NK cell separation and purification device

Abstract

The utility model discloses an NK (Natural Killer) cell separation and purification device, which belongs to the technical field of cell purification and is characterized by comprising magnetic bead separation equipment, a pretreatment mechanism is arranged at the top of the magnetic bead separation equipment, and a screening mechanism is movably connected to the bottom of the pretreatment mechanism; a three-dimensional dynamic pretreatment system can be additionally arranged before magnetic bead sorting, the sample quality is effectively improved, the follow-up operation difficulty is reduced, firstly, an anticoagulant premixing mechanism in a mixing barrel can prevent sample blood coagulation, and impurities are reduced; secondly, the anticoagulant is in full contact with the sample in a three-dimensional mixing mode of combining rotation of the main shaft and swinging of the hydraulic rod, so that the mixing uniformity is improved, and local concentration deviation is avoided; meanwhile, the heating plate adjusts the temperature through autorotation, maintains the cell activity and reduces cell damage caused by temperature fluctuation, in addition, the hydraulic rod controls the swing amplitude by adopting differentiated stretching and retracting rates, and the cell structure is prevented from being damaged by violent oscillation.

Description

TECHNICAL FIELD

[0001] The utility model relates to cell purification technical field, especially NK cell separation and purification device. BACKGROUND

[0002] NK cell separation and purification have important significance, NK cells play a key role in immune surveillance, tumor immunity and other aspects, and accurate research on its function and use in immunotherapy and the like depend on high-purity NK cells, and the purity and activity are difficult to be taken into account in traditional separation methods such as density gradient centrifugation, with the development of technology, immunomagnetic bead method, flow cytometry and the like are gradually applied, the immunomagnetic bead method can separate NK cell surface markers by using specific antibodies, and the operation is relatively simple; flow cytometry can accurately sort according to the cell multi-parameter characteristics, but the cost is higher, these technologies are continuously improved, aiming at improving the purity, activity and yield of NK cell separation, and providing better support for related research and clinical application.

[0003] In the prior art, antibodies are combined with NK cells, which stimulates NK cells and may affect the accuracy of function experiments; on the other hand, negative sorting method can be used, in which the magnetic beads and antibodies do not contact NK cells, reducing the influence on the function of NK cells, but the non-specific combination of magnetic beads and cells is inevitable and the operation is complex, so that the separation purity of the magnetic bead sorting method is limited, and the separation purity is also limited by the activity and specificity of the antibody, which has high requirements for the production process, transportation, storage and use conditions of the antibody; the separation effect is closely related to the manufacturing process of the magnet and the stability of the magnetic field, high-quality magnets need to be imported and have high prices, and each magnet can only separate one sample each time, and the above factors limit the high-throughput development of experiments;

[0004] In view of the above problems, the existing patent (publication number: CN215757259U) proposes an NK cell separation and purification device, which is provided with six chambers connected in series at the head and tail through a microfluidic chip, the chamber at the head is connected with a sample inlet groove, and the chamber at the tail is connected with an enrichment groove, the separation device for peripheral blood NK cells provided by the utility model separates non-target cells from pre-separated PBMCs, so as to realize the purification of target cells, the technical process belongs to the negative enrichment method of cells, so the target cells are not contacted during the whole separation process and are not stimulated, so the influence on the functional activity of the target cells is minimized, the magnetic field separation is not needed, the process is simplified, the one-time microfluidic chip device has less manual operation, low manufacturing cost, and can separate 25mL of whole blood sample at most once, and the separation throughput is large.

[0005] In view of the above problems, the existing patent gives a solution, but the sample is not pretreated before separation and purification, so that the sample quality is poor and the internal impurities are more, which increases the difficulty of subsequent separation and purification.

[0006] To this end, an NK cell separation and purification device is proposed. Content of the utility model

[0007] The utility model discloses a kind of NK cell separation and purification devices, can solve the quality caused by existing not being pretreated, more internal impurities, make subsequent separation, the problem of increasing difficulty of purification.

[0008] To achieve the above object, the utility model provides the following technical scheme: an NK cell separation and purification device, including magnetic bead sorting equipment, pretreatment mechanism is arranged at the top of the magnetic bead sorting equipment, and the bottom of the pretreatment mechanism is movably connected with screening mechanism.

[0009] The pretreatment mechanism includes a mixing barrel, a support ring frame, a first servo motor, a main shaft and a swing mixing assembly, the mixing barrel is arranged at the top of the magnetic bead sorting equipment, the support ring frame is movably connected with the top and the bottom of the inside of the mixing barrel, the first servo motor is fixedly connected with the inside of the top support ring frame, the main shaft is fixedly connected with the output end of the first servo motor, the main shaft is rotatably connected with the inside of the bottom support ring frame, and the swing mixing assembly is movably connected with the outside of the main shaft.

[0010] Preferably, the screening mechanism includes a screening barrel, a sliding support column, a sieve plate, a first telescopic support column, a first spiral spring, a second telescopic support column and a second spiral spring.

[0011] Preferably, the screening barrel is fixedly connected with the bottom of the mixing barrel, the screening barrel is fixedly connected with the top of the magnetic bead sorting equipment, the sliding support column is fixedly connected with the left side and the right side of the inside of the mixing barrel, and the sieve plate is slidably connected with the inside of the sliding support column.

[0012] Preferably, the top first telescopic support column is fixedly connected with the top of the top sieve plate, the top first telescopic support column is fixedly connected with the top of the inside of the sliding support column, the first spiral spring is fixedly connected with the outside of the top first telescopic support column, the second telescopic support column is fixedly connected with the bottom of the top sieve plate, the second telescopic support column is fixedly connected with the top of the bottom sieve plate, the second spiral spring is fixedly connected with the outside of the second telescopic support column, the bottom first telescopic support column is fixedly connected with the bottom of the bottom sieve plate, the bottom first telescopic support column is fixedly connected with the bottom of the inside of the sliding support column, and the first spiral spring is fixedly connected with the outside of the bottom first telescopic support column.

[0013] Preferably, the swing mixing assembly includes a bearing rod, a second servo motor, a heating plate and a hydraulic rod.

[0014] Preferably, the bearing rod is rotatably connected on both sides of the main shaft, the front second servo motor is fixedly connected on the front side of the front bearing rod, the rear second servo motor is fixedly connected on the rear side of the rear bearing rod, the heating plate is fixedly connected on the output end of the second servo motor, the top hydraulic rod is rotatably connected on the top of the bearing rod, the bottom hydraulic rod is rotatably connected on the bottom of the bearing rod, and the hydraulic rod is rotatably connected on the outside of the main shaft.

[0015] Preferably, the inductive valve is movably connected to the bottom of the inside of the mixing barrel.

[0016] Preferably, the outside of the mixing barrel is fixedly connected with a support, and the support is fixedly connected to the top of the magnetic bead sorting equipment.

[0017] Compared with the prior art, the application has the beneficial effects that:

[0018] 1. The pretreatment mechanism can add a three-dimensional dynamic pretreatment system before magnetic bead sorting, effectively improve sample quality and reduce subsequent operation difficulty. Firstly, the anticoagulant premixing mechanism in the mixing barrel can prevent sample coagulation and reduce impurity generation. Secondly, the three-dimensional mixing mode combining main shaft rotation and hydraulic rod oscillation enables the anticoagulant to fully contact with the sample, improves mixing uniformity and avoids local concentration deviation. Meanwhile, the heating plate adjusts temperature through self-rotation, maintains cell activity, reduces cell damage caused by temperature fluctuation. In addition, the hydraulic rod controls oscillation amplitude by using differential extension rate, avoids violent shaking and damages cell structure. The pretreatment process significantly reduces impurities such as coagulation blocks and cell fragments in the sample through the synergistic effect of anticoagulation, temperature control and dynamic mixing, improves sample purity, provides high-quality raw materials for subsequent magnetic bead sorting, reduces efficiency loss and equipment loss caused by impurity interference in the separation and purification process, and finally improves the quality and reliability of the overall operation.

[0019] 2. The screening mechanism can realize sample pretreatment through a double-screening-plate vibration screening structure. The sample itself flow rate and gravity drive the screening plate to move without the need for an additional power source, which is energy-saving and environmentally friendly. Secondly, the double-screening-plate filters in stages. The large-aperture screening plate separates large-particle impurities first, and the small-aperture screening plate further refines the screening, improving impurity removal efficiency. The elastic support and the spiral spring combination convert impact kinetic energy into irregular vibration, avoiding screening hole blockage and enhancing screening uniformity. The pretreated sample has a significantly reduced impurity content, providing a pure matrix for subsequent magnetic bead sorting and improving NK cell capture specificity. This design solves the problem of low efficiency of traditional pretreatment relying on manual operation through automatic mechanical vibration screening, effectively reduces impurity interference in the sample, reduces separation and purification difficulty, and improves the stability and product quality of the overall process. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1The utility model discloses a whole structural drawing of NK cell separation and purification device,

[0021] Figure 2 The utility model discloses a partial structural drawing of NK cell separation and purification device,

[0022] Figure 3 The utility model discloses a whole structural drawing of pretreatment mechanism,

[0023] Figure 4 The utility model discloses a whole structural drawing of swing mixing subassembly,

[0024] Figure 5 The utility model discloses a whole structural drawing of screening mechanism.

[0025] In the drawing, 1, magnetic bead sorting equipment, 2, pretreatment mechanism, 21, mixing bucket, 22, support ring frame, 23, first servo motor, 24, main shaft, 25, swing mixing subassembly, 25a, bearing rod, 25b, second servo motor, 25c, heating plate, 25d, hydraulic rod, 3, screening mechanism, 31, screening bucket, 32, sliding support column, 33, screen plate, 34, first telescopic support, 35, first spiral spring, 36, second telescopic support, 37, second spiral spring, 4, inductive valve, 5, support. DETAILED DESCRIPTION

[0026] The technical scheme in the embodiments of the utility model will be described clearly and completely below with the drawings in the embodiments of the utility model. Obviously, the described embodiments are only part of the embodiments of the utility model and not all the embodiments. Based on the embodiments in the utility model, all other embodiments obtained by those skilled in the art without creative work belong to the scope of the utility model.

[0027] Please refer to Figures 1-5 The utility model provides technical scheme:

[0028] A NK cell separation and purification device, including magnetic bead sorting equipment 1, the top of magnetic bead sorting equipment 1 is provided with pretreatment mechanism 2, and the bottom of pretreatment mechanism 2 is movably connected with screening mechanism 3;

[0029] Pretreatment mechanism 2 includes mixing bucket 21, support ring frame 22, first servo motor 23, main shaft 24 and swing mixing subassembly 25, mixing bucket 21 is set at the top of magnetic bead sorting equipment 1, support ring frame 22 movably connects the top and bottom of the inside of mixing bucket 21, first servo motor 23 is fixedly connected to the inside of top support ring frame 22, main shaft 24 is fixedly connected to the output end of first servo motor 23, main shaft 24 is rotatably connected to the inside of bottom support ring frame 22, and swing mixing subassembly 25 is movably connected to the outside of main shaft 24.

[0030] In the embodiment, before separating and purifying the NK cell sample, the sample is no longer directly introduced into the magnetic bead sorting device 1, but is first injected into the mixing barrel 21 at the top of the liquid inlet of the magnetic bead sorting device 1. According to the number of samples to be separated and purified, the anticoagulant is added in a suitable proportion, the first servo motor 23 is started, the main shaft 24 on the center support ring frame 22 of the mixing barrel 21 is rotated, the heating plate 25c on the outside of the main shaft 24 is rotated, the sample and the anticoagulant are fully mixed, and the three-dimensional mixing is realized through the swing mixing assembly 25, which further enhances the mixing effect of the sample and the reagent, improves the sample quality, and is beneficial to the subsequent separation and purification operation.

[0031] Specifically, as shown in Figure 1 , Figure 2 , Figure 5 , the screening mechanism 3 includes a screening barrel 31, a sliding support column 32, a screen plate 33, a first telescopic support 34, a first spiral spring 35, a second telescopic support 36, and a second spiral spring 37.

[0032] Specifically, as shown in Figure 1 , Figure 2 , Figure 5 , the screening barrel 31 is fixedly connected to the bottom of the mixing barrel 21, the screening barrel 31 is fixedly connected to the top of the magnetic bead sorting device 1, the sliding support column 32 is fixedly connected to the left and right sides of the inside of the mixing barrel 21, and the screen plate 33 is slidingly connected to the inside of the sliding support column 32.

[0033] Specifically, as shown in Figure 1 , Figure 2 , Figure 5 , the top first telescopic support 34 is fixedly connected to the top of the top screen plate 33, the top first telescopic support 34 is fixedly connected to the top of the inside of the sliding support column 32, the first spiral spring 35 is fixedly connected to the outside of the top first telescopic support 34, the second telescopic support 36 is fixedly connected to the bottom of the top screen plate 33, the second telescopic support 36 is fixedly connected to the top of the bottom screen plate 33, the second spiral spring 37 is fixedly connected to the outside of the second telescopic support 36, the bottom first telescopic support 34 is fixedly connected to the bottom of the bottom screen plate 33, the bottom first telescopic support 34 is fixedly connected to the bottom of the inside of the sliding support column 32, and the first spiral spring 35 is fixedly connected to the outside of the bottom first telescopic support 34.

[0034] In the embodiment, the mixing is completed by mixing, the bottom induction valve 4 of the mixing barrel 21 is opened, the sample falls into the screening barrel 31, the screening barrel 31 is provided with two groups of sieve plates 33, the high and low positions are arranged according to different hole diameters, and are slidably connected with the sliding support column 32; when the sample falls, the flow rate and gravity impact the top sieve plate 33, the top sieve plate 33 is pressed to stretch the first telescopic strut 34 and the first spiral spring 35 between the top sieve plate 33 and the inner wall of the sliding support column 32, and simultaneously compresses the second telescopic strut 36 and the second spiral spring 37 between the top sieve plate 33 and the bottom sieve plate 33; the bottom sieve plate 33 is also pressed, the sample continues to impact the bottom sieve plate 33 after passing through the top sieve plate 33, the bottom sieve plate 33 compresses the first telescopic strut 34 and the first spiral spring 35 between the bottom sieve plate 33 and the inner wall of the sliding support column 32, and when the elastic potential energy accumulated by the first telescopic strut 34 and the second telescopic strut 36 and the springs is released, the sieve plate 33 vibrates irregularly, the screening is realized in combination with the flow rate of the sample and the gravity impact, the impurities are removed, the specific capture capability of the magnetic beads for the NK cells is improved, and the extraction and purification quality is improved; and then the screened sample enters the magnetic bead sorting equipment 1 for separation and purification.

[0035] Specifically, as shown in Figure 1 、 Figure 2 , the swing mixing assembly 25 comprises a bearing rod 25a, a second servo motor 25b, a heating plate 25c and a hydraulic rod 25d.

[0036] Specifically, as shown in Figure 5 、 Figure 3 , the bearing rod 25a is rotatably connected on both sides of the main shaft 24, the front second servo motor 25b is fixedly connected on the front side of the front bearing rod 25a, the rear second servo motor 25b is fixedly connected on the rear side of the rear bearing rod 25a, the heating plate 25c is fixedly connected on the output end of the second servo motor 25b, the top hydraulic rod 25d is rotatably connected on the top of the bearing rod 25a, the bottom hydraulic rod 25d is rotatably connected on the bottom of the bearing rod 25a, and the hydraulic rod 25d is rotatably connected on the outer side of the main shaft 24.

[0037] In the embodiment, when the main shaft 24 rotates, the outer side of the upper and lower two groups of hydraulic rods 25d work alternately, one group is stretched out and the other group is retracted, so as to reciprocally pull the bearing rod 25a of the bearing heating plate 25c and the second servo motor 25b up and down, so as to achieve swing mixing; the extension and retraction rates of the top and bottom hydraulic rods 25d are different, the slower side controls the swing frequency and speed, so as to avoid damage to the sample cells due to too large swing amplitude; at the same time, the heating plate 25c can rotate around the shaft center by means of the second servo motor 25b, and can also adjust its temperature according to the required temperature in the mixing barrel 21, so as to maintain the cell activity; this way combines the rotation mixing of the main shaft 24, the swing mixing and the self-rotation mixing of the heating plate 25c, and generates a three-dimensional mixing effect, so as to ensure that the medicament and the sample are fully contacted, and improve the quality and efficiency of subsequent operations.

[0038] Specifically, as shown in Figure 4As shown, the bottom of the inside of the mixing barrel 21 is movably connected with the induction valve 4.

[0039] Specifically, as shown in Figure 3 、 Figure 4 Figure 2 Figure 1 Figure 2 The outside of the mixing barrel 21 is fixedly connected with the support 5, and the support 5 is fixedly connected to the top of the magnetic bead sorting device 1.

[0040] In this embodiment: the induction valve 4 can close the bottom of the mixing barrel 21 when the mixing barrel 21 is working, so as to avoid the sample from directly entering the screening barrel 31 without being processed, and the support 5 can reinforce and support the mixing barrel 21.

[0041] Working principle: before the NK cell sample is separated and purified, the sample is no longer directly introduced into the magnetic bead sorting equipment 1, but is first injected into the mixing barrel 21 at the top of the liquid inlet of the magnetic bead sorting equipment 1, according to the number of samples to be separated and purified, the appropriate proportion of anticoagulant is added, the first servo motor 23 is started, the main shaft 24 on the support ring frame 22 in the center of the mixing barrel 21 is rotated, the heating plate 25c outside the main shaft 24 rotates, which promotes the sample and the anticoagulant to mix fully, when the main shaft 24 rotates, the upper and lower two groups of hydraulic rods 25d on the outside of the main shaft 24 work alternately, one group extends and the other group retracts, the bearing rod 25a bearing the heating plate 25c and the second servo motor 25b is pulled up and down reciprocatingly, the swinging mixing is realized, the extension and retraction rates of the top and bottom hydraulic rods 25d are different, the swinging frequency and speed are controlled through the slower side to prevent the sample cells from being damaged due to the too large swinging amplitude, at the same time, the heating plate 25c can rotate around the shaft center through the second servo motor 25b, and adjust its temperature according to the required temperature in the mixing barrel 21 to maintain the cell activity, this way combines the rotation mixing of the main shaft 24, the swinging mixing and the rotation mixing of the heating plate 25c, forms a three-dimensional mixing effect, ensures that the medicament and the sample are fully contacted, improves the quality and efficiency of the subsequent operation, after the mixing is completed, the inductive valve 4 at the bottom of the mixing barrel 21 is opened, the sample falls into the screening barrel 31, the screening barrel 31 is provided with two groups of screens 33 with different hole diameters, which are located at the higher and lower positions of the sliding support column 32 and are in sliding connection with the sliding support column 32, when the sample falls from the mixing barrel 21, the flow rate and gravity of the sample will impact the top screen 33, so that the top screen 33 is pressed downward, the first telescopic strut 34 between the top screen 33 and the inner wall of the sliding support column 32 and the first spiral spring 35 outside the first telescopic strut 34 are stretched, and the second telescopic strut 36 between the top screen 33 and the bottom screen 33 and the second spiral spring 37 outside the second telescopic strut 36 are compressed, the bottom screen 33 is also pressed to a certain extent, after the sample passes through the top screen 33, the sample continues to impact the bottom screen 33, so that the bottom screen 33 is compressed downward, the first telescopic strut 34 between the bottom screen 33 and the inner wall of the sliding support column 32 and the first spiral spring 35 outside the first telescopic strut 34 are released, when the elastic potential energy accumulated in the first telescopic strut 34, the second telescopic strut 36, the first spiral spring 35 and the second spiral spring 37 is released, the screen 33 will vibrate irregularly, which cooperates with the impact of the flow rate and gravity of the sample to realize the vibration screening, effectively removes the impurities in the sample, improves the specific capture ability of the magnetic beads to the NK cells, improves the quality of extraction and purification, and the screened sample then enters the magnetic bead sorting equipment 1 for separation and purification.

[0042] The above is only a preferred embodiment of the present application, and is not intended to limit the present application, any modification, equivalent replacement and improvement made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. An NK cell isolation and purification device comprising a magnetic bead sorting apparatus (1), characterized in that: The top of the magnetic bead sorting equipment (1) is provided with a pretreatment mechanism (2), the bottom of the pretreatment mechanism (2) is movably connected with a screening mechanism (3); The pretreatment mechanism (2) comprises a mixing barrel (21), a support ring frame (22), a first servo motor (23), a main shaft (24) and a swing mixing assembly (25), the mixing barrel (21) is arranged at the top of the magnetic bead sorting equipment (1), the top and bottom of the inner side of the mixing barrel (21) are movably connected with the support ring frame (22), the inner side of the top support ring frame (22) is fixedly connected with the first servo motor (23), the output end of the first servo motor (23) is fixedly connected with the main shaft (24), the inner side of the bottom support ring frame (22) is rotatably connected with the main shaft (24), and the outer side of the main shaft (24) is movably connected with the swing mixing assembly (25).

2. The NK cell isolation and purification device of claim 1, wherein: The screening mechanism (3) comprises a screening barrel (31), a sliding support column (32), a screen plate (33), a first telescopic support column (34), a first spiral spring (35), a second telescopic support column (36) and a second spiral spring (37).

3. The NK cell isolation and purification device of claim 2, wherein: The screening barrel (31) is fixedly connected at the bottom of the mixing barrel (21), the screening barrel (31) is fixedly connected at the top of the magnetic bead sorting equipment (1), the left and right sides of the inner side of the mixing barrel (21) are fixedly connected with the sliding support column (32), and the inner side of the sliding support column (32) is slidably connected with the screen plate (33).

4. The NK cell isolation and purification device of claim 2, wherein: The top of the top first telescopic support column (34) is fixedly connected with the top of the top screen plate (33), the top of the inner side of the sliding support column (32) is fixedly connected with the top first telescopic support column (34), the outer side of the top first telescopic support column (34) is fixedly connected with the first spiral spring (35), the bottom of the top screen plate (33) is fixedly connected with the second telescopic support column (36), the top of the bottom screen plate (33) is fixedly connected with the second telescopic support column (36), the outer side of the second telescopic support column (36) is fixedly connected with the second spiral spring (37), the bottom of the bottom screen plate (33) is fixedly connected with the bottom first telescopic support column (34), the bottom of the inner side of the sliding support column (32) is fixedly connected with the bottom first telescopic support column (34), and the outer side of the bottom first telescopic support column (34) is fixedly connected with the first spiral spring (35).

5. The NK cell isolation and purification device of claim 1, wherein: The swing mixing assembly (25) comprises a bearing rod (25a), a second servo motor (25b), a heating plate (25c) and a hydraulic rod (25d).

6. The NK cell isolation and purification device of claim 5, wherein: The two sides of the main shaft (24) are rotatably connected with the bearing rod (25a), the front side of the front side bearing rod (25a) is fixedly connected with the front side second servo motor (25b), the rear side of the rear side bearing rod (25a) is fixedly connected with the rear side second servo motor (25b), the output end of the second servo motor (25b) is fixedly connected with the heating plate (25c), the top of the bearing rod (25a) is rotatably connected with the top hydraulic rod (25d), the bottom of the bearing rod (25a) is rotatably connected with the bottom hydraulic rod (25d), and the outer side of the main shaft (24) is rotatably connected with the hydraulic rod (25d).

7. The NK cell isolation and purification device of claim 1, wherein: The bottom of the inside of the mixing barrel (21) is movably connected with an induction valve (4).

8. The NK cell isolation and purification device of claim 1, wherein: The outside of the mixing barrel (21) is fixedly connected with a support (5), and the support (5) is fixedly connected to the top of the magnetic bead sorting equipment (1).

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