Cell treatment centrifugal cup and cell treatment method

Abstract

The invention relates to a cell treatment centrifugal cup which comprises a cup cover and a cup body, a cup head for liquid to enter and exit is arranged on the cup cover, and a plurality of impeller turbulent flow blocks are evenly arranged at the bottom of the cup body at intervals. The impeller turbulent flow block comprises a bottom face and a protruding portion, the bottom face and the bottom of the cup body are fixedly connected or integrally formed, the bottom radius of the cup body extends upwards to form a datum plane L, the protruding portion is symmetrical relative to the datum plane L and extends in the vertical direction and the radius direction, and the outer contour of the bottom face comprises two side portions, a first end portion and a second end portion. The first end is close to the center of the cup body, and the second end is far away from the center of the cup body; the two side parts sequentially form an expansion part and a contraction part from the first end part to the second end part, the projection of the raised part on the reference plane L forms a ridge line, and the ridge line sequentially forms an ascending part and a descending part from the first end part to the second end part.

Description

Technical Field

[0001] The present invention relates to the technical field of cell medical equipment, and in particular to a centrifugal cup for mass cell processing and a cell processing method. Background Art

[0002] Cell therapy refers to the transplantation or infusion of normal or bioengineered human cells into the patient's body. The newly introduced cells can replace damaged cells. It is mainly stem cell therapy or immune cell therapy. Since cells require special process flows 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 and wash a large number of cultured cells, and finally make a single-suspended, highly active cell suspension for treatment, thereby ultimately achieving robust and low-cost production of cell drugs. However, how to prepare cells into single-suspended cell preparations, minimize cell damage caused by shear forces during the process, and improve cell survival rates are all difficult problems in large-scale cell preparation processes.

[0003] The use of microcarriers for the cultivation of mesenchymal stem cells (MSCs) can achieve large-scale production of MSCs. When harvesting cells, the microcarriers are lysed with a lysis buffer. At this time, the cells are in a clumping state. The cells need to be processed into a single suspension state and the impurities such as the culture medium, microcarrier lysate, and lysis buffer remaining in the cell fluid need to be washed away.

[0004] Currently, most of the 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 are unable to effectively suspend adherent cells cultured on microcarriers, especially when processing large-scale cells (more than 10 billion). Summary of the invention

[0005] In order to solve the above problems, the present invention provides a cell processing centrifugal cup, including a cup cover and a cup body. A cup head is arranged on the cup cover, and a liquid inlet and outlet and a corresponding liquid passage are arranged on the cup head. A plurality of impeller spoilers are evenly spaced at intervals along the circumferential direction at the bottom of the cup body; the impeller spoiler includes a bottom surface and a ridge, and the bottom surface is fixedly connected to the bottom of the cup body or integrally formed, and extends upward with the bottom radius of the cup body to form a reference plane L, and the ridge is symmetrical with respect to the reference plane L and extends in the vertical direction and the radial direction. The outer contour of the bottom surface includes two side parts, a first end, and a second end, wherein the first end is close to the center of the cup body, and the second end is far from the center of the cup body; the two side parts form an expansion part and a contraction part in sequence from the first end to the second end, the contraction parts on both sides are smoothly connected at the end away from the cup body to form the second end, and the expansion part is smoothly connected at the end close to the center of the cup body to form the first end, The projection of the raised portion on the reference plane L forms a ridge line, and the ridge line forms a rising portion and a descending portion in sequence from the first end to the second end, and the raised portion smoothly transitions from the ridge line to the two side portions.

[0006] Furthermore, the included angle between the expansion portion and the reference plane L is α, the projected length of the expansion portion on the reference plane L is C, and the projected length of the contraction portion on the reference plane L is S, wherein 5°≤α≤30°, 1≤C / S≤4.

[0007] Furthermore, the distance between the first end and the center of the cup body is 5-10 mm, and the distance between the second end and the peripheral wall of the cup body is 1-5 mm.

[0008] Furthermore, the bottom of the cup body is inclined upward from the center to the periphery, and the inclination angle is γ1. The 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. The projection of the descending portion on the horizontal plane is S1, wherein 5°≤γ1≤15°, 15°≤γ2≤45°, 1≤C1 / S1≤4, and γ2>γ1.

[0009] Further, C=C1, S=S1.

[0010] Furthermore, the distance between the upper edge of the cup body and the lowest point of its bottom is the cup body height H, and the distance between the highest point of the ridge and the lowest point of the bottom of the cup body is the ridge height h, where 1 <h / H≤6。

[0011] Furthermore, at least two impeller spoilers are arranged along the circumferential direction at the bottom of the cup body, and all the impeller spoilers are evenly distributed around the center.

[0012] The present invention also provides a cell treatment method, comprising the following steps: 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 above centrifuge cup.

[0013] Further, in step S2, the cleaning operation and the mixing operation are repeated 2-5 times.

[0014] Furthermore, the cells are stem cells, immune cells, VERO cells, 293 cells, CHO cells or HEK293 cells.

[0015] The present invention adopts a centrifugal cup with an impeller structure, which has good microcarrier cell processing ability when processing cells, can process cell clusters into a single suspension state, and keep the cells at a high viability. At the same time, during operation, the centrifugal cup adopts an intermittent breaking method, relying on the turbulent effect of the impeller structure in the centrifugal cup on the liquid when the centrifuge starts and stops, breaking the cells from the clumping state to a single suspension state, solving the problem of cell clumping and meeting the requirements for the single suspension state of cell preparations in clinical applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 It is a schematic diagram of a centrifugal cup with a non-disturbed flow structure; Figure 2 is a schematic diagram of a centrifugal cup provided with a Y-shaped baffle; Figure 3 It is a schematic diagram of the structure of the centrifugal cup of the present invention; Figure 4 It is a schematic diagram of the internal structure of the centrifugal cup of the present invention; Figure 5 It is a top view of the internal structure of the centrifugal cup of the present invention; Figure 6 is a cross-sectional view of a centrifugal cup of the present invention; Figure 7 This is a comparison of MSC cell single suspension in centrifugal cups with different structures and impeller spoiler structure; Figure 8 This is the case of a pulsator structure centrifugal cup processing 20 billion MSC cells in single suspension; Fig. 9 It is a centrifugal cup with a pulsator structure that can handle 30 billion 3T3 cells in single suspension. DETAILED DESCRIPTION

[0017] The centrifuge cup is a key device for processing cells, which includes a cup cover, a cup body, etc. In order to achieve uniform mixing and breaking of cells, a specific design of the centrifuge cup is usually required. Figure 1The centrifugal cup is a conventional cup body without any blocking structure. The liquid inside has only laminar flow and cannot form turbulence, so the mixing and breaking effect is not good. Due to the periodic forward and reverse rotation and pause, the liquid will form a longitudinal vortex effect under the alternating centrifugal force.

[0018] See also Figure 2 There is a Y-shaped push plate in the centrifuge cup. Since the push plate has a large area and a long radial length, the liquid in the cross section will form a small regional vortex, and since the outermost edge is far away from the cup wall, there is still laminar flow. Its mixing effect is better than that of the unstructured cup, but the turbulence is not strong enough, and the dispersion effect is improved but not enough. When the centrifuge cup is started and stopped, the cells will collide with the push plate due to its large area, thus affecting cell viability.

[0019] See also Figure 3-6 , is a structural diagram of a centrifugal cup of the present invention, which includes a cup cover 1 and a cup body 2. A cup head 3 is arranged on the cup cover 1, and a liquid inlet and outlet and a corresponding liquid passage are arranged on the cup head 3, which are used to introduce liquid into the centrifugal cup or discharge liquid from the centrifugal cup. This technology belongs to the prior art and will not be described in detail.

[0020] The bottom of the cup body 2 is evenly spaced along the circumferential direction with a plurality of impeller spoilers 4. Usually, the number of impeller spoilers does not exceed 10. Figure 4-6 In the embodiment, four impeller spoilers 4 are arranged along the circumferential direction at the bottom of the cup body, and the interval angle between two adjacent impeller spoilers 4 is 90 degrees. The structure of the impeller spoiler is described in detail below.

[0021] See also Figure 5-6, the base surface L is formed by extending upward with the bottom radius of the cup body 2, the impeller spoiler 4 includes a bottom surface and a raised portion, the bottom surface is fixedly connected to the bottom of the cup body 2 or formed in one piece, the raised portion is symmetrical with respect to the base surface L, and extends in 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 away from the center of the cup body; the two side portions 41 have an expansion portion 411 and a contraction portion 412 respectively. The two side portions form an expansion portion 411 and a contraction portion (412) in sequence from the first end portion 42 to the second end portion 43, the projection length of the expansion portion 411 on the base surface L is C, and the projection length of the contraction portion 412 on the base surface L is S, wherein 5°≤α≤30°, 1≤C / S≤4. The contraction portion 412 contracts toward the reference plane L from the connection point with the expansion portion 411, and the contraction portions 412 on both sides are smoothly connected at the end away from the cup body to form a second end portion 43, and the expansion portion 411 is smoothly connected at the end close to the center of the cup body to form a first end portion 42, the first end portion 42 is 5-10 mm away from the center of the cup body, and the second end portion 43 is 1-5 mm away from the peripheral wall of the cup body.

[0022] See also Figure 6 The bottom of the cup body 2 is tilted upward from the center to the periphery, and the tilt angle is γ1. The projection of the raised portion on the reference plane L forms a ridge line M. The ridge line M includes an ascending portion 44 and a descending portion 45. The projection length of the ascending portion 44 on the horizontal plane is C1, and the projection of the descending portion 45 on the horizontal plane is S1. The angle between the ascending portion 44 and the horizontal plane is γ2. The descending portion 45 extends from the connection point with the ascending portion 44 to the bottom surface, wherein γ2>γ1, 5°≤γ1≤15°, 15°≤γ2≤45°, and 1≤C1 / S1≤4. Preferably, C1=C, S1=S.

[0023] The distance between the upper edge of the cup body and the lowest point of its bottom is the cup body height H, and the distance between the highest point of the ridge and the lowest point of the bottom of the cup body is the ridge height h, where 1 <h / H≤6。

[0024] The working process of the centrifugal cup of the present invention is as follows: ① Continuous flow concentration process: When the centrifuge cup is in the centrifugal state, the cleaning liquid is passed into the centrifuge cup. When the cleaning liquid fills the centrifuge cup, the cleaning liquid is stopped and the cell sample liquid is started to be passed into the centrifuge cup. The cells are attached to the cup wall due to the centrifugal force. At the same time, the centrifugal supernatant is discharged from the centrifuge cup synchronously. 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 passed into the centrifuge cup.

[0025] ② Washing and mixing: After the concentration is completed, the cleaning liquid is introduced into the centrifuge cup again while the centrifuge cup is in the centrifugal state, and the supernatant is discharged simultaneously. This process will dilute and take away the liquid in the centrifuge cup. After a certain volume of cleaning liquid is continuously replaced, the cleaning operation is completed, and then the centrifugal operation of the centrifuge cup is paused, and the centrifuge cup is mixed by periodically rotating forward and reverse. At this time, the impeller structure at the bottom of the centrifuge cup can form a turbulent flow when the centrifuge cup is started and stopped, so as to break up the clumped cells. The above-mentioned cleaning and mixing operations are repeated many times to complete the cleaning and mixing steps. Multiple washings are performed to achieve the effect of removing impurities such as culture medium, carrier, lysate, cell debris, etc. After the cells in the centrifuge cup are washed, the supernatant is discharged while in the centrifugal state.

[0026] ③ Resuspension process: stop the centrifugal operation of the centrifuge cup, introduce the cell freezing solution into the centrifuge cup, and mix the cell suspension again; ④ Discharge the cell suspension: After quantifying the volume in the cup, discharge the cell suspension from the bottom channel into the product bag.

[0027] In the present invention, the centrifuge cup adopts an intermittent breaking method, relying on the turbulence effect of the impeller structure in the centrifuge cup on the liquid when the centrifuge starts and stops, to break up the cells from a clumping state to a single suspension state, thereby solving the problem of cell clumping and meeting the requirements for the single suspension state of cell preparations in clinical applications.

[0028] The centrifuge cup of the present invention is suitable for stem cells (such as MSC cells), immune cells, VERO cells, 293 cells and other adherent cells and CHO, HEK293 and other suspension cells.

[0029] The following is an experimental comparison of the treatment effects of different centrifuge cup structures on microcarrier-adherent cells.

[0030] Microcarriers were used in bioreactors to culture 3T3 and MSC cells. 3T3 cells were cultured to 5.0E+09~1E+10, and MSC cells were cultured to 2.0E+09~4.0E+09. The microcarriers were lysed with lysis buffer to obtain cell samples. After the cell treatment, concentration and washing were completed, the cells were counted, and the treatment effects of cups with different structures were compared by comparing the cell single suspension and viability.

[0031] In the following text, the non-structured centrifuge cup corresponds to Figure 1 , the Y-type baffle corresponds to Figure 2 The impeller structure is Figure 4-6 .

[0032] No structure, Y-shaped baffle, impeller structure (processing MSC cells. The cleaning parameters are: use 600 ml of washing solution each time, wash 4 times. The mixing parameters are: mixing acceleration 1500 rpm / s, mixing centrifugal speed (1500 rpm), mixing deceleration 1500rpm / s, and the number of mixing times for each washing is 30 times.

[0033] For single cell suspension, see Figure 7 The single suspension effect of cells in the cup without perturbation structure is poor, which is inferior to the impeller structure and cannot complete the treatment of microcarrier adherent cells. The cup with Y-shaped baffle structure and impeller structure can make MSC cells single suspended.

[0034] Comparison of cell viability in the Y-type baffle structure: no structure (98.03%) > impeller (96.43%) > Y-type baffle (86.28%). Although both the Y-type baffle and impeller structures can process cells into a single suspension state, the cell viability is reduced after the Y-type baffle treatment. This may be because when the centrifuge cup is started and stopped, the cells will collide with the push plate due to its large area, and multiple collisions affect cell viability. The impeller structure has a small area and does not produce violent contact with the cells. It basically only makes the cells suspended by turbulence, which can better maintain cell viability.

[0035] The results of treating different cells with different structures showed that the impeller structure had better microcarrier cell processing ability, could process cell clusters into a single suspension state, and could keep the cells at a high viability.

[0036] The following is the experimental data of the impeller structure centrifugal cup processing 20 billion MSC cells: Microcarriers were used to culture MSC cells in a bioreactor, and MSC cells were cultured to 2.0E+10. The microcarriers were lysed with a lysis solution to obtain a cell sample. A cup with an impeller structure was used to concentrate and wash 1.0E+10 MSC cells on a cell processing device. The washing parameters were: 600 ml of washing solution was used each time, and the washing was repeated 5 times. The mixing parameters were: mixing acceleration 1500 rpm / s, mixing centrifugal speed (1500 rpm), mixing deceleration 1500 rpm / s, and the mixing number was 20 times for each washing.

[0037] Figure 8 The cell suspension was single-cell suspension. In addition, the cell viability was 96.00%, the recovery rate was 90.06%, and the total number of live cells was 2.05E+10.

[0038] The following is the experimental data of the impeller structure centrifugal cup processing 30 billion 3T3 cells: Microcarriers were used in the bioreactor to culture 3T3 cells until 3.0E+10 3T3 cells were cultured, and the microcarriers were lysed with lysis buffer to obtain cell samples. A cup with an impeller structure was used to concentrate and wash 3.0E+10 3T3 cells on the cell processing device.

[0039] 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 mixing number of each washing is 20 times.

[0040] Fig. 9 In addition, the viability of 3T3 cells was 96.50%, the recovery rate was 96.85%, and the total number of live cells was 2.80E+10.

[0041] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A cell 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, and the ridge line forms a rising portion (44) and a descending portion (45) in sequence 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.

2. The centrifugal cup according to claim 1, characterized in that: The included angle between the expansion portion (411) and the reference plane L is α, 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, wherein 5°≤α≤30°, and 1≤C / S≤4.

3. The centrifugal cup according to claim 2, 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.

4. The centrifuge cup according to any one of claims 1 to 3, characterized in that: The bottom of the cup body (2) is inclined upward from the center to the periphery, and the inclination angle is γ1. The 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. The projection of the descending portion on the horizontal plane is S1, wherein 5°≤γ1≤15°, 15°≤γ2≤45°, 1≤C1 / S1≤4, and γ2>γ1.

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

6. The centrifuge cup according to any one of claims 1 to 3, characterized in that: The distance between the upper edge of the cup body and the lowest point of its bottom is the cup body height H, and the distance between the highest point of the ridge and the lowest point of the bottom of the cup body is the ridge height h, where 1 <h / H≤6。 7. The centrifuge cup according to any one of claims 1 to 3, 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.

8. 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 1-7.

9. The method according to claim 8, characterized in that In step S2, the washing operation and the mixing operation are repeated 2-5 times.

10. The method according to any one of claims 8 to 9, characterized in that: The cells are stem cells, immune cells, VERO cells, 293 cells, CHO cells or HEK293 cells.

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