Integrated cell treatment apparatus, method, and storage medium

Abstract

The invention discloses integrated cell treatment equipment, an integrated cell treatment method and a storage medium. The integrated cell treatment equipment comprises a controller as well as a separation module, a sorting module, a liquid changing module, a culture module and a subpackaging module which are in one-to-one correspondence and are sequentially connected, the controller is used for controlling the separation module to perform centrifugal treatment on the sample liquid and inputting the prepared first cell liquid into the sorting module; controlling a sorting module to perform magnetic sorting on the first cell sap, and inputting a second cell sap containing immunomagnetic cells obtained by sorting into a liquid changing module; controlling a liquid changing module to carry out cell transfection and centrifugal replacement on the second cell liquid to obtain a third cell liquid, and inputting the third cell liquid into a culture module; controlling a culture module to culture the third cell sap to obtain a target cell sap; and controlling a sub-packaging module to sub-package the target cell sap into a sub-packaging container. According to the invention, the labor cost is reduced, the treatment time is shortened, and the cell treatment efficiency and accuracy are improved.

Description

Technical Field

[0001] The present invention relates to the technical field of biological treatment, and particularly relates to an integrated cell processing device, method, and storage medium. Background Art

[0002] Currently, cell therapy is a key area of focus in the international medical forefront. Cell sorting, cell activation, cell transfection, cell culture, cell aliquoting, etc. are key steps in cell therapy. In the above operation processes of the prior art, all require manual operation on devices distributed in different areas of a sterile environment, and each step requires professionals to perform high - requirement operations in a clean room (a dedicated infrastructure with a clean room and all necessary instruments must be in place, qualified, and functioning properly to ensure sterility and aseptic sealing). The operation threshold is high. Moreover, due to the high - skill and time requirements for operators in the clean room for manufacturing these complex products (non - compliant operations will lead to errors or failures in cell processing results), the failure rate may be very high. Thus, the site cost and labor cost consumed are extremely high, and the above - mentioned cell processing process in the prior art is extremely inefficient. Therefore, how to reduce labor costs and improve cell processing efficiency is an urgent problem for those skilled in the art. Summary of the Invention

[0003] Embodiments of the present invention provide an integrated cell processing device, method, and storage medium to solve the problems of high labor costs and low cell processing efficiency in the prior art.

[0004] An integrated cell processing method includes a controller, and a separation module, a sorting module, a liquid - changing module, a culture module, and an aliquoting module that are in one - to - one correspondence and connected in sequence; the separation module, the sorting module, the liquid - changing module, the culture module, and the aliquoting module are all communicatively connected to the controller;

[0005] The controller is configured to:

[0006] Control the separation module to perform centrifugation on a sample liquid, and input the prepared first cell liquid into the sorting module;

[0007] Control the sorting module to perform magnetic sorting on the first cell liquid, and input the sorted second cell liquid containing immunomagnetic cells into the liquid - changing module;

[0008] Control the liquid - changing module to perform cell transfection and centrifugal replacement on the second cell liquid, obtain a third cell liquid, and input it into the culture module;

[0009] Control the culture module to culture the third cell liquid to obtain a target cell liquid;

[0010] Control the dispensing module to dispense the target cell solution into a dispensing container.

[0011] An integrated cell processing method, characterized in that the integrated cell processing method is executed by the above-mentioned integrated cell processing device, and the integrated cell processing method includes:

[0012] Control the separation module to perform centrifugation on the sample solution, and input the prepared first cell solution into the sorting module;

[0013] Control the sorting module to perform magnetic sorting on the first cell solution, and input the sorted second cell solution containing immunomagnetic cells into the liquid changing module;

[0014] Control the liquid changing module to perform cell transfection and centrifugal replacement on the second cell solution, obtain a third cell solution and input it into the culture module;

[0015] Control the culture module to culture the third cell solution to obtain a target cell solution;

[0016] Control the dispensing module to dispense the target cell solution into a dispensing container.

[0017] A computer-readable storage medium stores a computer program, and when the computer program is executed by a processor, the above-mentioned integrated cell processing method is implemented.

[0018] In the above-mentioned integrated cell processing device, method and storage medium, the integrated cell processing device includes a separation module, a sorting module, a liquid changing module, a culture module and a dispensing module that are in one-to-one correspondence and connected in sequence; and the above-mentioned modules are all communicatively connected to the controller, and thus the following steps can be sequentially executed through the controller: control the separation module to perform centrifugation on the sample solution, and input the prepared first cell solution into the sorting module; control the sorting module to perform magnetic sorting on the first cell solution, and input the sorted second cell solution containing immunomagnetic cells into the liquid changing module; control the liquid changing module to perform cell transfection and centrifugal replacement on the second cell solution, obtain a third cell solution and input it into the culture module; control the culture module to culture the third cell solution to obtain a target cell solution; control the dispensing module to dispense the target cell solution into a dispensing container.

[0019] In the present invention, the above-mentioned modules and the controller form an integrated cell processing device, and each module in the integrated cell processing device sequentially executes a plurality of steps. The cell processing procedures (centrifugation, magnetic sorting, cell transfection, centrifugal displacement, culturing, and aliquoting) are orderly connected in one-to-one correspondence. The entire cell processing process does not require manual participation and only needs to be automatically carried out by the integrated cell processing device, which is easy to ensure its aseptic and cleanliness requirements, thereby reducing labor costs, shortening the processing time, and improving the cell processing efficiency. At the same time, the one-to-one automatic processing throughout the integrated cell processing device is not prone to failures or mistakes, so that the accuracy of the cell processing process can be improved, and the finally obtained cell solution meets the standards. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for the description of the embodiments of the present invention will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0021] Figure 1 is a schematic block diagram of an integrated cell processing device in an embodiment of the present invention;

[0022] Figure 2 is a flowchart of an integrated cell processing method in an embodiment of the present invention;

[0023] Figure 3 is a schematic diagram of the controller of the integrated cell processing device in an embodiment of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0024] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts fall within the scope of protection of the present invention.

[0025] In one embodiment, as Figure 1 shown, an integrated cell processing device is provided, including a controller 10 and a separation module 20, a sorting module 30, a liquid replacement module 40, a culture module 50, and an aliquoting module 60 that are connected in sequence and in one-to-one correspondence; the separation module 20, the sorting module 30, the liquid replacement module 40, the culture module 50, and the aliquoting module 60 are all communicatively connected to the controller 10; wherein, each module in the above-mentioned controller 10 can be implemented in whole or in part by software, hardware, and their combinations. Understandably, as Figure 3As shown, the controller 10 includes a processor, a memory, a network interface, and a database connected via a system bus. Each module of the controller 10 can be embedded in or independent of the processor in hardware form, or stored in the memory in software form to facilitate the processor to call and execute the operations corresponding to each of 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 integrated cell processing method in the above embodiments. The network interface is used to communicate with an external terminal via a network connection. When the computer program is executed by the processor, it executes an integrated cell processing method corresponding to the integrated cell processing device.

[0026] In the present invention, the above-mentioned separation module 20, sorting module 30, liquid change module 40, culture module 50, dispensing module 60, and controller 10 can be integrated in one housing, or one or several of the modules can be integrated in one housing according to requirements, while other modules are integrated in other housings.

[0027] Specifically, as Figure 1 and Figure 2 shown, the controller 10 is used to execute the following integrated cell processing method:

[0028] S100, control the separation module 20 to perform centrifugation on the sample liquid, and input the prepared first cell liquid into the sorting module 30; it can be understood that the sample liquid can refer to a peripheral blood sample, and the first cell liquid can refer to PBMC (Peripheral Blood Mononuclear Cell) liquid or T cell liquid, etc.; among them, PBMC refers to the cells with a single nucleus in peripheral blood, and most of the PBMC are lymphocytes, including B cells and T cells. The separation module 20 refers to a device used to centrifuge the sample liquid to separate certain specific cells. Specifically, the above-mentioned separation module 20 can be connected to the sorting module 30 through the first connection module, and then transfer the first cell liquid obtained by centrifugation of the separation module 20 to the sorting module 30 through the first connection module.

[0029] Furthermore, the separation module 20 includes a first centrifugation device; controlling the separation module 20 to perform centrifugation on the sample liquid and input the prepared first cell liquid into the sorting module 30 includes:

[0030] Obtain the centrifugation parameters, and control the first centrifugation device to rotate at a preset speed according to the centrifugation parameters to perform centrifugation on the sample liquid to obtain an intermediate product cell liquid. Specifically, perform density gradient centrifugation on the sample liquid in the first centrifugation device at the preset speed. Herein, the preset speed can be 2000 rpm to 2500 rpm (Revolutions Per Minute, revolutions per minute); and during the above centrifugation process, first add the sample liquid and an appropriate separation liquid, such as a polysaccharide of sucrose (for example: Ficoll solution), to the first centrifugation device, and then use the density gradient centrifugation method to centrifuge and layer the sample liquid. In this way, the sample liquid will be layered according to density and cell size, separating out a red blood cell layer, a separation liquid layer (such as a separation liquid mainly composed of Ficoll), a PBMC layer, and a supernatant layer (mainly composed of plasma and platelets). After that, extract the intermediate product cell liquid from the liquid outlet near or located in the PBMC layer, and the rotation speed of the first centrifugation device can be continuously adjusted to fully extract the intermediate product cell liquid (such as PBMC liquid or T cell liquid) in the first centrifugation device. Further, after obtaining the above intermediate product cell liquid, the gradient centrifugation method can be continued. Use an intermediate product container to hold the intermediate product cell liquid, and then use the intermediate product container as a transfer to continuously purify the obtained intermediate product cell liquid.

[0031] Add magnetic beads and an antigen to the intermediate product cell liquid so that after the target cells in the intermediate product cell liquid bind to the magnetic beads and the antigen, a first cell liquid containing immunomagnetic beads cells is obtained, and the first cell liquid is input into the sorting module 30. That is to say, in this step, the antigen and magnetic beads (wherein, the magnetic beads can be micron-sized magnetic beads or nano-sized magnetic beads) are transported to the intermediate product container so that the target cells (such as T cells) in the purified intermediate product cell liquid bind to the magnetic beads and the antigen to obtain immunomagnetic beads cells (that is, the target cells in the first cell liquid are magnetically labeled through magnetism to generate immunomagnetic cells, also known as magnetic bead incubation. The above immunomagnetic beads cells can be positive immunocytes or negative immunocytes). Among them, the intermediate product cell liquid can be mixed to promote magnetic bead incubation. After completing the magnetic bead incubation, the intermediate product cell liquid after magnetic bead incubation (that is, the first cell liquid) is transmitted to the sorting module 30 through the first connection module.

[0032] S200, control the sorting module 30 to perform magnetic sorting on the first cell liquid, and input the sorted second cell liquid containing immunomagnetic cells into the liquid exchange module 40; the immunomagnetic cells refer to the target cells labeled with magnetism. It can be understood that the second cell liquid refers to the cell liquid containing immunomagnetic cells; the immunomagnetic cells have specific targeting properties, and the sorting module 30 can include a magnetic sorting device for screening the immunomagnetic cells in the first cell liquid through magnetism.

[0033] The sorting module 30 includes a magnetic sorting device; controlling the sorting module 30 to perform magnetic sorting on the first cell solution and inputting the sorted second cell solution containing immunomagnetic cells into the solution changing module 40 includes:

[0034] Controlling the magnetic sorting device to magnetically screen out the immunomagnetic cells bound with magnetic beads from the first cell solution and inputting the second cell solution containing the screened immunomagnetic cells into the solution changing module 40. Specifically, the above magnetic sorting device can be connected to the first centrifugal device through the first connection module, and then transfer the first cell solution obtained after centrifugal treatment by the first centrifugal device to the magnetic sorting device through the first connection module. Then, perform magnetic sorting on the immunomagnetic cells in the first cell solution through the magnetic sorting column of the magnetic sorting device, that is, utilize the affinity between the specific antibody or other molecules on the surface of the magnetic beads of the immunomagnetic cells and the cell surface molecules to magnetically adsorb and separate the immunomagnetic cells from the first cell solution. After that, a washing operation can be performed on the immunomagnetic cells (the immunomagnetic cells can be flushed into other containers after the washing operation), and then the immunomagnetic cells are mixed with a resuspension solution or buffer solution to obtain the second cell solution. Specifically, the sorting module 30 can be connected to the solution changing module 40 through the second connection module, and then transfer the second cell solution obtained after magnetic sorting by the sorting module 30 to the solution changing module 40 through the second connection module.

[0035] S300, controlling the solution changing module 40 to perform cell transfection and centrifugal replacement on the second cell solution, obtaining a third cell solution and inputting it into the culture module 50; specifically, the third cell solution refers to the cell solution obtained after the second cell solution performs cell transfection and centrifugal replacement. The solution changing module 40 refers to the module that performs cell transfection and centrifugal replacement on the second cell solution. Centrifugal replacement can refer to the process of replacing the solution (such as culture medium or cryopreservation solution, etc.) in the second cell solution. A culture medium is an artificial nutrient for culturing and propagating microorganisms, cells, and tissues, composed of various nutrients, including carbohydrates, nitrogen sources, minerals, vitamins, etc.

[0036] Further, the liquid changing module 40 includes a transfection unit and a replacement unit; in one embodiment, the transfection unit is configured to centrifuge the second cell solution and the transfection reagent (including transfection substances or viral vectors) in the centrifuge container after the second cell solution and the transfection reagent are respectively input into the centrifuge container, so that the transfection reagent enters the immunomagnetic cells to complete cell transfection. Among them, the above centrifugation process can be high-speed centrifugation (for example, centrifugation at a high speed of 2000 rpm to 2500 rpm). Furthermore, through the high-speed centrifugation method, the contact area and contact probability between the transfection reagent and the cells can be increased, thereby improving the transfection efficiency and effect.

[0037] Specifically, controlling the liquid changing module 40 to perform cell transfection on the second cell solution specifically means: inputting the second cell solution obtained by magnetic sorting into the centrifuge container of the second centrifuge device of the transfection unit through the second connection module, and then adding a transfection reagent (transfection substance or viral vector) to the centrifuge container of the second centrifuge device, so that exogenous DNA or RNA is introduced onto the nuclei of the immunomagnetic beads cells in the second cell solution through the transfection reagent.

[0038] Understandably, in another embodiment, controlling the liquid changing module 40 to perform cell transfection on the second cell solution specifically means: the transfection unit is configured to apply an electric field to the electroporation chamber after the second cell solution and the transfection reagent are respectively input into the electroporation chamber, so that the transfection reagent enters the immunomagnetic cells through the electric field to complete cell transfection; that is, inputting the second cell solution obtained by magnetic sorting into the electroporation chamber of the transfection unit through the second connection module, adding a transfection reagent to the electroporation chamber, and applying an electric field to the electroporation chamber to generate transient pores in the cell membrane, so that the transfection reagent carrying exogenous DNA or RNA binds to the immunomagnetic beads cells in the activated second cell solution, thereby completing cell transfection.

[0039] Further, the replacement unit is configured to replace the initial centrifugation in the second cell solution after cell transfection with a first renewal solution to complete centrifugation replacement and obtain a third cell solution. That is, after controlling the liquid changing module 40 to perform cell transfection on the second cell solution, it is also necessary to control the liquid changing module 40 to perform centrifugation replacement on the second cell solution, which specifically may refer to replacing the initial solution (such as water or physiological saline) in the second cell solution with a first renewal solution (such as a culture medium or a cryopreservation solution) to provide necessary nutrients for the cell culture process.

[0040] In a further embodiment, the replacement unit includes a first connection pipeline, a second connection pipeline, a replacement container for containing the second cell solution after cell transfection, a first container for containing waste liquid, and a second container for containing a first renewal solution; the controller 10 is further configured to: control a preset installation device to connect the first connection pipeline between the replacement container and the first container, and connect the second connection pipeline between the replacement container and the second container; preferably, before starting to execute step S100 (that is, before controlling the separation module 20 to perform centrifugation on the sample liquid), it is necessary to first complete the assembly of the replacement unit of the integrated cell processing device, so that during the subsequent cell processing through this integrated cell processing device, there is no need to assemble the device midway, and only the integrated cell processing device that has been assembled once needs to be started and run.

[0041] The replacement unit is further configured to output the initial solution in the second cell solution after cell transfection from the replacement container to the first container through the first connection pipeline, and input the first renewal solution in the second container into the replacement container through the second connection pipeline, so as to complete centrifugal replacement and obtain a third cell solution. That is, during the execution of step S300, when performing centrifugal replacement, the initial solution (such as water or physiological saline) in the second cell solution in the replacement container is output to the first container through the first connection pipeline (no longer needed), and is replaced with the first renewal solution (such as a culture medium or a cryopreservation solution) in the second container that is input into the replacement container through the second connection pipeline, so as to provide necessary nutrients and the like for the cell culture process corresponding to the immunomagnetic bead cells through the first renewal solution replaced into the replacement container. Among them, the first renewal solution is input into the replacement container under high-speed centrifugation to replace the non-target liquid (initial solution) after layering. In order to culture cells with the first renewal solution subsequently, setting the first renewal solution as a culture medium is one way.

[0042] In an embodiment, the integrated cell processing device includes at least one connection and transmission module; the controller is further configured to: control the connection and transmission module to connect the separation module and the sorting module, or / and connect the sorting module and the liquid replacement module, or / and connect the liquid replacement module and the culture module, or / and connect at least one input port of the culture module and the dispensing module. That is, the connection and transmission module can be one or more, and through the above connection and transmission module, the connection between the input ports of the separation module, the sorting module, the liquid replacement module, the culture module, and the dispensing module can be realized.

[0043] In a specific embodiment, the connection and transfer module includes one or more of a first connection module, a second connection module, a third connection module, and a fourth connection module; the controller 10 is further configured to: control a preset installation device to connect the separation module 20 and the sorting module 30 through the first connection module, connect the sorting module 30 and the liquid exchange module 40 through the second connection module, connect the liquid exchange module 40 and the culture module 50 through the third connection module, and connect the culture module 50 and at least one input port of the sub-packaging module 60 through the fourth connection module. Wherein, the preset installation device can be a robotic arm, a sterile tube connecting machine, a heat sealer, etc., and the first connection module, the second connection module, the third connection module, and the fourth connection module can all be liquid transfer instruments and / or connecting pipelines installed by the preset installation device.

[0044] That is, in the present invention, before starting to execute step S100 (that is, before controlling the separation module 20 to perform centrifugation on the sample liquid), it is necessary to first complete the connection and assembly between all modules of the integrated cell processing device (including the separation module 20, the sorting module 30, the liquid exchange module 40, the culture module 50, and the sub-packaging module 60). In this way, while enabling the entire cell processing process to always work in a sterile environment, it is also possible to avoid mid-course equipment assembly during the subsequent cell processing using this integrated cell processing device. Only the integrated cell processing device that has been assembled once needs to be started and run, and then cell processing can be achieved through this integrated cell processing device.

[0045] Specifically, the above-mentioned separation module 20 can be connected to the sorting module 30 through the first connection module, and then transfer the first cell liquid obtained after centrifugation of the separation module 20 to the sorting module 30 through the first connection module. The above-mentioned sorting module 30 can be connected to the liquid exchange module 40 through the second connection module, and then transfer the second cell liquid obtained after magnetic sorting of the sorting module 30 to the liquid exchange module 40 through the second connection module. The above-mentioned liquid exchange module 40 can be connected to the culture module 50 through the third connection module, and then transfer the third cell liquid obtained after cell transfection and centrifugal replacement of the liquid exchange module 40 to the culture module 50 through the third connection module.

[0046] Wherein, the sub-packaging module 60 includes a plurality of input ports; after the culture in the culture module 50 is completed and the target cell liquid is obtained, the controller 10 obtains preset sub-packaging parameters, and determines at least one target port from all the input ports of the sub-packaging module 60 according to the preset sub-packaging parameters, and then controls the preset installation device to connect the fourth connection module between the culture module 50 and the target port.

[0047] S400, control the culture module 50 to culture the third cell fluid to obtain a target cell fluid; wherein, the target cell fluid refers to the cell fluid finally obtained after culturing and propagating the target cells corresponding to the immunomagnetic bead cells in the third cell fluid.

[0048] In one embodiment, the culture module 50 includes a culture chamber having a culture space meeting preset culture conditions and a culture container disposed in the culture space; that is, cell culture must be carried out in an environment meeting preset culture conditions. Therefore, in this embodiment, the culture space in the culture chamber meeting the preset culture conditions can achieve the best culture effect, wherein the preset culture conditions can be set according to the specific cells to be cultured.

[0049] Further, the controller 10 is further configured to: before controlling the culture module to culture the third cell fluid, connect the liquid exchange module 40 and the culture container through the connection and transmission module (specifically, through the third connection module). Further, before starting to execute step S100 (that is, before controlling the separation module 20 to perform centrifugation on the sample fluid), it is necessary to connect the above-mentioned liquid exchange module 40 to the culture container in the culture chamber through the third connection module, so as to transfer the third cell fluid obtained after cell transfection and centrifugal replacement by the liquid exchange module 40 to the culture container through the third connection module in subsequent steps; at the same time, it is ensured that during the subsequent cell processing by this integrated cell processing device, there is no need to assemble the device midway, and only need to start and run the integrated cell processing device that has been assembled once, thereby realizing cell processing through this integrated cell processing device.

[0050] Further, step S400, that is, controlling the culture module 50 to culture the third cell fluid to obtain a target cell fluid, includes: after confirming that the third cell fluid has been completely input into the culture container, culturing the third cell fluid in the culture container under the preset culture conditions; that is, after confirming that the third cell fluid has been completely input into the culture container, the target cells corresponding to the immunomagnetic bead cells in the third cell fluid can be cultured in the culture chamber.

[0051] Monitor the culture progress of the third cell fluid in the culture container in real time; the culture progress is determined according to the culture duration, or according to the target density and the actual cell density of the third cell fluid in the culture container collected by a preset density collection device; it is understandable that the culture progress can be characterized by the culture duration or the cell density in the culture container. When the culture progress is characterized by the cell density, the preset density collection device can be a device for monitoring the actual cell density in the third cell fluid in the culture container. For example, the preset density collection device can be a photographing device. By photographing and monitoring the magnified image of the third cell fluid in the culture container, the actual cell density can be further analyzed based on the magnified image. Each culture progress is also associated with a target density. When the actual cell density reaches the target density, the current culture progress can be determined as the culture progress corresponding to the target density.

[0052] After receiving the liquid change instruction, perform a liquid change operation on the third cell fluid; the liquid change instruction is automatically generated at the liquid change node or generated after triggering a preset button. Each liquid change node is associated with a culture progress; among them, the liquid change instruction can be generated after triggering a preset button on the integrated cell processing device or a smart device communicatively connected to the integrated cell processing device. The liquid change instruction can also be automatically generated at the liquid change node (the liquid change node is set according to the culture progress corresponding to different target cells and their specific culture plans. Different target cells and different amounts of target cells to be finally cultured in the culture plan require the liquid change node to be set to be associated with different culture progress). Specifically, since the culture process may be very long, it is necessary to timely replace the solution in the third cell fluid during the culture process to continuously provide necessary nutrients for cell culture. In this embodiment, each time a liquid change instruction is received (such as each liquid change node), a liquid change needs to be performed to provide the necessary substances for the cell culture process.

[0053] When it is confirmed that the culture progress is greater than or equal to the preset completion progress, it is confirmed that the culture of the target cell fluid is completed. That is, when it is confirmed that the culture progress is greater than or equal to the preset completion progress, it means that the target cells in the third cell fluid already meet the requirements. At this time, it can be confirmed that the third cell fluid contained in the culture container is the cultured target cell fluid that is completed.

[0054] In a further embodiment, the culture module 50 further includes a third connecting pipeline, a fourth connecting pipeline, a third container for containing waste liquid, and a fourth container for containing a second renewal solution; the controller 10 is further configured to: control the preset installation device to connect the third connecting pipeline between the culture container and the third container, and connect the fourth connecting pipeline between the culture container and the fourth container; that is, before starting to execute step S100 (that is, before controlling the separation module 20 to perform centrifugation on the sample liquid), the culture module 50 needs to be installed completely, so as to ensure the aseptic performance of the entire integrated cell processing device and enable the integrated cell processing device that has been assembled once to be started and run during the subsequent cell processing process without the need to assemble the device midway, and then realize cell processing through the integrated cell processing device.

[0055] Further, after receiving the liquid change instruction, the operation of changing the liquid of the third cell liquid includes:

[0056] After reaching the liquid change node, output the current culture medium in the culture container to the third container through the third connecting pipeline, and input the second renewal solution in the fourth container into the culture container through the fourth connecting pipeline, so as to continue culturing the third cell liquid in the culture container with the second renewal solution; that is, when reaching the liquid change node, it is necessary to output the current culture medium (which may be the first renewal solution or a solution renewed again after the first renewal solution, such as a culture medium or a cryopreservation solution) in the culture container to the third container through the third connecting pipeline, and then replace the current culture medium with the second renewal solution in the fourth container input into the culture container through the fourth connecting pipeline (such as a culture medium or a cryopreservation solution, and the second renewal solution may be the same as or different from the previous first renewal solution or other renewal solutions), so as to continuously provide necessary nutrients and the like for the culture process of the target cells corresponding to the immunomagnetic bead cells through the second renewal solution replaced into the culture container.

[0057] S500, control the dispensing module 60 to dispense the target cell liquid into a dispensing container.

[0058] In one embodiment, the dispensing module 60 includes a demagnetization unit, a cleaning unit and a dispensing unit; step S500, that is, the control of the dispensing module 60 to dispense the target cell liquid into a dispensing container, includes:

[0059] Control the target cell fluid to flow through the magnetic separation space of the demagnetization unit, so that the immunomagnetic cells bound with magnetic beads in the target cell fluid are adsorbed in the magnetic separation space by the magnetic field generated by the magnetic field generating device disposed opposite to the magnetic separation space, and the remaining target cell fluid containing the immunomagnetic cells not bound with magnetic beads is transported to the cleaning unit for cleaning; that is, in this embodiment, the magnetic separation space may refer to the accommodation space in a container (such as a liquid bag) having a bend. Specifically, after obtaining the target cell fluid in step S400, the target cell fluid is transferred to the magnetic separation space of the demagnetization unit (including a container having a magnetic separation space and a magnetic field generating device) through the fourth connection module and the target port, so that the target cell fluid flows through the magnetic separation space. Then, a magnetic field is applied to the magnetic separation space by the magnetic field generating device to adsorb the immunomagnetic bead cells with magnetic beads in the target cell fluid on the inner wall of the magnetic separation space of the container, and other non-magnetic target cells will flow into the cleaning unit along with the remaining target cell fluid for cleaning. In this way, the target cell fluid after removing the immunomagnetic bead cells can be obtained. A control switch may be provided at the inlet end of the container to control the flow rate of the target cell fluid flowing into the magnetic separation space. Pressure may also be applied to the container to increase the contact area between the container and the platform, so that the immunomagnetic bead cells are more easily adsorbed in the magnetic separation space. It can be understood that a cleaning solution is injected into the cleaning unit (such as another liquid bag) to clean the target cell fluid flowing into the cleaning unit, that is, the cleaning unit is controlled to shake or rotate at a preset centrifugal speed, so that the cell metabolites are dissolved in the cleaning solution, and then the cleaning solution and the target cell fluid are located at different positions in the cleaning unit. The cleaning solution may be a mixture of physiological saline and 0.5% HSA (human serum albumin). It can be understood that when the immunomagnetic bead cells are bound with micron magnetic beads, they must undergo the previous demagnetization treatment (removing the immunomagnetic bead cells in the target cell fluid) in the demagnetization unit before entering the cleaning unit for the next step. However, for the immunomagnetic bead cells bound with nano magnetic beads, it is not necessary to undergo the step of demagnetization treatment (removing the immunomagnetic bead cells in the target cell fluid) in the demagnetization unit. Instead, after obtaining the target cell fluid in step S400, the target cell fluid can be directly transferred to the cleaning unit through the fourth connection module and the target port for the next step, because the nano magnetic beads are smaller and less harmful to the human body.

[0060] The target cell solution after cleaning is transported to the sub-packaging unit for centrifugal extraction operation, so as to extract impurities from the target cell solution and obtain a concentrated cell solution; in this embodiment, it is necessary to connect the cleaning unit and the sub-packaging unit before step S100. Furthermore, in this step, an extraction operation is performed on the stratified cleaning solution and the target cell solution, so as to remove the cleaning solution and other non-cell impurities, and obtain a concentrated cell solution containing only T cells and / or T cell subsets. In another embodiment, during the centrifugal extraction operation, the target cell solution with a high concentration can be separated to the upper layer, and the cleaning solution and impurities with a low concentration are separated in the lower layer, and the cleaning solution and impurities are discharged through the drain port, so as to obtain a concentrated cell solution containing only T cells and / or T cell subsets.

[0061] A suspension is input into the concentrated cell solution to dilute the concentrated cell solution, and the diluted cell solution is sub-packaged into the sub-packaging containers. Specifically, after obtaining the concentrated cell solution, the concentrated cell solution is first sampled and detected, that is, the cell density of the concentrated cell solution is detected to ensure the cell survival rate and cell activity in the concentrated cell solution sub-packaged into the preparation bag, that is, to ensure the quality of the cells in the concentrated cell solution. And it is judged whether the cell density in the concentrated cell solution is greater than or equal to a preset density threshold; wherein, the preset density threshold can be set according to the density and quantity of the cells required for cell therapy.

[0062] Specifically, after sampling and detecting the concentrated cell solution to determine whether the concentrated cell solution meets the preset density threshold, when the concentrated cell solution meets the preset density threshold, a suspension is input into the sub-packaging module 60 containing the concentrated cell solution, and the cell solution after the suspension is input is sub-packaged into at least one preparation bag. If the concentrated cell solution does not meet the preset density threshold, a cryopreservation solution is added to the concentrated cell solution, and the concentrated cell solution can be directly sub-packaged into at least one preparation bag. Among them, the amount of the cryopreservation solution can be set according to the volume of the concentrated cell solution.

[0063] That is to say, in this embodiment, the immunomagnetic beads cells are efficiently removed through the demagnetization unit. The cleaning of the target cell solution and the extraction of the metabolites in the target cell solution are realized through the cleaning unit and the sub-packaging unit, and then the preparation and sub-packaging of the finally required concentrated cell solution are realized.

[0064] In the present invention, the above-mentioned modules and the controller 10 constitute an integrated cell processing device. In this integrated cell processing device, the modules sequentially execute multiple steps, and the cell processing procedures (centrifugation, magnetic sorting, cell transfection, centrifugal displacement, culturing, and aliquoting) are orderly connected in one-to-one correspondence. The entire cell processing process does not require manual participation and only needs to be automatically performed by the integrated cell processing device, which is easy to ensure the aseptic and clean requirements, thereby reducing the labor cost, shortening the processing time, and improving the cell processing efficiency. At the same time, the one-to-one automatic processing throughout the integrated cell processing device is not prone to failures or mistakes, so the accuracy of the cell processing process can be improved, making the finally obtained cell solution meet the standards.

[0065] In one embodiment, as Figure 2 shown, there is provided an integrated cell processing method. The integrated cell processing method is executed by the above-mentioned integrated cell processing device, and the integrated cell processing method includes:

[0066] S100, controlling the separation module 20 to perform centrifugation on the sample solution, and inputting the first cell solution prepared thereby into the sorting module 30;

[0067] S200, controlling the sorting module 30 to perform magnetic sorting on the first cell solution, and inputting the second cell solution containing immunomagnetic cells obtained by sorting into the liquid replacement module 40;

[0068] S300, controlling the liquid replacement module 40 to perform cell transfection and centrifugal displacement on the second cell solution, obtaining a third cell solution and inputting it into the culturing module 50;

[0069] S400, controlling the culturing module 50 to culture the third cell solution to obtain a target cell solution;

[0070] S500, controlling the aliquoting module 60 to aliquot the target cell solution into aliquot containers.

[0071] This integrated cell processing method corresponds one-to-one to the integrated cell processing device in the above embodiment. For the specific limitations of the integrated cell processing method, reference can be made to the limitations of the integrated cell processing device in the foregoing text, which will not be elaborated herein.

[0072] In the integrated cell processing method of the present invention, through each module in the integrated cell processing device, multiple steps are sequentially executed, and the cell processing processes (centrifugation, magnetic sorting, cell transfection, centrifugal displacement, culture, and aliquoting) are orderly connected in one-to-one correspondence. The entire cell processing process does not require manual participation and only needs to be automatically performed by the integrated cell processing device, which is easy to ensure its aseptic and cleanliness requirements, thereby reducing labor costs, shortening the processing time, and improving the cell processing efficiency. At the same time, the one-to-one automatic processing of each module of the integrated cell processing device is not prone to failures or mistakes, so the accuracy of the cell processing process can be improved, making the finally obtained cell solution meet the standards.

[0073] It should be understood that the magnitudes of the sequence numbers of the steps in the above embodiments do not mean the order of execution. The order of execution of each process should be determined by its function and internal logic, and should not constitute any limitation to the implementation process of the embodiments of the present invention.

[0074] In one embodiment, a computer-readable storage medium is provided. The computer-readable storage medium stores a computer program, and when the computer program is executed by a processor, the above-mentioned integrated cell processing method is implemented.

[0075] Those of ordinary skill in the art can understand that all or part of the processes of implementing the methods in the above embodiments can be completed by instructing relevant hardware through a computer program. 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 methods. Among them, any reference to a memory, storage, database, or other medium used in the embodiments provided in the present application can include non-volatile and / or volatile memories. Non-volatile memories can include read-only memory (ROM), programmable ROM (PROM), electrically programmable ROM (EPROM), electrically erasable programmable ROM (EEPROM), or flash memory. Volatile memories can include random access memory (RAM) or external cache memory. By way of 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 (DDR SDRAM), enhanced SDRAM (ESDRAM), synchronous link (Synchlink) DRAM (SLDRAM), memory bus (Rambus) direct RAM (RDRAM), direct memory bus dynamic RAM (DRDRAM), and memory bus dynamic RAM (RDRAM), etc.

[0076] Those skilled in the art can clearly understand that, for the convenience and conciseness of description, only the above-mentioned division of each functional unit and module is used as an example. In actual applications, the above-mentioned functions can be assigned to different functional units and modules according to needs, that is, the internal structure of the device is divided into different functional units or modules to complete all or part of the functions described above.

[0077] The above-described embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements on some of the technical features; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate 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. An integrated cell processing device, characterized in that, It includes a controller, as well as a separation module, a sorting module, a liquid replacement module, a culture module, and a sub-packaging module that are in one-to-one correspondence and connected in sequence; the separation module, the sorting module, the liquid replacement module, the culture module, and the sub-packaging module are all communicatively connected to the controller; The controller is configured to: Control the separation module to perform centrifugation on the sample liquid and input the prepared first cell liquid into the sorting module; Control the sorting module to perform magnetic sorting on the first cell liquid and input the sorted second cell liquid containing immunomagnetic cells into the liquid replacement module; The immunomagnetic cells refer to target cells labeled with magnetism; Control the liquid replacement module to perform cell transfection and centrifugal replacement on the second cell liquid to obtain a third cell liquid and input it into the culture module; Control the culture module to culture the third cell liquid to obtain a target cell liquid; Control the sub-packaging module to sub-package the target cell liquid into a sub-packaging container.

2. The integrated cell processing device according to claim 1, wherein The liquid replacement module includes a transfection unit and a replacement unit; The transfection unit is configured to, after the second cell liquid and the transfection reagent are respectively input into a centrifugation container, perform centrifugation on the liquid in the centrifugation container so that the transfection reagent binds to the immunomagnetic cells; or the transfection unit is configured to, after the second cell liquid and the transfection reagent are respectively input into an electroporation chamber, apply an electric field to the electroporation chamber, and through the electric field, enable the transfection reagent to enter the immunomagnetic cells to complete cell transfection; The replacement unit is configured to replace the initial centrifugation in the second cell liquid after cell transfection with a first renewal solution to complete centrifugal replacement and obtain a third cell liquid.

3. The integrated cell processing device according to claim 2, characterized in that, The replacement unit includes a first connection pipeline, a second connection pipeline, a replacement container for containing the second cell liquid after cell transfection, a first container for containing waste liquid, and a second container for containing the first renewal solution; The controller is further configured to: control a preset installation device to connect the first connection pipeline between the replacement container and the first container, and connect the second connection pipeline between the replacement container and the second container; The replacement unit is further configured to output the initial solution in the second cell liquid after cell transfection from the replacement container to the first container through the first connection pipeline, and input the first renewal solution in the second container into the replacement container through the second connection pipeline to complete centrifugal replacement and obtain a third cell liquid.

4. The integrated cell processing device according to claim 1, characterized in that, The integrated cell processing device includes at least one connection and transmission module; The controller is further configured to: Control the connection and transmission module to connect the separation module and the sorting module, or / and connect the sorting module and the liquid replacement module, or / and connect the liquid replacement module and the culture module, or / and connect at least one input port of the culture module and the sub-packaging module.

5. The integrated cell processing device according to claim 4, wherein The culture module includes a culture chamber having a culture space meeting preset culture conditions and a culture container provided in the culture space; The controller is further configured to: Before controlling the culture module to culture the third cell solution, connect the liquid changing module and the culture container through the connection and transmission module; Controlling the culture module to culture the third cell solution to obtain a target cell solution includes: After confirming that all of the third cell solution has been input into the culture container, culturing the third cell solution in the culture container under the preset culture conditions; Real-time monitoring of the culture progress of the third cell solution in the culture container; the culture progress is determined according to the culture duration, or according to the target density and the actual cell density of the third cell solution in the culture container collected by a preset density collection device; After receiving a liquid changing instruction, performing a liquid changing operation on the third cell solution; the liquid changing instruction is automatically generated at a liquid changing node or generated after triggering a preset button, and each liquid changing node is associated with a culture progress; When it is confirmed that the culture progress is greater than or equal to a preset completion progress, it is confirmed that the culture of the target cell solution is completed.

6. The integrated cell processing device according to claim 5, wherein The culture module further includes a third connection pipeline, a fourth connection pipeline, a third container for containing waste liquid, and a fourth container for containing a second renewal solution; The controller is further configured to: Control a preset installation device to connect the third connection pipeline between the culture container and the third container, and connect the fourth connection pipeline between the culture container and the fourth container; After receiving the liquid changing instruction, performing a liquid changing operation on the third cell solution includes: After receiving the liquid changing instruction, outputting the current culture medium in the culture container to the third container through the third connection pipeline, and inputting the second renewal solution in the fourth container into the culture container through the fourth connection pipeline, so as to continue culturing the third cell solution in the culture container with the second renewal solution.

7. The integrated cell processing device according to claim 1, characterized in that, The separation module includes a first centrifugation device; controlling the separation module to perform centrifugation on a sample solution and input the prepared first cell solution into the sorting module includes: Obtaining centrifugation parameters, and controlling the first centrifugation device to rotate at a preset speed according to the centrifugation parameters to perform centrifugation on the sample solution to obtain an intermediate product cell solution; Adding magnetic beads and an antigen to the intermediate product cell solution so that the target cells in the intermediate product cell solution bind to the magnetic beads and the antigen, obtaining a first cell solution containing immunomagnetic beads cells, and inputting the first cell solution into the sorting module; The sorting module includes a magnetic sorting device; controlling the sorting module to perform magnetic sorting on the first cell solution and input the sorted second cell solution containing immunomagnetic cells into the liquid changing module includes: Controlling the magnetic sorting device to magnetically screen out the immunomagnetic cells that have bound magnetic beads from the first cell solution, and inputting the second cell solution containing the screened immunomagnetic cells into the liquid changing module.

8. The integrated cell processing device according to claim 7, wherein, The dispensing module includes a demagnetization unit, a cleaning unit, and a dispensing unit; Controlling the dispensing module to dispense the target cell solution into a dispensing container includes: Control the target cell fluid to flow through the magnetic separation space of the demagnetization unit, so that the immunomagnetic cells bound with magnetic beads in the target cell fluid are adsorbed in the magnetic separation space by the magnetic field generated by the magnetic field generating device oppositely arranged with the magnetic separation space, and the remaining target cell fluid containing immunomagnetic cells not bound with magnetic beads is transported to the cleaning unit for cleaning; Transport the target cell fluid after cleaning to the sub-packaging unit for centrifugal extraction operation, so as to obtain concentrated cell fluid after removing impurities from the target cell fluid; Input a suspension into the concentrated cell fluid to dilute the concentrated cell fluid, and sub-pack the diluted cell fluid into the sub-packaging containers.

9. An integrated cell processing method, characterized in that, The integrated cell processing method is executed by the integrated cell processing device according to any one of claims 1 to 8, and the integrated cell processing method includes: Control the separation module to perform centrifugal treatment on the sample fluid, and input the first cell fluid prepared into the sorting module; Control the sorting module to perform magnetic separation on the first cell fluid, and input the second cell fluid containing immunomagnetic cells obtained by sorting into the liquid replacement module; Control the liquid replacement module to perform cell transfection and centrifugal replacement on the second cell fluid, obtain the third cell fluid and input it into the culture module; Control the culture module to culture the third cell fluid to obtain the target cell fluid; Control the sub-packaging module to sub-pack the target cell fluid into the sub-packaging containers.

10. A computer-readable storage medium, characterized in that, The computer-readable storage medium stores a computer program, and when the computer program is executed by a processor, the integrated cell processing method according to claim 9 is implemented.

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