Cell processing centrifugal cup and high-throughput cell concentration and cleaning device

Abstract

A cell processing centrifugal cup, comprising a cover body (1) and a cup body (2), the cover body (1) being used for closing an upper opening of the cup body (2), and the cup body (2) comprising an upper cup body (21) and a lower cup body (22) which are integrally formed. The centrifugal cup is internally provided with a circular sleeve (7); the upper end of the circular sleeve (7) is fixedly connected to a lower portion of the cover body (1), and the lower end thereof extends into the lower cup body (22) and is suspended; and a plurality of stirring paddles (6) are mounted outside the circular sleeve (7). The upper cup body (21) is of a cylindrical structure or a circular truncated cone structure having a large top and a small bottom; the inner wall surface of the lower cup body (22) gradually inclines towards the center from top to bottom; and the inner wall surface of the lower cup body (22) is smooth or is provided with at least one protrusion (221), the protrusion (221) being circumferentially arranged along the inner wall surface of the lower cup body (22). The centrifugal cup uses the cup body structure the inner wall of which has a certain degree of inclination, which, compared with existing straight cylindrical cups, helps to gather cells, reduces mutual pressing between cells and improves activity of the cells. The inner wall surface of the lower cup body is provided with a plurality of protrusions, and the plurality of stirring paddles are mounted outside the circular sleeve, so that aggregated cells can be gently dispersed into single suspension cells within a short time period.

Description

A cell processing centrifuge cup and a high-throughput cell concentration and cleaning device Technical Field

[0001] This invention relates to the field of cell processing technology, and in particular to a cell processing centrifuge cup and a high-throughput cell concentration and cleaning device. Background Technology

[0002] In the field of cell processing, cell solutions need to be concentrated, typically using centrifuge cups to concentrate low-concentration cell solutions into high-concentration solutions for subsequent cell processing. Existing cylindrical centrifuge cups are limited by their size and outlet location, making large-scale batch processing necessitate continuously increasing the cup volume. This increases the demands on centrifugation equipment, adds inconvenience, and significantly increases costs. Furthermore, during prolonged concentration, cells adhere to the inside of the centrifuge cup, severely impacting cell viability. For samples initially containing numerous cell clumps, existing centrifugation devices offer no effective solution; in fact, prolonged centrifugation and cell adhesion exacerbate clumping, failing to meet the requirement for single-suspension cell preparations. Summary of the Invention

[0003] This invention provides a cell processing centrifuge cup, comprising a lid and a cup body. The lid is used to seal the upper opening of the cup body. The cup body includes an integrally formed upper cup body and a lower cup body. A circular sleeve is provided inside the centrifuge cup. The upper end of the circular sleeve is fixedly connected to the lower part of the lid body, and the lower end extends into the lower cup body and is suspended. Multiple stirring paddles are installed on the outside of the circular sleeve. The upper cup body is cylindrical or frustum-shaped with a larger upper part and a smaller lower part. The inner wall surface of the lower cup body gradually slopes towards the center from top to bottom. The inner wall surface of the lower cup body is smooth or has at least one protrusion, which is arranged circumferentially along the inner wall surface of the lower cup body.

[0004] Furthermore, the centrifuge cup also includes a cup head, which is disposed above the cover body. The cup head is provided with a first interface, a second interface, and a third interface. A central tube is disposed inside the circular sleeve. One end of the central tube is connected to the third interface, and the other end extends into the bottom of the lower cup body and is suspended in the air. The first interface is connected to the interior of the circular sleeve. The second interface is connected to the space between the circular sleeve and the upper cup body.

[0005] Furthermore, a horizontal plate is provided inside the circular sleeve, dividing the interior of the circular sleeve into an upper space and a lower space. A through hole is provided in the center of the horizontal plate, through which the central tube passes, and the diameter of the through hole is larger than the diameter of the central tube.

[0006] Furthermore, the cup head and lid body are also provided with a first channel and a second channel. One end of the first channel is connected to the first interface and the other end is connected to the upper space. One end of the second channel is connected to the second interface and the other end is connected to the space between the circular sleeve and the upper cup body.

[0007] Furthermore, the axis of the central tube, the axis of the through hole, and the axis of the circular sleeve coincide with the axis of the cup body.

[0008] Furthermore, the distance from the edge of the stirring paddle to the center of the circular sleeve is r, and the radius of the lower end of the upper cup is R, where: 1 / 3 <r / R<1 / 2。

[0009] Furthermore, the overall internal height of the cup body is H, the height of the stirring paddle is h, and the distance from the upper edge of the stirring paddle to the upper edge of the cup body is D, where: 1 / 4 <h / H<1;1 / 10<D / H<2 / 3。

[0010] Furthermore, the angle between the generatrix of the upper cup body and the outer surface of the horizontal plane is α, where 45°≤α≤90°, and the angle of inclination between the inner wall surface of the lower cup body and the horizontal plane is β, where 20°≤β≤45°, and α>β.

[0011] The present invention also provides a high-throughput cell concentration and cleaning device, including the cell processing centrifuge cup described above.

[0012] In this invention, a cup structure with a certain incline on the inner wall is adopted. Compared with existing straight-cylinder cups, this is beneficial for guiding cell aggregation, reducing mutual compression between cells, and improving cell activity. Multiple stirring paddles are installed on the outside of the circular sleeve. For samples initially containing many cell clusters or cells that have severely adhered to the centrifuge wall, this allows for a gentler dispersal of clustered cells into single-suspension cells in a short time. Simultaneously, multiple protrusions are provided on the inner wall of the lower cup, and multiple stirring paddles are installed on the outside of the circular sleeve. For samples initially containing many cell clusters or cells that have severely adhered to the centrifuge wall, the protrusions of the lower cup and the stirring paddles in the centrifuge cup interlayer can achieve the effect of dispersing clustered cells into single-suspension cells in a shorter time, suitable for cell dispersion that is more resistant to shear forces.

[0013] In addition, the three-channel structure design solves the problem of cells remaining attached to the centrifuge cup during long-term concentration, thus better ensuring cell viability. Attached Figure Description

[0014] Figure 1 is a schematic diagram of the centrifuge cup;

[0015] Figure 2 is a cross-sectional view of the centrifuge cup;

[0016] Figure 3a shows the state of cell dispersion without a stirring paddle;

[0017] Figure 3b shows the state of the cells being dispersed with a stirring paddle. Detailed Implementation

[0018] Referring to Figure 1, the centrifuge cup of the present invention includes a lid 1 and a cup body 2. The lid 1 is used to close the upper opening of the cup body 2. The cup body 2 includes an integrally formed upper cup body 21 and a lower cup body 22. The upper cup body 21 is a cylindrical or frustum-shaped structure with a larger upper part and a smaller lower part. The inner wall surface of the lower cup body 22 gradually slopes towards the center from top to bottom. The outer side of the lower cup body 22 forms a cylindrical base 222. The inner wall surface of the lower cup body 22 is smooth or has at least one protrusion 221. The protrusion 221 is arranged circumferentially along the inner wall surface of the lower cup body 22.

[0019] The angle between the generatrix of the upper cup body 21 and the horizontal plane is α, where 45°≤α≤90°, and the angle between the inner wall surface of the lower cup body 22 and the horizontal plane is β, where 20°≤β≤45°, and α>β.

[0020] The centrifuge cup has a circular sleeve 7 inside, the axis of which coincides with the axis of the cup body 2. The upper end of the circular sleeve 7 is fixedly connected to the lower part of the cover 1, and the lower end extends into the lower cup body 22 and is suspended in the air. A horizontal plate 71 is provided inside the circular sleeve 7, dividing the interior of the circular sleeve 7 into an upper space 711 and a lower space 712. A through hole 72 is provided at the center of the horizontal plate 71, and the axis of the through hole 72 coincides with the axis of the cup body 2.

[0021] Multiple stirring paddles 6 are installed on the outside of the circular sleeve 7. The stirring paddles 6 are vertical plates, or elephant ear-shaped, ribbon-shaped, turbine-shaped, spiral-shaped, anchor-shaped, etc., and are located below the horizontal plate 71.

[0022] As one aspect of the invention, by installing multiple stirring paddles on the outside of the circular sleeve 7, the cell dispersal effect can be gently and effectively improved. This effect does not depend on whether the lower cup has protrusions, i.e., the inner wall of the lower cup can be smooth. Test results showed that: in the circular sleeve group without stirring paddles, the clumped cells could not be dispersed at all, with a clumping rate of 86.99%, far exceeding the standard requirement for clumping rate (≤30%). Under the microscope, many cells were still observed to be clumped together (Figure 3a). In the circular sleeve group with stirring paddles, the cell dispersal effect was better, with a lower clumping rate of about 17.36%. Under the microscope, the cells were all in a single suspension state (Figure 3b).

[0023] The distance from the edge of the stirring paddle 6 to the center of the circular sleeve 7 is r, and the radius of the lower end of the upper cup body is R, where 1 / 3 < r / R < 1 / 2. The overall height inside the cup body 2 is H, and the height of the stirring paddle is h, where 1 / 4 < h / H < 1; the distance from the upper edge of the stirring paddle to the upper edge of the cup body 2 is D, where 1 / 10 < D / H < 2 / 3.

[0024] The centrifuge cup further includes a cup head 9, which is arranged above the cover body 1. The cup head is provided with a first interface 91, a second interface 92, and a third interface 93. A central tube 83 is arranged inside the circular sleeve 7. The upper end of the central tube 83 is connected to the cup cover 1, and the lower end passes through the through hole 72 on the horizontal plate 71 and the circular sleeve and extends into the bottom of the lower cup body 22 and is suspended, 1 - 10 millimeters away from the bottom of the lower cup body 22. The axis of the central tube 83 coincides with the axis of the cup body 2.

[0025] The central tube 83 is communicated with the third interface 93, and the diameter of the through hole 72 is larger than the diameter of the central tube 83; a first channel 81 and a second channel 82 are also arranged inside the cup head 9 and the cover body 1. One end of the first channel 81 is communicated with the first interface 91, and the other end is communicated with the upper space 711. One end of the second channel 82 is communicated with the second interface 92, and the other end is communicated with the space between the circular sleeve 7 and the upper cup body 21. The first interface 91, the second interface 92, and the third interface 93 are communicated with the first channel 81, the second channel 82, and the central tube 83 in an internal sleeve manner, which belongs to the well-known technology in the field and will not be elaborated here.

[0026] The working process of the centrifuge cup is as follows:

[0027] I. Processing process of a large number of cells (cell quantity 3E+10 - 10E+10)

[0028] ① Filling the centrifuge cup with cleaning liquid: Open the first channel and the third channel, close the second channel, rotate the centrifuge cup, enter the centrifugation process, pump the cleaning liquid into the centrifuge cup through the third channel. After the cleaning liquid fills the centrifuge cup, stop pumping the cleaning liquid.

[0029] ② Continuous flow concentration process: Pump the cell sample liquid into the centrifuge cup through the third channel. Due to the centrifugal force, the cells gradually rise along the bottom slope and adhere to the cup wall, and at the same time, the centrifugation supernatant is discharged from the centrifuge cup through the first channel. This process continues until the cell thickness increases to a certain value, then open the second channel, and the cells are simultaneously discharged from the second channel to the intermediate product bag. The three channels are opened and working simultaneously, continuously concentrating the cell sample liquid until all the cell samples are concentrated. At this time, all the cells are pumped into the intermediate product bag.

[0030] ③ Emptying the centrifuge cup and pumping the cell suspension into the centrifuge cup: Stop the centrifugation operation and drain all the supernatant in the centrifuge cup through the third channel to the waste bag outside the centrifuge cup until the centrifuge cup is empty. Close the tubing of the second channel, while keeping the first and third channels open, and pump all the cell suspension in the intermediate product bag back into the centrifuge cup through the third channel.

[0031] ④ Washing process: The centrifuge cup rotates, entering the centrifugation process. Due to centrifugation, the cells gradually adhere to the wall of the centrifuge cup. Then, the cell washing solution is pumped into the centrifuge cup through the third channel, and the supernatant after centrifugation is discharged into the waste bag through the first channel. This process will remove the original liquid in the centrifuge cup. After continuously pumping in a set volume of washing solution and simultaneously pumping out the same volume of centrifugation supernatant, the first centrifugation washing work is completed.

[0032] ⑤ Mixing process: Pause the centrifugation operation and mix the centrifuge cup by repeatedly rotating it in both forward and reverse directions. At this time, the convex shape at the bottom of the centrifuge cup and the vertical stirring paddle inside the centrifuge cup can increase the mixing intensity and help break up clumped cells.

[0033] ⑥ Repeat the washing and mixing process: Repeat steps ④ to ⑤ above multiple times to remove impurities such as culture medium from the initial cell sample solution.

[0034] ⑦ Drain the supernatant: After the final wash and mixing, drain as much of the supernatant as possible through the first channel, stop the centrifugation operation, and the cell washing work is now complete.

[0035] ⑧ The process of replacing the washing solution with cryopreservation solution: After the cells in the centrifuge cup are washed, the centrifuge cup contains cells and a small amount of washing solution. The cell cryopreservation solution is pumped into the centrifuge cup through the third channel. The centrifuge cup is then mixed by rotating it in both forward and reverse directions to homogenize the cell suspension. After homogenization, the centrifuge cup is rotated, and after centrifugation for a certain period of time, the supernatant is discharged into the waste bag through the first channel. This step is used to remove residual washing solution from the cells.

[0036] ⑨ Cell resuspension process: The cell cryopreservation solution is pumped back into the centrifuge cup through the third channel, the cell suspension is mixed again, and the volume of the cell suspension in the centrifuge cup is quantified. The cell suspension is then discharged from the third channel into the product bag. The cell sample in the product bag is removed for subsequent work.

[0037] For the processing of large batches of cells, due to the large number of cells and the long process of cell sample concentration, in order to better ensure cell viability, the opening of the second channel 82 that connects to the inside of the centrifuge cup is close to the inner edge of the upper cup 21. This facilitates the continuous discharge of cells from the centrifuge cup to the intermediate product bag during the sample concentration process. During the subsequent cell washing and resuspension process, the cells remain inside the centrifuge cup.

[0038] II. Processing procedure for small batches of cells (cell count 1E+10~3E+10)

[0039] ① Filling the centrifuge cup with cleaning solution: Open the first and third channels, close the second channel, the centrifuge cup rotates and enters the centrifugation process. The cleaning solution is pumped into the centrifuge cup from the third channel. After the centrifuge cup is full of cleaning solution, stop pumping the cleaning solution.

[0040] ② Continuous flow concentration process: The cell sample solution is pumped into the centrifuge cup from the third channel. Due to centrifugation, the cells gradually rise along the bottom slope and adhere to the cup wall. At the same time, the supernatant is discharged from the centrifuge cup from the first channel. The concentration of the cell sample solution continues until all the cell sample is concentrated. At this time, all the cells are in the centrifuge cup.

[0041] ③ Drain the supernatant: After the cell sample solution is concentrated, since the number of cells in the centrifuge cup is small at this time, in order to drain the supernatant as much as possible, open the second channel and drain the supernatant in the centrifuge cup to the waste bag outside the centrifuge cup through the second channel.

[0042] ④ Cleaning process: While the centrifuge cup is in centrifugation mode, close the tubing of the second channel, while keeping the first and third channels open. Pump the cell washing solution into the centrifuge cup through the third channel. The supernatant after centrifugation is discharged from the first channel to the waste bag. This process will remove the original liquid in the centrifuge cup. After continuously pumping in a set volume of washing solution and simultaneously pumping out the same volume of centrifugation supernatant, the first centrifugation cleaning work is completed.

[0043] ⑤ Mixing process: Pause the centrifuge operation and mix the centrifuge cup by repeatedly rotating it in both forward and reverse directions. At this time, the protrusions at the bottom of the centrifuge cup and the vertical baffle inside the centrifuge cup can increase the mixing intensity and help break up clumped cells.

[0044] ⑥ Repeat the washing and mixing process: Repeat steps ④ to ⑤ above multiple times to remove impurities such as culture medium from the initial cell sample solution.

[0045] ⑦ Drain the supernatant: After the final wash and mixing, since the number of cells in the centrifuge cup is still relatively small, in order to drain as much supernatant as possible and maintain the centrifugation state, open the second channel and drain the supernatant from the centrifuge cup into the waste bag outside the centrifuge cup. Close the second channel and stop the centrifugation operation. This completes the cell washing process.

[0046] ⑧ The process of replacing the washing solution with cryopreservation solution: After the cells in the centrifuge cup are washed, the centrifuge cup contains cells and a small amount of washing solution. The cell cryopreservation solution is pumped into the centrifuge cup through the third channel. The centrifuge cup is then mixed by rotating it in both forward and reverse directions to homogenize the cell suspension. After mixing, the centrifuge cup is rotated and centrifuged for a certain period of time. Since the number of cells in the centrifuge cup is still small at this point, in order to drain as much supernatant as possible, the second channel is opened, and the supernatant in the centrifuge cup is drained through the second channel to the waste bag outside the centrifuge cup. This step is used to remove residual washing solution from the cells.

[0047] ⑨ Cell resuspension process: The cell cryopreservation solution is pumped back into the centrifuge cup through the third channel, the cell suspension is mixed again, and the volume of the cell suspension in the centrifuge cup is quantified. The cell suspension is then discharged from the third channel into the product bag. The cell sample in the product bag is removed for subsequent work.

[0048] When processing small batches of cell samples, due to the smaller number of cells and shorter processes such as cell sample concentration, it is more efficient to complete cell washing without constantly removing cells from the centrifuge cup. In this case, the opening connecting the second channel 82 to the inside of the centrifuge cup can be located between the circular sleeve 7 and the upper cup body 21. During the cell sample concentration, washing, mixing, and resuspending processes, the cells remain inside the centrifuge cup.

[0049] In another aspect of this invention, a cup structure with a certain incline on the inner wall is adopted. Compared with existing straight-walled cups, this is beneficial for guiding cell aggregation, reducing mutual compression between cells, and improving cell activity. Simultaneously, multiple protrusions are provided on the inner wall of the lower cup, and multiple stirring paddles are installed on the outside of the circular sleeve. For samples initially containing many cell clusters or cells that have severely adhered to the centrifuge wall, the protrusions of the lower cup and the vertical or other shaped stirring paddles on the centrifuge cup interlayer can achieve the effect of breaking up clustered cells into single suspended cells in a shorter time, making it suitable for cell dispersion that is more resistant to shear forces.

[0050] In addition, the three-channel structure design solves the problem of cells remaining attached to the centrifuge cup during long-term concentration, thus better ensuring cell viability.

[0051] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A cell processing centrifuge cup comprising a lid (1), a cup body (2), the lid (1) for closing an upper opening of the cup body (2), characterized in that, The cup body (2) comprises an integrally formed upper cup body (21) and lower cup body (22), The centrifugal cup is internally provided with a circular sleeve (7), the upper end of the circular sleeve (7) is fixedly connected with the lower part of the cover body (1), the lower end extends into the lower cup body (22) and is suspended; the outside of the circular sleeve (7) is provided with a plurality of stirring paddles (6); The upper cup body (21) is cylindrical or has a circular truncated cone structure with a large upper part and a small lower part, the inner wall surface of the lower cup body (22) gradually inclines to the center from top to bottom, and the inner wall surface of the lower cup body (22) is smooth or provided with at least one protrusion (221), the protrusion (221) is arranged along the circumferential direction of the inner wall surface of the lower cup body (22).

2. The cell processing centrifuge cup of claim 1, wherein, The centrifugal cup further comprises a cup head (9) arranged above the cover body (1), the cup head (9) is provided with a first interface (91), a second interface (92) and a third interface (93), the circular sleeve (7) is internally provided with a central pipe (83), one end of the central pipe (83) is in communication with the third interface (93), the other end extends into the bottom of the lower cup body (22) and is suspended; the first interface (91) is in communication with the inside of the circular sleeve (7); the second interface (92) is in communication with the space between the circular sleeve (7) and the upper cup body (21).

3. The cell processing centrifuge cup of any of claims 1-2, wherein, The circular sleeve (7) is internally provided with a horizontal plate (71), which divides the inside of the circular sleeve (7) into an upper space (711) and a lower space (712), the center of the horizontal plate (71) is provided with a through hole (72), the central pipe (83) passes through the through hole (72), and the diameter of the through hole (72) is greater than the diameter of the central pipe (83).

4. The cell processing centrifuge cup of any of claims 1-3, wherein, The cup head (9) and the cover body (1) are further provided with a first channel (81) and a second channel (82), one end of the first channel (81) is in communication with the first interface (91), the other end is in communication with the upper space (711), one end of the second channel (82) is in communication with the second interface (92), the other end is in communication with the space between the circular sleeve (7) and the upper cup body (21).

5. The cell processing centrifuge cup of claim 4, wherein, The axis of the central pipe (83), the axis of the through hole (72) and the axis of the circular sleeve (7) coincide with the axis of the cup body (2).

6. The cell processing centrifuge cup of claim 1, wherein, The distance between the edge of the stirring paddle (6) and the center of the circular sleeve (7) is r, the radius of the lower end of the upper cup body is R, and 1 / 3 < r / R < 1 / 2.

7. The cell processing centrifuge cup of claim 1, wherein, The total height of the inside of the cup body (2) is H, the height of the stirring paddle is h, and the distance between the upper edge of the stirring paddle and the upper edge of the cup body (2) is D, wherein 1 / 4 < h / H < 1 and 1 / 10 < D / H < 2 / 3.

8. The cell processing centrifuge cup of claim 1, wherein, The outer included angle between the generatrix of the upper cup body (21) and the horizontal plane is α, and 45° ≤ α ≤ 90°, the inclination angle between the inner wall surface of the lower cup body (22) and the horizontal plane is β, and 20° ≤ β ≤ 45°, and α > β.

9. A high throughput cell concentration and washing apparatus, characterized by, It comprises the cell processing centrifugal cup as claimed in any one of claims 1-8.

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