Blood treatment method

Abstract

The invention provides a blood treatment method, which relates to the field of biology and medical treatment, and comprises the following steps: concentrating and diluting a blood sample through centrifugal equipment to remove impurities except blood cells in the blood sample to obtain cell sap; the cell sap and the magnetic beads are mixed through centrifugal equipment, so that target cells in the cell sap are combined with the magnetic beads; and separating the target cells from the non-target cells through magnetic separation equipment to obtain target cell sap. The blood sample is concentrated and diluted through centrifugal equipment, so that impurities except blood cells in the blood sample are removed; the cell sap and the magnetic beads are mixed through centrifugal equipment, so that target cells in the cell sap are combined with the magnetic beads; target cells and non-target cells are separated through the magnetic separation equipment, extracellular fluid in target cell fluid is replaced with a culture medium through the centrifugal equipment, then the purpose of directly extracting the cells for culture in single blood sampling is achieved, the harvesting efficiency and the recovery rate of the cells to be cultured are improved, and the phenomena of cell agglomeration and conjunctiva are avoided.

Description

Technical Field

[0001] The present invention relates to the fields of biology and medical technology, and particularly to a blood treatment method. Background Art

[0002] Currently, in the field of cell therapy (CAR-T), the density gradient method (commonly known as PBMC separation) is mostly used to separate the buffy coat from red blood cells and plasma with Ficoll. The disadvantage of this method is that the cells need to go through a high-speed centrifugation separation process, and the final experimental results are related to many factors such as the equipment state, and phenomena such as cell clumping and membrane formation may occur. In the reinfusion treatment, cell clumping and membrane formation are not allowed. Summary of the Invention

[0003] The present invention provides a blood treatment method to solve the problem of cell clumping and membrane formation in the existing technology during cell extraction.

[0004] The present invention provides a blood treatment method, including:

[0005] Concentrating and diluting a blood sample by a centrifugation device to remove impurities other than blood cells in the blood sample, obtaining a cell solution;

[0006] Mixing the cell solution with magnetic beads by the centrifugation device to bind target cells in the cell solution to the magnetic beads;

[0007] Separating the target cells from non-target cells by a magnetic sorting device, obtaining a target cell solution;

[0008] Replacing the extracellular fluid in the target cell solution with a culture medium by the centrifugation device.

[0009] According to a blood treatment method provided by an embodiment of the present invention, the step of concentrating and diluting the blood sample by the centrifugation device includes:

[0010] Inputting a first predetermined amount of the blood sample into a centrifugation container of the centrifugation device, and adding a magnetic bead sorting buffer solution to dilute the blood sample to a first predetermined volume;

[0011] Controlling the centrifugation device to rotate at a first rotation speed for a first predetermined time and perform liquid extraction to concentrate the diluted blood sample, obtaining the cell solution of a second predetermined volume;

[0012] Controlling the centrifugation device to rotate at a second rotation speed and perform liquid extraction to concentrate the cell solution to a second predetermined volume, wherein the second rotation speed is less than the first rotation speed;

[0013] Inputting the cell solution into a liquid bag.

[0014] According to a blood processing method provided by an embodiment of the present invention, before the step of controlling the centrifuge device to rotate at a second rotation speed and perform liquid extraction, the method further includes:

[0015] Input a second predetermined amount of the magnetic bead sorting buffer solution into the centrifuge container to dilute the cell solution to a third predetermined volume;

[0016] Control the centrifuge device to rotate at a first rotation speed and perform liquid extraction to concentrate the cell solution to a first predetermined volume.

[0017] According to a blood processing method provided by an embodiment of the present invention, the step of mixing the cell solution with magnetic beads by the centrifuge device includes:

[0018] Input the magnetic beads with antibodies in the magnetic bead bag and the cell solution in the liquid bag into the centrifuge container respectively;

[0019] Incubate at a predetermined temperature for a second predetermined time;

[0020] Input a third predetermined amount of the magnetic bead sorting buffer solution into the centrifuge container;

[0021] Control the centrifuge device to rotate at a second rotation speed and perform liquid extraction to concentrate the cell solution to a second predetermined volume;

[0022] Input a resuspension solution into the centrifuge container to make the volume of the cell solution reach a fourth predetermined volume.

[0023] According to a blood processing method provided by an embodiment of the present invention, before the step of incubating at a predetermined temperature for a second predetermined time, the method further includes:

[0024] Input the magnetic bead sorting buffer solution into the magnetic bead bag for rinsing, and input the rinsed magnetic bead sorting buffer solution into the centrifuge container.

[0025] According to a blood processing method provided by an embodiment of the present invention, the step of separating the target cells from the non-target cells by a magnetic separation device includes:

[0026] In the case of determining the presence of a magnetic field, input the target cell solution combined with magnetic beads into a sorting column, so that the target cells are adsorbed on the sorting column and the non-target cells are transported to a waste liquid bag;

[0027] Switch the pipeline so that the pipeline after discharging the non-target cells is switched to be connected to the input pipeline of the centrifuge device;

[0028] In the case of determining the removal of the magnetic field, the magnetic bead sorting buffer is input into the sorting column to wash out the target cells from the sorting column and transport them to an input pipeline of the centrifugation device.

[0029] According to a blood processing method provided by an embodiment of the present invention, before the step of transporting the target cell solution combined with magnetic beads to the sorting column, it further includes:

[0030] Connect the output pipeline of the centrifugation device to the input pipeline of the magnetic sorting device.

[0031] According to a blood processing method provided by an embodiment of the present invention, the step of replacing the extracellular fluid in the target cell solution with a culture medium by the centrifugation device includes:

[0032] Control the centrifugation device to rotate at a first rotation speed and perform liquid extraction to concentrate the target cell solution to a second predetermined volume;

[0033] Input a fourth predetermined amount of the culture medium into the centrifugation container;

[0034] Control the centrifugation device to rotate at a first rotation speed and perform liquid extraction to concentrate the target cell solution to a second predetermined volume;

[0035] Input a fifth predetermined amount of the culture medium into the centrifugation container;

[0036] Transport the mixture of the target cell solution and the culture medium to a preparation bag.

[0037] According to a blood processing method provided by an embodiment of the present invention, before the step of inputting a fourth predetermined amount of the culture medium into the centrifugation container, it includes:

[0038] Input a sixth predetermined amount of the washing magnetic bead sorting buffer into the centrifugation container;

[0039] Control the centrifugation device to rotate at a first rotation speed and perform liquid extraction to concentrate the target cell solution to a second predetermined volume.

[0040] According to a blood processing method provided by an embodiment of the present invention, after the step of transporting the mixture of the target cell solution and the culture medium to the preparation bag, it further includes:

[0041] Input a fifth predetermined amount of the culture medium into the centrifugation container to wash the centrifugation container;

[0042] Transport the culture medium in the centrifugation container to the preparation bag so that the liquid volume in the preparation bag reaches a fourth predetermined volume.

[0043] The blood processing method provided by the embodiments of the present invention concentrates and dilutes a blood sample through a centrifugation device to remove impurities other than blood cells in the blood sample; mixes the cell fluid with magnetic beads through the centrifugation device so that target cells in the cell fluid bind to the magnetic beads; separates the target cells from non-target cells through a magnetic sorting device, and replaces the extracellular fluid in the target cell fluid with a culture medium through the centrifugation device, thereby achieving the purpose of directly extracting cells for culture in apheresis, improving the harvesting efficiency and recovery rate of cells to be cultured, and avoiding the phenomena of cell aggregation and cell membrane formation. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0045] Figure 1 It is a flowchart of the blood processing method provided by the embodiments of the present invention;

[0046] Figure 2 It is a flowchart of the steps of concentrating and diluting a blood sample through a centrifugation device provided by the embodiments of the present invention;

[0047] Figure 3 It is a flowchart of the steps of mixing the cell fluid with magnetic beads through a centrifugation device provided by the embodiments of the present invention;

[0048] Figure 4 It is a flowchart of the steps of separating target cells from non-target cells through a magnetic sorting device provided by the embodiments of the present invention;

[0049] Figure 5 It is a flowchart of the steps of replacing the extracellular fluid in the target cell fluid with a culture medium through a centrifugation device provided by the embodiments of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0050] The following further describes in detail the embodiments of the present invention in conjunction with the drawings and embodiments. The following embodiments are used to illustrate the present invention, but cannot be used to limit the scope of the present invention.

[0051] In the description of the embodiments of the present invention, it should be noted that the orientation or positional relationship indicated by the terms "center", "longitudinal", "transverse", "upper", "lower", "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer", etc. is based on the orientation or positional relationship shown in the drawings. It is only for the convenience of describing the embodiments of the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and thus should not be construed as a limitation to the embodiments of the present invention. In addition, the terms "first", "second", and "third" are only used for descriptive purposes and cannot be construed as indicating or implying relative importance.

[0052] In the description of the embodiments of the present invention, it should be noted that unless otherwise clearly specified and defined, the terms "connected" and "connected" should be understood in a broad sense. For example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be directly connected or indirectly connected through an intermediate medium. For those of ordinary skill in the art, the specific meanings of the above terms in the embodiments of the present invention can be understood according to specific circumstances.

[0053] In the embodiments of the present invention, unless otherwise clearly specified and defined, the first feature being "on" or "under" the second feature can be that the first and second features are in direct contact, or the first and second features are indirectly in contact through an intermediate medium. Moreover, the first feature being "above", "over", and "on top of" the second feature can be that the first feature is directly above or obliquely above the second feature, or merely indicates that the first feature has a higher horizontal height than the second feature. The first feature being "under", "beneath", and "underneath" the second feature can be that the first feature is directly below or obliquely below the second feature, or merely indicates that the first feature has a lower horizontal height than the second feature.

[0054] In the description of this specification, the description referring to terms such as "one embodiment", "some embodiments", "example", "specific example", or "some examples" means that the specific features, structures, materials, or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the embodiments of the present invention. In this specification, the schematic descriptions of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described can be combined in a suitable manner in any one or more embodiments or examples. In addition, without contradiction, those skilled in the art can combine and combine the different embodiments or examples described in this specification and the features of different embodiments or examples.

[0055] As Figure 1 shown, the blood treatment method includes:

[0056] Step S100: Concentrate and dilute the blood sample by a centrifugation device to remove impurities other than blood cells in the blood sample, obtaining cell fluid.

[0057] Step S200: Mix the cell fluid with magnetic beads by a centrifugation device so that target cells in the cell fluid bind to the magnetic beads.

[0058] Step S300: Separate the target cells from non-target cells by a magnetic sorting device, obtaining target cell fluid.

[0059] Step S400: Replace the extracellular fluid in the target cell fluid with a culture medium by a centrifugation device.

[0060] The blood processing method provided by the embodiment of the present invention concentrates and dilutes the blood sample by a centrifugation device to remove impurities other than blood cells in the blood sample; mixes the cell fluid with magnetic beads by a centrifugation device so that target cells in the cell fluid bind to the magnetic beads; separates the target cells from non-target cells by a magnetic sorting device, and replaces the extracellular fluid in the target cell fluid with a culture medium by a centrifugation device, thereby achieving the purpose of directly extracting cell culture in apheresis, improving the harvesting efficiency and recovery rate of cells to be cultured, and avoiding the phenomena of cell aggregation and cell membrane formation.

[0061] In an embodiment of the present invention, the step of concentrating and diluting the blood sample by a centrifugation device includes:

[0062] Step S110: Input a first predetermined amount of blood sample into a centrifugation container of the centrifugation device, and add a magnetic bead sorting buffer solution to dilute the blood sample to a first predetermined volume.

[0063] Since the impurity components in plasma will affect the subsequent binding of magnetic beads to target cells, the main purpose of concentrating and diluting the blood sample by a centrifugation device is to wash the blood sample. Washing can remove other impurities outside blood cells and avoid affecting the subsequent incubation effect.

[0064] It should be noted here that the magnetic bead sorting buffer solution is the magnetic bead sorting buffer solution. Of course, the specific type of the magnetic bead sorting buffer solution is not limited thereto, and it can also be other types of liquids.

[0065] In this embodiment, the first predetermined amount is 50 ml - 150 ml, the blood sample is an apheresis sample, and the first predetermined amount of blood sample is input into the centrifugation container through an I / O port of the centrifugation container. After injecting the blood sample, the magnetic bead sorting buffer solution is also injected into the centrifugation container. The magnetic bead sorting buffer solution is mixed with the blood sample, diluting the blood sample to the first predetermined volume. In this embodiment, the first predetermined volume is 250 ml. Of course, the size of the first predetermined volume is not limited thereto and is specifically determined according to actual needs.

[0066] When injecting the blood sample and the magnetic bead sorting buffer, hang the single-donor blood sample bag and the magnetic bead sorting buffer bag on different weighing hooks of the centrifugation device respectively, and monitor the amount of liquid injected from the liquid bags into the centrifuge container through the weighing hooks. During the injection process, the rotation of the peristaltic pump drives the liquid flow in the pipeline, and the injection pipeline is switched by switching the control valve. For example, the combination of the control valve and the three-way pipe can switch the source of the liquid bag injection.

[0067] Step S120: Control the centrifugation device to rotate at the first rotation speed for the first predetermined time and perform liquid extraction to concentrate the diluted blood sample and obtain cell liquid with a second predetermined volume.

[0068] During the operation of the centrifugation device, the rotation of the centrifuge container separates the cell liquid from other liquids, thereby concentrating the blood sample. In this embodiment, the first rotation speed is 1800 rpm. Of course, the magnitude of the first rotation speed is not limited thereto, and the optimal value is specifically selected according to the concentration effect. In this embodiment, the first predetermined time is 360 s. Of course, the magnitude of the first predetermined time is not limited thereto, and it can also be 350 s, 340 s or a shorter time. The first predetermined time is specifically determined according to the concentration effect and the rotation speed of the centrifuge container. The higher the rotation speed of the centrifuge container, the shorter the first predetermined time.

[0069] When the centrifuge container is at a rotation speed of 1800 rpm, the peristaltic pump extracts liquid through another I / O port. The liquid extraction can be performed by the same peristaltic pump or different peristaltic pumps. Through the I / O port, liquid can be extracted at a position close to the supernatant at the boundary between the supernatant layer and the PBMC layer, and the extracted supernatant is transported to the waste liquid bag until no more liquid can be extracted (for accurate monitoring, the outlet bubble sensor has a 30 s delay. Since the magnetic bead separation buffer contains protein, there will be bubbles in the discharged liquid, so a delay needs to be set). By using the above concentration method, the cell liquid in the centrifuge container can be concentrated to 90 ml. The remaining cell liquid in the centrifuge container contains a mixture of red blood cells + PBMC + magnetic bead sorting buffer. In this embodiment, the second predetermined volume is 10 ml.

[0070] Step S130: Control the centrifugation device to rotate at the second rotation speed and perform liquid extraction to concentrate the cell liquid to the second predetermined volume, where the second rotation speed is less than the first rotation speed.

[0071] It should be noted here that controlling the centrifugal device to rotate at the second rotational speed and perform liquid extraction can be completed once or multiple times. When the centrifugal device rotates at the second rotational speed and performs liquid extraction once, the centrifugal container rotates continuously until the liquid in the centrifugal container is concentrated to the second predetermined volume. Of course, the centrifugal device can also rotate at the second rotational speed and perform liquid extraction multiple times. For example, in an embodiment of the present invention, controlling the centrifugal device to rotate at the second rotational speed and perform liquid extraction includes the following steps:

[0072] Step S131, controlling the centrifugal device to rotate at the second rotational speed and perform liquid extraction to concentrate the cell liquid to 70 ml;

[0073] Step S132, controlling the centrifugal device to rotate at the second rotational speed and perform liquid extraction to concentrate the cell liquid to 40 ml;

[0074] Step S133, controlling the centrifugal device to rotate at the second rotational speed and perform liquid extraction to concentrate the cell liquid to 10 ml;

[0075] It should be noted here that the running time of each step in the above concentration process can be the same or different, and is specifically determined according to the concentration effect.

[0076] Step S140, inputting the cell liquid into the liquid bag.

[0077] The concentrated cell liquid is input into the liquid bag for storage, preparing for the subsequent incubation process.

[0078] In an embodiment of the present invention, before the step of inputting the cell liquid into the liquid bag, the steps of concentrating and diluting the blood sample by the centrifugal device further include:

[0079] Supplying magnetic bead sorting buffer solution to the cell liquid according to the cell density to adjust the volume of the cell liquid;

[0080] It should be noted here that the purpose of adjusting the volume of the cell liquid is to make the volume of the cell liquid match the capacity of the liquid bag.

[0081] In an embodiment of the present invention, before the step of controlling the centrifugal device to rotate at the second rotational speed and perform liquid extraction, the steps of concentrating and diluting the blood sample by the centrifugal device further include:

[0082] Inputting a second predetermined amount of magnetic bead sorting buffer solution into the centrifugal container to dilute the cell liquid to a third predetermined volume;

[0083] The purpose of adding a second predetermined amount of magnetic bead sorting buffer into the centrifuge container is to dilute the cell solution and prepare for subsequent re-concentration. In this embodiment, the second predetermined amount is 160 ml. Of course, the size of the second predetermined amount is not limited thereto, and it can also be 170 ml, 180 ml or other volumes. The second predetermined amount is specifically determined according to the volume of the cell solution and the third predetermined volume.

[0084] Control the centrifuge equipment to rotate at the first rotation speed and perform liquid extraction to concentrate the cell solution to the first predetermined volume.

[0085] The purpose of performing the above steps is to repeatedly dilute and extract the cell solution, thereby obtaining a purer cell solution and minimizing the supernatant in the cell solution. Of course, in order to further improve the purity of the cell solution, the above two steps can be repeatedly executed multiple times.

[0086] The following Figure 2 describes a specific embodiment of the present invention. As Figure 2 shown, the steps of concentrating and diluting the blood sample by the centrifuge equipment include:

[0087] Step S110: Input a first predetermined amount of blood sample into the centrifuge container of the centrifuge equipment, and add magnetic bead sorting buffer to dilute the blood sample to the first predetermined volume;

[0088] Step S120: Control the centrifuge equipment to rotate at the first rotation speed for a first predetermined time and perform liquid extraction to concentrate the diluted blood sample to obtain a cell solution with a second predetermined volume;

[0089] Step S130: Input a second predetermined amount of magnetic bead sorting buffer into the centrifuge container to dilute the cell solution to the third predetermined volume;

[0090] Step S140: Control the centrifuge equipment to rotate at the first rotation speed and perform liquid extraction to concentrate the cell solution to the first predetermined volume.

[0091] Step S150: Control the centrifuge equipment to rotate at the second rotation speed and perform liquid extraction to concentrate the cell solution to the second predetermined volume;

[0092] Step S160: Input magnetic bead sorting buffer into the cell solution for liquid supplementation according to the cell density to adjust the volume of the cell solution;

[0093] Step S170: Input the cell solution into the liquid bag.

[0094] In an embodiment of the present invention, the steps of mixing the cell solution with magnetic beads by the centrifuge equipment include the following steps:

[0095] Step S210: Input the magnetic beads with antibodies in the magnetic bead bag and the cell solution in the liquid bag into the centrifuge container respectively;

[0096] Specifically, implementing the above steps includes the following steps: Hang the magnetic bead bag on the centrifuge device, and the proportion of the magnetic beads with antibodies is 20 microliters / E07; Inject the magnetic beads with antibodies in the magnetic bead bag into the centrifuge container. According to the volume of cells to be input (generally within 10 ml), the centrifuge device injects the cell solution in the liquid bag into the centrifuge container.

[0097] It should be noted here that the magnetic bead bag is a CD4 magnetic bead bag for storing CD4 magnetic beads. In this embodiment, the magnetic beads with antibodies are CD4 magnetic beads.

[0098] Step S220: Incubate for a second predetermined time at a predetermined temperature;

[0099] The purpose of performing the incubation process is to enable the target cells to fully contact the magnetic beads, so that the magnetic sorting device can select the target cells bound to the magnetic beads from them. In this embodiment, the predetermined temperature is 4°C. Of course, the predetermined temperature is not limited thereto, and the optimal value is specifically obtained according to the experimental results. The length of the second predetermined time is specifically determined according to factors such as the amount of the cell solution and the temperature.

[0100] Step S230: Input a third predetermined amount of magnetic bead sorting buffer into the centrifuge container;

[0101] The purpose of inputting a third predetermined amount of magnetic bead sorting buffer into the centrifuge container is to dilute the cell solution again. In this embodiment, the third predetermined amount is 140 ml.

[0102] Step S240: Control the centrifuge device to rotate at a second rotational speed and perform liquid pumping to concentrate the cell solution to a second predetermined volume;

[0103] The purpose of concentrating the cell solution again is to further improve the purity of the cell solution.

[0104] It should be noted here that the specific value of the second predetermined volume here and the second predetermined volume in the above steps may be the same or different.

[0105] Step S250: Input a resuspension solution into the centrifuge container to make the volume of the cell solution reach a fourth predetermined volume.

[0106] The purpose of inputting a resuspension solution (4 times the concentrated volume) into the centrifuge container to make the volume of the cell solution reach a fourth predetermined volume is to make the volume of the cell solution equal to the capacity of the preparation bag. In this embodiment, the fourth predetermined volume is 50 ml. Of course, the size of the fourth predetermined volume is not limited thereto, and it is specifically determined according to the size of the preparation bag.

[0107] In one embodiment of the present invention, before the step of incubating for a second predetermined time at a predetermined temperature, the step of mixing the cell lysate with magnetic beads by a centrifugation device further includes:

[0108] Input a magnetic bead sorting buffer into the magnetic bead bag for rinsing, and input the rinsed magnetic bead sorting buffer into a centrifugation container.

[0109] The purpose of inputting the magnetic bead sorting buffer into the magnetic bead bag to clean the magnetic bead bag is to clean the cell lysate and magnetic beads on the inner surface of the magnetic bead bag, and to clean the cell lysate and magnetic beads in the pipeline so that they completely enter the centrifugation container. Generally, the total incubation volume does not exceed 15 ml.

[0110] The following combines Figure 3 Describe a specific embodiment of the present invention, as Figure 3 shown, the step of mixing the cell lysate with magnetic beads by a centrifugation device includes the following steps:

[0111] Step S210, input the magnetic beads with antibodies in the magnetic bead bag and the cell lysate in the liquid bag into the centrifugation container respectively;

[0112] Step S220, input a magnetic bead sorting buffer into the magnetic bead bag for rinsing, and input the rinsed magnetic bead sorting buffer into the centrifugation container.

[0113] Step S230, incubate for a second predetermined time at a predetermined temperature;

[0114] Step S240, input a third predetermined amount of magnetic bead sorting buffer into the centrifugation container;

[0115] Step S250, control the centrifugation device to rotate at a second rotational speed and perform liquid extraction to concentrate the cell lysate to a second predetermined volume;

[0116] Step S260, input a resuspension solution into the centrifugation container so that the volume of the cell lysate reaches a fourth predetermined volume.

[0117] In one embodiment of the present invention, the step of separating target cells from non-target cells by a magnetic separation device includes:

[0118] Step S310, in the case of determining the presence of a magnetic field, input the target cell lysate combined with magnetic beads into a sorting column so that the target cells are adsorbed on the sorting column and the non-target cells are transported to a waste liquid bag;

[0119] Since the target cells are bound to the magnetic beads, when the target cells pass through the sorting column, under the action of the magnetic field, the target cells are adsorbed to the inner wall of the sorting column. Since the non-target cells cannot bind to the magnetic beads, the non-target cells are not affected by the magnetic field when passing through the sorting column, and the non-target cells directly pass through the sorting column and enter the waste liquid bag. In this way, the target cells are left in the sorting column, while the non-target cells enter the waste liquid bag, realizing the separation of the target cells and the non-target cells.

[0120] Step S320, switch the pipeline so that the pipeline after discharging the non-target cells is switched to be connected to the input pipeline of the centrifugation device;

[0121] The purpose of switching the pipeline is to prepare for inputting the magnetic bead sorting buffer solution into the magnetic sorting device.

[0122] Furthermore, in order to prevent the non-target cells remaining in the pipeline from affecting the purity of the target cells, in an embodiment of the present invention, before the step of inputting the magnetic bead sorting buffer solution into the sorting column, the step of separating the target cells and the non-target cells by the magnetic sorting device further includes:

[0123] Replace the new consumable pipeline, or clean the pipeline of the centrifugation device.

[0124] It should be noted here that cleaning the pipeline of the centrifugation device requires flushing the entire consumable pipeline, and the cleaning liquid is the magnetic bead sorting buffer solution.

[0125] Step S330, when it is determined that the magnetic field is removed, input the magnetic bead sorting buffer solution into the sorting column to wash out the target cells from the sorting column and transport them to an input pipeline of the centrifugation device.

[0126] In an embodiment of the present invention, before the step of transporting the target cell solution bound with magnetic beads to the sorting column, the step of separating the target cells and the non-target cells by the magnetic sorting device further includes:

[0127] Connect the output pipeline of the centrifugation device to the input pipeline of the magnetic sorting device.

[0128] By connecting the centrifugation device and the magnetic sorting device, the target cell solution bound with magnetic beads in the centrifugation container can be transported to the magnetic sorting device, and then the magnetic sorting device sorts the target cell solution bound with magnetic beads.

[0129] The following combines Figure 4 Describe a specific embodiment of the present invention. As Figure 4 shown, the steps of separating the target cells and the non-target cells by the magnetic sorting device include:

[0130] Step S310, connect the output pipeline of the centrifugation device to the input pipeline of the magnetic sorting device;

[0131] Step S320, in the case of determining the presence of a magnetic field, input the target cell solution combined with magnetic beads into the sorting column, so that the target cells are adsorbed on the sorting column, and the non-target cells are transported to the waste liquid bag;

[0132] Step S330, switch the pipeline so that the pipeline after draining the non-target cells is switched to be connected to the input pipeline of the centrifugation device;

[0133] Step S340, replace the new consumable pipeline, or clean the pipeline of the centrifugation device;

[0134] Step S350, in the case of determining the removal of the magnetic field, input the magnetic separation magnetic bead sorting buffer solution into the sorting column to wash the target cells out of the sorting column and transport them to an input pipeline of the centrifugation device.

[0135] In an embodiment of the present invention, the step of replacing the extracellular fluid in the target cell solution with a culture medium by a centrifugation device includes:

[0136] Step S410, control the centrifugation device to rotate at a first rotational speed and perform liquid extraction to concentrate the target cell solution to a second predetermined volume;

[0137] The purpose of performing the above steps is to reduce the magnetic separation magnetic bead sorting buffer solution in the cell solution and prepare for subsequent replacement with a culture medium.

[0138] Step S420, input a fourth predetermined amount of culture medium into the centrifugation container;

[0139] In this embodiment, the fourth predetermined amount is 135 ml. Of course, the size of the fourth predetermined amount is not limited thereto, and is specifically determined according to the amount of the cell solution and the size of the centrifugation container.

[0140] Step S430, control the centrifugation device to rotate at a first rotational speed and perform liquid extraction to concentrate the target cell solution to a second predetermined volume;

[0141] Inputting a culture medium into the centrifugation container and then concentrating the cell solution can achieve replacing the magnetic separation magnetic bead sorting buffer solution in the cell solution with a culture medium for direct culturing.

[0142] Step S440, input a fifth predetermined amount of culture medium into the centrifugation container;

[0143] The purpose of inputting a fifth predetermined amount of culture medium into the centrifugation container is to dilute the cell solution so that the cell solution and the culture medium are mixed in proportion. In this embodiment, the fifth predetermined amount is 20 ml. Of course, the size of the fifth predetermined amount is not limited thereto, and is specifically determined according to the capacity of the preparation bag.

[0144] Step S450, transfer the mixture of the target cell solution and the culture medium into the preparation bag.

[0145] In one embodiment of the present invention, before the step of inputting the fourth predetermined amount of the culture medium into the centrifuge container, the step of replacing the extracellular fluid in the target cell solution with the culture medium by the centrifugation device includes:

[0146] Input the sixth predetermined amount of the washing magnetic bead sorting buffer into the centrifuge container;

[0147] Control the centrifugation device to rotate at the first rotation speed and perform liquid extraction to concentrate the target cell solution to the second predetermined volume.

[0148] The purpose of performing the above two steps is to use the washing magnetic bead sorting buffer to wash the magnetic bead sorting buffer in the centrifuge container to avoid residual magnetic bead sorting buffer in the cell solution.

[0149] In one embodiment of the present invention, after the step of transferring the mixture of the target cell solution and the culture medium into the preparation bag, the step of replacing the extracellular fluid in the target cell solution with the culture medium by the centrifugation device further includes:

[0150] Input the fifth predetermined amount of the culture medium into the centrifuge container to rinse the centrifuge container;

[0151] Transfer the culture medium in the centrifuge container into the preparation bag so that the liquid volume in the preparation bag reaches the fourth predetermined volume.

[0152] Although the mixture of the target cell solution and the culture medium is transferred into the preparation bag in the above step, there is still the mixture of the target cell solution and the culture medium in the centrifuge container and the pipeline. Therefore, by inputting the culture medium into the centrifuge container, the centrifuge container and the pipeline can be rinsed, so that the target cell solution completely enters the preparation bag, improving the harvesting efficiency and recovery rate of the target cells.

[0153] In this embodiment, the amount of the mixture of the target cell solution and the culture medium transferred into the preparation bag is 30 ml, the capacity of the centrifuge container after washing is 20 ml, and the total volume of the liquid input into the preparation bag twice is 50 ml.

[0154] The following combines Figure 5 Describe a specific embodiment of the present invention, as Figure 5 shown,

[0155] The step of replacing the extracellular fluid in the target cell solution with the culture medium by the centrifugation device includes:

[0156] Step S410, control the centrifugation device to rotate at the first rotation speed and perform liquid extraction to concentrate the target cell solution to the second predetermined volume;

[0157] Step S420: Input a sixth predetermined amount of washing magnetic bead sorting buffer into the centrifuge container;

[0158] Step S430: Control the centrifuge equipment to rotate at the first rotation speed and perform liquid extraction to concentrate the target cell solution to a second predetermined volume.

[0159] Step S440: Input a fourth predetermined amount of culture medium into the centrifuge container;

[0160] Step S450: Control the centrifuge equipment to rotate at the first rotation speed and perform liquid extraction to concentrate the target cell solution to a second predetermined volume;

[0161] Step S460: Input a fifth predetermined amount of culture medium into the centrifuge container;

[0162] Step S470: Transport the mixed solution of the target cell solution and the culture medium to the preparation bag.

[0163] Step S480: Input a fifth predetermined amount of culture medium into the centrifuge container to rinse the centrifuge container;

[0164] Step S490: Transport the culture medium in the centrifuge container to the preparation bag so that the liquid volume in the preparation bag reaches the fourth predetermined volume.

[0165] The blood treatment method provided by the embodiment of the present invention concentrates and dilutes the blood sample through the centrifuge equipment to remove impurities other than blood cells in the blood sample; makes the cell solution and magnetic beads mix through the centrifuge equipment so that the target cells in the cell solution bind to the magnetic beads; separates the target cells from the non-target cells through the magnetic separation equipment, and replaces the extracellular fluid in the target cell solution with the culture medium through the centrifuge equipment; compared with the existing extraction methods, the blood treatment method provided by the embodiment of the present invention realizes the purpose of directly extracting cell culture in single blood collection, improves the harvesting efficiency and recovery rate of the cells to be cultured, and avoids the phenomenon of cell aggregation and conjunctiva. Using the blood treatment method provided by the present invention, the whole process takes 4 hours, can process a maximum of 250 ml of samples, and the maximum cell processing amount is 8E10.

[0166] Finally, it should be noted that the above 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 make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A blood treatment method, characterized in that, Comprising: Concentrating and diluting a blood sample by means of a centrifugal device to remove impurities other than blood cells in the blood sample, thereby obtaining a cell solution; Mixing the cell solution with magnetic beads by means of the centrifugal device, so that target cells in the cell solution bind to the magnetic beads; Separating the target cells from non-target cells by means of a magnetic separation device to obtain a target cell solution; Replacing the extracellular fluid in the target cell solution with a culture medium by means of the centrifugal device.

2. The blood treatment method according to claim 1, characterized in that, The step of concentrating and diluting the blood sample by means of the centrifugal device includes: Inputting a first predetermined amount of the blood sample into a centrifugal container of the centrifugal device and adding a magnetic bead sorting buffer solution to dilute the blood sample to a first predetermined volume; Controlling the centrifugal device to rotate at a first rotational speed for a first predetermined time and perform liquid extraction to concentrate the diluted blood sample, thereby obtaining the cell solution with a second predetermined volume; Controlling the centrifugal device to rotate at a second rotational speed and perform liquid extraction to concentrate the cell solution to a second predetermined volume, wherein the second rotational speed is less than the first rotational speed; Inputting the cell solution into a liquid bag.

3. The blood treatment method according to claim 2, wherein, Before the step of controlling the centrifugal device to rotate at the second rotational speed and perform liquid extraction, it further includes: Inputting a second predetermined amount of the magnetic bead sorting buffer solution into the centrifugal container to dilute the cell solution to a third predetermined volume; Controlling the centrifugal device to rotate at the first rotational speed and perform liquid extraction to concentrate the cell solution to a first predetermined volume.

4. The blood treatment method according to claim 2 or 3, characterized in that The step of mixing the cell solution with magnetic beads by means of the centrifugal device includes: Respectively inputting magnetic beads with antibodies in a magnetic bead bag and the cell solution in the liquid bag into the centrifugal container; Incubating at a predetermined temperature for a second predetermined time; Inputting a third predetermined amount of the magnetic bead sorting buffer solution into the centrifugal container; Controlling the centrifugal device to rotate at the second rotational speed and perform liquid extraction to concentrate the cell solution to a second predetermined volume; Inputting a resuspension solution into the centrifugal container to make the volume of the cell solution reach a fourth predetermined volume.

5. The blood treatment method according to claim 4, wherein Before the step of incubating at the predetermined temperature for the second predetermined time, it further includes: Inputting the magnetic bead sorting buffer solution into the magnetic bead bag for rinsing, and inputting the rinsed magnetic bead sorting buffer solution into the centrifugal container.

6. The blood treatment method according to any one of claims 1 to 3, characterized in that, The step of separating the target cells from non-target cells by means of the magnetic separation device includes: In the case of determining the presence of a magnetic field, inputting the target cell solution combined with magnetic beads into a sorting column, so that the target cells are adsorbed on the sorting column and the non-target cells are transported to a waste liquid bag; Switching the pipeline so that the pipeline after discharging the non-target cells is switched to be communicated with the input pipeline of the centrifugal device; In the case of determining the removal of the magnetic field, inputting a magnetic bead sorting buffer solution into the sorting column to wash out the target cells from the sorting column and transport them to an input pipeline of the centrifugal device.

7. The blood treatment method according to claim 6, wherein Before the step of transporting the target cell solution combined with magnetic beads to the sorting column, it further includes: Connecting the output pipeline of the centrifugal device to the input pipeline of the magnetic separation device.

8. The blood treatment method according to claim 2 or 3, characterized in that, The step of replacing the extracellular fluid in the target cell solution with a culture medium by means of the centrifugation device includes: Controlling the centrifugation device to rotate at a first rotational speed and perform liquid extraction to concentrate the target cell solution to a second predetermined volume; Inputting a fourth predetermined amount of the culture medium into the centrifugation container; Controlling the centrifugation device to rotate at a first rotational speed and perform liquid extraction to concentrate the target cell solution to a second predetermined volume; Inputting a fifth predetermined amount of the culture medium into the centrifugation container; Transporting the mixed solution of the target cell solution and the culture medium to a preparation bag.

9. The blood treatment method according to claim 8, characterized in that Before the step of inputting the fourth predetermined amount of the culture medium into the centrifugation container, it includes: Inputting a sixth predetermined amount of a washing magnetic bead sorting buffer solution into the centrifugation container; Controlling the centrifugation device to rotate at a first rotational speed and perform liquid extraction to concentrate the target cell solution to a second predetermined volume.

10. The blood treatment method according to claim 8, characterized in that After the step of transporting the mixed solution of the target cell solution and the culture medium to the preparation bag, it further includes: Inputting a fifth predetermined amount of the culture medium into the centrifugation container to rinse the centrifugation container; Transporting the culture medium in the centrifugation container to the preparation bag so that the liquid volume in the preparation bag reaches a fourth predetermined volume.

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