Cell processing method and cell harvesting device

Abstract

The invention relates to a cell treatment method which comprises the following steps: S1, filling a centrifugal cup with a cleaning solution, namely rotating the centrifugal cup to keep a centrifugal state, introducing the cleaning solution into the centrifugal cup, and stopping pumping the cleaning solution after the centrifugal cup is filled with the cleaning solution; s2, continuous flow concentration: pumping the cell sample liquid into a centrifugal cup, enabling cells to be attached to the wall of the centrifugal cup under the action of centrifugal force, and discharging supernate out of the centrifugal cup; s3, cleaning the cell sample: pumping the cleaning solution into the centrifugal cup, after the centrifugal cup is filled with the cleaning solution, continuously pumping the cleaning solution with a set volume and synchronously pumping out a centrifugal supernatant with the same volume, and maintaining for a certain time; s4, scattering the cell sample: stopping the centrifugal state of the centrifugal cup, periodically rotating the centrifugal cup forwards and backwards, and scattering clustered cells in the centrifugal cup into single suspension cells; s5, cell resuspension: introducing the cell cryopreservation liquid into a centrifugal cup, and uniformly mixing the centrifugal cup in a positive and negative rotation manner to uniformly mix the cell suspension; s6, collecting the cells.

Description

[0001] This application is a divisional application of a Chinese invention patent application with an application number of 202411489245.5, an application date of October 24, 2024, and an invention title of "A Cell Processing Method and a Cell Harvesting Device". Technical Field

[0002] The present invention relates to the technical field of cell medical devices, and particularly to a cell processing method and a cell harvesting device. Background Art

[0003] Cell therapy refers to the process of applying cells derived from autologous or allogeneic human sources after in vitro manipulation and inputting them into the human body for disease treatment. To ensure the treatment effect, cells are often cultured and then concentrated into a single-suspended and highly active cell suspension for treatment. When cell culture is completed, a large number of cells need to be concentrated and washed to remove excess culture medium, microcarriers, microcarrier lysate, digestive juice, metabolic waste liquid, etc. during the culture process. In the current biomedical industry, when harvesting cells cultured on a large scale, automated machines are mostly used for the concentration and washing of cell suspensions. However, due to the inconsistent initial cell sample states, problems such as the interruption of the concentration process often occur during the operation of automated machines, seriously affecting the stability of continuous-flow concentration and washing operations. At the same time, during continuous-flow centrifugation, cells will adhere tightly to the inner wall of the device, forming a thick layer of cell clumps. How to prepare cells into a single-suspended cell preparation, 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

[0004] The present invention provides a cell processing method, including the following steps:

[0005] Step S1: Fill the centrifuge cup with cleaning liquid: Keep the centrifuge cup rotating to maintain a centrifugal state, pump the cleaning liquid into the centrifuge cup, and stop pumping the cleaning liquid after the centrifuge cup is filled with the cleaning liquid;

[0006] Step S2: Continuous-flow concentration: Pump the cell sample liquid into the centrifuge cup. Under the action of centrifugal force, the cells adhere to the centrifuge cup wall, and the supernatant is discharged from the centrifuge cup;

[0007] Step S3: Wash the cell sample: Pump the cleaning liquid into the centrifuge cup. After the centrifuge cup is filled with the cleaning liquid, continuously pump a certain volume of cleaning liquid and simultaneously pump out the same volume of centrifugal supernatant, and maintain for a certain period of time until the pumping of the preset volume of cleaning liquid is completed;

[0008] Step S4: Disperse the cell sample: Stop the centrifugal state of the centrifuge cup, rotate the centrifuge cup periodically forward and backward, and use an intermittent dispersion method to disperse the clumped cells in the centrifuge cup into single-suspended cells;

[0009] Step S5: Cell resuspension: Feed the cell cryopreservation solution into the centrifuge cup, and mix the centrifuge cup in a forward and reverse rotation manner to mix the cell suspension;

[0010] Step S6: Collect cells: Drain the cell suspension from the centrifuge cup.

[0011] Further, in Step S2, before the cell sample solution is pumped into the centrifuge cup, continuously mix the cell sample to keep the cell sample suspended in the cell sample solution, and the mixed cell sample solution is pumped into the centrifuge cup.

[0012] Further, before Step S5, the above Steps S3 to S4 are repeated multiple times to remove impurities in the cell sample solution.

[0013] Further, after the end of Step S1 and / or Step S2 and / or Step S3, a protein-free solution is pumped into the centrifuge cup to rinse the inlet pipeline.

[0014] Further, 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.

[0015] The present invention also provides a cell harvesting device, including a machine base, an operation panel, and a hanging rack. A centrifuge cup accommodation cavity is provided inside the machine base. Multiple hooks for hanging liquid storage bags are provided on the hanging rack. Using the above cell processing method, the centrifuge cup is located in the centrifuge cup accommodation cavity. The liquid storage bags include a cleaning solution bag, a resuspension solution bag, and a cell product bag. The cleaning solution is pre-placed in the cleaning solution bag, the cell cryopreservation solution is pre-placed in the resuspension solution bag, and the cell suspension is collected in the cell product bag after being drained from the centrifuge cup.

[0016] Further, the liquid storage bag further includes a cell sample bag, and the cell sample solution is pre-placed in the cell sample bag.

[0017] Further, the cell harvesting device further includes a mixing device. The mixing device includes a support plate. Hooks are provided on the support plate. A push plate parallel to the lower part of the support plate is provided. The hooks are used to hang the cell sample bag, and the cell sample solution is pre-placed in the cell sample bag. The push plate can move relative to the support plate to squeeze the cell sample bag.

[0018] Further, multiple 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.

[0019] Further, it is characterized in that the centrifuge cup includes a cup body, a cup lid, and a cup head. The cup head is provided with a liquid inlet and a liquid inlet / outlet or a liquid outlet, and a plurality of baffles are further arranged in the cup body.

[0020] In the present invention, by adopting a mixing device, cells can be kept evenly suspended in the storage bag, so that the sample can be mixed while the concentration operation is carried out on the cell harvesting device. During the gap of sample concentration, some cells will deposit in the tiny liquid inlet channel of the centrifuge cup, and long-term accumulation is likely to cause blockage of the centrifuge cup. In the present invention, during the gap of concentrated liquid inlet, a protein-free solution is used to flush the liquid inlet pipeline of the centrifuge cup to continuously ensure the smoothness of the pipeline, jointly solving the problem of automatic concentration interruption. The centrifuge cup adopts an intermittent dispersion method, relying on the resistance of multiple baffles in the centrifuge cup opposite to the centrifugal force, to disperse the cells from the agglomerated state into a single suspension state, solving the problem of cell agglomeration and meeting the requirements for the single suspension state of cell preparations in clinical applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 is a structural diagram of the cell harvesting device;

[0022] Figure 2 is a schematic structural diagram of the centrifuge cup;

[0023] Figure 3 is a schematic diagram of the liquid pipeline connection of the cell harvesting device;

[0024] Figure 4 is a schematic structural diagram of the mixing device;

[0025] Figure 5 is a cell processing flow chart. DETAILED DESCRIPTION OF THE INVENTION

[0026] See Figure 1 , the cell harvesting device of the present invention includes a machine base 1, an operation panel 3, a hanging rack 5, and a moving rack 6. A centrifuge cup accommodating cavity 2 is arranged in the machine base 1. A plurality of hooks for hanging storage bags are arranged on the hanging rack 5. The 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 arranged on one side of the machine base 1. The machine base 1 is arranged on the moving rack 6.

[0027] A centrifuge cup 21 is arranged in the centrifuge cup accommodating cavity 2. See Figure 2 , the centrifuge cup 21 of the present invention includes a cup body 211, a cup lid 212, and a cup head 213. The cup head 213 is provided with a liquid inlet 2131 and a liquid inlet / outlet 2132. A plurality of baffles 214 are further arranged in the cup body 211.

[0028] 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 communicated with the centrifuge cup 21 through the liquid pipeline. The ultrasonic sensor is used to judge 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 liquid. Figure 3 As shown in the connection schematic diagram of the liquid storage bag and the centrifuge cup through the liquid pipeline, 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.

[0029] The liquid inlet 2131 of the centrifuge cup is connected to the pinch valve 319. The cell sample is pumped in through the pinch valves 314 and 319, and the cell washing liquid 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. The resuspension liquid is pumped in through the pinch valves 313, 3110 and 3113. When discharging the supernatant in the centrifuge cup, it is discharged to the waste liquid bag 13 through the pinch valves 3113 and 3111 or 3112. When discharging the cell suspension in the centrifuge cup, 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 2 channels in the upper part of the centrifuge cup 21, namely the liquid inlet 2131 and the liquid inlet and outlet 2132, which are used for sample pumping in and discharging. Among them, three sampling pipe orifices extend out on the channel of the liquid inlet and outlet 2132.

[0030] For the cell sample bag 9, as Figure 1 shown, it is hung on the hanger 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. During the process of gradually pumping the sample in the cell sample bag into the centrifuge cup, the problem of blocking the centrifuge cup will occur. In the present invention, a mixing device is proposed to solve this technical problem.

[0031] See Figure 4 , the mixing device includes a support plate 41. 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 hook 42 and abuts against the support plate 41. A push plate 43 parallel to it is arranged 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. A 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.

[0032] When the push plate 43 moves towards the support plate 41 in the present invention, the lower part of the cell sample bag 9 will be squeezed, thereby mixing the cells inside the cell sample bag 9. Since the upward squeezing force and the downward gravity of the cells in the cell sample bag reach equilibrium, the cells 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. Specifically, Figure 3 in [description of a specific part], it is connected to the pinch valve 314, so that the sample can be mixed while the concentration operation is carried out on the cell harvesting device, and adjusting the strength of the mixing device will not cause the interruption of the automatic concentration.

[0033] See Figure 5 , the working process of the cell harvesting device of the present invention is as follows:

[0034] S1: Fill the centrifuge cup with cleaning liquid: The centrifuge cup rotates to maintain the centrifugal state, and the cleaning liquid is introduced into the centrifuge cup. After the centrifuge cup is filled with the cleaning liquid, stop pumping the cleaning liquid.

[0035] Specifically: The centrifuge cup rotates to maintain the centrifugal state. Open the pinch valves 312, 3110 and 3113, and pump the cleaning liquid in the cleaning liquid bag 7 into the centrifuge cup from the liquid inlet 2131. After the centrifuge cup is filled with the cleaning liquid (about 250 ml), stop pumping the cleaning liquid, close the pinch valve 312, and open the pinch valve 313 to pump the protein-free solution (such as normal saline) in the resuspension liquid bag 8 into the centrifuge cup, and rinse the liquid inlet pipeline with about 10 - 30 ml.

[0036] S2: Continuous flow concentration: Pump the cell sample liquid in the cell sample bag into the centrifuge cup. Under the action of centrifugal force, the cells adhere to the inner 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.

[0037] After the concentration is completed, pump a protein-free solution (such as normal saline) into the centrifuge cup, and rinse the liquid inlet pipeline with about 10 - 30 ml.

[0038] By closing the pinch valves 313 and 3110, opening the pinch valves 314, 319 and 3111, and keeping the pinch valve 3113 open, pump the cell sample liquid in the cell sample bag 9 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 liquid inlet 2131 of the centrifuge cup. At the same time, the centrifugal supernatant passes through the liquid inlet and outlet 2132 of the centrifuge cup and is discharged from the centrifuge cup to the waste liquid bag 13 through the pinch valves 3113 and 3111. Continuously concentrate the cell sample liquid until all the cell samples are concentrated. Then close the pinch valves 314 and 319, and keep discharging the centrifugal supernatant until the supernatant cannot be discharged (due to the structural design of the centrifuge cup, at this time, there is about 70 ml of remaining liquid in the centrifuge cup). Close the pinch valve 3111, open the pinch valves 313 and 3110, and pump the protein-free solution (such as physiological saline) in the resuspension bag 8 into the centrifuge cup to rinse the liquid inlet pipeline for about 10 - 30 ml, and then close 313 and 3110.

[0039] During the continuous flow concentration process, as the initial sample quantity increases, the concentration time becomes longer. Under normal circumstances, due to gravity, the cells in the cell sample bag 9 gradually accumulate at the bottom of the liquid storage bag. During the process of gradually pumping the sample in the cell sample bag 9 into the centrifuge cup, the accumulated thick cell liquid may be pumped into the centrifuge cup at the same time, so it is very easy to cause the problem of blocking the centrifuge cup. In the present invention, the cell sample bag 9 is suspended on the mixing device. When the push plate 43 moves towards the support plate 41, it will squeeze the lower part of the cell sample bag 9, thereby mixing the cells inside the cell sample bag 9. The cells in the cell sample bag 9 reach an equilibrium due to the upward squeezing force and the downward gravity, so they can remain evenly suspended in the liquid storage bag. While mixing the sample, the concentration operation is carried out on the cell harvesting device, and at the same time, adjusting the force of the mixing device will not cause the interruption of automatic concentration.

[0040] Furthermore, the cleaning solution contains rich protein components, and the initial cell sample contains cell clumps of different sizes. At the end of the cleaning solution or cell sample liquid inlet process, the cell clumps in this liquid are very likely to adhere to the inner wall of the tiny liquid inlet pipeline of the centrifuge cup, and long-term accumulation is likely to cause blockage of the centrifuge cup. In this method, at the end of the concentrated liquid inlet, a protein-free solution is used to rinse the liquid inlet pipeline of the centrifuge cup to continuously ensure the smoothness of the pipeline, and it is used in combination with the mixing device to jointly solve the problem of automatic concentration interruption.

[0041] S3: Clean the cell sample: Pump the cleaning solution into the centrifuge cup, continuously pump a certain volume of cleaning solution and simultaneously pump out the same volume of centrifugal supernatant, and maintain for a certain period of time until the preset volume of cleaning solution is pumped in.

[0042] Maintain the centrifugal state of the centrifuge cup, open the pinch valves 312 and 319, pump the cell washing solution in the washing solution 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 liquid inlet and outlet 2132 out of the centrifuge cup into the waste liquid bag 13. This process will carry away the original liquid in the centrifuge cup. After continuously pumping in the set volume of washing solution and simultaneously 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 (such as physiological saline) in the resuspension bag 8 into the centrifuge cup. Rinse the liquid inlet pipeline with about 10 - 30 ml and then close the pinch valves 313 and 3110.

[0043] Step S4: Disperse the cell sample: Stop the centrifugal state of the centrifuge cup. The centrifuge cup rotates forward and backward periodically, and the agglomerated cells in the centrifuge cup are dispersed into single-suspended cells by an intermittent dispersion method.

[0044] Repeat the above steps S3 and S4 multiple times to achieve the effect of removing impurities such as the culture medium in the cell sample solution.

[0045] After long-term and continuous centrifugal adhesion, the adhesion between cells is serious. The present invention adopts an intermittent left-right dispersion method, relying on the resistance of multiple baffles in the centrifuge cup opposite to the centrifugal force, to disperse the cells from the agglomerated state into a single-suspended state, solve the problem of cell agglomeration, and meet the requirements for the single-suspended state of cell preparations in clinical applications.

[0046] Step S5: Cell resuspension: Replace the liquid in the resuspension bag 8 with cell cryopreservation solution. Open the pinch valve 313, and pump the cell cryopreservation solution in the resuspension bag 8 into the centrifuge cup through the liquid inlet and outlet, and mix the cell suspension in the centrifuge cup by rotating forward and backward.

[0047] Step S6: Collect the cells. Open the pinch valves 3113, 315 and 316 or 317 or 318, and discharge the cell suspension from the liquid inlet and outlet 2132 into the product bag.

[0048] From the cell processing data, it can be seen that when the cell harvesting device is running, the initial cell sample is suspended on the mixing device, and the cells can be well mixed without affecting the cell harvesting process. The cell viability of the harvested cells is 93.68%, which is greater than 90%, meeting the process use requirements.

[0049] Under different dispersion and mixing parameters, the comparison of cell dispersion and mixing effects is as follows:

[0050]

[0051] Test group 1, the maximum speed is 300 rpm, the cell viability is 94%, greater than 90%, meeting the requirements. The cell aggregation rate is 45%, higher than 30%, indicating that this parameter cannot break up the cell aggregates, not meeting the requirements. It is necessary to increase the intensity of dispersion and mixing.

[0052] Test group 2, the maximum speed is 500 rpm, the acceleration is 100 rpm. During multiple dispersion and mixing processes, the cell precipitate could not be completely broken up, and visible cell aggregates existed. 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. It was observed that the cell aggregates disappeared, and the final cell aggregation rate was 19.72%, less than 30%, meeting the requirements.

[0053] Test group 3, the maximum speed is 500 rpm, the acceleration is 300 rpm, the cell viability is 96.81%, greater than 90%, meeting the requirements. The cell aggregation rate is 20.01%, less than 30%, meeting the requirements. This indicates that this parameter can well break up and mix the precipitated cells and aggregated cells to make the cells single-suspended.

[0054] In the above embodiments, the centrifuge cup has a liquid inlet 2131 and a liquid inlet / outlet 2132. In step S1, the cleaning liquid and the protein-free solution in the resuspension liquid bag are pumped into the centrifuge cup through the liquid inlet / outlet 2132. Those skilled in the art can understand that the liquid inlet / outlet 2132 can also be only for liquid outlet, that is, the liquid inlet / outlet is a liquid outlet, which means the centrifuge cup has a liquid inlet and a liquid outlet. In this way, in step S1, the cleaning liquid and the protein-free solution in the resuspension liquid bag are pumped into the centrifuge cup through the liquid inlet 2131, without passing through the original liquid inlet / outlet 2132.

[0055] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it; 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 cell treatment method, characterized in that: The following steps are involved: 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; 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; 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; 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; 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; Step S6: Collect cells: discharge the cell suspension from the centrifuge cup.

2. The method according to claim 1, characterized in that 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.

3. The method according to claim 1 or 2, characterized in that: Before step S5, the above steps S3 to S4 are repeated multiple times to remove impurities in the cell sample solution.

4. The method according to claim 1 or 2, characterized in that: 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.

5. The method according to claim 1 or 2, characterized in that: 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.

6. A cell harvesting device, comprising a base (1), an operating panel (3), and a rack (5), wherein the base (1) is provided with a centrifuge cup accommodating chamber (2), and the rack (5) is provided with a plurality of hooks for hanging liquid storage bags, characterized in that: The cell processing method according to any one of claims 1 to 5 is adopted, wherein 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.

7. The cell harvesting device according to claim 6, characterized in that: The liquid storage bag also includes a cell sample bag (9), and the cell sample liquid is pre-placed in the cell sample bag (9).

8. The cell harvesting device according to claim 6, characterized in that: The cell harvesting device further comprises a mixing device, the mixing device comprising a support plate (41), a hook (42) being arranged on the support plate (41), a push plate (43) being arranged parallel to the support plate (41) at the lower part of the support plate (41), the hook (42) being used to hang a cell sample bag (9), the cell sample liquid being pre-placed in the cell sample bag (9), and the push plate (43) being movable relative to the support plate (41) so as to squeeze the cell sample bag (9).

9. The cell harvesting device according to claim 8, characterized in that: 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). 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).

10. The cell harvesting device according to claim 6, characterized in that: 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).

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