Centralized and distributed migration body extraction device and migration body extraction method

Abstract

The invention discloses a centralized distributed migration body extraction device and a migration body extraction method, relates to the technical field of migration body extraction, and aims to reduce the space occupied by the extraction device and improve the extraction efficiency of migration bodies. The extraction device comprises a box body and a filter connected to the side part of the box body; the inner space of the box body is divided into an upper space and a lower space by a first partition plate in the box body; the upper space forms a centrifugal chamber; the centrifugal chamber comprises a damping device arranged on the upper surface of the first partition plate, a centrifugal motor assembly arranged on the damping device and a centrifugal disc connected with the centrifugal motor assembly. A second partition plate is arranged in the lower space in the vertical direction, and the lower space is divided into a culture chamber and a cold control chamber by the second partition plate; the culture chamber and the cold control chamber are arranged in parallel. The method is suitable for extracting the migration body.

Description

Technical Field

[0001] The present invention relates to the technical field of migratory body extraction, and in particular to a centralized distributed migratory body extraction device and a migratory body extraction method. Background Art

[0002] Migrasomes are vesicle-like structures formed at the tip or bifurcation of the contractile filaments left behind at the tail during cell migration. The formation of this structure is driven by the assembly of large domains rich in tetraspanins, so molecules associated with tetraspanin-rich domains (such as integrins) are highly enriched in migrasomes. Knocking down or knocking out tetraspanins that promote migrasome formation impairs the formation of migrasomes. Migrasomes play an important role in intercellular communication. They are rich in signaling molecules (such as chemokines, cytokines, and angiogenic factors) and can play an auxiliary role in targeting signaling molecules to precise locations, which has a significant impact on physiological processes that require the integration of spatial and chemical information. Migrasomes are also involved in mitochondrial quality control, maintaining the homeostasis of mitochondria in cells by removing damaged mitochondria. In addition, migrasomes also play a key role in the coagulation process and are an essential component of the coagulation system.

[0003] The study of migratory bodies is of great significance to cell biology. When studying migratory bodies, they need to be extracted, but the existing advance methods have the following problems: multiple different devices are needed in the extraction process. These multiple different devices are independent of each other, which not only takes up a large space, but also requires the extraction of migratory bodies to be operated back and forth between various devices, which wastes time and is inefficient. Summary of the invention

[0004] In view of the deficiencies of the prior art, the present invention provides a centralized distributed migration body extraction device and a migration body extraction method, which can reduce the space occupied by the extraction device and help improve the extraction efficiency of the migration body.

[0005] To achieve the above objectives, an embodiment of the present invention provides a centralized distributed migration body extraction device, comprising a box body with an opening at the top and a filter connected to the side of the box body; wherein a first partition is arranged in the horizontal direction inside the box body, and the first partition divides the internal space of the box body into an upper space and a lower space; the upper space forms a centrifugal chamber; the centrifugal chamber comprises a shock absorbing device arranged on the upper surface of the first partition, a centrifugal motor assembly arranged on the shock absorbing device, and a centrifugal disk connected to the centrifugal motor assembly; a plurality of centrifuge tube placement grooves are arranged on the centrifuge disk, each centrifuge tube placement groove is provided with a centrifuge tube bearing seat, and an angle adjustment mechanism for adjusting the pitch angle of the centrifuge tube bearing seat; a second partition is arranged in the vertical direction in the lower space, and the second partition divides the lower space into a culture chamber and a cold control chamber; the culture chamber is arranged parallel to the cold control chamber.

[0006] Optionally, the centrifugal motor assembly includes a mounting box and a centrifugal motor mounted in the mounting box; the bottom of the mounting box extends outward to form a horizontal shock-absorbing plate; the shock-absorbing device includes an annular shock-absorbing groove provided on the upper surface of the first partition plate, and a plurality of evenly arranged shock-absorbing springs are fixedly connected to the bottom of the annular shock-absorbing groove; the shock-absorbing plate is supported on the shock-absorbing springs.

[0007] Optionally, a guide groove is vertically provided on the inner side wall of the annular shock-absorbing groove; more than two guide sliders are symmetrically connected to the side of the shock-absorbing plate, and the free ends of the guide sliders are arranged in the guide groove and are slidably connected to the vertical groove bottom of the guide groove.

[0008] Optionally, the filter includes an outer shell, in which an upper filter membrane, a middle filter membrane and a lower filter membrane are arranged in sequence from top to bottom; an opening is provided at the waist of the outer shell, a filter membrane frame is provided at the opening, the filter membrane frame can move up and down relative to the opening, and the middle filter membrane is arranged on the filter membrane frame; the filter membrane frame is connected to a vibration rod, so that the filter membrane frame can be driven to vibrate up and down by the up and down vibration of the vibration rod.

[0009] Optionally, a first through hole is opened on the side wall of the box body, and a second through hole is opened on the side wall of the annular shock-absorbing groove; the second through hole corresponds to the first through hole in the horizontal direction, and the second through hole corresponds to the first guide slider, and the aperture of the second through hole is smaller than the end size of the free end of the first guide slider; the source end of the vibration rod passes through the first through hole, the second through hole and is connected to the free end of the first guide slider; the outer diameter of the vibration rod is smaller than the apertures of the first through hole and the second through hole.

[0010] Optionally, a through hole is formed on the first partition plate, and the through hole connects the cold control chamber and the centrifugal chamber.

[0011] Optionally, a groove is provided on the outer side of the bottom of the box body, and a suction cup is provided in the groove; an air pump accommodating groove and a first air path are provided at the bottom of the box body, an air pump is provided in the air pump accommodating groove, a first end of the first air path is connected to the air suction port of the air pump, and a second end of the first air path is connected to the adsorption cavity of the suction cup; a needle-shaped pore is also provided at the bottom of the box body, and the needle-shaped pore connects the first air path with the culture chamber.

[0012] The present invention also provides a method for extracting a migrating body using any of the centralized distributed migrating body extraction devices described above, comprising the following steps: S1. Cultivating cells in a culture medium in a culture dish, and removing the supernatant of the culture medium in the culture dish with a pipette; S2, using a pipette to add 0.25% trypsin-ethylenediaminetetraacetate to the culture dish, and allowing the trypsin-ethylenediaminetetraacetate to completely cover all cells in the culture dish, and then placing the culture dish in a cell culture incubator at a temperature of 37° and a carbon dioxide content of 5% for 8-12 hours; S3. After the culture in the cell culture incubator is completed, the culture dish is taken out from the cell culture incubator, and the cell suspension containing trypsin-ethylenediaminetetraacetate is transferred to the first centrifuge tube using a pipette; S4, placing the first centrifuge tube in a centrifuge for a first centrifugation, extracting the supernatant in the first centrifuge tube after the first centrifugation by a pipette, and transferring the supernatant to a second centrifuge tube for a second centrifugation, and transferring the supernatant in the second centrifuge tube after the second centrifugation to a third centrifuge tube for a third centrifugation by a pipette; S5, removing the supernatant in the third centrifuge tube after the third centrifugation by a pipette, filtering the supernatant with a filter to form a sterile phosphate buffered saline solution for re-suspending, and transferring the re-suspended solution into an EP tube by a pipette for the fourth centrifugation; S6. Remove the supernatant in the EP tube after the fourth centrifugation by pipetting, filter the migration body precipitate through a filter, resuspend it in sterile phosphate buffered saline solution, and store it in a freezer at -80°.

[0013] Optionally, in the above step S1, when removing the supernatant of the culture medium, rinse with 2 ml of sterile phosphate buffered saline, and the number of rinses is at least 3 times; In the above S3 step, use a sterile Pasteur tube to draw 2 ml of phosphate-buffered saline solution into the culture dish, blow it several times with a 1 ml pipette to blow off at least part of the cells and migratory bodies, and transfer the phosphate-buffered saline solution containing the cells to the first centrifuge tube through the pipette, repeat adding 2 ml of phosphate-buffered saline solution to continue rinsing, blow and rinse for a total of 3 times, and transfer all the phosphate-buffered saline solution containing the cells to the first centrifuge tube.

[0014] Optionally, in the above steps S4 and S5, the first centrifuge tube is centrifuged at 1000 rpm at room temperature for 5 minutes, the second centrifuge tube is centrifuged at a temperature of 4°, a centrifugal force of 4000G, and a centrifugal force of 20 minutes, the third centrifuge tube is centrifuged at a temperature of 4°, a centrifugal force of 20000G, and a centrifugal force of 30 minutes, and the EP tube is centrifuged at a temperature of 4°, a centrifugal force of 20000G, and a centrifugal force of 30 minutes.

[0015] The centralized distributed migration body extraction device and migration body extraction method provided by the present invention include a box body with an opening at the top and a filter connected to the side of the box body; wherein a first partition is arranged in the horizontal direction inside the box body, and the first partition divides the internal space of the box body into an upper space and a lower space; the upper space forms a centrifugal chamber; the centrifugal chamber includes a shock absorbing device arranged on the upper surface of the first partition, a centrifugal motor assembly arranged on the shock absorbing device, and a centrifugal disc connected to the centrifugal motor assembly; a plurality of centrifugal tube placement slots are arranged on the centrifugal disc, and a centrifugal tube bearing seat is arranged in each centrifugal tube placement slot, and An angle adjustment mechanism is used to adjust the pitch angle of the centrifuge tube supporting seat; a second partition is provided in the lower space along the vertical direction, and the second partition divides the lower space into a culture chamber and a cold control chamber; the culture chamber is arranged in parallel with the cold control chamber, so that the culture chamber, cold control chamber, centrifugal chamber and filter used in the extraction process of the migration body are concentratedly distributed, which overcomes the disadvantages of multiple different equipment (culture equipment, cold control equipment, centrifugal equipment, etc.) in the prior art that are independent of each other and occupy a large space, and is also conducive to reducing the turnover operation time of the extraction of the migration body between different extraction links, thereby improving the extraction efficiency of the migration body. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 It is a cross-sectional structural schematic diagram of an embodiment of a centralized distributed migration body extraction device of the present invention; Figure 2 It is a schematic diagram of the coordinated installation of the installation box and the shock absorbing groove of the present invention; Figure 3 It is a partial structural schematic diagram of a centrifugal disk, a centrifugal tube supporting seat and an angle adjustment mechanism according to an embodiment of the present invention; Figure 4 A cross-sectional schematic diagram of a centrifugal disk in one embodiment of the present invention; Figure 5 This is a structural schematic diagram of a centrifuge tube supporting seat in one embodiment of the present invention; Figure 6 This is a schematic diagram of the internal structure of a pull-out box in an embodiment of the present invention; Figure 7 It is a top view of a drawer box in one embodiment of the present invention. DETAILED DESCRIPTION

[0017] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0018] refer to Figure 1 and Figure 2 An embodiment of the present invention provides a centralized distributed migration body extraction device, including a box body 1 with an opening on the top and a filter 2 connected to the side of the box body 1.

[0019] A first partition 10 is horizontally arranged inside the box body 1 , and the first partition 10 divides the internal space of the box body 1 into an upper space and a lower space.

[0020] The upper space forms a centrifugal chamber 11. The centrifugal chamber 11 includes a shock absorbing device 111 disposed on the upper surface of the first partition 10, a centrifugal motor assembly 112 disposed on the shock absorbing device 111, and a centrifugal disc 113 connected to the centrifugal motor assembly 112; the centrifugal disc 113 is provided with a plurality of centrifugal tube placement grooves 114, each centrifugal tube placement groove 114 is provided with a centrifugal tube bearing seat 115, and an angle adjustment mechanism 116 for adjusting the pitch angle of the centrifugal tube bearing seat 115.

[0021] A second partition 12 is vertically arranged in the lower space, and the second partition 12 divides the lower space into a culture chamber 13 and a cold control chamber 14 ; the culture chamber 13 and the cold control chamber 14 are arranged in parallel.

[0022] During centrifugation operation, the centrifuge tube 15 can be placed in the centrifuge tube support seat 115, and the pitch angle of the centrifuge tube support seat 115 can be adjusted using the angle adjustment mechanism 116, and then the motor 1121 in the centrifuge motor assembly 112 rotates to centrifuge the contents in the centrifuge tube 15.

[0023] When the motor 1121 in the centrifugal motor assembly 112 rotates, vibration is usually generated. The centrifugal chamber 11 is arranged above the culture chamber 13 and the cold control chamber 14, which can reduce the adverse effects of the vibration generated when the motor 1121 rotates on the culture chamber 13 and the cold control chamber 14. The position setting of the centrifugal chamber 11, in conjunction with the setting of the shock absorbing device 111, can greatly reduce the adverse effects of the vibration generated when the motor 1121 rotates on the culture chamber 13 and the cold control chamber 14.

[0024] In the embodiment of the present invention, the culture chamber 13, cold control chamber 14, centrifugal chamber 11 and filter 2 used in the process of extracting the migration body are centrally distributed, which overcomes the disadvantage that multiple different equipment (culture equipment, cold control equipment, centrifugal equipment, etc.) in the prior art are independent of each other and occupy a large space. It is also beneficial to reduce the turnover operation time of the extraction of the migration body between different extraction links, so as to improve the extraction efficiency of the migration body.

[0025] See also Figure 2 In some embodiments, the centrifugal motor assembly 112 includes a mounting box 1122 and a motor 1121 mounted in the mounting box 1122 ; the bottom of the mounting box 1122 extends outward to form a horizontal shock absorbing plate 1123 .

[0026] The shock absorbing device 111 includes an annular shock absorbing groove 1111 arranged on the upper surface of the first partition plate 10 , and a plurality of evenly arranged shock absorbing springs 1112 are fixedly connected to the bottom of the annular shock absorbing groove 1111 ; the shock absorbing plate 1123 is supported on the shock absorbing springs 1112 .

[0027] The horizontal shock-absorbing plate 1123 extending outward from the bottom of the mounting box 1122 can, on the one hand, lower the center of gravity of the centrifugal motor assembly 112 and enhance the stability of the motor when it is working; on the other hand, it can transmit the vibration generated by the motor when it is working to the shock-absorbing spring 1112, which absorbs the vibration.

[0028] The centrifugal motor 1121 can be controlled to have different rotation speeds through a motor controller.

[0029] In some embodiments, a guide groove 1113 is vertically provided on the inner side wall of the annular damping groove 1111; two or more guide sliders 1114 are symmetrically connected to the side of the damping plate 1123, and the free ends of the guide sliders 1114 are arranged in the guide groove 1113 and are slidably connected to the vertical groove bottom of the guide groove 1113. One end of the guide slider 1114 is connected to the side of the damping plate 1123, and the other end (free end) can slide up and down along the guide groove 1113 and is slidably connected to the vertical groove bottom of the guide groove 1113, so that it can guide the up and down vibration of the damping plate 1123, and can also play a role in limiting and centering the damping plate 1123, preventing the damping plate 1123 from vibrating in the horizontal direction, thereby increasing the stability of the centrifugal operation when the motor rotates.

[0030] See also Figure 1 In some embodiments, the filter 2 includes a shell 21, in which an upper filter membrane 22, a middle filter membrane 23 and a lower filter membrane 24 are sequentially arranged from top to bottom; an opening is provided at the waist of the shell 21, and a filter membrane frame 25 is provided at the opening, and the filter membrane frame 25 can move up and down relative to the opening, and the middle filter membrane 23 is arranged on the filter membrane frame 25; the filter membrane frame 25 is connected to a vibration rod 26, so that the filter membrane frame 25 can be driven to vibrate up and down by the up and down vibration of the vibration rod 26.

[0031] The filter 2 is provided with three layers of filter membranes, namely, upper, middle and lower layers. The middle filter membrane 23 is provided between the upper filter membrane 22 and the lower filter membrane 24, and the middle filter membrane 23 is a filter membrane that can vibrate up and down.

[0032] When the middle filter membrane 23 vibrates from top to bottom, negative pressure is generated between the middle filter membrane 23 and the upper filter membrane 22, and positive pressure is generated between the middle filter membrane 23 and the lower filter membrane 24. This can increase the speed at which the filtrate passes through the upper filter membrane 22 and the lower filter membrane 24.

[0033] When the middle filter membrane 23 vibrates from bottom to top, positive pressure is generated between the middle filter membrane 23 and the upper filter membrane 22, and negative pressure is generated between the middle filter membrane 23 and the lower filter membrane 24. This can generate a force to move particulate matter attached to the upper surfaces of the upper filter membrane 22 and the lower filter membrane 24 away from the upper surfaces of the upper filter membrane 22 and the lower filter membrane 24, thereby improving the pore permeability of the upper filter membrane 22 and the lower filter membrane 24.

[0034] The static spacing distance between the middle filter membrane 23 and the upper filter membrane 22 is smaller than the static spacing distance between the middle filter membrane 23 and the lower filter membrane 24, which is beneficial to provide a larger penetration time and space for the liquid between the middle filter membrane 23 and the lower filter membrane 24. In one example, the static spacing distance between the middle filter membrane 23 and the upper filter membrane 22 is 5 mm, and the static spacing distance between the middle filter membrane 23 and the lower filter membrane 24 is 10 mm.

[0035] Each layer of filter membrane can be a 100-mesh filter membrane.

[0036] When the current batch of extracts is centrifuged, the other batches of extracts can be filtered at the same time. That is, the centrifugation and filtering operations can be performed simultaneously.

[0037] In some embodiments, a first through hole is opened on the side wall of the box body 1, and a second through hole is opened on the side wall of the annular shock-absorbing groove 1111; the second through hole corresponds to the first through hole in the horizontal direction, and the second through hole corresponds to the first guide slider 1114, and the aperture of the second through hole is smaller than the end size of the free end of the first guide slider 1114.

[0038] The source end of the vibration rod 26 passes through the first through hole and the second through hole and is connected to the free end of the first guide slider 1114; the outer diameter of the vibration rod 26 is smaller than the apertures of the first through hole and the second through hole.

[0039] The source end of the vibration rod 26 passes through the first through hole and the second through hole and is connected to the free end of the first guide slider 1114, that is, the source end of the vibration rod 26 passes through the first through hole and the second through hole, and is indirectly connected to the shock-absorbing plate 1123 at the bottom of the installation box 1122 through the first guide slider 1114. In this way, on the one hand, the vibration of the shock-absorbing plate 1123 can be transmitted to the filter membrane frame 25 and the middle filter membrane 23 on the filter membrane frame 25 through the vibration rod 26, so as to achieve auxiliary vibration absorption of the shock-absorbing plate 1123. On the other hand, the vibration of the shock-absorbing plate 1123 is transmitted to the filter membrane frame 25 and the middle filter membrane 23 on the filter membrane frame 25 through the vibration rod 26, so as to drive the middle filter membrane 23 to vibrate up and down. In this way, while assisting the vibration absorption of the shock-absorbing plate 1123, the vibration energy of the shock-absorbing plate 1123 can be effectively utilized to improve the pore transmittance of the upper filter membrane 22 and the lower filter membrane 24.

[0040] See also Figure 1 and Figure 2In some embodiments, a through hole h1 is formed on the first partition 10, and the through hole h1 connects the cold control chamber 14 and the centrifugal chamber 11, so as to facilitate unified adjustment of the temperature in the cold control chamber 14 and the centrifugal chamber 11, such as controlling the temperature to 4° so that the temperature in the centrifugal chamber 11 meets the centrifugal requirements.

[0041] See also Figure 1 In some embodiments, a groove 117 is provided on the outer side of the bottom of the box body 1 , and a suction cup 118 is provided in the groove 117 .

[0042] An air pump receiving groove and a first air path 119 are provided at the bottom of the box body 1, an air pump 120 is provided in the air pump receiving groove, a first end of the first air path 119 is connected to an air suction port of the air pump 120, and a second end of the first air path 119 is connected to an adsorption cavity of the suction cup 118; a needle-shaped pore (not shown in the figure) is also provided at the bottom of the box body 1, and the needle-shaped pore connects the first air path 119 with the culture chamber 13.

[0043] When in use, the box 1 is placed on the table, and the air pump 120 is started. The air pump 120 is used to extract the gas in the adsorption cavity of the suction cup 118 through the first air path 119, thereby reducing the air pressure in the adsorption cavity of the suction cup 118. The suction cup 118 is firmly adsorbed on the table by using the air pressure difference inside and outside the suction cup 118, thereby firmly fixing the box 1 on the table to prevent the device from sliding on the table when centrifuged. At the same time, the air pump 120 is used to suck the gas in the culture chamber 13 through the first air path 119 and the needle-shaped pores to form a slightly flowing airflow in the culture chamber 13, thereby forming a good culture environment in the culture chamber 13.

[0044] See also Figure 4 In some embodiments, a connecting groove 1131 corresponding to the centrifugal motor 1121 is provided at the bottom of the centrifugal disk 113, a connecting hole 1132 fixedly connected to the output shaft of the centrifugal motor 1121 is provided at the inner top of the connecting groove 1131, and a plurality of uniform centrifuge tube placement grooves 114 are provided at the top edge of the connecting groove 1131.

[0045] See also Figure 3 and Figure 5 The centrifuge tube supporting seat 115 includes a rectangular fixed cylinder 1151 with an open top, and the bottom of the fixed cylinder 1151 is installed in the centrifuge tube placement groove 114 through a movable shaft 1152.

[0046] A fixed rubber block 1153 is provided on the inner wall of one side of the fixed cylinder 1151, a movable plate 1154 is provided in the fixed cylinder 1151, a movable rubber block 1155 is provided on the movable plate 1154, and the test tube 15 is installed between the movable rubber block 1155 and the fixed rubber block 1153, and a fixing bolt 1156 is provided on the outer side of the fixed cylinder 1151, the inner end of the fixing bolt 1156 is movably connected to the movable plate 1154, and the fixing bolt 1156 is threadedly matched with the fixed cylinder 1151.

[0047] The angle adjustment mechanism 116 includes an electric telescopic rod 1161 and a T-shaped guide groove 1113 which is opened on the outside of the fixing tube 1151 and extends along the length direction of the fixing tube 1151 .

[0048] The bottom of the electric telescopic rod 1161 is installed at the bottom of the centrifuge tube placement groove 114, and the top of the electric telescopic rod 1161 is movably connected with a T-shaped slider 1162. The slider 1162 slides in cooperation with the guide groove 1113. When the electric telescopic rod 1161 is extended or retracted, the top of the electric telescopic rod 1161 and the slider 1162 can rotate flexibly, so that the electric telescopic rod 1161 can drive the slider 1162 to move in the guide groove 1113, thereby adjusting the inclination angle of the centrifuge tube support seat 115.

[0049] The cold control room 14 and the culture room 13 are respectively provided with a first temperature controller and a second temperature controller, and the culture room 13 can be connected to the carbon dioxide storage tank. One side of the culture room 13 is open, and a pull-out box 30 is installed in the culture room 13.

[0050] See also Figure 6 and Figure 7 The culture dish 40 is installed in the drawer box 30 through the limiting assembly. The interior of the drawer box 30 is divided into two culture areas 702a and 702b by a partition plate 701.

[0051] The limiting assembly in the culture area 702a includes a limiting spring 801 fixedly connected to the partition plate 701, a movable arc-shaped limiting block 802 arranged at the end of the limiting spring 801, and an arc-shaped limiting block 803 fixedly connected to the inner wall of the pull-out box 30. The arc-shaped limiting block 803 corresponds to the arc-shaped limiting block 802, and a culture dish 40 is fixed between the two.

[0052] The upper and lower surfaces of the outer end of the pull-out box 30 are provided with rubber sealing pads 703, and the outer end of the pull-out box 30 is provided with a built-in handle 704. In this way, the limit spring 801 squeezes the limit block 802, and the culture dish 40 is fixed by the arc-shaped limit block 802 to prevent the culture dish 40 from shaking back and forth in the culture area 702a. The rubber sealing pad 703 can make the seal between the pull-out box 30 and the opening of the culture chamber 13 more tight, and prevent the gas in the culture chamber 131 from escaping. The built-in handle 704 facilitates the pulling and pulling of the pull-out box 30. A one-way valve (not shown in the figure) that opens inward is provided on the front side wall of the pull-out box 30 to cooperate with the air intake of the air pump 120.

[0053] As a preferred embodiment, temperature detection devices are respectively provided in the centrifugal chamber 11 and the culture chamber 13 , and a carbon dioxide concentration detection device is also provided in the culture chamber 13 .

[0054] The temperature controller can adjust the temperature inside the centrifugal chamber 11 and the culture chamber 13. The exhaust port of the carbon dioxide storage tank is provided with a solenoid valve, and the discharge of the carbon dioxide storage tank is controlled by the solenoid valve. At the same time, the temperature detection device and the carbon dioxide concentration detection device can always detect the temperature and carbon dioxide content inside the culture chamber 13102.

[0055] See also Figure 1 The bottom of the box 1 is also provided with a winding chamber 105, in which a power cord 1052 is installed through a coil spring 1051, and the power cord 1052 is connected to the outside. The coil spring 1051 can be used to wind the power cord 1052 in the winding chamber 105, so as to avoid the excessively long power cord 1052 from being tangled.

[0056] A top cover is movably installed on the top of the installation box 112 to facilitate installation of the centrifugal motor 1121 into the installation box 112, and the output end of the centrifugal motor 1121 passes through the center of the top cover.

[0057] See also Figure 1 In this embodiment, a sealing cover 45 is provided on the top of the box body 1, and the sealing cover 45 is fixedly connected to the box body 1 by a buckle. When in use, the sealing cover 45 can be tightly combined with the box body 1 by the buckle to seal the box body 1, which helps to protect the liquid in the test tube 15 from escaping and ensures that the temperature in the box body 1 reaches a certain balance. A rubber layer 41 is provided on the inner side of the sealing cover 40. The rubber layer 41 prevents the test tube 15 from being accidentally thrown out and directly hitting the sealing cover 45, which helps to protect the test tube 15 from being damaged and prevents the liquid in the test tube 15 from splashing on the sealing cover 45 and corroding the sealing cover.

[0058] The method of using the migration body extraction device in the present invention is as follows: When cells need to be cultured, the culture dish 40 containing cells is fixed in the pull-out box 30 by means of the limiting assembly, and the pull-out box 30 is pushed into the culture chamber 13, the second temperature controller and the carbon dioxide storage tank are started, the temperature in the culture chamber 13 is adjusted by means of the second temperature controller, and the carbon dioxide content in the culture chamber 13 is adjusted by means of the carbon dioxide storage tank, so as to meet the requirements for cell culture; when centrifugation is required, the test tube 15 containing the cell suspension is placed between the two rubber blocks, the fixing bolt 1156 is rotated, and the moving plate 1154 is pushed to move by means of the cooperation between the fixing bolt 1156 and the fixing cylinder 1151, until the two rubber blocks tightly clamp and fix the test tube, and the electric telescopic rod 1 is started. 161, the electric telescopic rod 1161 and the slider 1162 are used to adjust the inclination angle of the test tube to facilitate the centrifugal treatment of the test tube, the sealing cover is sealed with the box body by a buckle, the first temperature controller is started, and the temperature in the centrifugal chamber 11 is controlled to 4° or other suitable temperature by the first temperature controller to meet the centrifugal temperature requirement, and the centrifugal motor 1121 is started at the same time. The centrifugal motor 1121 drives the centrifugal disk 113 to rotate, and drives the test tube to rotate rapidly through the centrifugal disk 113 to achieve the centrifugal effect. The present invention can limit and fix test tubes of different sizes, and can adjust the inclination angle of the test tube to facilitate centrifugation. At the same time, cells can be cultured in the culture chamber 13, thereby expanding the scope of use of the device.

[0059] The embodiment of the present invention further provides a method for extracting a migrating body using any of the aforementioned centralized distributed migrating body extraction devices, comprising the following steps: S1. Cultivating cells in a culture medium in a culture dish, and removing the supernatant of the culture medium in the culture dish with a pipette; S2, using a pipette to add 0.25% trypsin-ethylenediaminetetraacetate to the culture dish, and allowing the trypsin-ethylenediaminetetraacetate to completely cover all cells in the culture dish, and then placing the culture dish in a cell culture incubator at a temperature of 37° and a carbon dioxide content of 5% for 8-12 hours; S3. After the culture in the cell culture incubator is completed, the culture dish is taken out from the cell culture incubator, and the cell suspension containing trypsin-ethylenediaminetetraacetate is transferred to the first centrifuge tube using a pipette; S4, placing the first centrifuge tube in a centrifuge for a first centrifugation, extracting the supernatant in the first centrifuge tube after the first centrifugation by a pipette, and transferring the supernatant to a second centrifuge tube for a second centrifugation, and transferring the supernatant in the second centrifuge tube after the second centrifugation to a third centrifuge tube for a third centrifugation by a pipette; S5, using a pipette to remove the supernatant in the third centrifuge tube after the third centrifugation, filtering the supernatant with a filter to form a sterile phosphate buffered saline solution for re-suspending, and transferring the re-suspended solution into an EP (Eppendorf) tube by a pipette for the fourth centrifugation; S6. Remove the supernatant in the EP tube after the fourth centrifugation by pipetting, filter the migration body precipitate through a filter, resuspend it in sterile phosphate buffered saline solution, and store it in a freezer at -80°.

[0060] This method embodiment has the beneficial effects corresponding to the aforementioned centralized distributed migration body extraction device. In addition, the migration body extraction method provided by the present invention, after removing the supernatant of the culture medium in the culture dish with a pipette, 0.25% trypsin-ethylenediaminetetraacetate is added to the culture dish using a pipette, and the trypsin-ethylenediaminetetraacetate is completely covered with all cells in the culture dish, and then the culture dish is placed in a cell culture incubator with a temperature of 37° and a carbon dioxide content of 5% for 8-12 hours, which can reduce damage to the cells; after the culture in the cell culture incubator is completed, the culture dish is taken out of the cell culture incubator, and the cell suspension containing trypsin-ethylenediaminetetraacetate is subjected to multiple differential centrifugation to gradually remove other types of organelles and particles, so that the extracted migration body has good specificity.

[0061] In this step, as a preferred embodiment, when the migratory body is filtered through the filter, multiple layers of filter membranes are arranged in the filter, and the migratory body is filtered by the multiple layers of filter membranes to improve the filtering effect and reduce the number of filtering operations. Each layer of filter membrane can be a 100-mesh filter membrane.

[0062] See also Figure 1 In one embodiment, the filter 2 is provided with three layers of filter membranes, namely, upper, middle and lower layers. The middle filter membrane 23 is provided between the upper filter membrane 22 and the lower filter membrane 24, and the middle filter membrane 23 is a filter membrane that can vibrate up and down.

[0063] When the middle filter membrane 23 vibrates from top to bottom, negative pressure is generated between the middle filter membrane 23 and the upper filter membrane 22, and positive pressure is generated between the middle filter membrane 23 and the lower filter membrane 24. This can increase the speed at which the filtrate passes through the upper filter membrane 22 and the lower filter membrane 24.

[0064] When the middle filter membrane 23 vibrates from bottom to top, positive pressure is generated between the middle filter membrane 23 and the upper filter membrane 22, and negative pressure is generated between the middle filter membrane 23 and the lower filter membrane 24. This can generate a force to move particulate matter attached to the upper surfaces of the upper filter membrane 22 and the lower filter membrane 24 away from the upper surfaces of the upper filter membrane 22 and the lower filter membrane 24, thereby improving the pore permeability of the upper filter membrane 22 and the lower filter membrane 24.

[0065] In some embodiments, in the above step S1, when removing the supernatant of the culture medium, 2 ml of sterile phosphate buffered saline is used for washing, and the number of washing times is at least 3 times to effectively remove other substances such as exosomes and extracellular vesicles.

[0066] In some embodiments, in the above-mentioned step S2, after the culture dish is cultured in a cell culture incubator at a temperature of 37° and a carbon dioxide content of 5% for 8-12 hours, the culture dish is taken out and observed under a microscope to see whether the cells shrink into a bright spot shape, and whether the cells fall like falling snow when the outer wall of the culture dish is tapped. If the cells are observed to shrink into a bright spot shape, and the cells fall like falling snow when the outer wall of the culture dish is tapped, it is determined that the culture in the cell culture incubator is completed, so that the completion of the culture can be known in time.

[0067] In the above step S3, 2 ml of phosphate buffered saline solution is drawn into the culture dish using a sterile Pasteur tube, blown several times with a 1 ml pipette to blow off at least some of the cells and migratory bodies, and the phosphate buffered saline solution containing the cells is transferred to the first centrifuge tube by a pipette, and 2 ml of phosphate buffered saline solution is repeatedly added to continue washing, and the washing is blown and washed three times in total, and the phosphate buffered saline solution containing the cells is transferred to the first centrifuge tube. Repeated use of 2 ml of phosphate buffered saline solution for multiple washings can further remove exosomes and exosomes.

[0068] In some embodiments, in the above steps S4 and S5, the first centrifuge tube is centrifuged at room temperature at 1000 rpm for 5 minutes, the second centrifuge tube is centrifuged at 4°C, 4000G, and 20 minutes, the third centrifuge tube is centrifuged at 4°C, 20000G, and 30 minutes, and the EP tube is centrifuged at 4°C, 20000G, and 30 minutes. Through multi-stage differential centrifugation, cells and other influencing substances can be gradually removed.

[0069] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A centralized and distributed mobile body extraction device, characterized in that: A box body including a top opening and a filter connected to a side of the box body; Wherein, a first partition is arranged in the horizontal direction inside the box body, and the first partition divides the internal space of the box body into an upper space and a lower space; The upper space forms a centrifugal chamber; the centrifugal chamber comprises a shock absorbing device arranged on the upper surface of the first partition, a centrifugal motor assembly arranged on the shock absorbing device, and a centrifugal disc connected to the centrifugal motor assembly; the centrifugal disc is provided with a plurality of centrifugal tube placement grooves, each centrifugal tube placement groove is provided with a centrifugal tube bearing seat, and an angle adjustment mechanism for adjusting the pitch angle of the centrifugal tube bearing seat; A second partition is arranged in the lower space along the vertical direction, and the second partition divides the lower space into a cultivation chamber and a cold control chamber; the cultivation chamber is arranged in parallel with the cold control chamber.

2. The centralized and distributed mobile body extraction device according to claim 1, characterized in that: The centrifugal motor assembly comprises a mounting box and a centrifugal motor mounted in the mounting box; the bottom of the mounting box extends outward to form a horizontal shock absorbing plate; The shock absorbing device comprises an annular shock absorbing groove provided on the upper surface of the first partition plate, and a plurality of evenly arranged shock absorbing springs are fixedly connected to the bottom of the annular shock absorbing groove; The damping plate is supported on the damping spring.

3. The centralized distributed migration body extraction device according to claim 2, characterized in that: A guide groove is vertically provided on the inner side wall of the annular shock-absorbing groove; The side of the shock-absorbing plate is symmetrically connected to more than two guide sliding blocks, and the free ends of the guide sliding blocks are arranged in the guide groove and are slidably connected to the vertical groove bottom of the guide groove.

4. The centralized and distributed mobile body extraction device according to claim 2, characterized in that: The filter comprises a shell, in which an upper filter membrane, a middle filter membrane and a lower filter membrane are sequentially arranged from top to bottom; An opening is provided at the waist of the shell, a filter membrane frame is provided at the opening, the filter membrane frame can move up and down relative to the opening, and the middle filter membrane is provided on the filter membrane frame; The filter membrane frame is connected to a vibration rod, so that the filter membrane frame is driven to vibrate up and down through the up and down vibration of the vibration rod.

5. The centralized and distributed migratory body extraction device according to claim 4, characterized in that: A first through hole is formed on the side wall of the box body, and a second through hole is formed on the side wall of the annular shock absorbing groove; The second through hole corresponds to the first through hole in the horizontal direction, and the second through hole corresponds to the first guide slider, and the aperture of the second through hole is smaller than the end size of the free end of the first guide slider; The source end of the vibration rod passes through the first through hole and the second through hole and is connected to the free end of the first guide slider; the outer diameter of the vibration rod is smaller than the aperture of the first through hole and the second through hole.

6. The centralized distributed migration body extraction device according to claim 1, characterized in that: A through hole is provided on the first partition plate, and the through hole connects the cold control chamber and the centrifugal chamber.

7. The centralized and distributed migratory body extraction device according to claim 1, characterized in that: The outer side of the bottom of the box body is provided with a groove, and a suction cup is provided in the groove; An air pump receiving groove and a first air path are provided at the bottom of the box body, an air pump is provided in the air pump receiving groove, a first end of the first air path is connected to an air suction port of the air pump, and a second end of the first air path is connected to an adsorption cavity of the suction cup; The bottom of the box body is also provided with a needle-shaped hole, and the needle-shaped hole connects the first gas path with the culture chamber.

8. A method for extracting a migrating body using the centralized distributed migrating body extraction device according to any one of claims 1 to 7, characterized in that: The following steps are involved: S1. Cultivating cells in a culture medium in a culture dish, and removing the supernatant of the culture medium in the culture dish with a pipette; S2, using a pipette to add 0.25% trypsin-ethylenediaminetetraacetate to the culture dish, and allowing the trypsin-ethylenediaminetetraacetate to completely cover all cells in the culture dish, and then placing the culture dish in a cell culture incubator at a temperature of 37° and a carbon dioxide content of 5% for 8-12 hours; S3. After the culture in the cell culture incubator is completed, the culture dish is taken out from the cell culture incubator, and the cell suspension containing trypsin-ethylenediaminetetraacetate is transferred to the first centrifuge tube using a pipette; S4, placing the first centrifuge tube in a centrifuge for a first centrifugation, extracting the supernatant in the first centrifuge tube after the first centrifugation by a pipette, and transferring the supernatant to a second centrifuge tube for a second centrifugation, and transferring the supernatant in the second centrifuge tube after the second centrifugation to a third centrifuge tube for a third centrifugation by a pipette; S5, removing the supernatant in the third centrifuge tube after the third centrifugation by a pipette, filtering the supernatant with a filter to form a sterile phosphate buffered saline solution for re-suspending, and transferring the re-suspended solution into an EP tube by a pipette for the fourth centrifugation; S6. Remove the supernatant in the EP tube after the fourth centrifugation by pipetting, filter the migration body precipitate through a filter, resuspend it in sterile phosphate buffered saline solution, and store it in a freezer at -80°.

9. The method for extracting migratory bodies according to claim 8, characterized in that: In the above step S1, when removing the supernatant of the culture medium, rinse with 2 ml of sterile phosphate buffered saline, and the number of rinses is at least 3 times; In the above S3 step, use a sterile Pasteur tube to draw 2 ml of phosphate-buffered saline solution into the culture dish, blow it several times with a 1 ml pipette to blow off at least part of the cells and migratory bodies, and transfer the phosphate-buffered saline solution containing the cells to the first centrifuge tube through the pipette, repeat adding 2 ml of phosphate-buffered saline solution to continue rinsing, blow and rinse for a total of 3 times, and transfer all the phosphate-buffered saline solution containing the cells to the first centrifuge tube.

10. The method for extracting migratory bodies according to claim 8, characterized in that: In the above steps S4 and S5, the first centrifuge tube is centrifuged at 1000 rpm for 5 minutes at room temperature, the second centrifuge tube is centrifuged at 4°C, a centrifugal force of 4000G, and a centrifugal force of 20 minutes, the third centrifuge tube is centrifuged at 4°C, a centrifugal force of 20000G, and a centrifugal force of 30 minutes, and the EP tube is centrifuged at 4°C, a centrifugal force of 20000G, and a centrifugal force of 30 minutes.

Images