Cell sample processing method, device, system and medium

Abstract

The invention discloses a cell sample treatment method, device and system and a medium. The method comprises the following steps: enabling a cell sample to flow into a centrifugal container; performing centrifugal treatment on the cell sample in the centrifugal container to obtain initial cell sap; performing antigen binding and magnetic separation on the initial cell sap to separate out enriched cell sap; activating the enriched cell sap by inputting a regulator to obtain activated cell sap; performing genetic modification on the activated cell sap to obtain target cell sap; and sub-packaging the target cell sap into a plurality of preparation bags. According to the full-closed cell sample treatment device, a full-closed full-flow cell sample treatment process integrating cell sorting, cell magnetic sorting, cell activation, cell genetic modification and a cell preparation is realized, the requirement of working in a sterile environment is met, and external pollution of the cell sample in the treatment process can be avoided. Furthermore, high-efficiency, standardized and large-scale production and preparation of the target cell sap are realized.

Description

Technical Field

[0001] The present invention relates to the field of biological processing technology, and in particular to a cell sample processing method, device, system and medium. Background Art

[0002] At present, cell therapy is a key development field in the international medical frontier. Cell sorting, cell magnetic sorting, cell activation, cell genetic modification, and cell preparation are key steps in cell therapy. However, the existing cell sorting, cell magnetic sorting, cell activation, and cell genetic modification operations are all performed manually on equipment distributed in different areas. These operations need to be performed in a sterile environment, and each step requires high-level operation by professionals. The operation threshold is high, resulting in high site and labor costs, long cycles, and extremely low efficiency. Summary of the invention

[0003] The embodiments of the present invention provide a cell sample processing method, device, system and medium to solve the problems of high cost, long cycle and low efficiency in cell therapy in the prior art.

[0004] A cell sample processing method, comprising:

[0005] The cell sample is flowed into a centrifuge container;

[0006] Centrifuging the cell sample in the centrifugal container to obtain an initial cell fluid;

[0007] Performing antigen binding and magnetic separation on the initial cell fluid to separate the enriched cell fluid;

[0008] Activating the enriched cell fluid by inputting a regulator to obtain an activated cell fluid;

[0009] genetically modifying the activated cell fluid to obtain target cell fluid;

[0010] The target cell solution is divided into a plurality of preparation bags.

[0011] A cell modification treatment device, comprising:

[0012] A sample inflow module, used for flowing the cell sample into a centrifugal container;

[0013] A cell separation module, used for centrifuging the cell sample in the centrifugal container to obtain an initial cell fluid;

[0014] A cell enrichment module, used for performing antigen binding and magnetic separation on the initial cell fluid to separate the enriched cell fluid;

[0015] A cell activation module, used to activate the enriched cell fluid by inputting a regulator to obtain an activated cell fluid;

[0016] A genetic modification module, used for genetically modifying the activated cell fluid to obtain a target cell fluid;

[0017] The preparation packaging module is used to package the target cell solution into multiple preparation bags.

[0018] A cell sample processing method system comprises a centrifugal container and a controller connected to the centrifugal container, wherein the controller is used to execute the cell sample processing method.

[0019] A computer-readable storage medium stores a computer program, and when the computer program is executed by a processor, the cell sample processing method described above is implemented.

[0020] The above-mentioned cell sample processing method, device, system and medium realize the flow of cell samples into a centrifugal container; centrifugal treatment of the cell sample in the centrifugal container to obtain initial cell fluid; antigen binding and magnetic separation of the initial cell fluid to separate enriched cell fluid; activation of the enriched cell fluid by inputting a regulator to obtain activated cell fluid; genetic modification of the activated cell fluid to obtain target cell fluid; and packaging the target cell fluid into multiple preparation bags.

[0021] The present invention realizes the enrichment of cell fluid by centrifuging the cell sample in a centrifugal container, and realizing antigen binding and magnetic separation after centrifugation in the centrifugal container, and activating the enriched cells in the centrifugal container by a regulator, and then genetically modifying the activated cell fluid to obtain the target cell fluid, and finally preparing the target cell fluid from the centrifugal container into each preparation bag, realizing a fully enclosed and full-flow cell sample processing process integrating cell sorting, cell magnetic sorting, cell activation, cell genetic modification, and cell preparation, ensuring the requirements of working in a sterile environment, and being able to avoid external contamination of the cell sample during the processing, and improving the quality of cell culture. The above steps are completed in an integrated manner, avoiding the scattered settings between the equipment, reducing the occupied space, reducing manual intervention, thereby reducing the site cost and labor cost. And then reducing the cycle of the cell preparation, realizing the automation and standardization of the target cell fluid preparation process, and improving the efficiency of the cell preparation of the target cell fluid. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings required for use in the description of the embodiments of the present invention will be briefly introduced below. Obviously, the accompanying drawings in the following description are only some embodiments of the present invention. For ordinary technicians in this field, other accompanying drawings can be obtained based on these accompanying drawings without paying creative labor.

[0023] Figure 1 is a flow chart of a cell sample processing method according to an embodiment of the present invention;

[0024] Figure 2 is a flow chart of step S20 of a cell sample processing method according to an embodiment of the present invention;

[0025] Figure 3 is a principle block diagram of a cell sample processing device in one embodiment of the present invention;

[0026] Figure 4 Schematic diagram of a cell sample processing method system in one embodiment of the present invention. DETAILED DESCRIPTION

[0027] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0028] In one embodiment, if Figure 1 As shown, a cell sample processing method is provided, comprising the following steps:

[0029] S10. Flow the cell sample into a centrifuge container.

[0030] It can be understood that initially, the cell sample is contained in one or more cell sample liquid bags, and the cell sample in the cell sample liquid bag is transported to the centrifugal container along the input pipeline through a non-contact conveying device (preferably a peristaltic pump near the liquid inlet). The non-contact conveying device can be arranged in a peristaltic pump near the liquid outlet, and the cell sample in the cell sample liquid bag is allowed to flow into the centrifugal container by generating negative pressure in the centrifugal container. The input pipeline connects the cell sample liquid bag and the centrifugal container, and the opening of the cell sample flowing into the input pipeline can also be controlled by a switch valve. The input pipeline can include a filter and a drip bucket, etc.

[0031] Furthermore, the cell sample processing method provided in this embodiment is implemented by an integrated cell sample processing device, and the entire device is placed in a sterile environment; a centrifugal container is installed on the device, the centrifugal container is in a sealed sterile environment, and an input pipe is connected to the centrifugal container, and the other end of the input pipe can be connected to a collection bag (the collection bag is used to store solutions such as cell fluid or other reagents). In this way, it can be ensured that the cell sample is input into the centrifugal container, and the regulator is input into the centrifugal container in the subsequent steps in a sealed sterile environment, and the quality of the cell fluid or the reagent itself will not be damaged due to environmental influences, thereby improving the quality of cell culture.

[0032] S20, centrifuging the cell sample in the centrifugal container to obtain an initial cell fluid.

[0033] Understandably, the cell sample is a sample including one or more of T cells, T cell subsets and / or T cell progenitor cells. The initial cell fluid refers to PBMC fluid or T cell fluid. The centrifugal container is a container for holding the cell sample, including but not limited to a centrifuge cup, a piston centrifuge cylinder, a centrifuge tank or a centrifuge bottle.

[0034] The PBMC fluid is a cell fluid containing peripheral blood mononuclear cells (PBMC). PBMC refers to cells with a single nucleus in peripheral blood, including lymphocytes, monocytes, dendritic cells (DC) and a small number of other cells. Most of the PBMCs are lymphocytes, including B cells and T cells.

[0035] Specifically, the cell sample is flowed into the centrifugal container in the integrated device through the liquid inlet of the centrifugal container (opened when in use and closed when out of use), and an appropriate sucrose polymer (e.g., Ficoll solution) can be added at the same time, and the centrifugal container is controlled to perform centrifugal treatment at a preset speed. Through density gradient centrifugation, the cell sample is stratified according to density and cell size, and the red blood cell layer (mainly composed of red blood cells), the separation layer (mainly composed of the separation liquid of Ficoll), the PBMC layer (mainly composed of mononuclear cells) and the supernatant layer (mainly composed of plasma and blood cells) are separated. The intermediate product cell fluid is extracted from the liquid outlet near or located at the PBMC layer. The rotation speed can be continuously adjusted during the process to fully extract the intermediate product cell fluid in the centrifugal container. In one embodiment, the rotation speed is continuously reduced to make the boundaries of each layer tilt, that is, the vertical plane is tilted, so that the boundary of the PBMC layer can cover the liquid outlet. The liquid outlet is arranged below the centrifugal container, so as to fully extract the intermediate product cell fluid. The intermediate product cell fluid may include liquid from the liquid separation layer, the PBMC layer and the supernatant layer. The intermediate product cell fluid is pumped into the centrifugal container by a peristaltic pump near the liquid outlet or a peristaltic pump near the liquid inlet. The cell fluid is transferred to the intermediate product bag through the transfer pipeline, and then the cleaning liquid is introduced to clean the centrifugal container and the pipeline. The flow direction of the cleaning liquid can be cleaned according to the design requirements of the pipeline. A switch valve (or a control valve) is set in the pipeline, and the opening and closing of different positions in the pipeline path are controlled by the switch valve to allow the cleaning liquid to clean each pipeline path and flush the unnecessary components in the centrifugal container into the waste liquid bag. The cleaning liquid can be a mixture of physiological saline and 0.5% HSA (human serum albumin). After cleaning, the intermediate product in the intermediate product bag is flushed out by a peristaltic pump. When the product cell fluid flows back into the centrifugal container, the peristaltic pump can be reversed for transmission (for example, the peristaltic pump rotates clockwise during extraction and counterclockwise during reflow), and then the centrifugal container is controlled to rotate centrifugally to re-stratify the intermediate product cell fluid in the centrifugal container, and the cleaning fluid is input to make the components of the liquid separation layer and the supernatant layer combine with the cleaning fluid (hydrophilic combination) to form a layer, so that the liquid containing the above components after the combination can be extracted from the liquid outlet to the waste liquid bag, and the rotation speed of the centrifugal container is continuously adjusted to leave the liquid of the PBMC layer, and the liquid is determined as the initial cell fluid.

[0036] In another embodiment, non-T cells can also be labeled, and then the rotation of the centrifugal container is controlled to stratify the labeled cell samples by centrifugal force, and then extracted into an intermediate product bag. The liquid in the intermediate product bag is then returned to the centrifugal container, and the liquid is washed and then extracted. During the extraction process, filtration or pipeline adsorption is added for the labeled non-T cells to obtain an initial cell fluid containing T cells, and finally the obtained initial cell fluid is returned to the centrifugal container.

[0037] The integrated device is a device that integrates cell sorting, cell magnetic sorting, cell activation, cell genetic modification, and cell preparation. The centrifugal container includes a liquid inlet and a liquid outlet. Both the liquid inlet and the liquid outlet can flow in and out of liquid, that is, both can flow in and extract liquid. In one embodiment, the liquid inlet of the centrifugal container is arranged above the container, and the liquid outlet of the centrifugal container is arranged below the container.

[0038] S30, performing antigen binding and magnetic separation on the initial cell fluid to separate the enriched cell fluid.

[0039] It can be understood that the enriched cell fluid refers to a cell fluid enriched with T cells.

[0040] Specifically, the antigen liquid and the magnetic beads are transported to a centrifugal container, wherein the antigen liquid is a liquid containing antigen molecules, for example, antigen binding molecules CD2, CD3, CD4, CD8, CD25, CD28, CD27, CD45RA, CD45RO, CD62L, CD95, CD127, CD137, α / βTCR, γ / δTCR, CCR7, PD-1 or Lag3, etc. The magnetic beads can be micron-sized magnetic beads (magnetic beads with a particle size of 1 to 5 microns) or nano-sized magnetic beads (magnetic beads with a particle size of 5-100 nanometers). In the process of transporting the antigen liquid and the magnetic beads to the centrifugal container, the antigen liquid and the magnetic beads can be flowed into the same pipeline and then passed through the liquid inlet. The liquid flows into a centrifugal container, which can rotate back and forth at a specific speed to allow the initial cell fluid to bind to antigens. The antigen binding is to bind antigen molecules and magnetic beads to cells in the initial cell fluid, and the initial cell fluid after antigen binding is extracted from the centrifugal container to the magnetic separation column near the outlet through a peristaltic pump near the outlet. The pipeline between the outlet and the magnetic separation column can pass through the peristaltic pump, and then magnetic separation is achieved through the magnetic separation column, that is, the initial cell fluid is subjected to immunomagnetic cell separation by magnetic bead sorting, that is, the affinity of specific antibodies or other molecules on the surface of magnetic beads with cell surface molecules is utilized to separate specific types of immunomagnetic cells bound to magnetic beads, thereby separating the enriched cell fluid. The initial cell fluid can also be labeled by optical excitation, that is, the specific binding of fluorescent markers to cell surface molecules is used to extract the initial cell fluid after specific binding in the centrifugal container out of the centrifugal container, and then the specifically bound cells are transported through a specific channel according to the single cell channel. The specifically bound cells can be transported through a funnel-shaped channel to narrow the channel so that the specifically bound cells emit fluorescent signals under optical excitation for sorting, and enriched cells are separated by lasers, detection channels, filters, etc. The laser can excite the wavelength of the labeled cells, and then the wavelength is detected by the detector and filter, and then the wavelength presented by the required fluorescent element is selected, and the detected cells are harvested according to different labels, thereby harvesting enriched cell fluid.

[0041] In one embodiment, magnetic beads and antigens can be combined with T cells so that the T cells are marked with magnetism. Then, the cell fluid containing the marked T cells in the centrifugal container is transferred to the intermediate liquid bag by a peristaltic pump, and the intermediate liquid bag is moved to the variable magnetic device, and an external magnetic field is applied to the variable magnetic device, that is, the T cells bound to the magnetic beads and antigens in the liquid of the intermediate liquid bag are adsorbed by the variable magnetic device, and the variable magnetic device is controlled to swing or vibrate by a swing parameter (which can be set according to actual conditions to make the variable magnetic device swing at different angles) or a vibration parameter to make the T cells bound to the magnetic beads and antigens adsorbed by the variable magnetic device evenly distributed, and at the same time, other substances in the intermediate liquid bag except the adsorbed T cells bound to the magnetic beads and antigens are suspended in the liquid. Then, a waste liquid discharge operation is performed to make the liquid in the intermediate liquid bag except the adsorbed T cells bound to the magnetic beads and antigens flow into the waste liquid bag. And a washing operation is performed on the T cells bound to the magnetic beads and antigens in the intermediate liquid bag. Finally, a resuspension solution or buffer is injected into the intermediate liquid bag to separate the enriched cell fluid, and then the separated enriched cell fluid is returned to the centrifuge container. Among them, the cell removal magnetic beads can be performed after subsequent cell culture to obtain the final cells. The variable magnetic device can change the strength of the magnetic field, and can also make the magnetic separation column have magnetic and non-magnetic conversion by moving the magnet.

[0042] S40, activating the enriched cell fluid by inputting a regulator to obtain activated cell fluid.

[0043] It can be understood that the activated cytosol refers to the cytosol after the T cells are activated.

[0044] Specifically, the regulator is placed in a regulator liquid bag, and the regulator can be injected into the centrifugal container through an inlet and outlet in the centrifugal container by a peristaltic pump. The regulator is a solvent that can activate T cells. The regulator activates T cells by requiring the participation of a dual signal system. For example, in addition to the first signal provided by MHC / antigen peptide-TCR (T cell antigen receptor, T cell receptor), the second signal must be provided by the costimulatory molecule to activate the T cells. The T cell receptor is stimulated by the regulator to receive the costimulatory signal, thereby activating the T cell, and the activated cell fluid can be obtained. The B7 protein on the APC (Antigen-Presenting Cell, antigen presenting cell) can provide the costimulatory signal necessary for activation and produce the following costimulatory effect after binding to the CD28 receptor of the enriched cell fluid containing T cells. The B7 protein binds to the CTLA-4 (cytotoxic T lymphocyte-associated protein 4) receptor to promote the deactivated T cells.

[0045] S50, genetically modifying the activated cell fluid to obtain target cell fluid.

[0046] Understandably, the genetic modification can be obtained by a variety of methods, such as viral and non-viral vectors, mRNA (electroporation technology), genome editing tools, etc. Among them, in the viral vector method, the retroviral vector (RV) is a widely used viral vector, and the lentiviral vector (lentiviral vector) contains a cis-acting element central polypurine tract (cPPT), which enables it to effectively infect static T cells. The genetic modification can transfer the structure of CAR into the activated cell fluid (activated T cells) through genetic engineering, and finally obtain the target cell fluid, which is called a targeted CAR-T (Chimeric Antigen Receptor T-Cell Immunotherapy) cell. Among them, the activated cell fluid can be genetically modified by one of viral vectors, transposons, nanoparticles, liposomes, and electroporation-mediated transfection.

[0047] S60, packaging the target cell solution into a plurality of preparation bags.

[0048] Specifically, after the target cell solution, the target cell solution is divided into a preparation by a peristaltic pump, and the target cell solution is divided into a plurality of preset preparation bags by a peristaltic pump. The target cell solution can also be accurately divided into a plurality of preparation bags by introducing a quantitative tube, and the preparation bags are squeezed to be emptied.

[0049] In one embodiment, during the process of packaging the target cell fluid into each of the preparation bags, the centrifugal container can be rotated left and right to mix the target cell fluid so that it will not precipitate, thereby ensuring the activity of the T cells, and then packaged into each of the preparation bags. The packaging process is to package the liquid in the centrifugal container into a corresponding volume in each preparation bag according to the preparation requirements of the preparation bag.

[0050] Among them, all pipeline paths in the present invention can be equipped with sensors to monitor the pipelines according to needs, for example, a bubble sensor is used to monitor the flow direction of the liquid in the pipeline and the flow rate in the pipeline, a pressure sensor is used to monitor the pressure in the pipeline, a flow sensor is used to monitor the transmission flow rate, a photoelectric sensor is used to detect the capacity of the liquid in the centrifugal container, etc. All pipeline paths in the present invention can be equipped with switch valves to open and close the pipelines according to needs, for example, a solenoid valve can be used to open and close the input / output of each liquid bag.

[0051] In one embodiment, the target cell fluid in the centrifugal container is quantitatively prepared by a quantitative tube to pack the quantitatively prepared target cell fluid into each preparation bag. The quantitative preparation process is to respectively provide a bubble sensor / liquid sensor on the inlet pipe and the outlet pipe of the quantitative tube, and monitor the time when the liquid enters and flows out of the quantitative tube by the bubble sensor / liquid sensor, which is defined as the initial time and the final time. According to the fixed capacity of the quantitative tube and the time difference between the two times, the constant flow rate of the liquid flowing through the peristaltic pump with a constant speed during this period can be determined. The liquid can be accurately packed into each preparation bag by this constant flow rate, because the capacity of the preparation bag divided by the constant flow rate is the working time for packing each preparation bag. The time of entering the preparation bag is sensed by the sensor provided on each preparation bag, so as to improve the accuracy of the packing amount of each preparation bag. Before the bubble sensor / liquid sensor detects the moment when the liquid enters and flows out of the quantitative tube, the liquid entering the quantitative tube can be debubbled. Initially, the inlet of the quantitative tube can be located below the outlet of the quantitative tube, and the bubbles in the quantitative tube can be emptied by the principle of rising bubbles. After the quantitative tube is filled, the quantitative tube can be set so that the inlet of the quantitative tube is higher than the outlet of the quantitative tube to keep the quantitative tube filled with liquid.

[0052] The present invention achieves enrichment of cell fluid by centrifuging the cell sample in a centrifugal container, and realizing antigen binding and magnetic separation after centrifugation in the centrifugal container, and activating the enriched cells in the centrifugal container by a regulator, and then genetically modifying the activated cell fluid to obtain the target cell fluid, and finally preparing the target cell fluid from the centrifugal container into each preparation bag, realizing a fully closed and full-flow cell sample processing process integrating cell sorting, cell magnetic sorting, cell activation, cell genetic modification, and cell preparation, ensuring the requirements of working in a sterile environment, and being able to avoid external contamination of the cell sample during the processing process. And then the automation and standardization of the target cell fluid preparation process are realized, reducing the site cost and labor cost, and realizing the efficient, standardized, and large-scale production and preparation of the target cell fluid.

[0053] In one embodiment, if Figure 2 As shown, in the step S20, centrifuging the cell sample in the centrifugal container to obtain an initial cell fluid includes:

[0054] S201, controlling the centrifugal container to rotate at a preset speed so that the cell sample in the centrifugal container can be centrifuged and layered; the cell sample includes at least one of T cells, T cell subsets and T cell progenitor cells.

[0055] S202, separating the initial cell fluid from the cell sample after centrifugation and stratification.

[0056] It is understandable that the preset rotation speed is the rotation speed of the centrifuge container set in advance. The initial cell fluid is PBMC fluid or T cell fluid. The cell sample includes at least one of T cells, T cell subsets and T cell progenitor cells.

[0057] Specifically, after the cell sample flows into the centrifugal container, the centrifugal container is controlled to rotate at a preset speed. Due to the different densities and sizes of each cell, different cells are stratified into different cell layers by centrifugation, and then the cell layer containing the PBMC fluid or the T cell fluid is separated, that is, the initial cell fluid is separated from the cell sample after centrifugal stratification. Among them, after the cell sample completes stable stratification, the centrifugal container is controlled to stop rotating, thereby avoiding cell damage to the cells in the cell sample during an excessively long centrifugation process. That is, in this embodiment, the separation of the initial cell fluid is achieved by centrifugal stratification at a preset speed, thereby improving the efficiency of separating the initial cell fluid.

[0058] In another embodiment, the step S20, that is, centrifuging the cell sample in the centrifugal container to obtain an initial cell fluid, includes:

[0059] S203, labeling the cells in the cell sample to obtain labeled cells and unlabeled cells.

[0060] S204, performing centrifugation to remove labels from all the labeled cells and all the unlabeled cells to obtain the initial cell fluid.

[0061] It is understood that non-marked cells refer to T cells, T cell subsets and / or T cell progenitor cells, and marked cells refer to other cells such as B cells.

[0062] Specifically, after the cell sample flows into the centrifugal container, the cells in the cell sample are labeled, that is, a preset labeling liquid is added to the centrifugal container, and the centrifugal container is controlled to rotate so that different labeled antigens in the preset labeling liquid label different cells in the cell sample, for example, B cells can be labeled by CD19, and inhibitory cells can be marked by CD25 and CD14 respectively for removal, so that all cells in the cell sample are divided into labeled cells and unlabeled cells. Further, all labeled cells and all unlabeled cells are centrifuged for labeling removal, that is, the centrifugal container is controlled to rotate and centrifuge so that the labeled cells and all unlabeled cells are layered, that is, because the mass of the labeled cells is heavier than that of the unlabeled cells, the two types of cells are separated by centrifugation. Then, the unlabeled cells in the specific cell layer are separated, and a small amount of labeled cells are adsorbed in the pipeline by a magnet to obtain the initial cell fluid. Among them, it is also possible to distinguish by the labeling of T cells, and compared with the labeling of other cells, labeled T cells are easier to distinguish. It is only necessary to centrifuge the labeled T cells and adsorb the labeled T cells to obtain the initial cell fluid. By labeling the cells in the cell sample, T cells and non-T cells in the cell sample can be distinguished. By centrifuging the labeled cell sample, T cell fluid can be separated and the initial cell fluid can be obtained.

[0063] In one embodiment, the step S30, that is, performing antigen binding and magnetic separation on the initial cell fluid to separate the enriched cell fluid, includes:

[0064] S301, adding magnetic beads and antigens to a centrifugal container containing the initial cell fluid, so that the magnetic beads and antigens are combined with the initial cells in the initial cell fluid to obtain a mixed cell fluid.

[0065] It can be understood that the mixed cell fluid refers to the cell fluid after the magnetic beads and antigens are coupled to the initial cells.

[0066] Specifically, after the initial cell fluid is separated, magnetic beads and antigens are added to the centrifugal container containing the initial cell fluid, and the antigens are directly or indirectly coupled to the surface of the magnetic beads to form a magnetic bead complex with affinity, so that the magnetic bead complex can bind to the T cells in the initial cell fluid, that is, the centrifugal container is rotated to evenly mix the initial cell fluid and the magnetic bead complex, so that the magnetic bead complex can bind to the T cells, that is, the antigen is directly or indirectly coupled to the magnetic bead complex, specifically recognized by the T cell surface antibody, and combined with the T cell surface antibody, so that the magnetic bead complex is directly connected to the cell to form a magnetic marker, thereby obtaining a mixed cell fluid.

[0067] In one embodiment, micron-sized magnetic beads and antigens are added to a centrifugal container containing the initial cell fluid, and the antigens are directly or indirectly coupled to the surface of the micron-sized magnetic beads to form a micron-sized magnetic bead complex with affinity, so that the micron-sized magnetic beads are combined with the non-T cells in the initial cell fluid, that is, the centrifugal container is rotated to uniformly mix the initial cell fluid with the micron-sized magnetic bead complex, and the micron-sized magnetic bead complex and the non-T cells in the initial cell fluid are magnetically labeled, that is, the antigen is directly or indirectly coupled to the micron-sized magnetic bead complex, and is combined with the non-T cell surface antibody to form a magnetic label for the non-T cells, thereby obtaining a micron-sized mixed cell fluid. Among them, micron-sized magnetic beads and antigens can also be combined with T cells to achieve the acquisition of micron-sized mixed cell fluid, and the magnetic beads need to be removed later.

[0068] In another embodiment, after separating the initial cell fluid, nano-scale magnetic beads and antigens are added to the centrifugal container containing the initial cell fluid, and the antigen is directly or indirectly coupled to the surface of the nano-scale magnetic beads to form a nano-scale magnetic bead complex with affinity, so that the nano-scale magnetic beads are combined with the T cells in the initial cell fluid, that is, the centrifugal container is rotated to make the initial cell fluid and the nano-scale magnetic bead complex uniformly mixed, so that the nano-scale magnetic bead complex is combined with the T cells, that is, the antigen is directly or indirectly coupled to the nano-scale magnetic bead complex, and the specific recognition is carried out with the T cell surface antibody, and the nano-scale magnetic beads are combined with the T cell surface antibody, so that the nano-scale magnetic beads are directly connected to the T cells to form a magnetic label, thereby obtaining a nano-mixed cell fluid. Wherein, when the nano-magnetic beads and antigens are used for magnetic labeling, there is no need to separate the nano-magnetic beads subsequently. Understandably, nano-magnetic beads can also be used to carry out magnetic labeling with non-T cells to obtain a nano-mixed cell fluid.

[0069] S302, performing magnetic bead sorting on the mixed cell fluid to sort out the enriched cell fluid.

[0070] It can be understood that the enriched cell fluid refers to a cell fluid enriched with T cells.

[0071] Specifically, the mixed cell liquid is input into the liquid bag on the variable magnetic device by a peristaltic pump, and then, the T cells bound to the magnetic beads and antigens in the liquid of the liquid bag are adsorbed by the variable magnetic device by applying an external magnetic field, and the variable magnetic device is controlled to swing by setting appropriate swing parameters, so that the T cells bound to the magnetic beads and antigens adsorbed by the variable magnetic device are evenly distributed, and the adsorbed T cells bound to the magnetic beads and antigens are prevented from accumulating in large quantities. At the same time, other substances in the liquid bag except the adsorbed T cells bound to the magnetic beads and antigens are suspended in the liquid. Then, the waste liquid discharge operation is performed, that is, by opening the waste liquid discharge pipeline connected to the liquid bag, so that the liquid except the adsorbed T cells bound to the magnetic beads and antigens in the liquid bag flows into the waste liquid bag through the waste liquid discharge pipeline. Furthermore, the cleaning pipeline connected to the liquid bag is opened to perform a cleaning operation on the T cells bound to the magnetic beads and antigens in the liquid bag. Finally, a resuspension liquid or a buffer is injected into the liquid bag so that the T cells bound to the magnetic beads and antigens remain active, and the magnetic field is canceled or removed, thereby sorting out the enriched cell liquid.

[0072] In another embodiment, after applying an external magnetic field by a variable magnetic device to adsorb non-T cells bound to magnetic beads in the liquid of the liquid bag, reverse transmission is performed by a peristaltic pump, that is, the liquid in the liquid bag except the adsorbed non-T cells bound to magnetic beads and antigens is input into a centrifugal container by a peristaltic pump. Then, the magnetic field is canceled or removed, the waste liquid discharge pipeline connected to the waste liquid bag is opened, and the non-T cells bound to magnetic beads and antigens are flowed into the waste liquid bag through the waste liquid discharge pipeline. In this embodiment, since the non-T cells bound to magnetic beads and antigens are removed, the cell fluid input into the centrifugal container by the peristaltic pump is the enriched cell fluid. That is, in this embodiment, the T cell fluid is efficiently and stably separated and collected by magnetic bead sorting, and the T cell fluid is efficiently and stably captured.

[0073] In one embodiment, in step S40, that is, activating the enriched cell fluid by inputting a regulator to obtain the activated cell fluid, comprises:

[0074] S401, injecting the regulator into the centrifugal container containing the enriched cell fluid.

[0075] S402, activating the enriched cells in the enriched cell fluid by the co-stimulator in the regulator to obtain the activated cell fluid; the regulator is one or more of agonist antibodies, cytokines, recombinant co-stimulatory molecules and small drug inhibitors.

[0076] It is understood that the modulator is one or more of an agonistic antibody, a cytokine, a recombinant co-stimulatory molecule, and a small drug inhibitor.

[0077] Specifically, after obtaining the enriched cell fluid, the regulator can be injected into the enriched cell fluid of the centrifugal container through an inlet and outlet in the centrifugal container by a peristaltic pump to activate the cells in the enriched cell fluid, that is, the enriched cells in the enriched cell fluid are activated by the co-stimulator in the regulator, that is, the antigen binds to the antigen receptor on the surface of the T cell to generate a specific signal, that is, the first activation signal. For example, TCR / CD3 binds to the specific MHC (major histocompatibility complex, major tissue compatibility complex) molecule antigen peptide complex on the surface of the antigen presenting cell (APC) to generate a specific antigen stimulation signal. Then, the co-stimulator in the regulator interacts with the receptor on the surface of the T cell to produce a non-specific co-stimulatory signal, which is used to activate the cells in the enriched cell fluid to obtain the activated cell fluid.

[0078] In one embodiment, the step S402, that is, activating the enriched cells in the enriched cell fluid by the co-stimulator in the regulator to obtain the activated cell fluid, includes:

[0079] S4021. Using the co-stimulator in the regulator, the enriched cells in the enriched cell fluid produce a co-stimulatory effect to obtain active cell fluid.

[0080] S4022, sampling and testing the active cell fluid to obtain a test result.

[0081] S4023. When the detection result indicates that the enriched cells have been successfully activated, the active cell fluid is determined as the activated cell fluid.

[0082] It can be understood that active cell fluid refers to T cells that contain co-stimulatory effects. The detection result is used to characterize whether the enriched cells are successfully activated.

[0083] Specifically, after obtaining the enriched cell fluid, the co-stimulator in the regulator causes the enriched cells in the enriched cell fluid to produce a co-stimulatory effect, that is, the co-stimulator binds to the specific cell receptor on the surface of the enriched cell to produce a specific antigen stimulation signal, and the non-specific antigen stimulation signal is generated by the interaction of multiple pairs of co-stimulatory molecules on the surface of the enriched cell and the corresponding receptors of the T cells, wherein CD28 is the most important co-stimulatory molecule, and the non-specific antigen stimulation signal can fully activate the T cells, secrete cytokines and express cytokine receptors, thereby obtaining active cell fluid. Further, the active cell fluid is sampled and tested, that is, a small amount of liquid (for example, 3ml, etc.) is discharged from the discharge port of the centrifugal container into the sampling tube, that is, the cells in the sampling tube are immunologically tested, and the expression level of activation-related molecules on the cell surface in the sampling tube, such as CD69, CD25, etc., is detected to determine whether the cells are in an activated state. It is also possible to determine whether the cells are in an activated state by detecting cytokines secreted by cells in the sampling tube, such as IL-2, IFN-γ, etc., so that the test results can be obtained. When the test results indicate that the enriched cells have been successfully activated, the active cell fluid is determined as the activated cell fluid. That is, the acquisition of the active cell fluid is achieved. By sampling and testing the active cell fluid, the activation of the enriched cells is ensured, which provides a guarantee for the subsequent genetic modification of T cells.

[0084] In one embodiment, the step S401 includes:

[0085] The co-stimulator comprises at least one of an anti-CD3 antibody or fragment and an anti-CD28 antibody or fragment.

[0086] or / and

[0087] The co-stimulator comprises a nanomatrix, the nanomatrix comprising at least one of a matrix of mobile polymer chains and a matrix connected to the mobile polymer chains. Preferably, the co-stimulator is a biodegradable nanomatrix, and the nanomatrix comprises at least one of a matrix of mobile polymer chains and a matrix connected to the mobile polymer chains. Wherein the size of the nanomatrix is ​​1nm to 500nm. Wherein the same or different matrices of mobile polymer chains are connected to anti-CD3 and anti-CD28 antibodies or fragments. If the anti-CD3 and anti-CD28 antibodies or fragments are connected to different mobile polymer chain matrices, the nanomatrix is ​​fine-tuned to stimulate T cells.

[0088] In one embodiment, the step S50, i.e., genetically modifying the activated cell fluid to obtain the target cell fluid, includes:

[0089] S501, performing transduction treatment on the activated cell fluid to obtain the target cell fluid.

[0090] Specifically, retroviral vectors and lentiviral vectors are commonly used for viral transduction. Retrovirus integrates viral DNA (deoxyribonucleic acid, DeoxyriboNucleic Acid) into the cell DNA in the activated cell fluid to produce stable transfection. Lentiviral vector effectively integrates exogenous genes or exogenous RNA (ribonucleic acid, Ribonucleic Acid) into the chromosomes of cells in the activated cell fluid. Among them, the viral vector is added to the activated cell fluid in the centrifugal container through a pipeline so that the viral vector infects the cells in the activated cell fluid, that is, the viral surface envelope protein binds to the cell receptor and enters the cell. The RNA of the viral vector is reversely transcribed into double-stranded DNA in the cell. Then, the double-stranded DNA forms an integration complex with the integrase, and the integration complex enters the cell nucleus through the nuclear pore, and then the integration complex binds to the specific site of the T cell DNA to form a new complex. The integrase rearranges and connects the DNA sequence of the virus with the DNA sequence of the T cell, and the integrase is released therefrom to embed the viral genome into the DNA of the T cell, thereby obtaining the target cell fluid.

[0091] or

[0092] S502, performing transfection treatment on the activated cell fluid to obtain the target cell fluid.

[0093] It can be understood that cell transfection refers to the introduction of exogenous DNA or RNA and other substances into cells, such as transposons and transfection reagents.

[0094] Specifically, after obtaining the activated cell fluid, the activated cell fluid is transfected, that is, the exogenous gene is inserted into the chromosome of the cell, that is, the transfection reagent can be added to the activated cell fluid in the centrifugal container through a pipeline, and the exogenous DNA or RNA is introduced into the nucleus of the cell using the transfection reagent, for example, DNA and calcium chloride are mixed in phosphate buffered saline to form a calcium phosphate-DNA precipitate, and then it is dispersed on the cell by centrifugal rotation, and calcium phosphate promotes the binding of the condensed DNA in the coprecipitate to the cell surface, and the DNA enters the cell through endocytosis. Alternatively, cationic liposome-mediated transfection, that is, the positively charged head group and the negatively charged nucleic acid form a complex through electrostatic action, and enter the cell through the endocytosis of the cell, so as to achieve the subsequent genetic modification of the cells in the activated cell fluid, thereby obtaining the target cell fluid.

[0095] or

[0096] S503, performing an electroporation operation on the activated cell fluid to obtain the target cell fluid.

[0097] It can be understood that electroporation is a cell transfection method that generates transmembrane voltage on the cell membrane by applying an external pulsed electric field, thereby forming recoverable micropores on the cell membrane surface and introducing exogenous substances into the cell.

[0098] Specifically, after obtaining the activated cell fluid, the transfection substance is added to the activated cell fluid in the centrifugal container through the pipeline, and the activated cell fluid with the transfection substance is transferred to the electroporation cell through a peristaltic pump, and an electric field is applied to the electroporation cell to produce transient holes in the cell membrane. Alternatively, the electroporation cell can be a microfluidic chip, which is equivalent to separating various flow channels in the electroporation cell so that the cell membrane can be hit with holes by the electric field during each flow channel, so that the transfection substance carries exogenous DNA or RNA and combines with the cells in the activated cell fluid. Then, the electroporated cells are washed and transferred to a suitable liquid bag through a peristaltic pump to achieve genetic modification of the cells in the activated cell fluid, thereby obtaining the target cell fluid.

[0099] or

[0100] S504, performing mechanical transfection treatment on the activated cell fluid to obtain the target cell fluid.

[0101] Specifically, after obtaining the activated cell fluid, the activated cell fluid is subjected to mechanical transfection treatment, that is, the activated cell fluid is transferred to the extrusion device through a peristaltic pump, and the transfection material is transferred at the same time. The extrusion device mechanically squeezes the cells to squeeze the transfection material into the nuclei of the cells in the activated cell fluid, promotes the formation of the transduction complex, and achieves genetic modification of the cells in the activated cell fluid, thereby obtaining the target cell fluid.

[0102] In one embodiment, the step S501, i.e., performing transduction treatment on the activated cell fluid to obtain the target cell fluid, includes:

[0103] The activated cell fluid is transduced by a transduction enhancer to obtain the target cell fluid; the transduction enhancer includes one or more of a polycationic agent, poloxamer, poloxamer, and an adhesion molecule.

[0104] It is understood that the transduction enhancer includes one or more of a polycationic agent, a poloxamer, and an adhesion molecule. A polycationic agent is a molecule with a positive charge, for example, agents such as polybrene, protamine sulfate, poly-L-lysine, and peptides with a net positive charge. Adhesion molecules include fibronectin or modified fibronectin, or protein targeting domains, such as antibodies, antibody complexes, and the like.

[0105] Specifically, after obtaining the activated cell fluid, the activated cell fluid is transduced by a transduction enhancer, that is, the transduction enhancer contained in the liquid bag is input into a centrifugal container through a pipeline, and the centrifugal container is rotated so that the transduction enhancer is fully in contact with the transduction substance and the cell, thereby using the transduction enhancer to enhance the interaction between the transduction substance and the cells in the activated cell fluid, promoting the transduction substance to bind to the cell, and realizing that the transduction substance brings the genetic material into the activated cell, realizing genetic modification of the cells in the activated cell fluid, thereby obtaining the target cell fluid. Among them, the polycationic agent promotes the formation of cell transduction complexes by interacting with the negative charge on the cell membrane. Poloxamer can be used to prepare liposomes and other nanoparticles, and liposomes and nanoparticles can be used to transduce various molecules into cells. Poloxamer can also promote the formation of cell transduction complexes by interacting with the cell membrane. Adhesion molecules can mediate the interaction between the transduction complex and the cell membrane and promote the transduction complex to enter the cell. That is, in this embodiment, by using a transduction enhancer, the transduction efficiency of the activated cell fluid is improved, so that the transduction substance can enter the cell more easily, and the time of cell transduction is reduced.

[0106] It should be understood that the order of execution of the steps in the above embodiment does not necessarily mean the order of execution. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiment of the present invention.

[0107] In one embodiment, a cell modification treatment device is provided, which corresponds one-to-one to the cell modification treatment method in the above embodiment. Figure 3 As shown, the cell modification processing device includes a sample inflow module 10, a cell separation module 20, a cell enrichment module 30, a cell activation module 40, a genetic modification module 50 and a packaging preparation module 60. The functional modules are described in detail as follows:

[0108] The sample inflow module 10 is used to flow the cell sample into the centrifugal container;

[0109] The cell separation module 20 is used to centrifuge the cell sample in the centrifugal container to obtain an initial cell fluid;

[0110] A cell enrichment module 30 is used to perform antigen binding and magnetic separation on the initial cell fluid to separate the enriched cell fluid;

[0111] A cell activation module 40 is used to activate the enriched cell fluid by inputting a regulator to obtain an activated cell fluid;

[0112] A genetic modification module 50 is used to genetically modify the activated cell fluid to obtain a target cell fluid;

[0113] The preparation packaging module 60 is used to package the target cell solution into a plurality of preparation bags.

[0114] A cell sample processing method system comprises a centrifugal container and a controller connected to the centrifugal container, wherein the controller is used to execute the cell sample processing method.

[0115] For the specific definition of the cell sample processing method system, controller and its various units and modules, please refer to the definition of the cell sample processing method above, which will not be repeated here. Each module in the above controller can be implemented in whole or in part by software, hardware and their combination. It is understandable that Figure 4 As shown, the controller includes a processor, a memory, a network interface and a database connected via a system bus. Each module of the controller can be embedded in or independent of the processor in hardware form, or can be stored in the memory in software form, so that the processor can call and execute the operations corresponding to the above modules. Among them, the processor is used to provide computing and control capabilities. The memory includes a non-volatile storage medium and an internal memory. The non-volatile storage medium stores an operating system, a computer program and a database. The internal memory provides an environment for the operation of the operating system and the computer program in the non-volatile storage medium. The database is used to store the data used in the cell sample processing method in the above embodiment. The network interface is used to communicate with an external terminal through a network connection. When the computer program is executed by the processor, a cell sample processing method is implemented.

[0116] In one embodiment, a computer-readable storage medium is provided, wherein the computer-readable storage medium stores a computer program, and when the computer program is executed by a processor, the above-mentioned cell sample processing method is implemented.

[0117] Those skilled in the art can understand that all or part of the processes in the above-mentioned embodiment methods can be completed by instructing the relevant hardware through a computer program, and the computer program can be stored in a non-volatile computer-readable storage medium. When the computer program is executed, it can include the processes of the embodiments of the above-mentioned methods. Among them, any reference to memory, storage, database or other media used in the embodiments provided in this 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 and not limitation, RAM is available in many forms, such as static RAM (SRAM), dynamic RAM (DRAM), synchronous DRAM (SDRAM), double data rate SDRAM (DDRSDRAM), 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).

[0118] Those skilled in the art can clearly understand that for the convenience and simplicity of description, only the division of the above-mentioned functional units and modules is used as an example. In actual applications, the above-mentioned functions can be distributed and completed by different functional units and modules as needed, that is, the internal structure of the device can be divided into different functional units or modules to complete all or part of the functions described above.

[0119] The embodiments described above are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, a person skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features may be replaced by equivalents. Such modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention, and should all be included in the protection scope of the present invention.

Claims

1. A cell sample processing method, characterized in that: include: The cell sample is flowed into a centrifuge container; Centrifuging the cell sample in the centrifugal container to obtain an initial cell fluid; Performing antigen binding and magnetic separation on the initial cell fluid to separate the enriched cell fluid; Activating the enriched cell fluid by inputting a regulator to obtain an activated cell fluid; genetically modifying the activated cell fluid to obtain target cell fluid; The target cell solution is divided into a plurality of preparation bags.

2. The cell sample processing method according to claim 1, characterized in that: The step of centrifuging the cell sample in the centrifugal container to obtain an initial cell solution comprises: Controlling the centrifugal container to rotate at a preset speed so that the cell sample in the centrifugal container can be centrifuged and layered; the cell sample includes at least one of T cells, T cell subpopulations and T cell progenitor cells; The initial cell fluid is separated from the cell sample after centrifugation and stratification.

3. The cell sample processing method according to claim 1, characterized in that: The method of subjecting the initial cell fluid to antigen binding and magnetic separation to separate the enriched cell fluid comprises: Adding magnetic beads and antigens to a centrifugal container containing the initial cell fluid, so that the magnetic beads and antigens are combined with the initial cells in the initial cell fluid to obtain a mixed cell fluid; The mixed cell fluid is subjected to magnetic bead sorting to sort out the enriched cell fluid.

4. The cell sample processing method according to claim 1, characterized in that: The step of activating the enriched cell fluid by inputting a regulator to obtain the activated cell fluid comprises: injecting the regulator into a centrifuge container containing the enriched cell fluid; The enriched cells in the enriched cell fluid are activated by the co-stimulator in the regulator to obtain the activated cell fluid; the regulator is one or more of agonistic antibodies, cytokines, recombinant co-stimulatory molecules and small drug inhibitors.

5. The cell sample processing method according to claim 4, characterized in that: The step of activating the enriched cells in the enriched cell fluid by the co-stimulator in the regulator to obtain the activated cell fluid comprises: The co-stimulator in the regulator causes the enriched cells in the enriched cell fluid to produce a co-stimulatory effect, thereby obtaining active cell fluid; Sampling and testing the active cell fluid to obtain a test result; When the detection result indicates that the enriched cells have been successfully activated, the active cell fluid is determined as the activated cell fluid.

6. The cell sample processing method according to claim 5, characterized in that: The co-stimulator comprises at least one of an anti-CD3 antibody or fragment and an anti-CD28 antibody or fragment; or / and The co-stimulator includes a nanomatrix including at least one of a matrix of mobile polymer chains and a matrix attached to the mobile polymer chains.

7. The cell sample processing method according to claim 1, characterized in that: The step of genetically modifying the activated cell fluid to obtain the target cell fluid comprises: Performing transduction treatment on the activated cell fluid to obtain the target cell fluid; or performing transfection treatment on the activated cell fluid to obtain the target cell fluid; or Performing electroporation on the activated cell fluid to obtain the target cell fluid; or The activated cell fluid is subjected to mechanical transfection treatment to obtain the target cell fluid.

8. The cell sample processing method according to claim 7, characterized in that: The step of performing transduction treatment on the activated cell fluid to obtain the target cell fluid comprises: The activated cell fluid is transduced by a transduction enhancer to obtain the target cell fluid; the transduction enhancer includes one or more of a polycationic agent, poloxamer, poloxamer, and an adhesion molecule.

9. A cell sample processing device, characterized in that: include: A sample inflow module, used for flowing the cell sample into a centrifugal container; A cell separation module, used for centrifuging the cell sample in the centrifugal container to obtain an initial cell fluid; A cell enrichment module, used for performing antigen binding and magnetic separation on the initial cell fluid to separate the enriched cell fluid; A cell activation module, used to activate the enriched cell fluid by inputting a regulator to obtain an activated cell fluid; A genetic modification module, used for genetically modifying the activated cell fluid to obtain a target cell fluid; The preparation packaging module is used to package the target cell solution into multiple preparation bags.

10. A cell sample processing method system, characterized in that: It comprises a centrifugal container and a controller connected to the centrifugal container, and the controller is used to execute the cell sample processing method according to any one of claims 1 to 8.

11. A computer-readable storage medium storing a computer program, characterized in that: When the computer program is executed by a processor, the cell sample processing method according to any one of claims 1 to 8 is implemented.

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