification device is used to perform cell count identification on the cell image to determine the number of cells in the cell image, and then the image parameters corresponding to the cell image are obtained. The cell density is determined based on the number of cells and the image parameters. The image parameters are the image area and the image depth, and by obtaining the set image area and image depth, the image volume of the captured image is determined, and the cell density is obtained by dividing the number of cells by the image volume.

[0054] In one embodiment, step S20, that is, the step of performing a washing and concentration treatment on the post-culture cell solution in the cell culture vessel to obtain a concentrated cell solution, This includes performing a washing treatment on the post-culture cell solution in the cell culture vessel to obtain a mixed cell solution.

[0055] As can be understood, the reason for performing a washing treatment on the post-culture cell saturation beforehand in this embodiment is to preliminarily wash away impurities other than cells contained in the post-culture cell saturation, thereby obtaining a mixed cell saturation. In this embodiment, a buffer is added to the cell culture vessel containing the post-culture cell saturation, and the mixed cell saturation is obtained by performing a centrifugal washing operation on the buffer and the cell culture vessel containing the post-culture cell saturation.

[0056] Here, the buffer solution is used to wash the post-culture cell solution for impurities other than cells. The volume of buffer solution to be added is determined according to the volume of the post-culture cell solution. For example, a post-culture cell solution of 250 mL or less can be washed with 800 mL of buffer solution, and a post-culture cell solution of 250 mL to 500 mL can be washed with 1200 mL of buffer solution. Preferably, in this application, if the cell culture vessel contains a post-culture cell solution of 250 mL or less, washing with 400 mL of buffer solution is performed twice. By adding the buffer solution in installments, the washing effect of the buffer solution on the post-culture cell solution can be fully exerted each time, the washing effect for impurities other than cells can be enhanced, and consequently the purity of the mixed cell solution can be improved.

[0057] The mixed cell solution in the cell culture vessel is subjected to a concentration process to obtain the concentrated cell solution.

[0058] As can be understood, the mixed cell solution obtained after centrifugal washing of the cultured cell solution with buffer may not have a sufficiently high cell concentration and may contain small amounts of non-cell impurities. Therefore, after obtaining the mixed cell solution, a centrifugal extraction operation is performed on the cell culture vessel containing the mixed cell solution to separate and remove non-cell impurities from the mixed cell solution, thereby obtaining a concentrated cell solution containing only the target cells. In this way, during the centrifugal extraction operation, the higher concentration cell solution can be extracted to the upper layer, and the lower concentration cell solution or impurities can be extracted to the lower layer, thereby increasing the cell concentration and further removing non-cell impurities. At this point, the concentrated cell solution in the cell culture vessel will ultimately contain only T cells and / or T cell subsets.

[0059] In one embodiment, after step S20, that is, after performing a washing and concentration treatment on the post-culture cell solution in the cell culture vessel to obtain a concentrated cell solution, the method further... This includes performing a sampling test on the concentrated cell solution and determining whether or not the concentrated cell solution meets the pre-set formulation conditions.

[0060] To ensure understanding, after the cell culture medium in the cell culture vessel is washed and concentrated to obtain a concentrated cell solution, a sampling test is performed on the concentrated cell solution before dispensing it into multiple formulation bags. That is, density and activity measurements are performed on the concentrated cell solution to ensure the cell viability and activity in the concentrated cell solution dispensed into the formulation bags. In other words, the quality of the cells in the concentrated cell solution is ensured. The pre-set formulation conditions are that the cell density in the concentrated cell solution is above a pre-set density threshold, and the cell activity is above a pre-set activity threshold. Here, the pre-set density threshold and activity threshold can be set according to the cell density, number, and activity rate required for cell therapy.

[0061] If the concentrated cell solution satisfies the pre-set formulation conditions, the concentrated cell solution satisfying the pre-set formulation conditions is dispensed into multiple formulation bags.

[0062] Specifically, a sampling test is performed on the concentrated cell solution to determine whether or not it meets the pre-set formulation conditions. If the concentrated cell solution meets the pre-set formulation conditions, it is dispensed into multiple formulation bags. If the concentrated cell solution does not meet the pre-set formulation conditions, it can be subjected to a washing and concentration process again. After the washing and concentration process, it is determined again whether or not the concentrated cell solution meets the pre-set formulation conditions. If it does meet the pre-set formulation conditions, it is dispensed into multiple formulation bags.

[0063] As should be understood, the relative numbers of the steps in the above embodiment do not indicate the order of execution, and the execution order of each process should be determined by its function and underlying logic, and do not constitute any limitation on the implementation process of the embodiment of this application.

[0064] In one embodiment, a cell proliferation module is used to perform a cell proliferation treatment on the target cell medium in a cell culture vessel to obtain a post-culture cell medium. A washing and concentrating module for performing a washing and concentrating process on the post-culture cell solution in the cell culture vessel to obtain a concentrated cell solution, The present invention provides a cell preparation processing apparatus, which includes a first dispensing formulation module for dispensing the concentrated cell solution in a cell culture vessel into a plurality of formulation bags.

[0065] Furthermore, the cell proliferation module, An activation reagent delivery submodule for delivering an activation reagent to the cell culture vessel, wherein the activation reagent is one or more of the following: stimulating antibody CD3, stimulating antibody CD28, cytokine recombinant protein, costimulatory molecule, lectin, ionophore, synthetic molecule, antigen-presenting cell, artificial antigen-presenting cell, and feeder cell. The system includes a solution oscillating and mixing submodule for performing an oscillating and mixing operation on a cell culture vessel containing the cell solution to be cultured and the activation reagent, in order to obtain the post-culture cell solution.

[0066] Furthermore, the solution oscillating mixing submodule is During the agitation mixing process, a density measurement unit is used to measure the cell density of the cells in the cell culture vessel using a density acquisition device, and to determine the cell density of the cells in the cell culture vessel. The system includes a density comparison unit for obtaining the post-culture cell solution, which stops the agitation mixing operation when it detects that the cell density is above a preset density threshold.

[0067] Furthermore, the density measurement unit, The image acquisition device acquires images of the cells in the cell culture vessel and includes an image acquisition subunit for obtaining cell images, The cell identification device performs cell counting and identification on the cell image and includes a cell identification subunit for determining the number of cells in the cell image, The system includes a density conversion subunit for acquiring image parameters corresponding to the cell image and for determining the cell density based on the number of cells and the image parameters.

[0068] Furthermore, the washing and concentration module, A washing unit for performing a washing treatment on the post-culture cell solution in the cell culture vessel to obtain a mixed cell solution, The system includes a concentration unit for performing a concentration process on the mixed cell solution in the cell culture vessel to obtain the concentrated cell solution.

[0069] Furthermore, the cleaning unit, A buffer supply subunit for adding buffer to a cell culture vessel containing the cultured cell medium, The system includes a centrifugal washing subunit for performing a centrifugal washing operation on a cell culture vessel containing the buffer solution and the post-culture cell solution to obtain the mixed cell solution.

[0070] Furthermore, the concentration processing unit includes a centrifugal extraction subunit for performing a centrifugal extraction operation on a cell culture vessel containing a mixed cell solution to separate and remove impurities other than cells from the mixed cell solution, thereby obtaining a concentrated cell solution containing only the target cells. The target cells include T cells and / or a subset of T cells.

[0071] Furthermore, the first dispensing formulation module is A cryopreservation solution input unit for adding the cryopreservation solution to a culture vessel containing the concentrated cell solution, The system includes a dispensing compounding unit for mixing the cryopreservation solution and concentrated cell solution in the cell culture vessel and dispensing the mixture into a compounding bag.

[0072] Furthermore, the dispensing formulation unit is The system includes a dispensing subunit for quantitatively formulating the cryopreservation solution and concentrated cell solution in a cell culture vessel using a quantitative tube, and for dispensing the quantitatively formulated cryopreservation solution and concentrated cell solution into individual formulation bags.

[0073] Furthermore, the cell preparation processing device is A sampling inspection module for performing a sampling inspection on the concentrated cell solution and determining whether or not the concentrated cell solution meets pre-set formulation conditions, If the concentrated cell solution satisfies the pre-set formulation conditions, the system includes a second dispensing formulation module for dispensing the concentrated cell solution that satisfies the pre-set formulation conditions into a plurality of formulation bags.

[0074] A cell preparation processing system comprising a culture vessel and a controller connected to the culture vessel, wherein the controller is used to perform the cell preparation processing method.

[0075] Specific limitations of the cell preparation processing system, controller, and each of its units and modules can be found in the limitations relating to the cell preparation processing method described above and will not be repeated here. Each module in the controller can be implemented in whole or in part by software, hardware, or a combination thereof. As can be understood, as shown in Figure 3, the controller includes a processor, storage device, network interface, and database connected by a system bus. Each module of the controller may be built into the processor as hardware or independent of the processor, or may be stored in the storage device as software, so that the processor can call and execute the operations corresponding to each of the above modules. Here, the processor is used to provide computation and control capabilities. The storage device includes a readable storage medium and built-in memory. The readable storage medium stores an operating system, computer-readable instructions, and a database. The built-in memory provides an environment for the execution of the operating system and computer-readable instructions in the readable storage medium. The database is used to store data used in the cell preparation processing method in the above embodiment. The network interface is used to communicate with external terminals via a network. The cell preparation processing method is realized when the computer-readable instructions are executed by the processor. The readable storage medium provided in this embodiment includes a non-volatile readable storage medium and a volatile readable storage medium.

[0076] In one embodiment, one or more readable storage media storing computer-readable instructions are provided. The readable storage media provided in this embodiment include non-volatile readable storage media and volatile readable storage media. Computer-readable instructions are stored in the readable storage media, and when these computer-readable instructions are executed by one or more processors, one or more processors realize the cell preparation processing method.

[0077] Those skilled in the art will understand that all or part of the flows in the methods of the embodiments described above can be implemented by instructing the relevant hardware with computer-readable instructions. These computer-readable instructions can be stored in a non-volatile or volatile-readable storage medium, and at runtime, the computer-readable instructions can include the flows of the embodiments of the methods described above. Any references to storage devices, storage, databases, or other media used in the embodiments provided herein can include non-volatile and / or volatile storage devices. Non-volatile storage devices can include read-only memory (ROM), programmable ROM (PROM), electrically programmable ROM (EPROM), electrically erasable programmable ROM (EEPROM), or flash memory. Volatile storage devices can include random-access memory (RAM) or external cache memory. While not an exhaustive list, 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), synchlink DRAM (SLDRAM), rambus direct RAM (RDRAM), direct rambus dynamic RAM (DRDRAM), and rambus dynamic RAM (RDRAM).

[0078] Those skilled in the art will clearly understand that the above-described division of each functional unit and module is merely an example for the sake of convenience and simplicity. In actual applications, the above functions can be assigned to different functional units and modules as needed. That is, the internal structure of the device can be divided into different functional units or modules, and all or part of the above functions can be performed by these units or modules.

[0079] The embodiments described above are merely for illustrative purposes and are not limiting. Although the present application has been described in detail with reference to the embodiments described above, it is still possible to modify the technical solutions described in each of the embodiments described above, or to substitute equivalents for some of their technical features, as will be understood by those skilled in the art. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions in each embodiment of the present application, and should all be included within the scope of protection of the present application. [Explanation of Symbols]

[0080] 1. Sample bag, 2. Cleaning solution bag, 3. Replacement fluid bag, 4. Formulation bag, 5. Intermediate product bags, 6. Culture medium, 7. Shut-off valves, 8. Bubble sensor, 9. Pressure sensor, 10. Peristaltic pump, 11, quantitative tube, 12, Cell culture container

Claims

1. The process involves performing a cell proliferation treatment on the target cell medium in a cell culture vessel to obtain a post-culture cell medium, and The post-culture cell solution in the aforementioned cell culture vessel is subjected to a washing and concentration process to obtain a concentrated cell solution. A method for processing cell preparations, characterized by comprising dispensing the concentrated cell solution in a cell culture vessel into a plurality of preparation bags.

2. The step of performing a cell proliferation treatment on the cell culture medium in the aforementioned cell culture vessel to obtain a post-culture cell medium is: This includes adding an activation reagent to the cell culture vessel, The activation reagent is one or more of the following: stimulating antibody CD3, stimulating antibody CD28, cytokine recombinant protein, costimulatory molecule, lectin, ionophore, synthetic molecule, antigen-presenting cell, artificial antigen-presenting cell, and feeder cell. A method for processing a cell preparation according to claim 1, characterized by performing a shaking mixing operation on a cell culture vessel containing the cell solution to be cultured and the activation reagent to obtain the post-culture cell solution.

3. The step of performing a shaking and mixing operation on the cell culture vessel containing the cell medium to be cultured and the activation reagent to obtain the post-culture cell medium is as follows: During the agitation mixing process, the cell density of the cells in the cell culture vessel is measured using a density acquisition device to determine the cell density of the cells in the cell culture vessel. A method for processing a cell preparation according to claim 2, characterized in that, when it is detected that the cell density is equal to or greater than a predetermined density threshold, the shaking and mixing operation is stopped to obtain the post-culture cell solution.

4. The density acquisition device comprises an image acquisition device and a cell identification device. The step of measuring the cell density of the cells in the cell culture vessel using the density acquisition device and determining the cell density of the cells in the cell culture vessel is as follows: The image acquisition device acquires images of the cells in the cell culture vessel to obtain cell images, The cell identification device performs cell counting and identification on the cell image to determine the number of cells in the cell image. A method for processing a cell preparation according to claim 3, characterized by comprising obtaining image parameters corresponding to the cell image and determining the cell density based on the number of cells and the image parameters.

5. The step of performing a washing and concentration treatment on the post-culture cell solution in the cell culture vessel to obtain a concentrated cell solution is: The post-culture cell solution in the cell culture vessel is washed to obtain a mixed cell solution. A method for processing a cell preparation according to claim 1, characterized by comprising: performing a concentration process on the mixed cell solution in the cell culture vessel to obtain the concentrated cell solution.

6. The step of performing a washing treatment on the post-culture cell solution in the cell culture vessel to obtain a mixed cell solution is: Adding a buffer to the cell culture vessel containing the cultured cell solution, A method for processing a cell preparation according to claim 5, characterized by comprising: performing a centrifugal washing operation on a cell culture vessel containing the buffer solution and the post-culture cell solution to obtain the mixed cell solution.

7. The step of performing a concentration process on the mixed cell solution in the cell culture vessel to obtain the concentrated cell solution is: The process includes performing a centrifugal extraction operation on a cell culture vessel containing a mixed cell solution to separate and remove impurities other than cells from the mixed cell solution, thereby obtaining a concentrated cell solution containing only the target cells. The method for processing a cell preparation according to claim 5, characterized in that the target cells include T cells and / or a subset of T cells.

8. The step of dispensing the concentrated cell solution in the cell culture vessel into multiple formulation bags is: The cryopreservation solution is added to the culture vessel containing the concentrated cell solution, A method for processing a cell preparation according to claim 1, characterized by comprising mixing the cryopreservation solution and concentrated cell solution in the cell culture vessel and then dispensing the mixture into a preparation bag.

9. The step of mixing the cryopreservation solution and concentrated cell solution in the cell culture vessel and then dispensing it into a formulation bag is as follows: A method for processing a cell preparation according to claim 8, characterized by comprising quantitatively formulating the cryopreservation solution and concentrated cell solution in a cell culture vessel using a quantitative tube, and dispensing the quantitatively formulated cryopreservation solution and concentrated cell solution into each preparation bag.

10. The step of performing a washing and concentration treatment on the post-culture cell solution in the cell culture vessel to obtain a concentrated cell solution is: A sampling test is performed on the concentrated cell solution to determine whether or not the concentrated cell solution meets the pre-set formulation conditions. A method for processing a cell preparation according to claim 1, further comprising dispensing the concentrated cell solution that satisfies the pre-set formulation conditions into a plurality of preparation bags, if the concentrated cell solution satisfies the pre-set formulation conditions.

11. A cell proliferation module for performing cell proliferation treatment on the target cell medium in a cell culture vessel to obtain a post-culture cell medium, A washing and concentrating module for performing a washing and concentrating process on the post-culture cell solution in the cell culture vessel to obtain a concentrated cell solution, A cell preparation processing apparatus comprising: a first dispensing formulation module for dispensing the concentrated cell solution in a cell culture vessel into a plurality of formulation bags.

12. The aforementioned cell proliferation module is An activation reagent delivery submodule for delivering an activation reagent to the cell culture vessel, wherein the activation reagent is one or more of the following: stimulating antibody CD3, stimulating antibody CD28, cytokine recombinant protein, costimulatory molecule, lectin, ionophore, synthetic molecule, antigen-presenting cell, artificial antigen-presenting cell, and feeder cell. The apparatus for processing cell preparations according to claim 11, further comprising a solution oscillating mixing submodule for performing an oscillating mixing operation on a cell culture vessel containing the cell solution to be cultured and the activation reagent to obtain the post-culture cell solution.

13. The solution oscillating mixing submodule is, During the agitation mixing process, a density measurement unit is used to measure the cell density of the cells in the cell culture vessel using a density acquisition device, and to determine the cell density of the cells in the cell culture vessel. The apparatus for a cell preparation according to claim 12, further comprising: a density comparison unit for stopping the shaking and mixing operation and obtaining the post-culture cell solution when it is detected that the cell density is equal to or greater than a preset density threshold.

14. The density measurement unit is The image acquisition device acquires images of the cells in the cell culture vessel and includes an image acquisition subunit for obtaining cell images, The cell identification device performs cell counting and identification on the cell image and includes a cell identification subunit for determining the number of cells in the cell image, The apparatus for a cell preparation according to claim 13, further comprising a density conversion subunit for acquiring image parameters corresponding to the cell image and determining the cell density based on the number of cells and the image parameters.

15. The aforementioned washing and concentration module is A washing unit for performing a washing treatment on the post-culture cell solution in the cell culture vessel to obtain a mixed cell solution, The apparatus for a cell preparation according to claim 11, further comprising a concentration processing unit for performing a concentration process on the mixed cell solution in the cell culture vessel to obtain the concentrated cell solution.

16. The aforementioned cleaning unit is A buffer supply subunit for adding buffer to a cell culture vessel containing the cultured cell medium, The apparatus for processing cell preparations according to claim 15, further comprising a centrifugal washing subunit for performing a centrifugal washing operation on a cell culture vessel containing the buffer and the post-culture cell solution to obtain the mixed cell solution.

17. The apparatus for processing cell preparations according to claim 15, wherein the concentration processing unit comprises a centrifugal extraction subunit for performing a centrifugal extraction operation on a cell culture vessel containing a mixed cell solution to separate and remove impurities other than cells from the mixed cell solution, and to obtain a concentrated cell solution containing only target cells, wherein the target cells include T cells and / or a T cell subset.

18. A sampling inspection module for performing a sampling inspection on the concentrated cell solution and determining whether or not the concentrated cell solution meets pre-set formulation conditions, The cell preparation processing apparatus according to claim 11, further comprising a second dispensing formulation module for dispensing the concentrated cell solution that satisfies the pre-set formulation conditions into a plurality of formulation bags, provided that the concentrated cell solution satisfies the pre-set formulation conditions.

19. The system comprises a cell culture vessel and a controller connected to the cell culture vessel, the controller being used to perform the following cell preparation processing method, The method for processing the cell preparation is as follows: The process involves performing a cell proliferation treatment on the target cell medium in a cell culture vessel to obtain a post-culture cell medium, and The post-culture cell solution in the aforementioned cell culture vessel is subjected to a washing and concentration process to obtain a concentrated cell solution. A method for processing cell preparations, characterized by comprising dispensing the concentrated cell solution in a cell culture vessel into a plurality of preparation bags.

20. One or more readable storage media storing computer-readable commands, When the computer-readable command is executed by one or more processors, the step of causing the one or more processors to perform the following steps is: The process involves performing a cell proliferation treatment on the target cell medium in a cell culture vessel to obtain a post-culture cell medium, and The post-culture cell solution in the aforementioned cell culture vessel is subjected to a washing and concentration process to obtain a concentrated cell solution. One or more readable storage media storing computer-readable commands, characterized by comprising dispensing the concentrated cell solution in a cell culture vessel into a plurality of formulation bags.

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