A cell processing method and a cell harvesting device

Abstract

The invention relates to a cell processing method, comprising the following steps: S1: filling a centrifugal cup with a cleaning liquid: the centrifugal cup is rotated to maintain a centrifugal state, and the cleaning liquid is passed into the centrifugal cup. After the cleaning liquid fills the centrifugal cup, the cleaning liquid is stopped from being pumped in; S2: continuous flow concentration: a cell sample liquid is pumped into the centrifugal cup, and under the action of centrifugal force, the cells adhere to the wall of the centrifugal cup, and the supernatant is discharged from the centrifugal cup; S3: cleaning the cell sample: the cleaning liquid is pumped into the centrifugal cup, and after the cleaning liquid fills the centrifugal cup, a set volume of the cleaning liquid is continuously pumped in and a centrifugal supernatant of the same volume is synchronously pumped out, and the process is maintained for a certain period of time; S4: breaking up the cell sample: the centrifugal state of the centrifugal cup is stopped, and the centrifugal cup is periodically rotated forward and reversely to break up the clumped cells in the centrifugal cup into single suspended cells; S5: cell resuspending: a cell freezing liquid is passed into the centrifugal cup, and the centrifugal cup is mixed by rotating forward and reversely to mix the cell suspension; S6: collecting cells.

Description

Technical Field

[0001] The present invention relates to the technical field of cell medical devices, and in particular to a cell processing method and a cell harvesting device. Background Art

[0002] Cell therapy refers to the process of using autologous or allogeneic cells from humans to treat diseases after in vitro manipulation and then input into the human body. In order to ensure the therapeutic effect, it is often necessary to concentrate the cells into a single suspended, highly active cell suspension for treatment. When the cell culture is completed, a large number of cells need to be concentrated and washed to remove excess culture medium, microcarriers, microcarrier lysate, digestion fluid and metabolic waste fluid during the culture process. At present, in the biopharmaceutical industry, when harvesting cells from large-scale culture, most automated machines are used to concentrate and clean the cell suspension. However, due to the different states of the initial cell samples, the concentration process often breaks when the automated machine is running, which seriously affects the stability of continuous flow concentration and cleaning. At the same time, during the continuous flow centrifugation process, the cells will cling to the inner wall of the equipment to form a thick layer of cell clumps. How to prepare cells into single-suspended cell preparations, minimize cell damage caused by shear force during the process, and improve cell survival rate are all difficult problems in large-scale cell preparation processes. Summary of the invention

[0003] The present invention provides a cell treatment method, comprising the following steps:

[0004] Step S1: Filling the centrifuge cup with cleaning liquid: The centrifuge cup is rotated to maintain a centrifugal state, and the cleaning liquid is passed into the centrifuge cup. When the cleaning liquid fills the centrifuge cup, the pumping of the cleaning liquid is stopped;

[0005] Step S2: Continuous flow concentration: the cell sample liquid is pumped into the centrifuge cup. Under the action of centrifugal force, the cells adhere to the wall of the centrifuge cup, and the supernatant is discharged from the centrifuge cup;

[0006] Step S3: Washing the cell sample: pumping the washing liquid into the centrifuge cup, and after the washing liquid fills the centrifuge cup, continuously pumping in a certain volume of the washing liquid and synchronously pumping out the same volume of the centrifugal supernatant, and maintaining a certain time until the preset volume of the washing liquid is pumped in;

[0007] Step S4: breaking up the cell sample: stopping the centrifugal cup, rotating the centrifugal cup forward and reverse periodically, and using an intermittent breaking method to break up the clumped cells in the centrifugal cup into single suspended cells;

[0008] Step S5: cell resuspension: the cell freezing solution is passed into the centrifuge cup, and the centrifuge cup is mixed by forward and reverse rotation to mix the cell suspension;

[0009] Step S6: Collect cells: discharge the cell suspension from the centrifuge cup.

[0010] Furthermore, in step S2, before the cell sample liquid is pumped into the centrifuge cup, the cell sample is continuously mixed to keep the cell sample suspended in the cell sample liquid, and the mixed cell sample liquid is pumped into the centrifuge cup.

[0011] Furthermore, before step S5, the above steps S3 to S4 are repeated multiple times to remove impurities in the cell sample solution.

[0012] Further, after step S1 and / or step S2 and / or step S3 are completed, the protein-free solution is pumped into the centrifuge cup to flush the liquid inlet pipeline.

[0013] Furthermore, in step S4 and step S5, the maximum speed of the centrifuge cup is greater than 500 rpm, and the acceleration is greater than 300 rpm.

[0014] The present invention also provides a cell harvesting device, including a base, an operation panel, and a rack. The base is provided with a centrifuge cup accommodating cavity, and the rack is provided with a plurality of hooks for hanging a liquid storage bag. The above-mentioned cell processing method is adopted, the centrifuge cup is located in the centrifuge cup accommodating cavity, and the liquid storage bag includes a cleaning liquid bag, a resuspension liquid bag, and a cell product bag. The cleaning liquid is pre-placed in the cleaning liquid bag, and the cell freezing liquid is pre-placed in the resuspension liquid bag. After the cell suspension is discharged from the centrifuge cup, it is collected in the cell product bag.

[0015] Furthermore, the liquid storage bag also includes a cell sample bag, and the cell sample liquid is pre-placed in the cell sample bag.

[0016] Furthermore, the cell harvesting device also includes a mixing device, which includes a support plate, a hook is provided on the support plate, and a push plate parallel to the support plate is provided at the lower part of the support plate, the hook is used to hang a cell sample bag, the cell sample liquid is pre-placed in the cell sample bag, and the push plate can move relative to the support plate to squeeze the cell sample bag.

[0017] Further, it is characterized in that a plurality of push rods are arranged between the push plate and the support plate, one end of the push rod is fixedly connected to the push plate, and the other end passes through the support plate, and the driving mechanism drives the push rod to move relative to the support plate, so that the push plate can move relative to the support plate.

[0018] Furthermore, it is characterized in that the centrifugal cup includes a cup body, a cup cover, and a cup head, the cup head is provided with a liquid inlet and a liquid inlet and outlet or a liquid outlet, and a plurality of baffles are also provided in the cup body.

[0019] In the present invention, by using a mixing device, the cells can be kept evenly suspended in the liquid storage bag, so that the concentration operation can be performed on the cell harvesting device while the sample is mixed. During the gap of sample concentration, some cells will be deposited in the tiny liquid inlet channel of the centrifuge cup. Long-term accumulation can easily lead to blockage of the centrifuge cup. In the gap of concentrated liquid inlet, the present invention uses a protein-free solution to flush the centrifuge cup liquid inlet pipeline to continuously ensure the smooth flow of the pipeline and jointly solve the problem of automatic concentration interruption. The centrifuge cup adopts an intermittent breaking method, relying on the resistance of multiple baffles in the centrifuge cup against the centrifugal force, to break the cells from the clumping state to the single suspension state, solve the problem of cell clumping, and meet the requirements for the single suspension state of cell preparations in clinical applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 is a structural diagram of a cell harvesting device;

[0021] Figure 2 It is a schematic diagram of the centrifugal cup structure;

[0022] Figure 3 It is a schematic diagram of the liquid pipeline connection of the cell harvesting device;

[0023] Figure 4 1 is a schematic diagram of the structure of a mixing device;

[0024] Figure 5 It is a flow chart of cell processing. DETAILED DESCRIPTION

[0025] See also Figure 1 The cell harvesting device of the present invention comprises a base 1, an operation panel 3, a rack 5, and a mobile rack 6. The base 1 is provided with a centrifuge cup accommodating chamber 2. The rack 5 is provided with a plurality of hooks for hanging liquid storage bags. The liquid storage bags include: a cleaning liquid bag 7, a resuspension liquid bag 8, a cell sample bag 9, and a plurality of cell product bags (10, 11, 12). A waste liquid bag 13 is provided on one side of the base 1. The base 1 is provided on the mobile rack 6.

[0026] The centrifugal cup accommodating chamber 2 is provided with a centrifugal cup 21. Figure 2 The centrifugal cup 21 of the present invention comprises a cup body 211 , a cup cover 212 , and a cup head 213 . The cup head 213 is provided with a liquid inlet 2131 and a liquid inlet and outlet 2132 . A plurality of baffles 214 are also provided in the cup body 211 .

[0027] The operation panel is provided with a peristaltic pump, a plurality of pinch valves, an ultrasonic sensor, a pressure sensor and a liquid pipeline. The liquid storage bag is connected to the centrifuge cup 21 through the liquid pipeline. The ultrasonic sensor is used to determine the presence of liquid in the liquid pipeline. The pressure sensor is used to measure the pressure in the liquid pipeline. The peristaltic pump is used to drive the flow of the liquid. Figure 3The figure is a schematic diagram of the connection between the liquid storage bag and the centrifuge cup through the liquid pipeline, wherein the plurality of pinch valves (311, 312, 313, 314, 315, 316, 317, 318, 319, 3110, 3111, 3112, 3113, 3114) are located on the liquid pipeline and are used to control the flow of liquid in the liquid pipeline.

[0028] The liquid inlet 2131 of the centrifuge cup is connected to the pinch valve 319, and the cell sample is pumped in through the pinch valves 314 and 319, and the cell washing solution is pumped in through the pinch valves 312 and 319. The liquid inlet and outlet 2132 of the centrifuge cup is connected to the pinch valve 3113, and the resuspension is pumped in through the pinch valves 313, 3110 and 3113; when the supernatant in the centrifuge cup is discharged, it is discharged to the waste liquid bag 13 through the pinch valves 3113 and 3111 or 3112; when the cell suspension in the centrifuge cup is discharged, it is discharged to the cell product bag 10 or 11 or 12 through the pinch valves 3113, 315 and 316 or 317 or 318. There are two channels on the upper part of the centrifuge cup 21, the liquid inlet 2131 and the liquid inlet and outlet 2132, which are used for sample pumping and discharging, wherein three sampling tube openings extend from the liquid inlet and outlet 2132 channel.

[0029] For cell sample bag 9, Figure 1 As shown, the cell sample bag is hung on the rack 5, but under normal circumstances, the cells in the cell sample bag gradually accumulate at the bottom of the cell sample bag due to gravity, and the centrifuge cup may be blocked during the process of the sample in the cell sample bag being gradually pumped into the centrifuge cup. The present invention proposes a mixing device to solve this technical problem.

[0030] See also Figure 4 The mixing device includes a support plate 41, and a plurality of hooks 42 are arranged in parallel on the support plate. The upper part of the cell sample bag 9 is hung on the hooks 42 and abuts against the support plate 41. A push plate 43 parallel to the support plate 41 is provided at the lower part of the support plate 41. A plurality of push rods 44 are arranged between the push plate 43 and the support plate. One end of the push rod 44 is fixedly connected to the push plate 43, and the other end can pass through the support plate 41. The driving mechanism (not shown) drives the push rod 44 to move relative to the support plate 41, so that the push plate 43 can move relative to the support plate 41.

[0031] In the present invention, when the push plate 43 moves toward the support plate 41, it squeezes the lower part of the cell sample bag 9, thereby mixing the cells inside the cell sample bag 9. Since the upward squeezing force and the downward gravity are balanced, the cells in the cell sample bag can be kept evenly suspended in the liquid storage bag. Then, the cell sample bag 9 is connected to the cell harvesting device through a liquid pipeline. Figure 3 In the middle, it is connected to the pinch valve 314, so that the sample can be mixed while the concentration operation is performed on the cell harvesting device, and the adjustment of the strength of the mixing device will not cause the automatic concentration to be cut off.

[0032] See also Figure 5 The cell harvesting device of the present invention works as follows:

[0033] S1: Fill the centrifuge cup with cleaning liquid: The centrifuge cup rotates to maintain the centrifugal state, and the cleaning liquid is passed into the centrifuge cup. When the cleaning liquid fills the centrifuge cup, stop pumping in the cleaning liquid.

[0034] Specifically: the centrifuge cup rotates and maintains the centrifugal state, the pinch valves 312, 3110 and 3113 are opened, and the cleaning liquid in the cleaning liquid bag 7 is pumped into the centrifuge cup from the liquid inlet 2131. After the cleaning liquid fills the centrifuge cup (about 250 ml), the pumping of the cleaning liquid is stopped, the pinch valve 312 is closed, the pinch valve 313 is opened, and the protein-free solution (such as physiological saline) in the resuspension liquid bag 8 is pumped into the centrifuge cup, and about 10-30 ml of the liquid inlet pipeline is flushed.

[0035] S2: Continuous flow concentration: The cell sample liquid in the cell sample bag is pumped into the centrifuge cup. Under the action of centrifugal force, the cells adhere to the wall of the centrifuge cup, and the supernatant is discharged from the centrifuge cup. During this process, the mixing device squeezes the cell sample bag 9 to keep the cells suspended in the sample bag.

[0036] After concentration is completed, use a protein-free solution (such as saline) to pump into the centrifuge cup, about 10-30 ml to flush the liquid inlet pipe.

[0037] By closing the pinch valves 313 and 3110, opening the pinch valves 314, 319 and 3111, and keeping the pinch valve 3113 open, the cell sample liquid in the cell sample bag 9 is pumped into the centrifuge cup. At this time, the cell suspension in the cell sample bag 9 passes through the pinch valves 314 and 319 and is pumped into the centrifuge cup through the centrifuge cup liquid inlet 2131. At the same time, the centrifugal supernatant passes through the centrifuge cup liquid inlet and outlet 2132 through the pinch valves 3113 and 3111 and is discharged from the centrifuge cup to the waste liquid bag 13. The cell sample liquid is continuously concentrated until the cell sample is completely concentrated, and the pinch valves 314 and 319 are closed to keep discharging the centrifugal supernatant until the supernatant cannot be discharged (due to the structural design of the centrifuge cup, the remaining liquid in the centrifuge cup is about 70 ml at this time). Close the pinch valve 3111, open the pinch valves 313 and 3110, pump the protein-free solution (such as physiological saline) in the resuspension bag 8 into the centrifuge cup, about 10-30 ml to flush the liquid inlet pipe, and close 313 and 3110.

[0038] During the continuous flow concentration process, as the number of initial samples increases, the concentration time becomes longer. Under normal circumstances, the cells in the cell sample bag 9 gradually accumulate at the bottom of the liquid storage bag due to gravity factors. In the process of gradually pumping the sample in the cell sample bag 9 into the centrifuge cup, the accumulated cell concentrate will be pumped into the centrifuge cup at the same time, so it is very easy to cause the problem of clogging the centrifuge cup. The present invention suspends the cell sample bag 9 on the mixing device, and when the push plate 43 moves toward the support plate 41, it squeezes the lower part of the cell sample bag 9, thereby mixing the cells inside the cell sample bag 9. Since the cells in the cell sample bag 9 are balanced by the upward squeezing force and the downward gravity, they can remain evenly suspended in the liquid storage bag, and the concentration operation can be performed on the cell harvesting device while mixing the sample. At the same time, adjusting the strength of the mixing device will not cause the automatic concentration to be interrupted.

[0039] Furthermore, the cleaning fluid contains rich protein components, and the initial cell sample contains cell clumps of varying sizes. When the cleaning fluid or cell sample inlet process is completed, the cell clumps in the liquid are very likely to adhere to the inner wall of the tiny inlet pipe of the centrifuge cup. Long-term accumulation can easily lead to clogging of the centrifuge cup. At the end of the concentrated liquid inlet, the method uses a protein-free solution to flush the centrifuge cup inlet pipe to continuously ensure that the pipe is unobstructed. It is used in combination with a mixing device to jointly solve the problem of automatic concentration interruption.

[0040] S3: Washing the cell sample: Pump the washing solution into the centrifuge cup, continuously pump in a certain volume of the washing solution and synchronously pump out the same volume of the centrifugal supernatant, and maintain it for a certain period of time until the preset volume of the washing solution is pumped in.

[0041] Keep the centrifugal cup in the centrifugal state, open the pinch valves 312 and 319, pump the cell washing solution in the washing solution bag 7 into the centrifugal cup from the liquid inlet 2131, after about 150 ml, open the pinch valves 3113 and 3111, discharge the centrifugal supernatant from the centrifugal cup from the liquid inlet and outlet 2132 to the waste liquid bag 13. This process will take away the original liquid in the centrifugal cup. After continuously pumping in a set volume of washing solution and synchronously pumping out the same volume of centrifugal supernatant, close the pinch valves 312, 319 and 3111, open the pinch valves 313 and 3110, pump the protein-free solution (such as physiological saline) in the resuspension liquid bag 8 into the centrifugal cup, about 10-30 ml to flush the liquid inlet pipeline, and close the pinch valves 313 and 3110.

[0042] Step S4: breaking up the cell sample: stopping the centrifugal cup, rotating the centrifugal cup forward and reverse periodically, and using an intermittent breaking method to break up the clumped cells in the centrifugal cup into single suspended cells;

[0043] The above steps S3 and S4 are repeated multiple times to achieve the effect of removing impurities such as culture medium in the cell sample solution.

[0044] After long-term and continuous centrifugal adhesion, cells adhere severely to each other. The present invention adopts a left-right intermittent breaking method, relying on the resistance of multiple baffles in the centrifuge cup against the centrifugal force, to break up the cells from a clumping state to a single suspension state, thereby solving the problem of cell clumping and meeting the requirements for a single suspension state of cell preparations in clinical applications.

[0045] Step S5: cell resuspension: replace the liquid in the resuspension liquid bag 8 with the cell freezing liquid, open the pinch valve 313, pump the cell freezing liquid in the resuspension liquid bag 8 into the centrifuge cup through the liquid inlet and outlet, and mix the cell suspension by rotating the centrifuge cup forward and reversely;

[0046] Step S6: Collect cells. Open the pinch valves 3113, 315 and 316 or 317 or 318 to discharge the cell suspension from the liquid inlet and outlet 2132 to the product bag.

[0047] From the cell processing data, it can be seen that when the cell harvesting device is used, the initial cell sample is suspended on the mixing device, which can better mix the cells and will not affect the cell harvesting process. The viability of the harvested cells is 93.68%, which is greater than 90%, meeting the process requirements.

[0048] Comparison of cell disaggregation and mixing effects under different disaggregation and mixing parameters. The data are as follows:

[0049]

[0050] In test group 1, the maximum speed was 300 rpm, and the cell viability was 94%, which was greater than 90%, meeting the requirements. The cell clumping rate was 45%, which was higher than 30%, indicating that this parameter could not break up the cell clusters and did not meet the requirements. It was necessary to increase the intensity of breaking and mixing.

[0051] In test group 2, the maximum speed was 500 rpm and the acceleration was 100 rpm. During the multiple breaking and mixing processes, the cell pellet could not be completely opened, and there were visible cell clusters. When setting the parameters in the final cell resuspension step, the parameters were changed to a maximum speed of 500 rpm and an acceleration of 300 rpm. The disappearance of the cell clusters was observed, and the final cell agglomeration rate was 19.72%, less than 30%, which met the requirements.

[0052] In test group 3, the maximum speed was 500 rpm, the acceleration was 300 rpm, and the cell viability was 96.81%, which was greater than 90% and met the requirements. The cell clumping rate was 20.01%, which was less than 30%, which met the requirements. This shows that this parameter can well break up and mix the precipitated cells and clumped cells to make the cells single-suspended.

[0053] In the above embodiment, the centrifuge cup has a liquid inlet 2131 and a liquid inlet and outlet 2132. In step S1, the protein-free solution in the cleaning liquid and the resuspension liquid bag is pumped into the centrifuge cup through the liquid inlet and outlet 2132. It can be understood by those skilled in the art that the liquid inlet and outlet 2132 can also be used only for liquid discharge, and the liquid inlet and outlet are liquid outlets, that is, the centrifuge cup has a liquid inlet and a liquid outlet. In this way, in step S1, the protein-free solution in the cleaning liquid and the resuspension liquid bag is pumped into the centrifuge cup through the liquid inlet 2131 without having to pass through the original liquid inlet and outlet 2132.

[0054] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A cell harvesting device, comprising a base (1), an operating panel (3), a rack (5), a centrifugal cup, and a mixing device, wherein the base (1) is provided with a centrifugal cup accommodating chamber (2), the rack (5) is provided with a plurality of hooks for hanging liquid storage bags, and a waste liquid bag (13) is provided on one side of the base (1); It is characterized in that The centrifuge cup is located in the centrifuge cup accommodating chamber (2), the liquid storage bag comprises a cleaning liquid bag (7), a resuspension liquid bag (8), and a cell product bag, the cleaning liquid is pre-placed in the cleaning liquid bag (7), the cell freezing liquid is pre-placed in the resuspension liquid bag (8), and the cell suspension is discharged from the centrifuge cup and collected in the cell product bag; The centrifugal cup comprises a cup body (211), a cup cover (212), and a cup head (213); the cup head (213) is provided with a liquid inlet (2131) and a liquid inlet and outlet (2132) or a liquid outlet; and a plurality of baffles (214) are further provided in the cup body (211); The mixing device comprises a support plate (41), a hook (42) is arranged on the support plate (41), a push plate (43) parallel to the support plate (41) is arranged at the lower part of the support plate (41), the hook (42) is used to hang a cell sample bag (9), the cell sample liquid is pre-placed in the cell sample bag (9), and the push plate (43) can move relative to the support plate (41) to squeeze the cell sample bag (9); A plurality of push rods (44) are arranged between the push plate (43) and the support plate (41), one end of the push rod (44) is fixedly connected to the push plate (43), and the other end passes through the support plate (41), and the driving mechanism drives the push rod (44) to move relative to the support plate (41), so that the push plate (43) can move relative to the support plate (41); The operation panel is provided with a peristaltic pump, a plurality of pinch valves, an ultrasonic sensor, a pressure sensor and a liquid pipeline; the liquid storage bag, the cell sample bag (9) and the waste liquid bag (13) are connected to the centrifuge cup (21) via the liquid pipeline; the ultrasonic sensor is used to determine the presence of liquid in the liquid pipeline; the pressure sensor is used to measure the pressure in the liquid pipeline; the peristaltic pump is used to drive the flow of the liquid; the plurality of pinch valves are located on the liquid pipeline and are used to control the flow of the liquid in the liquid pipeline.

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