Blending device and cell harvesting equipment

Abstract

The utility model relates to a blending device and cell harvesting equipment, which comprises a support plate, a hook is arranged on the support plate, a push plate parallel to the support plate is arranged at the lower part of the support plate, the hook is used for hanging a cell sample bag, and the push plate can move relative to the support plate so as to extrude the cell sample bag. The cell harvesting equipment comprises a machine base, a hanging frame and the uniform mixing device, a centrifugal cup containing cavity is formed in the machine base, a plurality of liquid storage bags are hung on the hanging frame, and the centrifugal cup containing cavity is used for containing the centrifugal cups.

Description

Technical Field

[0001] The utility model relates to the technical field of cell medical devices, in particular to a mixing device and cell harvesting equipment using the same. Background Art

[0002] Cell therapy refers to the process of using cells from a person's own or allogeneic sources to be manipulated in vitro and then introduced into the human body for the treatment of diseases. 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 after culture. 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 liquid during the culture process. Currently, in the biopharmaceutical industry, when harvesting cells from large-scale culture, most automated machines are used to concentrate and wash the cell suspension. However, due to the different states of the initial cell samples, the concentration process often breaks down when the automated machine is running, seriously affecting the stability of the continuous flow concentration and washing work. At the same time, during the continuous flow centrifugation process, the cells will cling to the inner wall of the equipment, forming 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 the large-scale cell preparation process. Summary of the Invention

[0003] The utility model provides a mixing device, comprising a support plate, a hook is provided on the support plate, 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, and the push plate can move relative to the support plate to squeeze the cell sample bag.

[0004] Furthermore, a plurality of push rods are provided 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. 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.

[0005] The present invention also provides a cell harvesting device, comprising a machine base and a hanging rack, wherein a centrifuge cup accommodating cavity is provided in the machine base, a plurality of liquid storage bags are hung on the hanging rack, the centrifuge cup accommodating cavity is used to accommodate the centrifuge cup, and the device also comprises the above-mentioned mixing device.

[0006] Furthermore, the liquid storage bag includes a cleaning liquid bag, a resuspension liquid bag, and multiple cell product bags.

[0007] Furthermore, 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. The cup body is also provided with a plurality of baffles.

[0008] Furthermore, the base is arranged on a movable frame.

[0009] Furthermore, a waste liquid bag is provided on one side of the machine base, and the waste liquid bag is connected to the centrifuge cup.

[0010] Furthermore, the cell harvesting device also includes an operation panel, on which a peristaltic pump, multiple pinch valves, sensors and liquid pipelines are provided. The liquid storage bag is connected to the centrifuge cup through the liquid pipeline, and the peristaltic pump is used to drive the flow of liquid.

[0011] Furthermore, the machine base has an upper table, on which a peristaltic pump, a plurality of pinch valves, a sensor and a liquid pipeline are arranged, and the upper opening of the centrifuge cup accommodating cavity is also located on the upper table.

[0012] Furthermore, the sensor includes an ultrasonic sensor and / or a pressure sensor, the ultrasonic sensor is used to determine the presence of liquid in the liquid pipeline, and the pressure sensor is used to measure the pressure in the liquid pipeline.

[0013] In this utility model, the mixing device maintains a uniform suspension of cells in the liquid storage bag, allowing the sample to be mixed while the concentration operation is performed on the cell harvester, thus solving the problem of clogging the centrifuge cup. The centrifuge cup uses an intermittent breaking method, relying on the resistance of multiple baffles inside the centrifuge cup against the centrifugal force to break up the cells from a clumping state to a single suspension state, thus solving the problem of cell clumping and meeting the requirement for a single suspension state for cell preparations in clinical applications.

[0014] In addition, devices such as the peristaltic pump, pinch valve, sensor, and liquid pipeline are arranged on the upper surface of the base, which reduces the overall height of the cell harvesting device and is more conducive to the miniaturization of the device. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is a diagram of the cell harvesting equipment structure;

[0016] Figure 2 It is a schematic diagram of the centrifugal cup structure;

[0017] Figure 3 This is a schematic diagram of the liquid pipeline connection of the cell harvesting equipment;

[0018] Figure 4 1 is a schematic diagram of the structure of the mixing device;

[0019] Figure 5 It is a flow chart of cell processing;

[0020] Figure 6 is a structural diagram of another embodiment of a cell harvesting device. DETAILED DESCRIPTION

[0021] See also Figure 1The cell harvesting device of the present invention includes a base 1, an operating panel 3, a hanger 5, and a mobile rack 6. The base 1 is provided with a centrifuge cup accommodating chamber 2. The hanger 5 is provided with multiple 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 multiple cell product bags (10, 11, 12). A waste liquid bag 13 is provided on one side of the base 1. The base 1 is set on the mobile rack 6.

[0022] The centrifugal cup accommodating chamber 2 is provided with a centrifugal cup 21. Figure 2 The centrifugal cup 21 of the present invention includes 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 .

[0023] The control panel is provided with a peristaltic pump, multiple 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 3 The figure is a schematic diagram of the connection between the liquid storage bag and the centrifuge cup through the liquid pipeline. The multiple 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.

[0024] exist Figure 1 In the figure, the surface of the opening of the centrifuge cup accommodating chamber 2 is a horizontal plane, and the operation panel 3 has a certain angle with the horizontal plane. Although it is convenient for the user to operate, in order to ensure that the various liquid storage bags mounted on the rack 5 do not hinder the user's operation, the rack 5 needs to be designed to be higher, which makes the height of the entire device higher. Figure 6 Another embodiment of a cell harvesting device is provided. The base 1 has an upper surface. The peristaltic pump, multiple pinch valves, ultrasonic sensor, pressure sensor, and liquid pipelines on the operating panel are arranged on the upper surface. The upper opening of the centrifuge cup accommodating chamber 2 is also located on the upper surface. A display screen 18 is provided on the front of the base 1. This design reduces the overall height of the cell harvesting device, further facilitating its miniaturization.

[0025] For cell sample bag 9, Figure 1As shown, the cell sample bag is hung on a rack 5. However, under normal circumstances, the cells in the cell sample bag gradually accumulate at the bottom of the cell sample bag due to gravity. As the sample in the cell sample bag is gradually pumped into the centrifuge cup, the centrifuge cup may become clogged. Therefore, the present invention proposes a mixing device to solve this technical problem.

[0026] See also Figure 4 The mixing device includes a support plate 41, which is provided with a plurality of hooks 42 arranged in parallel. The upper part of the cell sample bag 9 is hung on the hook 42 and abuts against the support plate 41. A push plate 43 is provided at the lower part of the support plate 41 and is parallel to the support plate 41. A plurality of push rods 44 are provided 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.

[0027] When the push plate 43 moves toward the support plate 41, the lower part of the cell sample bag 9 is squeezed, thereby mixing the cells inside the cell sample bag 9. The cells in the cell sample bag are kept evenly suspended in the liquid storage bag because the upward squeezing force and the downward gravity are balanced. Then, the cell sample bag 9 is connected to the cell harvesting device through the 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 adjusting the strength of the mixing device will not cause the automatic concentration to stop.

[0028] The centrifuge cup's liquid inlet 2131 is connected to a pinch valve 319. Cell samples are pumped in through pinch valves 314 and 319, while cell wash solution is pumped in through pinch valves 312 and 319. The centrifuge cup's liquid inlet and outlet 2132 are connected to pinch valve 3113. Resuspension solution is pumped in through pinch valves 313, 3110, and 3113. When the supernatant is discharged from the centrifuge cup, it is discharged into waste liquid bag 13 through pinch valves 3113 and 3111, or 3112. When the cell suspension is discharged from the centrifuge cup, it is discharged into cell product bag 10, 11, or 12 through pinch valves 3113, 315, and 316, or 317, or 318. The centrifuge cup 21 has two channels on its upper portion: liquid inlet 2131 and liquid inlet and outlet 2132. These channels are used for pumping in and out samples. Three sampling ports extend from the liquid inlet and outlet 2132.

[0029] See also Figure 5 The working process of the cell harvesting device of the present invention is as follows:

[0030] 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. After the cleaning liquid fills the centrifuge cup, stop pumping in the cleaning liquid.

[0031] 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 centrifuge cup is filled with the cleaning liquid (about 250 ml), the pumping of the cleaning liquid is stopped, the pinch valve 312 is closed, and the pinch valve 313 is opened. The protein-free solution (e.g., physiological saline) in the resuspension liquid bag 8 is pumped into the centrifuge cup to flush the liquid inlet pipe with about 10-30 ml.

[0032] 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.

[0033] 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.

[0034] By closing pinch valves 313 and 3110, opening pinch valves 314, 319, and 3111, and keeping pinch valve 3113 open, the cell sample liquid in cell sample bag 9 is pumped into the centrifuge cup. At this point, the cell suspension in cell sample bag 9 passes through pinch valves 314 and 319 and is pumped into the centrifuge cup through the centrifuge cup liquid inlet 2131. Simultaneously, the centrifuged supernatant flows out of the centrifuge cup through the centrifuge cup liquid inlet and outlet 2132, through pinch valves 3113 and 3111, and into waste bag 13. The cell sample liquid concentration process continues until the cell sample is completely concentrated. Pinch valves 314 and 319 are then closed, and the supernatant continues to be discharged until no more supernatant can be discharged (due to the centrifuge cup's structural design, approximately 70 ml of liquid remains in the centrifuge cup at this point). 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.

[0035] 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. 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. 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 9 can be kept evenly suspended in the liquid storage bag. The concentration operation can be performed on the cell harvesting device while the sample is mixed. At the same time, adjusting the strength of the mixing device will not cause the automatic concentration to stop.

[0036] 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 is added, the cell clumps in the liquid are very likely to adhere to the inner wall of the tiny liquid inlet pipe of the centrifuge cup. Long-term accumulation can easily cause clogging of the centrifuge cup. At the end of the concentration liquid addition, this method uses a protein-free solution to flush the liquid inlet pipe of the centrifuge cup to continuously ensure that the pipe is unobstructed. When used in conjunction with a mixing device, the problem of flow interruption in automated concentration is solved.

[0037] S3: Washing the cell sample: Pump the washing solution into the centrifuge cup, continuously pumping in a certain volume of washing solution and synchronously pumping out the same volume of centrifugal supernatant, and maintain a certain time until the preset volume of washing solution is pumped in.

[0038] Keep the centrifuge cup in a centrifugal state, open the pinch valves 312 and 319, and pump the cell washing fluid in the washing fluid bag 7 into the centrifuge cup from the liquid inlet 2131. After about 150 ml, open the pinch valves 3113 and 3111, and discharge the centrifugal supernatant from the centrifuge cup from the liquid inlet and outlet 2132 to the waste liquid bag 13. This process will take away the original liquid in the centrifuge cup. After continuously pumping in a set volume of washing fluid 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, and pump the protein-free solution (e.g., physiological saline) in the resuspension fluid bag 8 into the centrifuge cup. About 10-30 ml of the solution is flushed into the liquid inlet pipe, and then close the pinch valves 313 and 3110.

[0039] Step S4: breaking up the cell sample: stopping the centrifugal cup, rotating the centrifuge cup periodically forward and reverse, and using an intermittent breaking method to break up the clumped cells in the centrifuge cup into single suspended cells;

[0040] Repeat the above steps S3 and S4 multiple times to remove impurities such as culture medium in the initial cell sample solution.

[0041] After long-term and continuous centrifugation, cells will adhere to each other seriously. 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, thus solving the problem of cell clumping and meeting the requirement for a single suspension state of cell preparations in clinical applications.

[0042] Step S5: Resuspending cells: Replace the liquid in the resuspension bag 8 with a cell freezing solution. Open the pinch valve 313 and pump the cell freezing solution in the resuspension bag 8 into the centrifuge cup through the liquid inlet and outlet. Mix the cell suspension by rotating the centrifuge cup forward and reverse.

[0043] 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 into the product bag.

[0044] The initial cell state indicates that, when the cell harvester is in operation, the initial cell sample is suspended on the mixing device, which allows for good cell mixing without affecting the cell harvesting process. The harvested cell viability is 93.68%, exceeding 90%, meeting the process requirements.

[0045] Comparison of cell disaggregation effects under different mixing parameters. The data are as follows:

[0046]

[0047]

[0048] In test group 1, the highest mixing speed was 300 rpm, and the cell viability was 94%, exceeding 90%, meeting the requirement. However, the cell clumping rate was 45%, exceeding 30%, indicating that this parameter was insufficient to break up the cell clumps and did not meet the requirement. Increasing the mixing intensity is necessary.

[0049] In Experimental Group 2, the cell pellet failed to fully disintegrate during multiple mixing cycles, with a high mixing speed of 500 rpm and a high mixing speed of 100 rpm. Visible cell clumps were present. When the mixing parameters were adjusted to a high mixing speed of 500 rpm and a high mixing speed of 300 rpm during the final cell resuspension step, the cell clumps disappeared, resulting in a final cell clumping rate of 19.72%, less than 30%, meeting the requirement.

[0050] In test group 3, with a maximum mixing speed of 500 rpm and a mixing acceleration of 300 rpm, the cell viability was 96.81%, exceeding 90%, meeting the requirement. The cell clumping rate was 20.01%, less than 30%, meeting the requirement. This indicates that these parameters effectively separate pelleted and clumped cells, allowing cells to be suspended in a single stream.

[0051] In the above embodiment, the centrifuge cup has a liquid inlet 2131 and a liquid inlet and outlet 2132. In step S1, the cleaning solution and the protein-free solution in the resuspension bag are pumped into the centrifuge cup through the liquid inlet and outlet 2132. Those skilled in the art will appreciate that the liquid inlet and outlet 2132 can also be used solely for liquid discharge, with the liquid inlet and outlet serving as the liquid outlet, meaning that the centrifuge cup has both a liquid inlet and a liquid outlet. Thus, in step S1, the protein-free solution in the cleaning solution and the resuspension bag is pumped into the centrifuge cup through the liquid inlet 2131, without having to pass through the original liquid inlet and outlet 2132.

[0052] 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 can still be modified, or some of the technical features thereof can 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 various embodiments of the present invention.

Claims

1. A mixing device, characterized in that: The invention comprises a support plate (41), a hook (42) is provided on 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), the hook (42) is used to hang a cell sample bag (9), and the push plate (43) can move relative to the support plate (41) to squeeze the cell sample bag (9).

2. The mixing device according to claim 1, characterized in that A plurality of push rods (44) are provided 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). 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).

3. A cell harvesting device, comprising a base (1) and a rack (5), wherein a centrifuge cup accommodating chamber (2) is provided in the base (1), a plurality of liquid storage bags are hung on the rack (5), and the centrifuge cup accommodating chamber (2) is used to accommodate a centrifuge cup (21), characterized in that: Also includes the mixing device according to any one of claims 1-2.

4. The cell harvesting device according to claim 3, characterized in that The liquid storage bag comprises a cleaning liquid bag (7), a resuspension liquid bag (8), and a plurality of cell product bags (10, 11, 12).

5. The cell harvesting device according to claim 3, characterized in that The centrifugal cup (21) 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. A plurality of baffles (214) are also provided in the cup body (211).

6. The cell harvesting device according to claim 3, characterized in that The machine base (1) is arranged on a movable frame (6).

7. The cell harvesting device according to claim 3, characterized in that A waste liquid bag (13) is provided on one side of the machine base (1), and the waste liquid bag (13) is communicated with the centrifuge cup (21).

8. The cell harvesting device according to claim 3, characterized in that The cell harvesting device further comprises an operation panel, on which a peristaltic pump, a plurality of pinch valves, a sensor and a liquid pipeline are arranged. The liquid storage bag is connected to the centrifuge cup (21) through the liquid pipeline, and the peristaltic pump is used to drive the flow of the liquid.

9. The cell harvesting device according to claim 3, characterized in that The machine base (1) has an upper table, on which a peristaltic pump, a plurality of pinch valves, a sensor and a liquid pipeline are arranged, and the upper opening of the centrifuge cup accommodating chamber (2) is also located on the upper table.

10. The cell harvesting device according to claim 8 or 9, characterized in that The sensor includes an ultrasonic sensor and / or a pressure sensor. The ultrasonic sensor is used to determine the presence of liquid in the liquid pipeline, and the pressure sensor is used to measure the pressure in the liquid pipeline.

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