Seed cell separation device and seed cell preparation equipment

Abstract

The invention provides a seed cell separation device and seed cell preparation equipment, and relates to the technical field of medical engineering.The seed cell separation device comprises a capsule shell, an installation part, a filter membrane, a vibration generation part and an acoustic wave transducer. An included angle is formed between the filter membrane and the vertical direction, so that a cell suspension flows along the membrane surface under the action of gravity to generate tangential shearing force, and a liquid inlet is higher than a liquid outlet, so that deposition and blockage of cells on the membrane surface are reduced, and the transmembrane pressure is reduced, so that the membrane pollution problem is relieved, and the filtration flux and the cell recovery rate are improved; by combining high-frequency vibration of the acoustic wave transducer, cell adsorption is reduced, cells on the filter membrane fall off mildly, mechanical damage is avoided, high motility and functionality of seed cells are guaranteed, the problems that in an existing separation technology, membrane pollution is serious, cell damage is large, and efficiency is low are solved, and efficient and mild separation treatment on the seed cells is achieved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of medical engineering, in particular to a seed cell separation device and a seed cell preparation equipment. BACKGROUND

[0002] The separation and extraction of seed cells (such as peripheral blood mononuclear cells PBMC) is a key step in immune cell storage, cell therapy and biomedical research.

[0003] At present, the commonly used seed cell separation methods include density gradient centrifugation, tangential flow filtration, rotary membrane filtration, enzymatic method, magnetic bead sorting and flow cytometry. However, these existing technologies all have significant defects: the density gradient centrifugation method is tedious, time-consuming (≥60 minutes), has low cell recovery rate (70-85%), large activity loss (15-20%), and open operation is easy to cause cross contamination; the conventional tangential flow filtration method has serious membrane adsorption (non-specific adsorption rate > 25%), high mechanical damage rate (≥10%), and low recovery efficiency; the rotary membrane filtration method has high equipment and consumable cost; the enzymatic method and the magnetic bead sorting method may have negative effects on cell function, and the operation conditions are harsh; the flow cytometry requires special equipment and technical personnel, and has high cost and cell damage.

[0004] Overall, the existing technologies generally have low separation efficiency, large cell damage, serious membrane pollution, insufficient automation, and high operation threshold, which are difficult to meet the needs of clinical large-scale, high-throughput, gentle and efficient separation of seed cells. SUMMARY

[0005] The present application aims to provide a seed cell separation device and a seed cell preparation equipment to alleviate the technical problem that the existing technologies are difficult to meet the needs of clinical large-scale, high-throughput, gentle and efficient separation of seed cells.

[0006] The present application aims to provide a seed cell separation device, which comprises a membrane cassette housing, a mounting piece, a filter membrane, a vibration generating piece and a sound wave transducer.

[0007] The membrane cassette housing has a processing space for filtering liquid.

[0008] The mounting piece is arranged in the membrane cassette housing.

[0009] The filter membrane is arranged in the mounting piece and forms an angle with the vertical direction, and the filter membrane separates the processing space into a suspension space and a waste liquid space.

[0010] The vibration generating piece is in transmission connection with the filter membrane to drive the vibration of the filter membrane.

[0011] The sound wave transducer is arranged in the membrane cassette housing and has an output end facing the filter membrane.

[0012] The filter membrane is at an obtuse angle with the horizontal direction, one side space is a suspension space, and the other side is a waste liquid space, the membrane cartridge shell is provided with a liquid inlet and a liquid outlet, the liquid inlet and the liquid outlet are both directed to the filter membrane and located in the suspension space, the liquid inlet is located above the liquid outlet, and the bottom of the membrane cartridge shell is provided with a waste liquid port in communication with the waste liquid space.

[0013] Further, the filter membrane is at an angle of greater than or equal to 60 degrees and less than or equal to 80 degrees with the horizontal direction.

[0014] Further, the seed cell separation device further comprises a rotating mechanism.

[0015] The rotating mechanism comprises a driving member, a mounting frame and a plurality of flexible connecting members, the driving member has a rotating end, the mounting frame is in transmission connection with the rotating end, and the flexible connecting members are arranged in the mounting frame.

[0016] The membrane cartridge shell is connected with the plurality of flexible connecting members to rotate with the mounting frame.

[0017] Further, the seed cell separation device further comprises a contact switch.

[0018] The contact switch is arranged on one side of the mounting frame, and when the membrane cartridge shell is rotated to the state that the liquid outlet and the liquid inlet are both directed downward, the mounting frame is in contact with the triggering end of the contact switch.

[0019] Further, the seed cell separation device further comprises a circulating mechanism.

[0020] The input end of the circulating mechanism is connected with the liquid outlet, and the output end is connected with the liquid inlet.

[0021] Further, the circulating mechanism further comprises a pressure detection member.

[0022] The pressure detection member is arranged at the input end of the circulating mechanism to detect the pressure of the input liquid.

[0023] The purpose of the present application is also to provide a seed cell preparation device, comprising: a pretreatment device, a waste liquid collection assembly, a sample collection assembly and a provided seed cell separation device.

[0024] The pretreatment device is used for performing sedimentation separation treatment on an initial sample, and the pretreatment device has a waste liquid discharge end and a suspension discharge end.

[0025] The liquid inlet of the seed cell separation device is connected with the suspension discharge end.

[0026] The sample collection assembly is connected with the liquid outlet of the seed cell separation device.

[0027] The waste liquid collection assembly is connected with the waste liquid discharge end and the waste liquid port.

[0028] Further, the pretreatment device comprises a separation container and a transmittance sensor.

[0029] The separation container has a separation space, and the separation container is used for introducing the initial sample and a sedimentation agent into the separation space.

[0030] The transmittance sensor is arranged at the waste liquid discharge end.

[0031] Further, the pretreatment device further comprises a first peristaltic pump and a bubble detector.

[0032] The first peristaltic pump is used for pumping the initial sample, and a discharge end of the first peristaltic pump is connected with an introduction end of the separation container.

[0033] The bubble detector is arranged at the introduction end of the separation container.

[0034] Further, the waste liquid collection assembly comprises a second peristaltic pump.

[0035] The second peristaltic pump is connected with the waste liquid discharge end and the waste liquid port of the pretreatment device.

[0036] Beneficial effects: Specifically, the filter membrane in the application is arranged at an angle with the vertical direction, so that the cell suspension flows along the membrane surface under the action of gravity, a tangential shear force is generated, the liquid inlet is higher than the liquid outlet, which is beneficial to the uniform flow of the cell suspension on the membrane surface, avoids local concentration polarization, reduces the deposition and blockage of cells on the membrane surface, reduces the transmembrane pressure, thereby reducing the membrane pollution problem, improving the filtration flux and cell recovery rate, and in the filtration process, the filter membrane is driven to vibrate by the vibration generator, combined with the high-frequency vibration of the acoustic wave transducer, the pollutants (such as platelets or fibrin) attached to the surface of the filter membrane are disturbed, cell adsorption is reduced, and at the same time, cell damage caused by high-pressure backwashing is avoided, the cell activity and integrity are ensured, after filtration, the waste liquid is discharged from the waste liquid port, and vibration and sound waves are combined again to make the cells on the filter membrane fall off gently, mechanical damage is avoided, the high activity and functionality of the seed cells are ensured, thereby effectively alleviating the problems of serious membrane pollution, great cell damage and low efficiency in the existing separation technology, and high-efficiency and gentle separation processing of seed cells is realized.

[0037] The seed cell preparation equipment provided by the application comprises the seed cell separation device provided by the application, and compared with the prior art, the seed cell separation device has the advantages described above, which will not be described here. BRIEF DESCRIPTION OF DRAWINGS

[0038] In order to more clearly illustrate the technical solutions in the specific embodiments of the present application or the prior art, the following will briefly introduce the drawings needed to be used in the description of the specific embodiments or the prior art. Obviously, the drawings described below are some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor based on these drawings.

[0039] Figure 1 The structural schematic diagram of the seed cell separation device provided for the first embodiment of the present application is shown in the figure. Figure 2 The bottom axial structural schematic diagram of the seed cell separation device provided for the first embodiment of the present application is shown in the figure. Figure 3 The structural schematic diagram of the membrane box shell in the seed cell separation device provided for the first embodiment of the present application is shown in the figure. Figure 4 The structural schematic diagram of the seed cell separation device provided for the first embodiment of the present application is shown in the figure. Figure 3 The half-partial structural schematic diagram of the seed cell separation device provided for the first embodiment of the present application is shown in the figure. Figure 5 The structural schematic diagram of the seed cell preparation equipment provided for the second embodiment of the present application is shown in the figure. Figure 6 The comparison diagram of the capture rate of seed cells of different material membrane materials in the third embodiment of the present application is shown in the figure. Figure 7 The comparison diagram of the capture rate of seed cells of different pore size membrane materials in the fourth embodiment of the present application is shown in the figure. Figure 8 The comparison diagram of the number of cells in the target sample prepared by the sedimentation method and the centrifugal method in the fifth embodiment of the present application is shown in the figure. Figure 9 The comparison diagram of the cell viability in the target sample prepared by the sedimentation method and the centrifugal method in the fifth embodiment of the present application is shown in the figure. Figure 10 The comparison diagram of the flow cytometry in the target sample prepared by the sedimentation method and the centrifugal method in the fifth embodiment of the present application is shown in the figure. Figure 11 The comparison diagram of the number of cells in the target sample prepared by the sedimentation method and the centrifugal method in the sixth embodiment of the present application is shown in the figure. Figure 12 The comparison diagram of the cell viability in the target sample prepared by the sedimentation method and the centrifugal method in the sixth embodiment of the present application is shown in the figure. Figure 13 The comparison diagram of the flow cytometry in the target sample prepared by the sedimentation method and the centrifugal method in the sixth embodiment of the present application is shown in the figure. Figure 14 The comparison diagram of the number of cells in the target sample prepared by the sedimentation method and the centrifugal method in the seventh embodiment of the present application is shown in the figure. Figure 15A comparison chart of cell viability in the target sample prepared by the sedimentation method and the centrifugal method in the seventh embodiment of the present application; Figure 16 A comparison chart of CD3- and flow CD56+ in the target sample prepared by the sedimentation method and the centrifugal method in the seventh embodiment of the present application; Figure 17 A comparison chart of flow CD3+ and flow CD8+ in the target sample prepared by the sedimentation method and the centrifugal method in the seventh embodiment of the present application; Figure 18 A comparison chart of cell viability in the target sample prepared by the sedimentation method and the centrifugal method in the eighth embodiment of the present application; Figure 19 A comparison chart of flow in the target sample prepared by the sedimentation method and the centrifugal method in the eighth embodiment of the present application; Figure 20 A comparison chart of CD3- and flow CD56+ in the target sample prepared by the sedimentation method and the centrifugal method in the eighth embodiment of the present application; Figure 21 A comparison chart of flow CD3+ and flow CD8+ in the target sample prepared by the sedimentation method and the centrifugal method in the eighth embodiment of the present application.

[0040] Icon: 100-seed cell separation device; 110-membrane cassette housing; 111-suspension space; 112-waste liquid space; 113-liquid inlet; 114-liquid outlet; 115-waste liquid outlet; 120-mounting member; 130-filter membrane; 140-vibration generating member; 150-acoustic transducer; 160-rotation mechanism; 161-driving member; 162-flexible connecting member; 163-bracket; 164-mounting frame; 165-contact switch; 200-circulation mechanism; 210-pressure detecting member; 220-circulation pump; 300-preprocessing device; 310-separation container; 320-transmittance sensor; 330-first peristaltic pump; 340-bubble detector; 400-waste liquid collection assembly; 410-second peristaltic pump; 510-sample bag; 520-sedimentation agent bag; 530-buffer agent bag; 540-cryopreservation solution bag. DETAILED DESCRIPTION

[0041] In order to make the objects, technical solutions and advantages of the embodiments of the present application clearer, the following will clearly and completely describe the technical solutions in the embodiments of the present application with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments of the present application. The components of the embodiments of the present application described and shown in the accompanying drawings can be arranged and designed in various different configurations.

[0042] Therefore, the following detailed description of the embodiments of the present application provided in the accompanying drawings is not intended to limit the scope of the claimed present application, but only represents selected embodiments of the present application. All other embodiments obtained by those of ordinary skill in the art based on the embodiments in the present application without creative labor are within the scope of protection of the present application.

[0043] It should be noted that: similar reference numerals and letters represent similar items in the following drawings, therefore, once an item is defined in one drawing, it does not need to be further defined and explained in subsequent drawings.

[0044] In the description of the present application, it should be noted that the terms "center", "upper", "lower", "left", "right", "vertical", "horizontal", "inner", "outer" and the like indicate the orientation or positional relationship based on the orientation or positional relationship shown in the drawings, or the orientation or positional relationship when the product of the present application is usually placed, and are only for the convenience of describing the present application and simplifying the description, and therefore cannot be understood as indicating or implying that the indicated device or element must have a particular orientation, be constructed and operated in a particular orientation, and therefore cannot be understood as limiting the present application. In addition, the terms "first", "second", "third" and the like are only used for differentiation and cannot be understood as indicating or implying relative importance.

[0045] In addition, the terms "horizontal", "vertical" and the like do not mean that the components must be absolutely horizontal or vertical, but can be slightly inclined. For example, "horizontal" only means that its direction is relatively more horizontal than "vertical", and does not mean that the structure must be completely horizontal, but can be slightly inclined.

[0046] In the description of the present application, it should also be noted that, unless otherwise explicitly specified and limited, the terms "arrangement", "installation", "connection", "connection" should be understood broadly, for example, it can be fixedly connected, or it can be detachably connected, or integrally connected; it can be mechanically connected, or it can be electrically connected; it can be directly connected, or it can be indirectly connected through an intermediate medium; it can be the communication inside two elements. For those of ordinary skill in the art, the specific meaning of the above terms in the present application can be understood according to the specific circumstances.

[0047] The present application will be further described in detail below through specific embodiments and in conjunction with the accompanying drawings.

[0048] Embodiment one Please refer to Figure 1 、 Figure 3 、 Figure 4 The seed cell separation device 100 provided by the embodiment comprises a membrane cassette shell, a mounting piece 120, a filter membrane 130, a vibration generating piece 140 and a sound wave transducer 150.

[0049] The membrane cassette shell 110 has a processing space for filtering liquid. The mounting piece 120 is arranged in the membrane cassette shell 110. The filter membrane 130 is arranged on the mounting piece 120 and forms an angle with the vertical direction, and the filter membrane 130 divides the processing space into a suspension space 111 and a waste liquid space 112. The vibration generating piece 140 is in transmission connection with the filter membrane 130 to drive the filter membrane 130 to vibrate. The sound wave transducer 150 is arranged on the membrane cassette shell 110 and the output end thereof faces the filter membrane 130.

[0050] In the embodiment, the side space of the filter membrane 130 which forms an obtuse angle with the horizontal direction is the suspension space 111, and the other side is the waste liquid space 112. The membrane cassette shell 110 is provided with a liquid inlet 113 and a liquid outlet 114, both of which face the filter membrane 130 and are located in the suspension space 111. The liquid inlet 113 is located above the liquid outlet 114. The bottom of the membrane cassette shell 110 is provided with a waste liquid outlet 115 which is in communication with the waste liquid space 112.

[0051] In the embodiment, the filter membrane 130 is arranged at an angle with the vertical direction, so that the cell suspension flows along the membrane surface under the action of gravity, a tangential shear force is generated, the deposition and blockage of cells on the membrane surface are reduced, the transmembrane pressure is reduced, and thus the membrane pollution problem is significantly alleviated, and the filtration flux and cell recovery rate are improved. In the embodiment, the liquid inlet 113 and the liquid outlet 114 are both located in the suspension space 111 above the filter membrane 130, and the liquid inlet 113 is higher than the liquid outlet 114, which is conducive to the formation of uniform flow of the cell suspension on the membrane surface, avoids local concentration polarization, and improves the separation precision and efficiency. The waste liquid outlet 115 is arranged at the bottom, which is convenient for timely discharge of impurities and waste liquid, and maintains the stability of the system.

[0052] During the filtration process, the filter membrane 130 is driven to vibrate by the vibration generating piece 140, and in combination with the high-frequency vibration of the sound wave transducer 150, the pollutants (such as platelets or fibrin) attached to the membrane surface can be effectively disturbed, dynamic cleaning is realized, non-specific adsorption of cells is reduced, cell damage caused by high-pressure backwashing is avoided, and the cell activity and integrity are ensured.

[0053] After filtration, the waste liquid is discharged from the waste liquid outlet 115, and the cells on the filter membrane 130 are gently detached in combination with vibration and sound waves, mechanical damage is avoided, the high activity and functionality of the seed cells are ensured, and the device is suitable for subsequent cell culture, cryopreservation and treatment applications.

[0054] The seed cell separation device 100 provided by the embodiment is compact in structure, can be integrated with a peristaltic pump, a sensor and a control system to realize fully-closed and automatic operation, reduces manual intervention and pollution risk, is suitable for high-throughput sample processing, meets clinical and industrial application requirements, effectively alleviates problems such as serious membrane pollution, great cell damage and low efficiency in existing separation technologies through the synergistic effect of the inclined structure of the filter membrane 130, vibration dissociation and acoustic cleaning, and realizes efficient and gentle separation and processing of the seed cells.

[0055] It should be noted that in the embodiment, the filter membrane 130 is made of polyether sulfone (PES) or polycarbonate (PC), and the pore size gradient distribution includes but is not limited to 3 μm, 4 μm, 5 μm and 8 μm. The ultrasonic wave generated by the acoustic transducer 150 is 1 MHz, and the pulse duty cycle is 50%. The mounting member 120 is a support net made of polypropylene (PP), and the filter membrane 130 is attached to the surface of the support net.

[0056] In the embodiment, the angle between the filter membrane 130 and the horizontal direction is greater than or equal to 60° and less than or equal to 80°.

[0057] When the angle is less than 60°, the membrane surface is too flat, the gravity component in the direction perpendicular to the membrane surface is increased, and the deposition and compaction of cells on the membrane surface are intensified, which is not conducive to the flushing of pollutants. When the angle is greater than 80°, the membrane surface is nearly vertical, the shear force generated by the tangential flow is weakened, and the flow is close to dead-end filtration, which is easy to cause rapid clogging.

[0058] In the embodiment, the angle between the filter membrane 130 and the horizontal direction is greater than or equal to 60° and less than or equal to 80° (preferably 70°±5°). Within this range, the flushing force of the tangential fluid and the auxiliary effect of the gravity are balanced, the optimal shear force can be generated to flush the membrane surface, and the deposition of cells or macromolecules is minimized, thereby effectively alleviating membrane pollution and clogging. Specifically, the angle between the filter membrane 130 and the horizontal direction in the embodiment is specifically 70°.

[0059] At this angle, the fluid can form a stable and uniform laminar flow along the membrane surface, and the transmembrane pressure required to achieve a high filtration rate is reduced. The lower transmembrane pressure means that the cells are subjected to less extrusion stress, thereby directly reducing the risk of mechanical damage and ensuring the high activity of the cells. At the same time, the smooth flow channel ensures the filtration flux, so that the processing capacity of the device can reach 1 L / hour, and the application requirement of high-throughput is met.

[0060] Please refer to Figure 1 , Figure 2 In the embodiment, the seed cell separation device 100 further comprises a rotating mechanism 160.

[0061] The rotating mechanism 160 comprises a driving member 161, a mounting frame and flexible connecting members 162. The driving member 161 has a rotating end, the mounting frame is in transmission connection with the rotating end, and the flexible connecting members 162 are arranged in the mounting frame.

[0062] In the embodiment, the rotating mechanism 160 is in transmission connection with the membrane cassette shell 110 through the mounting frame and the flexible connecting members 162 to realize the overturning of the membrane cassette shell 110. After the filtration is completed, the membrane cassette is driven to rotate (for example, to a horizontal state or an arbitrary angle within 180°) by the rotating mechanism 160, combined with buffer flushing and ultrasonic activation, so that the cells attached to the surface of the filter membrane 130 are gently detached. This physical overturning recovery method replaces the high-pressure backwashing commonly used in the traditional tangential flow filtration, avoids the damage of high shear force to the cells, ensures that the cells can still maintain a high activity rate in the recovery stage, and thus improves the final recovery efficiency and quality of the target seed cells as a whole.

[0063] In addition, the rotating mechanism 160, in operation, constitutes a cooperative dissociation structure with the vibration generator 140 and the acoustic transducer 150. During the overturning process or after being overturned to a specific angle, the vibration generator 140 and the acoustic transducer 150 are started, so that the combined force of the change of the direction of gravity and physical vibration can more effectively disturb and peel the cell layer on the membrane surface, realize automatic, efficient and gentle and lossless cell recovery, and improve the recovery efficiency.

[0064] Specifically, in the embodiment, the driving member 161 in the rotating mechanism 160 is a driving motor, the mounting frame comprises a support 163 and a mounting frame 164, the support 163 is used for supporting structure, the support 163 has two axially opposite shaft holes, each of which is provided with a bearing, and the mounting frame 164 is arranged between the two shaft holes and the two ends of the mounting frame 164 are respectively connected with the two bearings. The output end of the driving motor is arranged in the shaft hole on one side and is in transmission connection with the mounting frame 164 through the bearing to drive the mounting frame 164 to overturn. The plurality of flexible connecting members 162 are arranged in the mounting frame 164 along the circumferential direction of the mounting frame 164, and each of the plurality of flexible connecting members 162 is a connecting structure having one end connected with the inner wall of the mounting frame 164 and the other end connected with the membrane cassette shell 110, so as to avoid the vibration of the membrane cassette shell 110 being transmitted to the mounting frame.

[0065] Under this structure, the driving motor drives the membrane cartridge shell 110 to rotate, which can adjust the setting angle of the filter membrane 130, so that the filter membrane 130 is in the preferred angle range, and the membrane cartridge shell 110 can be turned over after filtration to make the liquid inlet 113 and the liquid outlet 114 face downward to discharge the separated cell suspension. In addition, under this transmission structure, the rotation stability and the accuracy of the turning angle are very excellent (such as turning at any angle within 180° as described in the document, and the rotation time is less than or equal to 5 seconds), and the flexible connection can not only avoid vibration transmission, but also reduce the rigid impact of the turning action on the membrane cartridge shell 110, which protects the internal components of the membrane cartridge shell 110, and realizes the "bag-in-bag-out" full sealing and one-key operation from sample input to cell cryopreservation, greatly reducing the operation complexity and human error risk.

[0066] In the present embodiment, the seed cell separation device 100 further comprises a contact switch 165.

[0067] The contact switch 165 is arranged on one side of the mounting frame, and when the membrane cartridge shell 110 is rotated to the state that the liquid outlet 114 and the liquid inlet 113 both face downward, the mounting frame contacts the triggering end of the contact switch 165.

[0068] In the present embodiment, the contact switch 165 is arranged on one side of the bracket 163 as a position detection component, and the detection end faces the mounting frame 164, so as to detect whether the membrane cartridge shell 110 has been rotated to the right position, i.e., the state that the liquid outlet 114 and the liquid inlet 113 both face downward (for example, the membrane surface originally inclined upward is turned over to face downward, which is beneficial for cell shedding). When the mounting frame is rotated and touches the triggering end of the contact switch 165, it is confirmed that the membrane cartridge shell 110 has been rotated to the right position. The in-place prompt is realized through the physical positioning feedback mechanism of hardware, which avoids the accumulated error that may exist in the simple dependence on the rotation time of the motor or the angle encoder, and ensures that the membrane cartridge can accurately and repeatedly reach the optimal recovery position each time.

[0069] In the present embodiment, the contact switch 165 is triggered to confirm that the membrane cartridge has safely reached the specified position before the sound wave transducer 150 is started, which avoids the liquid splashing, pressure abnormality or equipment damage that may be caused by starting the ultrasonic wave when the turning is not in place, and improves the safety level.

[0070] In combination Figure 5 In the present embodiment, the seed cell separation device 100 further comprises a circulating mechanism 200. The input end of the circulating mechanism 200 is connected with the liquid outlet 114, and the output end is connected with the liquid inlet 113.

[0071] In this embodiment, the circulation mechanism 200 connects the liquid outlet 114 and the liquid inlet 113 of the membrane cassette 110 to form a closed circulation loop, so that the cell suspension can flow through the inclined filter membrane 130 multiple times to realize multiple circulation filtration. Compared with single filtration, the circulation filtration mode realized by the circulation mechanism 200 significantly increases the effective contact opportunity of target cells (such as PBMC) with the filter membrane 130, and allows more sufficient washing, continuously discharges small-particle impurities such as red blood cells that are not intercepted by the membrane holes through the waste liquid outlet 115, and improves the capture efficiency of target seed cells and the purity of the final product.

[0072] In addition, the circulation mechanism 200 has a synergistic effect with the structure of the inclined filter membrane 130 and the vibration dissociation technology. The tangential flow of the liquid generates a continuous shear force on the surface of the filter membrane 130, which can flush the deposited cells or contaminants, effectively alleviate the problem of membrane flux decline caused by concentration polarization during filtration, and enable the entire separation process to maintain stable high flux at a lower transmembrane pressure, thereby shortening the total processing time (which can be shortened to 30 minutes).

[0073] In addition, when the circulation mechanism 200 extracts the filtrate from the suspension space 111, a filtration gap is generated at this time, and the suspension space 111 is empty. Buffer can be buffered into the suspension space 111 to dilute the suspension and maintain the optimal filtration environment, without stopping to inject the buffer, so as to realize reagent addition while ensuring the continuity and efficiency of the process.

[0074] Specifically, in this embodiment, the circulation mechanism 200 includes a circulation pipeline and a circulation pump 220. The two ends of the circulation pipeline are respectively connected to the liquid outlet 114 and the liquid inlet 113, and the circulation pump 220 is arranged on the circulation pipeline to extract liquid from the liquid outlet 114 and discharge into the liquid inlet 113.

[0075] In this embodiment, the circulation mechanism 200 further includes a pressure detection member 210. The pressure detection member 210 is arranged at the input end of the circulation mechanism 200 to detect the input liquid pressure.

[0076] Excessive transmembrane pressure is the main reason for the extrusion of cells and the occurrence of mechanical damage. Specifically, the pressure detection member 210 in this embodiment is arranged on the circulation pipeline and located between the liquid outlet 114 and the input end of the circulation pump 220, so that the pressure detection member 210 can dynamically monitor the outlet liquid pressure in the membrane cassette 110.

[0077] When the pressure detection member 210 detects that the filter membrane 130 is in an "overpressure state", protective measures (such as automatically reducing the pump speed or triggering a specific cleaning program) can be taken in time, so as to actively avoid the damage to the cells caused by high pressure, thereby improving the high activity rate of the seed cells.

[0078] And the pressure detection piece 210 can also work with the vibration generating piece 140. In the normal state, the vibration generating piece 140 maintains a low frequency to meet the requirements of flux and filtration time. When the overpressure state, the vibration generating piece 140 vibrates at a high frequency to actively intervene, reduce the viscous force between the cells and the membrane material, and dynamically remove the membrane surface pollutants to restore the flux.

[0079] Embodiment two As Figure 5 The seed cell preparation device provided in the embodiment includes a pretreatment device 300, a waste liquid collection assembly 400, a sample collection assembly, and the seed cell separation device 100 provided in the embodiment one.

[0080] The pretreatment device 300 is used for performing sedimentation separation treatment on the initial sample. The pretreatment device 300 has a waste liquid discharge end and a suspension discharge end. The liquid inlet 113 of the seed cell separation device 100 is connected with the suspension discharge end. The sample collection assembly is connected with the liquid outlet 114 of the seed cell separation device 100. The waste liquid collection assembly 400 is connected with the waste liquid discharge end and the waste liquid outlet 115.

[0081] In the embodiment, the initial blood sample is first subjected to sedimentation separation by the pretreatment device 300. The lower layer of waste liquid such as red blood cells is discharged into the waste liquid collection assembly 400 through the waste liquid discharge end, and the upper layer of suspension liquid containing target seed cells is directly introduced into the seed cell separation device 100 through the suspension discharge end for further filtration, so that the purified seed cell suspension is collected in the sample collection assembly (such as a cryopreservation bag).

[0082] The entire process in the embodiment can be connected through a disposable sterile closed pipeline, avoiding the cross contamination risk caused by open sample operation, and can be operated without a C-level laboratory environment. At the same time, without multiple tedious manual transfer steps (such as sedimentation, centrifugation, and washing), the total processing time is significantly shortened from ≥60 minutes of the traditional method to about 30 minutes, improving the efficiency and meeting the high flux demand.

[0083] In the embodiment, the pretreatment device 300 adopts the sedimentation method (such as using red blood cell sedimentation agent) for preliminary separation, avoiding the use of Ficoll reagent (phenol reagent), greatly saving the pretreatment cost, and being more gentle to cells. The suspension after preliminary separation by the pretreatment device 300 does not need to be subjected to secondary stress such as centrifugation, and is directly flowed into the inclined membrane separation device for purification. The cell loss and activity damage in the intermediate link are reduced, ensuring the highest possible recovery rate and cell activity from the sample to the final product, and the culture effect is equivalent to or even better than the centrifugation method.

[0084] And, the waste liquid collecting assembly 400 in the embodiment simultaneously receives the waste liquid (lower layer red blood cells) from the pretreatment device 300 and the waste liquid (filtered impurities) from the seed cell separating device 100. The management of multiple waste liquids makes the waste liquid treatment safer, more convenient, and facilitates the material balance accounting of the whole process, which meets the production specifications.

[0085] Under this structure, when the seed cells are prepared, the sample bag 510 is only connected, and the device can automatically complete all operations from sedimentation, filtration, washing to cell collection. This high integration and automation greatly reduces the technical requirements for the operator, reduces manual intervention, significantly reduces the labor cost and the risk of failure caused by operation errors.

[0086] In the embodiment, the pretreatment device 300 includes a separation container 310 and a transmittance sensor 320.

[0087] The separation container 310 has a separation space, the separation container 310 is used for introducing an initial sample and a sedimentation agent to the separation space, and a waste liquid discharge end and a suspension discharge end are both connected with the separation space. The transmittance sensor 320 is arranged at the waste liquid discharge end.

[0088] The transmittance sensor 320 in the embodiment is integrated at the waste liquid discharge end of the pretreatment device 300. Specifically, after the sample and the sedimentation agent are layered in the separation container 310, since there is a significant difference in transmittance between the lower layer of the sedimented red blood cell suspension and the upper layer of the suspension containing the target seed cells, the transmittance sensor 320 can detect in real time whether the liquid currently discharged by the separation container 310 is the waste liquid (red blood cells) or the target cell suspension.

[0089] When the change of the liquid transmittance is detected, indicating that the target cells will be discharged soon, the discharge to the waste liquid collecting assembly 400 is stopped, which greatly improves the separation accuracy, ensures that only the waste liquid is discharged without misdischarging the target cells, avoids the waste of the target cells, and guarantees the cell recovery rate.

[0090] In the embodiment, the pretreatment device 300 further includes a first peristaltic pump 330 and a bubble detector 340.

[0091] The first peristaltic pump 330 is used for pumping the initial sample, and the discharge end of the first peristaltic pump 330 is connected with the introduction end of the separation container 310. The bubble detector 340 is arranged at the introduction end of the separation container 310.

[0092] The first peristaltic pump 330 can pump and convey the initial sample (such as blood) from the sample bag 510 to the separation container 310, the conveying process is soft and without shearing, which avoids additional mechanical damage to the cells in the sampling stage.

[0093] The mixing of bubbles will interfere with the effect of sedimentation stratification, and may even affect the accuracy of subsequent sensor judgment, and potentially affect cell activity. The bubble detector 340 provided at the introduction end of the separation container 310 can detect the fluid state in the pipeline in real time. By judging the switching of the gas and liquid state input by the introduction end, the start and stop of the control of the first peristaltic pump 330 is realized, so as to prevent bubbles from entering the separation container 310 with the sample, so as to ensure that the sample entering the pretreatment device 300 is a continuous and stable liquid column.

[0094] In this embodiment, the combination of the first peristaltic pump 330 and the bubble detector 340 realizes automatic control of the sample introduction process, which can complete the quantitative and stable transportation from the sample bag 510 to the separation container 310 without manual intervention, and automatically stop the operation of the pump when bubbles are detected or the sample transportation is completed (gas enters the pipeline). Not only simplifies the operation, but also avoids the risk of pretreatment failure caused by improper human operation (such as introduction of bubbles and inaccurate transportation amount), improves the repeatability and reliability.

[0095] The first peristaltic pump 330, the bubble detector 340, the transmittance sensor 320 and the waste liquid peristaltic pump together constitute an automatic pretreatment system with complete functions and accurate control. From "when to start sampling, whether the sampling is normal" (controlled by the bubble detector 340) to "when to stop discharging waste liquid" (controlled by the transmittance sensor 320), the whole process realizes seamless automatic closed-loop management. It is ensured that the pretreatment stage can provide high-quality, bubble-free and clear stratification cell suspension feed for the seed cell separation device 100 at the back end.

[0096] In this embodiment, the waste liquid collection assembly 400 includes a second peristaltic pump 410.

[0097] The second peristaltic pump 410 is connected with the waste liquid discharge end and the waste liquid port 115 of the pretreatment device 300.

[0098] Specifically, the second peristaltic pump 410 in this embodiment can provide accurate and gentle negative pressure suction, avoid impact on the liquid level in the separation container 310 or the filtration process of the filter membrane 130 due to excessive or fluctuating suction, thereby maintaining the stability of the pretreatment sedimentation interface and the membrane surface fluid environment, and indirectly protecting the target cells.

[0099] The start and stop of the second peristaltic pump 410 are controlled by the feedback of the transmittance sensor 320, so as to ensure that the timing and rate of waste liquid discharge are in the optimal state, and the misdischarge of the target cells is avoided.

[0100] And, in this embodiment, by serving two different sources of waste liquid outlets (preprocessing device 300 and seed cell separation device 100) through the second peristaltic pump 410, the number of execution elements is reduced, the liquid circuit pipeline layout is simplified, thereby reducing the manufacturing cost, control complexity and potential failure points of the equipment.

[0101] In addition, when the seed cell separation device 100 discharges waste liquid, the second peristaltic pump 410 can provide a certain negative pressure suction to suck out small-particle impurities such as red blood cells from the upper cell suspension.

[0102] It should be noted that in this embodiment, sample bag 510, settling agent bag 520, buffer agent bag 530 and cryopreservation liquid bag 540 are provided to hold the initial sample, the settling agent, the buffer agent and the cryopreservation liquid respectively, and the sample bag 510, the settling agent bag 520, the buffer agent bag 530 and the cryopreservation liquid bag 540 are all connected through disposable sterile tubes, and the opening and closing of each disposable sterile tube is controlled through a plurality of pinch valves, thereby realizing the delivery and stoppage of the initial sample and each reagent.

[0103] Among them, the sample bag 510, the settling agent bag 520 and the buffer agent bag 530 are connected with the liquid inlet end of the separation container 310, and the buffer agent bag 530 is also connected with the cryopreservation liquid bag 540 and the suspension space 111 of the seed cell separation device 100. The liquid outlet 114 of the seed cell separation device 100 is also connected with the cryopreservation bag for storing the target cell suspension, so as to realize the collection and storage of the target cell suspension.

[0104] Embodiment three In this embodiment, the filter membrane 130 with the same pore size (5µm) but different materials is used as the filter membrane 130 in the seed cell separation device in Embodiment two to capture seed cells in fresh peripheral blood, so as to obtain the capture rate.

[0105] Specifically, in this embodiment, red blood cell settling solution and diluent are added to the peripheral blood, and after standing, the blood sample is stratified, and the cell suspension containing seed cells in the upper layer is collected and added into the filter membrane screening kit containing 5µm PA membrane material, 5µm PES membrane material, 5µm PTFE membrane material and 5µm PET membrane material, respectively. After pressure filtration, the seed cells on the membrane material are resuspended and counted using cell washing solution, and the capture rate of seed cells using Ficoll density gradient centrifugation is calculated.

[0106] Among them, the filter membrane 130 respectively adopts PA membrane material (high molecular thin film with polyamide (nylon) as base material), PES membrane material (polyether sulfone membrane), PTFE membrane material (polytetrafluoroethylene), and PET membrane material (polyester heat shrinkage film), and the capture rate is as follows Figure 6As shown, it can be seen that the PET film material is relatively more suitable for capturing seed cells.

[0107] Example Four In this embodiment, seed cells in fresh peripheral blood are captured by using nuclear pore membranes with different pore sizes (7 pm, 5 pm, and 2 pm) as filter membranes 130, and the capture rates are obtained.

[0108] Specifically, in this embodiment, red blood cell sedimentation solution and diluent are added to the peripheral blood, and after standing, the blood sample is stratified, and the cell suspension containing seed cells in the upper layer is collected and added to the filter membrane screening kit containing 7 pm PET film material, 5 pm PET film material, and 2 pm PET film material. After pressure filtration, the seed cells on the recovered membrane material are resuspended using a cell washing solution and counted, and the capture rates of seed cells using Ficoll density gradient centrifugation are calculated.

[0109] The capture rates of 7 pm PET film material, 5 pm PET film material, and 2 pm PET film material are as shown in Figure 7 As shown, it can be seen that the 2 pm PET film material can achieve efficient capture of seed cells.

[0110] Example Five In this embodiment, on the one hand, the seed cell preparation device provided in Example Two is used to harvest seed cells from a peripheral blood sample using a 2 pm PET film material filter membrane. The recovered seed cells are resuspended and counted using an NK cell culture medium, and the cells are cultured according to the conventional NK cell culture process.

[0111] The NK cell culture medium is a special culture medium specifically designed for in vitro culture, expansion, and maintenance of natural killer cells (NK cells) to support the proliferation, activation, and functional maintenance of NK cells to meet the needs of immunotherapy research and clinical applications.

[0112] In addition, the NK cells are harvested 5 times in a 14-day cycle, and the samples are detected for flow cytometry on day 0 and day 7, and for sterility, viability, and flow cytometry on day 14 to obtain the first data.

[0113] On the other hand, in this embodiment, seed cells are obtained by centrifugation of fresh peripheral blood and NK cells are cultured. The seed cells obtained by Ficoll density gradient centrifugation of the peripheral blood sample are counted, and the cells are cultured according to the conventional NK cell culture process. Seed cell inoculation and culture, and 5 times of liquid supplementation in a 14-day cycle to harvest NK cells are performed, and the samples are detected for flow cytometry on day 0 and day 7, and for sterility, viability, and flow cytometry on day 14 to obtain the second data.

[0114] The first data and the second data were compared in terms of cell number, viability, flow CD3- (non-T cells), and flow CD56+ (NK cells), respectively, to obtain the following data Figure 8 , Figure 9 , Figure 10 Data.

[0115] In the figure, the sedimentation method is the first data, i.e., the cell suspension data obtained by the seed cell preparation device provided in Example 2, and the centrifugation method is the second data. It can be seen that, compared with the centrifugation method, the seed cells obtained by the sedimentation prototype machine can also be cultured to obtain sufficient number, viability, and purity of NK cells.

[0116] Example Six In this embodiment, on the one hand, the seed cells of the peripheral blood sample were harvested by using the filter membrane of 2 pm PET membrane material through the seed cell preparation device provided in Example 2.

[0117] In this embodiment, the seed cells on the recovered filter membrane were resuspended and counted using the cell freezing solution, and the cells were frozen for 3 months (hereinafter referred to as 3M) according to the conventional freezing process. After 3M, the cells were resuspended and counted after 37°C water bath recovery and cell washing liquid washing, and the cells were cultured according to the conventional culture process of NK cells. The NK cells were harvested 5 times in a 14-day cycle, and the flow cytometry was detected on the 0th day and the 7th day, the sterility, viability, and flow cytometry were detected on the 14th day, to obtain the third data.

[0118] On the other hand, in this embodiment, the seed cells of the peripheral blood were also obtained by centrifugation method, and then frozen for 3M, and then cultured into NK cells. In this embodiment, the seed cells obtained from the peripheral blood sample using Ficoll density gradient centrifugation were counted, and the seed cells were frozen using the cell freezing solution. After the seed cells were frozen for 3M, the cells were resuspended and counted after 37°C water bath recovery and cell washing liquid washing, and the cells were cultured according to the conventional culture process of NK cells. The seed cells were inoculated and cultured, and the NK cells were harvested 5 times in a 14-day cycle, and the flow cytometry was detected on the 0th day and the 7th day, the sterility, viability, and flow cytometry were detected on the 14th day, to obtain the fourth data.

[0119] The third data and the fourth data were compared in terms of cell number, viability, flow CD3-, and flow CD56+ to obtain the following data Figure 11 , Figure 12 , Figure 13 Data.

[0120] In the figure, “sedimentation method-frozen for 3M” is the third data, i.e., the cell suspension data obtained by the seed cell preparation device provided in Example 2, and “centrifugation method-frozen for 3M” is the fourth data. It can be seen that, compared with the centrifugation method, the seed cells obtained by the sedimentation method and frozen for 3M can also be cultured to obtain sufficient number, viability, and purity of NK cells.

[0121] Example Seven In this example, on the one hand, the seed cell preparation device provided in Example Two is used to harvest seed cells from peripheral blood samples using a microporous filtration assembly of 2 pm PET membrane material. The seed cells on the recovered membrane material are resuspended and counted using CIK cell culture medium, and the cells are cultured according to the conventional CIK cell culture process. The CIK cells are harvested 5 times in a 14-day cycle, and on the 0th day and the 7th day, flow cytometry is performed for sampling and detection, and on the 14th day, sterility, viability, and flow cytometry are performed for sampling and detection, to obtain the fifth data.

[0122] On the other hand, fresh peripheral blood is centrifuged to obtain seed cells and culture CIK cells. The seed cells obtained from the peripheral blood sample using Ficoll density gradient centrifugation are counted, and the cells are cultured according to the conventional CIK cell culture process. Seed cell inoculation and culture and 5 times of liquid supplementing in a 14-day cycle are performed to harvest CIK cells, and on the 0th day and the 7th day, flow cytometry is performed for sampling and detection, and on the 14th day, sterility, viability, and flow cytometry are performed for sampling and detection, to obtain the sixth data.

[0123] The number of cells, viability, flow cytometry CD3-, flow cytometry CD56+, flow cytometry CD3+, and flow cytometry CD8+ in the fifth data and the sixth data are compared, respectively, to obtain the following data. Figure 14 、 Figure 15 、 Figure 16 、 Figure 17

[0124] Among them, Figures 14 to 17 The "sedimentation method" in the above data is the cell turbidity data obtained by the seed cell preparation device provided in Example Two, and the "centrifugation method" is the sixth data. It can be seen that, compared with the centrifugation method, the sedimentation method for obtaining seed cells can also culture sufficient number, viability, and purity of CIK cells.

[0125] Example Eight In this example, on the one hand, the seed cell preparation device provided in Example Two is used to harvest seed cells from peripheral blood samples using a filter membrane 130 of 2 pm PET membrane material, and the seed cells on the recovered membrane material are resuspended and counted using cell freezing solution, and the cells are frozen according to the conventional freezing process 3M. After 3M, the cells are resuspended and counted by washing with 37°C water bath and cell washing solution, and the cells are cultured according to the conventional CIK cell culture process. The CIK cells are harvested 5 times in a 14-day cycle, and on the 0th day and the 7th day, flow cytometry is performed for sampling and detection, and on the 14th day, sterility, mycoplasma, flow cytometry, and endotoxin are performed for sampling and detection, to obtain the seventh data.

[0126] ​In another aspect, in the present embodiment, the peripheral blood seed cells are obtained by centrifugation method, and then frozen for 3M before culturing CIK cells. The seed cells obtained from the peripheral blood sample using Ficoll density gradient centrifugation are counted, and the seed cells are frozen using cell freezing solution. After the seed cells are frozen for 3M, they are resuscitated in a 37℃ water bath, washed with cell washing solution, resuspended and counted. The cells are cultured according to the conventional CIK cell culture process, and the seed cells are inoculated and cultured, and the CIK cells are harvested within a 14-day period after 5 times of liquid supplement. The samples are taken on the 0th day and the 7th day for flow cytometry detection, and the samples are taken on the 14th day for sterility detection, mycoplasma detection, flow cytometry detection and endotoxin detection, to obtain the eighth data.

[0127] The cell number, viability, flow cytometry CD3- (non-T cells) and flow cytometry CD56+ (NK cells), flow cytometry CD3+ and flow cytometry CD8+ in the seventh data and the eighth data are compared, and the following data are obtained, respectively. Figure 18 、 Figure 19 、 Figure 20 、 Figure 21

[0128] Among them, Figures 18 to 21 The "sedimentation method" in the seventh data is the cell turbidity data obtained by the seed cell preparation device provided in Example 2, and the "centrifugation method" is the eighth data. It can be seen that, compared with the centrifugation method, the seed cells obtained by the sedimentation method can also culture CIK cells with sufficient number, viability and purity.

[0129] Finally, it should be noted that: the above examples are only used to illustrate the technical solutions of the present application, and not to limit them; although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that: it can still modify the technical solutions recorded in the foregoing examples, or make equivalent replacement for part or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the present embodiment.​

Claims

1. A seed cell separation device, characterized in that, include: The membrane housing (110) has a processing space for filtering liquids; The mounting component (120) is disposed inside the membrane housing (110); A filter membrane (130) is disposed on the mounting member (120) and forms an angle with the vertical direction. The filter membrane (130) divides the processing space into a suspension space (111) and a waste liquid space (112). A vibration generator (140) is connected to the filter membrane (130) to drive the filter membrane (130) to vibrate; An acoustic transducer (150) is disposed in the membrane housing (110) with its output end facing the filter membrane (130); The filter membrane (130) has a suspension space (111) on one side that forms an obtuse angle with the horizontal direction, and a waste liquid space (112) on the other side. The membrane housing (110) is provided with an inlet (113) and an outlet (114). Both the inlet (113) and the outlet (114) face the filter membrane (130) and are located in the suspension space (111). The inlet (113) is located above the outlet (114). The bottom of the membrane housing (110) is provided with a waste liquid outlet (115) that communicates with the waste liquid space (112).

2. The seed cell separation device according to claim 1, characterized in that, The angle between the filter membrane (130) and the horizontal direction is greater than or equal to 60° and less than or equal to 80°.

3. The seed cell separation device according to claim 1, characterized in that, The seed cell separation device (100) also includes a rotating mechanism (160); The rotating mechanism (160) includes a driving member (161), a mounting frame, and a flexible connector (162). The driving member (161) has a rotating end, the mounting frame is connected to the rotating end in a transmission manner, and there are multiple flexible connectors (162) disposed in the mounting frame. The outer shell (110) of the diaphragm is connected to a plurality of the flexible connectors (162) to rotate with the mounting frame.

4. The seed cell separation device according to claim 3, characterized in that, The seed cell separation device (100) also includes a contact switch (165); The contact switch (165) is located on one side of the mounting bracket. When the membrane housing (110) is rotated so that both the liquid outlet (114) and the liquid inlet (113) are facing downwards, the mounting bracket is connected to the trigger end of the contact switch (165).

5. The seed cell separation device according to claim 1, characterized in that, The seed cell separation device (100) also includes a circulation mechanism (200); The input end of the circulation mechanism (200) is connected to the liquid outlet (114), and the output end is connected to the liquid inlet (113).

6. The seed cell separation device according to claim 5, characterized in that, The circulation mechanism (200) also includes a pressure detection element (210); The pressure detection element (210) is located at the input end of the circulation mechanism (200) to detect the input liquid pressure.

7. A seed cell preparation device, characterized in that, include: The pretreatment device (300), the waste liquid collection assembly (400), the sample collection assembly, and the seed cell separation device (100) as described in any one of claims 1-6; The pretreatment device (300) is used to perform sedimentation separation treatment on the initial sample, and the pretreatment device (300) has a waste liquid discharge end and a suspension discharge end; The inlet (113) of the seed cell separation device (100) is connected to the suspension outlet; The sample collection component is connected to the liquid outlet (114) of the seed cell separation device (100); The waste liquid collection assembly (400) is connected to the waste liquid discharge end and the waste liquid port (115).

8. The seed cell preparation apparatus according to claim 7, characterized in that, The pretreatment device (300) includes: A separation container (310) has a separation space, the separation container (310) is used to introduce an initial sample and a settling agent into the separation space, and the waste liquid discharge end and the suspension discharge end are both connected to the separation space; A light transmittance sensor (320) is located at the waste liquid discharge end.

9. The seed cell preparation equipment according to claim 8, characterized in that, The pretreatment device (300) further includes: A first peristaltic pump (330) is used to aspirate the initial sample, and the discharge end of the first peristaltic pump (330) is connected to the inlet end of the separation container (310). A bubble detector (340) is located at the inlet end of the separation container (310).

10. The seed cell preparation apparatus according to claim 7, characterized in that, The waste liquid collection assembly (400) includes: The second peristaltic pump (410) is connected to the waste liquid discharge end of the pretreatment device (300) and the waste liquid port (115).

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