A cell processing centrifuge cup and a cell processing method

Abstract

The present invention relates to a cell processing centrifuge cup, which comprises a cup lid and a cup body. A cup head for liquid inlet and outlet is provided on the cup lid, and a plurality of impeller turbulence blocks are evenly spaced at the bottom of the cup body; the impeller turbulence blocks include a bottom surface and a raised portion. The bottom surface is fixedly connected or integrally formed with the bottom of the cup body, and a reference plane L is formed by extending upward along the radius of the bottom of the cup body. The raised portion is symmetric with respect to the reference plane L and extends along the vertical direction and the radial direction. The outer contour of the bottom surface includes two side portions, a first end portion, and a second end portion. The first end portion is close to the center of the cup body, and the second end portion is far from the center of the cup body; the two side portions sequentially form an expansion portion and a contraction portion from the first end portion to the second end portion. The projection of the raised portion on the reference plane L forms a ridge line, and the ridge line sequentially forms a rising portion and a falling portion from the first end portion to the second end portion.

Description

Technical Field

[0001] The present invention relates to the technical field of cell medical devices, and particularly to a centrifuge cup for processing a large number of cells and a cell processing method. Background Art

[0002] Cell therapy refers to transplanting or injecting normal or bioengineered human cells into a patient's body. The newly injected cells can replace damaged cells, mainly including stem cell therapy or immune cell therapy. Since cells require special technological processes for commercialization, large-scale production, transportation, and storage to ensure patient use, it is necessary to develop automated, closed, and large-scale cell production methods and devices to concentrate, wash a large number of cultured cells, and finally prepare a single-suspended and highly active cell suspension for treatment, thereby ultimately achieving robust and low-cost production of cell drugs. However, 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 large-scale cell preparation processes.

[0003] The use of microcarriers for the culture of mesenchymal stem cells (MSCs) can achieve large-scale production of MSCs. When harvesting cells, a lysis solution is used to lyse the microcarriers. At this time, the cells are in a clumped state and need to be processed into a single-suspended state, and impurities such as residual culture medium, microcarrier lysates, and lysis solution in the cell solution need to be washed away.

[0004] Most of the current cell processing devices on the market are designed for immune cell processing. The commonly used continuous flow method is to introduce cells into a centrifuge cup for processing. Most of the existing centrifuge cups cannot effectively suspend adherent cells cultured with microcarriers, especially when processing a large number of cells (more than 10 billion). Summary of the Invention

[0005] To solve the above problems, the present invention provides a cell processing centrifuge cup, including a cup lid and a cup body. A cup head is provided on the cup lid, and a liquid inlet / outlet and a corresponding liquid passage are provided on the cup head. A plurality of wave wheel turbulence blocks are evenly spaced along the circumference at the bottom of the cup body; each wave wheel turbulence block includes a bottom surface and a raised portion. The bottom surface is fixedly connected or integrally formed with the bottom of the cup body, and a reference plane L is formed by extending upward along the bottom radius of the cup body. The raised portion is symmetric with respect to the reference plane L and extends along the vertical direction and the radial direction.

[0006] The outer contour of the bottom surface includes two side portions, a first end portion, and a second end portion. The first end portion is close to the center of the cup body, and the second end portion is far from the center of the cup body; the two side portions sequentially form an expansion portion and a contraction portion from the first end portion to the second end portion. The contraction portions on both sides are smoothly connected at the end far from the cup body to form the second end portion, and the expansion portion is smoothly connected at the end close to the center of the cup body to form the first end portion.

[0007] The projection of the raised portion on the reference plane L forms a ridge line, which successively forms a rising portion and a falling portion from the first end to the second end, and the raised portion smoothly transitions from the ridge line to the two side portions.

[0008] Furthermore, the included angle between the expansion portion and the reference plane L is α, the projection length of the expansion portion on the reference plane L is C, and the projection length of the contraction portion on the reference plane L is S, where 5° ≤ α ≤ 30° and 1 ≤ C / S ≤ 4.

[0009] Furthermore, the distance from the first end to the center of the cup body is 5 - 10 mm, and the distance from the second end to the peripheral wall of the cup body is 1 - 5 mm.

[0010] Furthermore, the bottom of the cup body slopes upward from the center to the periphery, the inclination angle is γ1, the included angle between the rising portion 44 and the horizontal plane is γ2, the projection length of the rising portion 44 on the horizontal plane is C1, and the projection of the falling portion on the horizontal plane is S1, where 5° ≤ γ1 ≤ 15°, 15° ≤ γ2 ≤ 45°, 1 ≤ C1 / S1 ≤ 4, and γ2 > γ1.

[0011] Furthermore, C = C1 and S = S1.

[0012] Furthermore, the distance from the upper edge of the cup body to the lowest point of its bottom is the height H of the cup body, and the distance from the highest point of the raised portion to the lowest point of the bottom of the cup body is the height h of the raised portion, where 1 < h / H ≤ 6.

[0013] Furthermore, at least 2 wave wheel turbulence blocks are arranged circumferentially on the bottom of the cup body, and all the wave wheel turbulence blocks are evenly distributed around the center.

[0014] The present invention also provides a cell processing method, including the following steps,

[0015] S1: Cell concentration: When the centrifuge cup is in a centrifugal state, a cleaning solution is introduced into the centrifuge cup. After the cleaning solution fills the centrifuge cup, the introduction of the cleaning solution is stopped, and a cell sample solution is introduced into the centrifuge cup. The cells are attached to the cup wall under the action of centrifugal force. At the same time, the centrifugal supernatant is synchronously discharged from the centrifuge cup. This process continues until the cell concentration is completed;

[0016] S2: Cleaning and mixing: When the centrifuge cup is in a centrifugal state, the cleaning solution is introduced into the centrifuge cup again, and the supernatant is synchronously discharged. This process will dilute and carry away the liquid in the centrifuge cup. After continuously replacing a certain volume of the cleaning solution, the cleaning operation is completed. Then, the centrifugal operation of the centrifuge cup is paused, and the centrifuge cup is mixed by periodically rotating forward and backward. The wave wheel structure at the bottom of the centrifuge cup forms turbulence when the centrifuge cup starts and stops, so as to disperse the agglomerated cells; the above cleaning operation and mixing operation are repeated multiple times, and then the supernatant is discharged in the centrifugal state.

[0017] S3: Cell resuspension: Stop the centrifugation operation of the centrifuge cup, introduce the cell cryopreservation solution into the centrifuge cup, and drive the centrifuge cup to rotate forward and backward periodically for multiple times to mix the cell suspension evenly;

[0018] S4: Drain the cell suspension;

[0019] In the above steps, the centrifuge cup used is the above-mentioned centrifuge cup.

[0020] Further, in step S2, the washing operation and the mixing operation are repeated 2 - 5 times.

[0021] Further, the cells are stem cells, immune cells, VERO cells, 293 cells, CHO cells or HEK293 cells.

[0022] The present invention adopts a centrifuge cup with a wave wheel structure. When processing cells, it has good microcarrier cell processing ability, can process cell clusters into a single suspension state, and keep the cells with a high viability. At the same time, during the operation, the centrifuge cup adopts an intermittent dispersion method, relying on the turbulent flow effect of the wave wheel structure in the centrifuge cup on the liquid when the centrifuge starts and stops, to disperse the cells from the clustered state into a single suspension state, solve the problem of cell clustering, and meet the requirements for the single suspension state of cell preparations in clinical applications. Description of the Drawings

[0023] Figure 1 is a schematic diagram of a centrifuge cup without a turbulent flow structure;

[0024] Figure 2 is a schematic diagram of a centrifuge cup provided with a Y-shaped baffle;

[0025] Figure 3 is a schematic diagram of the centrifuge cup structure of the present invention;

[0026] Figure 4 is a schematic diagram of the internal structure of the centrifuge cup of the present invention;

[0027] Figure 5 is a top view of the internal structure of the centrifuge cup of the present invention;

[0028] Figure 6 is a sectional view of the centrifuge cup of the present invention;

[0029] Figure 7 is a comparison diagram of the single suspension situation of MSC cells in centrifuge cups with different structures and wave wheel turbulent flow structures;

[0030] Figure 8 is the single suspension situation of processing 20 billion MSC cells with a wave wheel structure centrifuge cup;

[0031] Figure 9 is the single suspension situation of processing 30 billion 3T3 cells with a wave wheel structure centrifuge cup. Detailed implementation mode

[0032] The centrifuge cup is a key device for processing cells, which includes a cup cover, a cup body, etc. In order to achieve the mixing and dispersion of cells, specific designs of the centrifuge cup are usually required. Refer to Figure 1 , the centrifuge cup is a conventional cup body without any blocking structure inside. The liquid inside only has laminar flow and cannot form turbulent flow, so the mixing and dispersion effect is not good. Due to the periodic forward and reverse rotation and pause, longitudinal eddy current effects will be formed when the liquid is under the alternating change of centrifugal force.

[0033] Refer to Figure 2 , a liquid pushing plate arranged in a Y shape is arranged in the centrifuge cup. Due to the large area and long radial length of the liquid pushing plate, small eddies will be formed in the cross-sectional liquid. And because the outermost edge is far from the cup wall, there is still laminar flow. Its mixing effect is better than that of the cup body without structure, but the turbulent flow is not strong enough, and the dispersion effect has been improved but is still not enough. When the centrifuge cup starts and stops, due to the large area of the liquid pushing plate, the cells will collide with it, thus affecting the cell viability.

[0034] Refer to Figures 3 - 6 , which is the structural diagram of the centrifuge cup of the present invention. It includes a cup cover 1 and a cup body 2. A cup head 3 is provided on the cup cover 1. The cup head 3 is provided with a liquid inlet and outlet and corresponding liquid passages for introducing liquid into the centrifuge cup or discharging liquid from the centrifuge cup. This technology belongs to the prior art and will not be elaborated here.

[0035] A plurality of wave wheel turbulence blocks 4 are evenly arranged at intervals along the circumference at the bottom of the cup body 2. Generally, the number of wave wheel turbulence blocks does not exceed 10. In Figures 4 - 6 , four wave wheel turbulence blocks 4 are arranged along the circumference at the bottom of the cup body, and the interval angle between two adjacent wave wheel turbulence blocks 4 is 90 degrees. The structure of the wave wheel turbulence block will be described in detail below.

[0036] Refer to Figures 5 - 6, a reference plane L is formed by extending upward from the bottom radius of the cup body 2. The impeller turbulence block 4 includes a bottom surface and a raised portion. The bottom surface is fixedly connected or integrally formed with the bottom of the cup body 2. The raised portion is symmetric with respect to the reference plane L and extends along the vertical direction and the radial direction. The outer contour of the bottom surface includes two side portions 41, a first end portion 42, and a second end portion 43. The first end portion 42 is close to the center of the cup body, and the second end portion 43 is far from the center of the cup body. The two side portions 41 respectively have an expansion portion 411 and a contraction portion 412. The two side portions sequentially form an expansion portion 411 and a contraction portion (412) from the first end portion 42 to the second end portion 43. The projection length of the expansion portion 411 on the reference plane L is C, and the projection length of the contraction portion 412 on the reference plane L is S, where 5° ≤ α ≤ 30°, and 1 ≤ C / S ≤ 4. The contraction portion 412 contracts from the connection point with the expansion portion 411 towards the reference plane L, and the two contraction portions 412 on both sides are smoothly connected at the end far from the cup body to form the second end portion 43. The expansion portion 411 is smoothly connected at the end close to the center of the cup body to form the first end portion 42. The distance from the first end portion 42 to the center of the cup body is 5 - 10 mm, and the distance from the second end portion 43 to the peripheral wall of the cup body is 1 - 5 mm.

[0037] See Figure 6 , the bottom of the cup body 2 slopes upward from the center to the periphery, and the inclination angle is γ1. The projection of the raised portion on the reference plane L forms a ridge line M. The ridge line M includes a rising portion 44 and a descending portion 45. The projection length of the rising portion 44 on the horizontal plane is C1, and the projection of the descending portion 45 on the horizontal plane is S1. The included angle between the rising portion 44 and the horizontal plane is γ2. The descending portion 45 extends from the connection point with the rising portion 44 towards the bottom surface, where γ2 > γ1, 5° ≤ γ1 ≤ 15°, 15° ≤ γ2 ≤ 45°, and 1 ≤ C1 / S1 ≤ 4. Preferably, C1 = C and S1 = S.

[0038] The distance from the upper edge of the cup body to the lowest point of its bottom is the height H of the cup body, and the distance from the highest point of the raised portion to the lowest point of the cup body bottom is the height h of the raised portion, where 1 < h / H ≤ 6.

[0039] The working process of the centrifuge cup of the present invention is as follows:

[0040] ① Continuous flow concentration process: Under the centrifugal state of the centrifuge cup, cleaning liquid is introduced into the centrifuge cup. After the cleaning liquid fills the centrifuge cup, the introduction of the cleaning liquid is stopped, and then the cell sample liquid is introduced into the centrifuge cup. The cells are attached to the cup wall under the centrifugal force. At the same time, the centrifugal supernatant is synchronously discharged from the centrifuge cup. This process continues until the cell concentration is completed, that is, the input amount of the cell sample liquid reaches the preset amount, and no new cell sample liquid is introduced into the centrifuge cup.

[0041] ② Washing and mixing: After concentration, while the centrifuge cup is in the centrifugal state, rinse solution is introduced into the centrifuge cup again, and the supernatant is discharged synchronously. This process will dilute and carry away the liquid in the centrifuge cup. After continuously replacing a certain volume of rinse solution, the washing operation is completed. Then, the centrifugal operation of the centrifuge cup is paused, and the centrifuge cup is mixed by periodically rotating forward and backward. At this time, the impeller structure at the bottom of the centrifuge cup can form a turbulent flow when the centrifuge cup starts and stops, dispersing the agglomerated cells. The above washing operation and mixing operation are repeated multiple times to complete the washing and mixing steps. Multiple washings are performed to achieve the effect of removing impurities such as culture medium, carrier, lysis solution, and cell debris. After the cells in the centrifuge cup are washed, the supernatant is discharged in the centrifugal state.

[0042] ③ Resuspension process: Stop the centrifugal operation of the centrifuge cup, introduce the cell cryopreservation solution into the centrifuge cup, and mix the cell suspension again;

[0043] ④ Discharging the cell suspension: After quantifying the volume in the cup, the cell suspension is discharged from the bottom channel into the product bag.

[0044] In the present invention, the centrifuge cup adopts an intermittent dispersion method. Relying on the turbulent flow effect of the impeller structure in the centrifuge cup on the liquid when the centrifuge starts and stops, the cells are dispersed 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.

[0045] The centrifuge cup of the present invention is applicable to adherent cells such as stem cells (e.g., MSC cells), immune cells, VERO cells, 293 cells, etc., as well as suspension cells such as CHO and HEK293.

[0046] Next, an experimental comparison is made on the treatment effects of different centrifuge cup structures on microcarrier adherent cells.

[0047] In a bioreactor, microcarriers are used to culture 3T3 and MSC cells. The 3T3 cells are cultured to 5.0E+09 - 1E+10, and the MSC cells are cultured to 2.0E+09 - 4.0E+09. The microcarriers are lysed using a lysis solution to obtain cell samples. After the cell treatment of concentration, washing is completed, cell counting is performed, and the treatment effects of different structured cups are compared by comparing the single suspension situation and viability of the cells.

[0048] The unstructured centrifuge cup in the following text corresponds to Figure 1 and the Y-shaped baffle corresponds to Figure 2 The impeller structure is Figures 4 - 6 .

[0049] Structureless, Y-shaped baffle, impeller structure (for processing MSC cells). Washing parameters: 600 ml of washing solution is used each time, and the washing is done 4 times. Mixing parameters: mixing acceleration 1500 rpm / s, mixing centrifugal speed (1500 rpm), mixing deceleration 1500 rpm / s, and the number of mixing times for each washing is 30 times.

[0050] For single cell suspension, refer to Figure 7 , the single cell suspension effect of the cup body without a turbulent flow structure is poor, inferior to the impeller structure, and it cannot complete the treatment of microcarrier adherent cells. The cup bodies with Y-shaped baffle structure and impeller structure can make MSC cells single cell suspension.

[0051] Comparison of cell viability of Y-shaped baffle structure: Structureless (98.03%) > Impeller (96.43%) > Y-shaped baffle (86.28%). Although both the Y-shaped baffle and impeller structures can process the cells into a single cell suspension state, after the treatment with the Y-shaped baffle, the cell viability decreases. This may be because when the centrifuge cup starts and stops, due to the large area of the liquid pushing plate, the cells will collide with it, and multiple collisions affect the cell viability. The impeller structure has a small area and does not have violent contact with the cells. It basically makes the cells single cell suspension only through the turbulent flow effect and can better maintain the cell viability.

[0052] The results of different structures for treating different cells show that the impeller structure has better microcarrier cell treatment ability, can process cell clusters into a single cell suspension state, and keep the cells with a high viability.

[0053] The following are the experimental data of the impeller structure centrifuge cup for treating 20 billion MSC cells:

[0054] In a bioreactor, microcarriers are used to culture MSC cells. The MSC cells are cultured to 2.0E+10, and the microcarriers are lysed with a lysis solution to obtain cell samples. The cup body with impeller structure is used to concentrate and wash 1.0E+10 MSC cells on a cell processing device. Washing parameters: 600 ml of washing solution is used each time, and the washing is done 5 times. Mixing parameters: mixing acceleration 1500 rpm / s, mixing centrifugal speed (1500 rpm), mixing deceleration 1500 rpm / s, and the number of mixing times for each washing is 20 times.

[0055] Figure 8 This is for the single cell suspension situation. In addition, the cell viability is 96.00%, the recovery rate is 90.06%, and the total number of live cells is 2.05E+10.

[0056] The following are the experimental data of the impeller structure centrifuge cup for treating 30 billion 3T3 cells:

[0057] Cultivate 3T3 cells to 3.0E+10 using microcarriers in a bioreactor, and lyse the microcarriers with lysis solution to obtain cell samples. Use a beaker with a paddle structure to concentrate and wash 3.0E+10 3T3 cells on a cell processing device.

[0058] The cleaning parameters are: use 600 ml of washing solution each time and wash 5 times. The mixing parameters are: mixing acceleration 1500 rpm / s, mixing centrifugal speed (1500 rpm), mixing deceleration 1500 rpm / s, and the number of mixing times for each washing is 20 times.

[0059] Figure 9 This is the case of single-cell suspension. In addition, the viability of 3T3 cells is 96.50%, the recovery rate is 96.85%, and the total number of live cells is 2.80E+10.

[0060] 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 processing centrifuge cup, comprising a cup cover (1), a cup body (2), a cup head (3) being arranged on the cup cover (1), and a liquid inlet and outlet and a corresponding liquid passage being arranged on the cup head (3), characterized in that: A plurality of impeller spoiler blocks (4) are evenly spaced along the circumferential direction at the bottom of the cup body (2); the impeller spoiler block (4) comprises a bottom surface and a raised portion, the bottom surface being fixedly connected to or integrally formed with the bottom of the cup body (2), and extending upward with the radius of the bottom of the cup body (2) to form a reference plane L, the raised portion being symmetrical with respect to the reference plane L and extending in the vertical direction and the radial direction, The outer contour of the bottom surface comprises two side portions (41), a first end portion (42), and a second end portion (43), wherein the first end portion (42) is close to the center of the cup body, and the second end portion (43) is far from the center of the cup body; the two side portions sequentially form an expansion portion (411) and a contraction portion (412) from the first end portion (42) to the second end portion (43), the contraction portions (412) on both sides are smoothly connected at an end away from the cup body to form the second end portion (43), and the expansion portion (411) is smoothly connected at an end close to the center of the cup body to form the first end portion (42). The projection of the raised portion on the reference plane L forms a ridge line, the ridge line sequentially forms a rising portion (44) and a descending portion (45) from the first end (42) to the second end (43), and the raised portion smoothly transitions from the ridge line to the two side portions; The included angle between the expansion portion (411) and the reference plane L is α, the projected length of the expansion portion (411) on the reference plane L is C, and the projected length of the contraction portion (412) on the reference plane L is S, wherein 5°≤α≤30°, 1≤C / S≤4; The projection length of the rising portion (44) on the horizontal plane is C1, and the projection length of the descending portion on the horizontal plane is S1, where 1≤C1 / S1≤4.

2. The centrifugal cup according to claim 1, characterized in that: The distance between the first end (42) and the center of the cup body is 5-10 mm, and the distance between the second end (43) and the peripheral wall of the cup body is 1-5 mm.

3. The centrifugal cup according to claim 1, characterized in that: The bottom of the cup body (2) is inclined upward from the center to the periphery, with an inclination angle of γ1, and the angle between the rising portion (44) and the horizontal plane is γ2, wherein 5°≤γ1≤15°, 15°≤γ2≤45°, and γ2>γ1.

4. The centrifugal cup according to claim 1, characterized in that: C=C1, S=S1.

5. The centrifuge cup according to any one of claims 1 to 4, characterized in that: At least two impeller spoiler blocks (4) are arranged along the circumferential direction at the bottom of the cup body, and all impeller spoiler blocks (4) are evenly distributed around the center.

6. A cell treatment method, characterized in that: The following steps are included: S1: Cell concentration: When the centrifuge cup is in the centrifugal state, the cleaning solution is introduced into the centrifuge cup. When the cleaning solution fills the centrifuge cup, the introduction of the cleaning solution is stopped and the cell sample solution is introduced into the centrifuge cup. The cells adhere to the cup wall due to the centrifugal force. At the same time, the supernatant is discharged from the centrifuge cup synchronously. This process continues until the cell concentration is completed. S2: cleaning and mixing: while the centrifugal cup is in a centrifugal state, the cleaning liquid is introduced into the centrifugal cup again, and the supernatant is discharged simultaneously. This process will dilute and take away the liquid in the centrifugal cup. After a certain volume of cleaning liquid is continuously replaced, the cleaning operation is completed, and then the centrifugal operation of the centrifugal cup is paused, and the centrifugal cup is mixed by periodically rotating forward and reversely. The impeller structure at the bottom of the centrifugal cup is used to form a turbulent flow when the centrifugal cup is started and stopped, so as to break up the clumped cells; the above cleaning and mixing operations are repeated many times, and then the supernatant is discharged in a centrifugal state; S3: Cell resuspension: Stop the centrifugal operation of the centrifuge cup, introduce the cell freezing solution into the centrifuge cup, and drive the centrifuge cup to periodically rotate forward and reverse multiple times to mix the cell suspension; S4: discharge the cell suspension; In the above steps, the centrifuge cup used is the centrifuge cup according to any one of claims 1-5.

7. The method according to claim 6, characterized in that In step S2, the washing operation and the mixing operation are repeated 2-5 times.

8. The method according to any one of claims 6-7, characterized in that: The cells are stem cells, immune cells, VERO cells, 293 cells, CHO cells or HEK293 cells.

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