A device for separating cell clusters in malignant pleural effusion and its usage method

Abstract

This invention discloses a cell cluster separation device and its usage method in malignant pleural effusion, belonging to the field of cell separation technology. By having two locking blocks approach each other and disengage from the locking groove, the locking state between the support base and the mounting base is released, allowing the support base and multiple centrifuge tubes to be removed from the centrifuge. When removing the centrifuge tubes, because the central axis of the tube rack and the support base is parallel, the centrifuge tubes can be directly pulled vertically upwards, facilitating the simultaneous removal of multiple centrifuge tubes. The downward movement of the conical seat can compress the two locking blocks, causing them to engage in the locking groove, thus achieving the purpose of locking the support base and the mounting base. Therefore, the modular design of the centrifuge, support base, and tube rack enables the rapid loading and unloading of batches of centrifuge tubes. The use of two sets of support bases and multiple tube racks for centrifugation allows for alternating loading and unloading of centrifuge tubes, shortening the time spent loading and unloading centrifuge tubes and facilitating the efficient and stable separation of cell clusters.

Description

Technical Field

[0001] This invention relates to the field of cell separation technology, specifically to a device for separating cell clusters from malignant pleural effusion and its method of use. Background Technology

[0002] In the biomedical engineering industry, malignant pleural effusion is a common complication in patients with malignant tumors. Its pathological characteristics include the accumulation of fluid in the pleural cavity, which usually contains tumor cells and their related cell clusters. Separating and analyzing the cell clusters in malignant pleural effusion not only helps to clarify the tumor diagnosis, but also provides important evidence for disease staging, treatment effect evaluation, and research on the mechanism of tumor invasion and metastasis.

[0003] Cell clump separation equipment is an important tool in the treatment of malignant pleural effusions, helping doctors and researchers to isolate tumor cells from the pleural effusion for further diagnosis and treatment.

[0004] Currently, common methods for separating cell clusters in malignant pleural effusion include, but are not limited to, centrifugation. Traditional centrifugation directly separates cells from liquid through a single centrifugation step. However, since cell clusters have similar densities to individual cells and fragments, the separation efficiency is low and cell clusters are easily lost. Especially when dealing with complex pleural effusion samples, existing methods are difficult to efficiently recover intact cell clusters. When using a centrifuge to separate cell clusters, centrifuge tubes containing samples must be placed one by one into the centrifuge and tilted in the centrifuge tank. This is inconvenient to operate and increases the workload. Furthermore, the integrated design of the centrifuge and centrifuge tank makes it difficult to quickly load and unload a batch of centrifuge tubes. Each time centrifuge tubes are loaded and unloaded, the centrifuge is not working and a lot of time is required, which is not conducive to the efficient and stable separation of cell clusters. Summary of the Invention

[0005] The purpose of this invention is to provide a device for separating cell clusters in malignant pleural effusion and its method of use, so as to solve the problem that the existing methods mentioned in the background art are difficult to efficiently recover intact cell clusters; when using a centrifuge to separate cell clusters, the centrifuge tubes containing the samples need to be placed in the centrifuge one by one and placed at an angle in the centrifuge tank, which is inconvenient to operate and increases the workload. Moreover, the integrated design of the centrifuge and centrifuge tank makes it difficult to achieve the purpose of rapid loading and unloading of batches of centrifuge tubes. During each loading and unloading of centrifuge tubes, the centrifuge is not working and a lot of time is required.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] A device for separating cell clusters from malignant pleural effusion includes a centrifuge with a storage compartment and a support base inside. A mounting base is connected to the bottom of the support base, and a centrifugal drive mechanism is mounted on the bottom of the mounting base. The centrifugal drive mechanism is installed on the bottom of the inner wall of the centrifuge. Two hinges are mounted on the back of the centrifuge, and a cover is mounted on the centrifuge via these hinges. Several tube supports are provided on the outer side of the support base, and two limiting components are installed within each tube support. A centrifuge tube is located in the middle of the two limiting components. Support rods are hinged to the lower part of the outer wall of the tube supports via pins. Adjustment components are hinged to the ends of several support rods that are close to each other, penetrating the support base and connected to the support base. The adjustment components are installed inside the support base. Sliding holes are provided on the outer side of the support base corresponding to the positions of the support rods, and the support rods slide through these sliding holes. A squeezing component is installed inside the adjustment components, and two locking components overlap at the bottom of the squeezing component. A square groove is provided at the bottom of the support base, and limiting grooves are provided on both sides of the square groove, with the locking components sliding within the limiting grooves.

[0008] As a further embodiment of the present invention, the mounting base is provided with a groove and a circular groove, the bottom of the support base overlaps in the groove, the square groove at the bottom of the support base is provided outside the rectangular protrusion at the top of the mounting base, and the inner walls of the circular groove are respectively provided with slots, and the locking component is engaged in the slots.

[0009] As a further embodiment of the present invention, the limiting component includes a clamping plate, the clamping plate being arc-shaped and having a guide plate fixed at its top. A plurality of limiting rods are fixed to the outer wall of the clamping plate, the limiting rods sliding through the tube frame. A first spring is sleeved on the limiting rod, the two ends of the first spring being fixed to the inner wall of the tube frame and the outer wall of the clamping plate, respectively. The centrifuge tube is located between two opposing clamping plates. A hinge seat is installed above the outer wall of the tube frame, and the hinge seat is connected to the outer wall of the support seat.

[0010] As a further embodiment of the present invention, the adjusting assembly includes a stud, with support bearings installed on both sides of the outer wall of the stud, the two support bearings being respectively snapped into the bottom and top of the inner wall of the support base, a nut being threadedly connected to the middle of the stud, a retaining sleeve being fixed to the outside of the nut, the end of the support rod being hinged to the outer wall of the retaining sleeve via a pin, and a turntable being fixedly connected to the top of the stud.

[0011] As a further embodiment of the present invention, a screw is threadedly connected to the edge of the turntable, the bottom end of the screw overlaps the top of the support base, and a rotating sleeve is installed on the top of the screw.

[0012] As a further embodiment of the present invention, the extrusion assembly includes a lead screw, which is disposed inside a stud. A rotating handle is fixed at the top of the lead screw. A threaded sleeve is connected to the external thread of the lead screw. The threaded sleeve is engaged with the bottom of the inner wall of the support base. A connecting bearing is provided at the bottom of the lead screw. A tapered seat is installed at the bottom of the connecting bearing. The outer wall of the tapered seat overlaps with two locking assemblies respectively.

[0013] As a further embodiment of the present invention, the locking component includes a locking block, which is slidably connected in a limiting groove. The locking block is engaged in the groove and its other side overlaps with a conical seat. A sliding rod is slidably inserted through the locking block. The sliding rod is fixed on both sides of the inner wall of the limiting groove. A second spring is sleeved on the sliding rod and fixed between the locking block and the inner wall of the limiting groove.

[0014] A method of using a device for separating cell clusters in malignant pleural effusion, the method comprising the following steps:

[0015] When removing centrifuge tubes from the storage compartment inside the centrifuge, the cover is opened to expose the centrifuge tubes and support base. By rotating the handle on the turntable, the lead screw is rotated. As the lead screw rotates inside the threaded sleeve, it moves upward. The lead screw then moves the conical seat upward through the connecting bearing. At the same time, two locking blocks slide on the outer wall of the conical seat. As the locking blocks disengage from the conical seat, the tension of the second spring causes the locking blocks to slide in the limiting groove, allowing the two locking blocks to approach each other and disengage from the locking groove on the inner wall of the circular groove. This releases the locking state between the support base and the mounting base. Lifting the support base allows it to be removed from the mounting base, and then the support base and multiple centrifuge tubes can be removed from the centrifuge. When removing the centrifuge tubes, since the central axis of the tube rack and the support base are parallel, the centrifuge tubes can be pulled vertically upward directly, allowing multiple centrifuge tubes to be removed simultaneously.

[0016] When installing centrifuge tubes inside the tube rack, the centrifuge tubes are inserted from the top of the tube rack, and the bottom end of the centrifuge tube contacts two guide plates. Because the two guide plates are combined into a Y-shaped design, the centrifuge tube can open the two guide plates and two clamping plates. After the centrifuge tube is inserted, the clamping plates are supported by the elastic force of the first spring, so that the clamping plates drive the limiting rod to move inside the tube rack, so that the two clamping plates come closer to each other and clamp and limit the centrifuge tube. The flexible clamping method can limit the centrifuge tube while preventing the centrifuge tube from being damaged by excessive pressure.

[0017] When placing the support base, tube rack, and centrifuge tubes inside the centrifuge, lift the support base so that the square groove at the bottom is engaged with the rectangular protrusion at the top of the mounting base. The groove limits the bottom of the support base, improving the stability of the installation. When locking the support base and the mounting base, rotate the handle in the opposite direction to make the lead screw rotate. During the rotation of the lead screw, the conical seat can be moved downward through the connecting bearing. The conical seat can squeeze the two locking blocks to move them away from each other until the locking blocks are engaged in the locking groove, thus achieving the purpose of locking the support base and the mounting base.

[0018] Before centrifugation, the centrifuge tubes are placed inside the centrifuge at a fixed angle. This ensures that the tubes maintain a fixed angle with the axis of rotation during centrifugation, helping sample particles slide along the tube wall to the bottom and form a precipitate that is more compact. By holding the rotating sleeve and rotating the turntable, the turntable drives the stud to rotate. The lead screw is located in the hole in the middle of the stud, so the stud does not rotate synchronously with the lead screw. During the rotation of the stud, the nut can move the clamping sleeve downwards. When the clamping sleeve moves downwards, it can push the support rod to deflect. The support rod then pushes the tube rack to deflect around the hinge seat. The sliding hole limits the support rod, improving the stability of the support rod driving the tube rack to deflect. As a result, multiple tube racks will drive multiple centrifuge tubes to deflect synchronously, placing the centrifuge tubes inside the centrifuge at a fixed angle. Next, the screw is tightened to move it downwards and make it contact the top of the support seat, thus locking the turntable and preventing it from rotating on its own during centrifugation.

[0019] When centrifuging centrifuge tubes containing malignant pleural effusion samples, close the lid and operate the centrifugation drive mechanism. The specific steps are as follows:

[0020] Step 1: Centrifuge at 1500 r / min for 10 minutes using the centrifuge drive mechanism, collect the precipitate at the bottom of the centrifuge tube, pour off the supernatant, and retain the precipitate for the next step;

[0021] Step 2: Add the precipitate to the corresponding volume of lysed red blood cell solution and incubate at room temperature for 15 minutes. After lysis, centrifuge at 1500 r / min for 5 minutes to remove lysed red blood cell fragments and other impurities, and collect the precipitate.

[0022] Step 3: Resuspend the precipitate obtained in Step 2 in 50 mL of PBS buffer, and then perform gradient centrifugation according to the following procedure:

[0023] Step 1 Centrifugation: Centrifuge at 800 r / min for 5 minutes, collect the precipitate, resuspend it in 50 mL PBS, and use it as the sample for the next centrifugation step. Collect the supernatant separately after centrifugation for use in free cell separation.

[0024] Second step centrifugation: Centrifuge the sample resuspended in the first step at 600 r / min for 5 minutes to obtain a new precipitate. Resuspend the precipitate in 50 mL PBS as the sample for the next centrifugation step.

[0025] The third step is centrifugation: the sample resuspended in the second step is centrifuged at 400 r / min for 5 minutes to obtain the bottom precipitate. After resuspending the precipitate, the centrifugation is repeated once at 400 r / min.

[0026] When removing centrifuge tubes from the centrifuge after centrifugation, open the cover and loosen the screws to disengage them from the support base. Rotate the turntable in the opposite direction using the rotating sleeve, causing the turntable to rotate the lead screw. This causes the nut to move the clamping sleeve upwards, which in turn pulls several support rods to deflect synchronously. This causes the support rods to pull the bottom of the tube rack closer to the support base until the tube rack and the centrifuge tubes are parallel to the central axis of the support base. After removing the support base from the centrifuge, the centrifuge tubes can be pulled vertically upwards.

[0027] Compared with the prior art, the beneficial effects of the present invention are:

[0028] 1. This invention rotates a screw by turning a handle on a turntable. As the screw rotates inside the threaded sleeve, it moves upwards, causing a conical seat to move upwards via a connecting bearing. Simultaneously, two locking blocks slide on the outer wall of the conical seat. During the disengagement of the locking blocks from the conical seat, the tension of a second spring causes the blocks to slide within a limiting groove, allowing them to approach each other and disengage from the groove on the inner wall of the circular groove. This releases the locking between the support and the mounting base, allowing the support to be lifted and removed from the mounting base. This enables the support and multiple centrifuge tubes to be removed from the centrifuge. When removing the centrifuge tubes, because the tube rack and the central axis of the support are parallel, the centrifuge tubes can be pulled vertically upwards, allowing multiple centrifuge tubes to be removed simultaneously. The support, tube rack, and centrifuge tubes are then placed... Inside the centrifuge, the support base is lifted, and the square groove at the bottom is engaged with the rectangular protrusion at the top of the mounting base. The groove limits the bottom of the support base, improving the stability of the setup. When locking the support base and the mounting base, the screw is rotated in the opposite direction by turning the handle. During the rotation of the screw, the conical seat moves downward through the connecting bearing. The conical seat can squeeze the two locking blocks away from each other until the locking blocks are engaged in the locking groove, thus achieving the purpose of locking the support base and the mounting base. Therefore, the modular design of the centrifuge, support base and tube rack enables the rapid loading and unloading of batch centrifuge tubes. Using two sets of support bases and multiple tube racks for centrifugation work allows for alternating loading and unloading of centrifuge tubes, shortening the time spent loading and unloading centrifuge tubes and facilitating the efficient and stable separation of cell clusters.

[0029] 2. This invention involves holding a rotating sleeve and rotating a turntable, which in turn causes the turntable to rotate the stud. The lead screw is located in a hole in the middle of the stud, so the stud does not rotate synchronously with the lead screw during rotation. During the rotation of the stud, the nut can move the clamping sleeve downwards. When the clamping sleeve moves downwards, it can push the support rod to deflect. The support rod then pushes the tube rack to deflect around the hinge seat. The sliding hole limits the support rod, improving the stability of the support rod driving the tube rack to deflect. As a result, multiple tube racks will drive multiple centrifuge tubes to deflect synchronously, so that the centrifuge tubes are placed inside the centrifuge at a fixed angle. This helps the sample particles slide along the tube wall to the bottom to form a precipitate, making the precipitate more compact. By adjusting the angle of the tube rack, the centrifugation effect is improved, and the centrifuge tubes are also kept parallel to the central axis of the support seat, making it convenient to pick up and put in multiple centrifuge tubes at the same time, thus improving the ease of operation.

[0030] 3. In this invention, when installing centrifuge tubes inside the tube rack, the centrifuge tubes are inserted from above the tube rack, with the bottom end of the centrifuge tube contacting two guide plates. Because the two guide plates are combined into a Y-shape, the centrifuge tube can easily spread the two guide plates and the two clamping plates. After the centrifuge tube is inserted, the clamping plates are supported by the elastic force of the first spring, causing the clamping plates to move the limiting rod inside the tube rack, so that the two clamping plates move closer to each other and clamp and limit the centrifuge tube. The flexible clamping method not only limits the centrifuge tube but also prevents the centrifuge tube from being damaged by excessive pressure, thus protecting the centrifuge tube.

[0031] 4. This invention employs a gradient centrifugation procedure when centrifuging centrifuge tubes containing malignant pleural effusion samples. By gradually reducing the centrifugal force and combining this with a resuspension method at each step, the high centrifugal force can be effectively avoided from damaging cell clusters, ensuring the integrity of the cell clusters and a high recovery rate. At the same time, the standardized operating procedure ensures the repeatability and scalability of the method. Attached Figure Description

[0032] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0033] Figure 1 This is a three-dimensional structural schematic diagram of the present invention;

[0034] Figure 2 This is a schematic diagram of a partial cross-section of the centrifuge of the present invention;

[0035] Figure 3 This is a schematic diagram of the connection between the support base and the mounting base of the present invention;

[0036] Figure 4 This is a schematic diagram of the cross-sectional structure of the pipe rack of the present invention;

[0037] Figure 5 For the present invention Figure 4 Enlarged structural diagram at point A;

[0038] Figure 6 This is a schematic diagram of the cross-sectional structure of the mounting base of the present invention;

[0039] Figure 7 This is a schematic diagram of the mounting base of the present invention;

[0040] Figure 8 This is a schematic diagram of the cross-section of the support base of the present invention;

[0041] Figure 9 For the present invention Figure 8 Enlarged structural diagram at point B;

[0042] Figure 10 This is a schematic diagram of the extrusion assembly of the present invention.

[0043] The attached diagram lists the components represented by each number as follows:

[0044] 1. Centrifuge; 2. Storage compartment; 3. Support base; 4. Mounting base; 5. Centrifugal drive mechanism; 6. Groove; 7. Circular groove; 8. Slot; 9. Tube rack; 10. Limiting assembly; 101. Clamping plate; 102. Guide plate; 103. Limiting rod; 104. First spring; 11. Support rod; 12. Adjusting assembly; 121. Stud; 122. Nut; 123. Sleeve; 124. Turntable; 125. Screw 126. Nail; 13. Rotating sleeve; 14. Sliding hole; 15. Support bearing; 16. Extrusion assembly; 17. Lead screw; 18. Rotating handle; 19. Threaded sleeve; 10. Connecting bearing; 11. Conical seat; 12. Locking assembly; 13. Locking block; 14. Sliding rod; 15. Second spring; 16. Limiting groove; 17. Square groove; 18. Machine cover; 19. Hinge; 20. Centrifuge tube; 21. Hinge seat. Detailed Implementation

[0045] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0046] Please see Figures 1-10 The present invention provides a technical solution:

[0047] A device for separating cell clusters in malignant pleural effusion includes a centrifuge 1, a storage compartment 2 on the centrifuge 1, a support base 3 inside the storage compartment 2, a mounting base 4 connected to the bottom of the support base 3, a centrifugal drive mechanism 5 installed at the bottom of the mounting base 4, the centrifugal drive mechanism 5 installed at the bottom of the inner wall of the centrifuge 1, and two hinges 20 installed on the back of the centrifuge 1, through which a cover 19 is installed.

[0048] Several tube racks 9 are provided on the outer side of the support base 3. A hinge seat 22 is installed on the upper part of the outer wall of the tube rack 9. The hinge seat 22 is rotatably connected to the top outer wall of the support base 3 by a pin. Two limiting components 10 are installed inside the tube rack 9. The limiting component 10 includes a clamping plate 101. The clamping plate 101 is arc-shaped and has a guide plate 102 fixed on its top. Several limiting rods 103 are fixed on the outer wall of the clamping plate 101. The limiting rods 103 slide through the tube rack 9. A first spring 104 is sleeved on the limiting rod 103. The two ends of the first spring 104 are fixed to the inner wall of the tube rack 9 and the outer wall of the clamping plate 101, respectively. The centrifuge tube 21 is located between two opposite clamping plates 101.

[0049] Because the two guide plates 102 are combined into a Y-shaped design, the centrifuge tube 21 can open the two guide plates 102 and the two clamping plates 101. The clamping plates 101 are supported by the elastic force of the first spring 104, so that the clamping plates 101 drive the limiting rod 103 to move within the tube frame 9, so that the two clamping plates 101 move closer to each other and clamp and limit the centrifuge tube 21. The flexible clamping method can limit the centrifuge tube 21 while preventing the centrifuge tube 21 from being damaged by excessive pressure.

[0050] Support rods 11 are hinged to the lower part of the outer wall of the pipe rack 9 via pins. Several support rods 11, with one end close to the others, pass through the support base 3 and are hinged to an adjusting assembly 12, which is installed inside the support base 3. As a further embodiment of the invention, the adjusting assembly 12 includes a stud 121. Support bearings 14 are respectively installed on both sides of the outer wall of the stud 121, and the two support bearings 14 are respectively engaged with the bottom and top of the inner wall of the support base 3. A nut 122 is threaded into the middle of the stud 121, and a retaining sleeve 123 is fixed to the outside of the nut 122. The end of the support rod 11 is hinged to the outer wall of the retaining sleeve 123 via a pin, and a turntable 124 is fixedly connected to the top of the stud 121. Sliding holes 13 are respectively opened on the outside of the support base 3 corresponding to the positions of the support rods 11, and the support rods 11 slide through and slide within the sliding holes 13.

[0051] During the rotation of the stud 121, the nut 122 can drive the sleeve 123 to move downward. When the sleeve 123 moves downward, it can push the support rod 11 to deflect. The support rod 11 then pushes the tube rack 9 to deflect around the hinge seat 22. As a result, multiple tube racks 9 will drive multiple centrifuge tubes 21 to deflect synchronously, which facilitates the synchronous adjustment of the angle of multiple centrifuge tubes 21.

[0052] An extrusion assembly 15 is provided through the interior of the adjustment assembly 12. The extrusion assembly 15 includes a lead screw 151, which is located in a hole opened in the middle of the stud 121. The lead screw 151 is provided through the interior of the stud 121, so the stud 121 will not drive the lead screw 151 to rotate synchronously during rotation. A rotating handle 152 is fixed at the top of the lead screw 151. A threaded sleeve 153 is connected to the external thread of the lead screw 151. The threaded sleeve 153 is engaged with the bottom of the inner wall of the support base 3. A connecting bearing 154 is provided at the bottom of the lead screw 151. A tapered seat 155 is installed at the bottom of the connecting bearing 154.

[0053] A screw 125 is threaded onto the edge of the turntable 124. The bottom end of the screw 125 overlaps the top of the support base 3, and a rotating sleeve 126 is installed on the top of the screw 125. By tightening the screw 125, it moves downward and contacts the top of the support base 3, thereby locking the turntable 124 and preventing it from rotating during centrifugation.

[0054] The bottom of the support base 3 has a square groove 18, and the two sides of the square groove 18 have limit grooves 17 respectively. The locking component 16 slides in the limit groove 17. The mounting base 4 has a groove 6 and a circular groove 7. The bottom of the support base 3 overlaps in the groove 6. The square groove 18 at the bottom of the support base 3 is located outside the rectangular protrusion at the top of the mounting base 4. The two sides of the inner wall of the circular groove 7 have slots 8 respectively. The locking component 16 is engaged in the slots 8.

[0055] By engaging the square groove 18 at the bottom of the support base 3 with the rectangular protrusion at the top of the mounting base 4, and limiting the bottom of the support base 3 through the groove 6, the erection stability is improved. Moreover, when the centrifugal drive mechanism 5 drives the mounting base 4 to rotate, it can ensure the stability of the mounting base 4 driving the support base 3 and the centrifugal tube 21 to rotate.

[0056] As a further embodiment of the present invention, the outer wall of the conical seat 155 is respectively connected to two locking components 16. The locking components 16 include a locking block 161, which is slidably connected in the limiting groove 17. The locking block 161 is engaged in the locking groove 8 and its other side is connected to the conical seat 155. A sliding rod 162 is slidably passed through the locking block 161. The sliding rod 162 is fixed on both sides of the inner wall of the limiting groove 17. A second spring 163 is sleeved on the sliding rod 162. The second spring 163 is fixed between the locking block 161 and the inner wall of the limiting groove 17.

[0057] Because the lead screw 151 is threaded into the threaded sleeve 153, and the threaded sleeve 153 is engaged with the bottom of the inner wall of the support base 3, the lead screw 151 can move downwards during rotation. This movement, via the connecting bearing 154, causes the tapered seat 155 to move downwards. The tapered seat 155 can press the two locking blocks 161 away from each other, facilitating synchronous adjustment of their positions. The tension of the second spring 163 causes the locking blocks 161 to slide on the limiting groove 17 and the slide rod 162, improving the stability of the horizontal movement of the locking blocks 161. This allows the two locking blocks 161 to approach each other and disengage from the locking groove 8 on the inner wall of the circular groove 7, facilitating the release of the locking state between the support base 3 and the mounting base 4. When the lead screw 151 rotates inside the connecting bearing 154, friction is generated between the tapered seat 155 and the locking blocks 161, preventing the tapered seat 155 from rotating synchronously with the lead screw 151, thus improving the stability of adjusting the locking blocks 161.

[0058] During operation, the modular design of the centrifuge 1, support base 3, and tube rack 9 enables the rapid loading and unloading of batches of centrifuge tubes 21. Two sets of support bases 3 and multiple tube racks 9 are used for centrifugation, allowing for alternating loading and unloading of centrifuge tubes 21, thus shortening the time spent loading and unloading centrifuge tubes 21 and facilitating the efficient and stable separation of cell clusters.

[0059] A method for using a device for separating cell clusters in malignant pleural effusion, the method comprising the following steps:

[0060] When centrifuge tubes 21 are removed from the storage compartment 2 inside centrifuge 1, the cover 19 is opened to expose centrifuge tubes 21 and support base 3. The screw 151 is rotated by rotating the handle 152 on the turntable 124. During the rotation of the screw 151 inside the threaded sleeve 153, it can move upward. The screw 151 then drives the conical seat 155 to move upward through the connecting bearing 154. At the same time, the two locking blocks 161 slide on the outer wall of the conical seat 155. During the process of the locking block 161 disengaging from the conical seat 155, the second spring 163 pulls the locking block 161 to slide in the limiting groove 17, so that the two locking blocks 161 can approach each other and disengage from the locking groove 8 on the inner wall of the circular groove 7, thereby releasing the locking state between the support seat 3 and the mounting seat 4. Lifting the support seat 3 can remove it from the mounting seat 4, and then the support seat 3 and multiple centrifuge tubes 21 can be removed from the centrifuge 1. When removing the centrifuge tubes 21, since the tube rack 9 and the central axis of the support seat 3 are parallel, the centrifuge tubes 21 can be directly pulled vertically upward, and multiple centrifuge tubes 21 can be removed simultaneously.

[0061] When installing centrifuge tubes 21 inside the tube rack 9, the centrifuge tubes 21 are inserted from above the tube rack 9, and the bottom end of the centrifuge tubes 21 contacts the two guide plates 102. Since the two guide plates 102 are combined into a Y-shaped design, the centrifuge tubes 21 can open the two guide plates 102 and the two clamping plates 101. After the centrifuge tubes 21 are inserted, the clamping plates 101 are supported by the elastic force of the first spring 104, so that the clamping plates 101 drive the limiting rod 103 to move inside the tube rack 9, so that the two clamping plates 101 move closer to each other and clamp and limit the centrifuge tubes 21. The centrifuge tubes 21 are limited by flexible clamping, while preventing the centrifuge tubes 21 from being damaged by excessive pressure.

[0062] When the support base 3, tube rack 9 and centrifuge tube 21 are placed inside the centrifuge 1, the support base 3 is lifted and the square groove 18 at the bottom is locked outside the rectangular protrusion at the top of the mounting base 4. The bottom of the support base 3 is limited by the groove 6 to improve the stability of the installation. When locking the support base 3 and the mounting base 4, the screw 151 is rotated by rotating the handle 152 in the opposite direction. During the rotation of the screw 151, the conical seat 155 can be moved downward through the connecting bearing 154. The conical seat 155 can squeeze the two locking blocks 161 to make them move away from each other until the locking blocks 161 are locked into the locking groove 8, so as to achieve the purpose of locking the support base 3 and the mounting base 4.

[0063] Before centrifugation, the centrifuge tube 21 is placed inside the centrifuge 1 at a fixed angle, so that the centrifuge tube 21 forms a fixed angle with the axis of rotation during centrifugation. This helps sample particles slide along the tube wall of the centrifuge tube 21 to the bottom to form a precipitate, making the precipitate more compact. Therefore, by holding the rotating sleeve 126 and rotating the turntable 124, the turntable 124 drives the stud 121 to rotate. The lead screw 151 is located in the hole in the middle of the stud 121, so the rotation of the stud 121 will not drive the lead screw 151 to rotate synchronously. During the rotation of the stud 121, the nut 122 can be driven... When the sleeve 123 moves downward, it can push the support rod 11 to deflect. The support rod 11 then pushes the tube rack 9 to deflect around the hinge seat 22. The support rod 11 is limited by the sliding hole 13, which improves the stability of the support rod 11 driving the tube rack 9 to deflect. As a result, multiple tube racks 9 will drive multiple centrifuge tubes 21 to deflect synchronously, so that the centrifuge tubes 21 are placed inside the centrifuge 1 at a fixed angle. Then, tighten the screw 125 to make it move downward and contact the top of the support seat 3, so as to lock the turntable 124 and prevent the turntable 124 from rotating during the centrifugation process.

[0064] When centrifuging centrifuge tube 21 containing malignant pleural effusion samples, close the lid 19 and control the centrifugation drive mechanism 5. The specific steps are as follows:

[0065] Step 1: Centrifuge the centrifuge drive mechanism 5 at 1500 r / min for 10 minutes, collect the precipitate at the bottom of the centrifuge tube 21, pour off the supernatant, and retain the precipitate for the next step;

[0066] Step 2: Add the precipitate to the corresponding volume of lysed red blood cell solution and incubate at room temperature for 15 minutes. After lysis, centrifuge at 1500 r / min for 5 minutes to remove lysed red blood cell fragments and other impurities, and collect the precipitate.

[0067] Step 3: Resuspend the precipitate obtained in Step 2 in 50 mL of PBS buffer, and then perform gradient centrifugation according to the following procedure:

[0068] Step 1 Centrifugation: Centrifuge at 800 r / min for 5 minutes, collect the precipitate, resuspend it in 50 mL PBS, and use it as the sample for the next centrifugation step. Collect the supernatant separately after centrifugation for use in free cell separation.

[0069] Second step centrifugation: Centrifuge the sample resuspended in the first step at 600 r / min for 5 minutes to obtain a new precipitate. Resuspend the precipitate in 50 mL PBS as the sample for the next centrifugation step.

[0070] The third step is centrifugation: the sample resuspended in the second step is centrifuged at 400 r / min for 5 minutes to obtain the bottom precipitate. After resuspending the precipitate, the centrifugation is repeated once at 400 r / min.

[0071] After centrifugation, when removing the centrifuge tube 21 from the centrifuge 1, open the cover 19 and loosen the screw 125 so that the screw 125 can disengage from the support base 3. Rotate the turntable 124 in the opposite direction through the rotating sleeve 126, so that the turntable 124 drives the lead screw 151 to rotate, so that the nut 122 can drive the clamp 123 to move upward. The clamp 123 then pulls several support rods 11 to deflect synchronously, so that the support rods 11 pull the bottom of the tube rack 9 close to the support base 3, until the tube rack 9 drives the centrifuge tube 21 to be parallel to the central axis of the support base 3. After removing the support base 3 from the centrifuge 1, the centrifuge tube 21 can be pulled vertically upward.

Claims

1. A cell mass separation device in malignant pleural effusion comprising a centrifuge (1) characterised in that: The centrifuge (1) is provided with a storage bin (2), the storage bin (2) is provided with a supporting seat (3), the bottom of the supporting seat (3) is connected with a mounting seat (4), the bottom of the mounting seat (4) is provided with a centrifugal driving mechanism (5), the centrifugal driving mechanism (5) is installed on the bottom of the inner wall of the centrifuge (1), the back of the centrifuge (1) is provided with two hinges (20), the centrifuge (1) is provided with a machine cover (19) through the two hinges (20), the outer side of the supporting seat (3) is provided with a plurality of pipe racks (9), the pipe rack (9) is provided with two limiting components (10), the middle part of the two limiting components (10) is provided with a centrifugal tube (21), the lower part of the outer wall of the pipe rack (9) is hinged with a supporting rod (11) through a pin shaft, the ends of the plurality of supporting rods (11) close to each other are penetrated through the supporting seat (3) and are hinged with an adjusting component (12), the adjusting component (12) is installed in the supporting seat (3), the positions corresponding to the supporting rods (11) of the supporting seat (3) are respectively provided with sliding holes (13), the supporting rods (11) are penetrated through and slide in the sliding holes (13), the inside of the adjusting component (12) is penetrated through and provided with an extrusion component (15), the bottom end of the extrusion component (15) is overlapped with two locking components (16), the bottom of the supporting seat (3) is provided with a square groove (18), the two sides in the square groove (18) are respectively provided with limiting grooves (17), the locking components (16) slide in the limiting grooves (17); The mounting seat (4) is provided with a groove (6) and a circular groove (7), the bottom of the supporting seat (3) is overlapped in the groove (6), the square groove (18) at the bottom of the supporting seat (3) is arranged outside the rectangular protrusion at the top of the mounting seat (4), the two sides of the inner wall of the circular groove (7) are respectively provided with clamping grooves (8), the locking components (16) are clamped in the clamping grooves (8); The adjusting component (12) comprises a threaded stud (121), the two sides of the outer wall of the threaded stud (121) are respectively provided with supporting bearings (14), the two supporting bearings (14) are respectively clamped in the bottom and the top of the inner wall of the supporting seat (3), the middle part of the threaded stud (121) is threadedly connected with a nut (122), the outer side of the nut (122) is fixedly provided with a clamping sleeve (123), the end of the supporting rod (11) is hinged with the outer wall of the clamping sleeve (123) through a pin shaft, the top end of the threaded stud (121) is fixedly connected with a rotating disc (124); The edge of the rotating disc (124) is threadedly connected with a screw (125), the bottom end of the screw (125) is overlapped on the top of the supporting seat (3), the top of the screw (125) is provided with a rotating sleeve (126); The extrusion assembly (15) comprises a lead screw (151) arranged through the inside of the stud (121), the top end of the lead screw (151) is fixed with a handle (152), the lead screw (151) is externally screwed with a threaded sleeve (153), the threaded sleeve (153) is clamped at the bottom of the inner wall of the support base (3), the bottom end of the lead screw (151) is provided with a connecting bearing (154), the bottom of the connecting bearing (154) is installed with a conical seat (155), and the outer wall of the conical seat (155) is respectively overlapped with two locking assemblies (16); The locking assembly (16) comprises a clamping block (161) which is slidingly connected in the limiting groove (17), the clamping block (161) is clamped in the clamping groove (8) and the other side is overlapped with the conical seat (155), the clamping block (161) is slidingly penetrated with a sliding rod (162), the sliding rod (162) is fixed on both sides of the inner wall of the limiting groove (17), the sliding rod (162) is externally sleeved with a second spring (163), and the second spring (163) is fixed between the clamping block (161) and the inner wall of the limiting groove (17).

2. A cell cluster separation device in malignant pleural effusion according to claim 1, characterized in that: The limiting assembly (10) comprises a clamping plate (101), the clamping plate (101) is arc-shaped and the top is fixed with a guide piece (102), the outer wall of the clamping plate (101) is fixed with a plurality of limiting rods (103), the limiting rods (103) are slidingly penetrated in the pipe frame (9), the limiting rods (103) are externally sleeved with first springs (104), and the two ends of the first springs (104) are respectively fixed on the inner wall of the pipe frame (9) and the outer wall of the clamping plate (101). The centrifugal tube (21) is located between the two opposite clamping plates (101), the hinge seat (22) is installed above the outer wall of the pipe frame (9), and the hinge seat (22) is connected with the outer wall of the support base (3).

3. A method of using a cell cluster separation device in malignant pleural effusion, the cell cluster separation device according to claim 2, wherein, The use method comprises the following steps: Step one: the centrifugal driving mechanism (5) is centrifuged at 1500 r / min for 10 minutes, the precipitate at the bottom of the centrifugal tube (21) is collected, the supernatant is poured, and the precipitate is reserved for the next operation; Step two: add the precipitate to the corresponding volume of lysed red blood cell solution, and react at room temperature for 15 minutes. After lysing is completed, centrifuge at 1500 r / min for 5 minutes to remove the red blood cell fragments and other impurities after lysis, and collect the precipitate; Step three: re-suspend the precipitate obtained in step two in 50 mL PBS buffer, and then perform gradient centrifugation according to the following centrifugal procedure: First step of centrifugation: centrifuge at 800 r / min for 5 minutes, collect the precipitate, re-suspend it in 50 mL PBS, and use it as the sample for the next step of centrifugation. The supernatant after centrifugation is collected separately for use in free cell separation; Second step of centrifugation: centrifuge the re-suspended sample in the first step at 600 r / min for 5 minutes to obtain a new precipitate, re-suspend the precipitate in 50 mL PBS again as the sample for the next step of centrifugation; Third centrifugation: centrifuge the resuspended sample at 400 r / min for 5 minutes to obtain the bottom precipitate, and repeat the centrifugation once at 400 r / min after resuspending the precipitate; When the centrifuge tube (21) is taken out from the centrifuge (1) after the centrifugation is completed, the cover (19) is opened, the screw (125) is loosened, the screw (125) can be separated from the support base (3), the rotating sleeve (126) is reversely rotated to rotate the rotating disc (124), the rotating disc (124) drives the screw rod (151) to rotate, the nut (122) drives the clamping sleeve (123) to move upwards, the clamping sleeve (123) pulls a plurality of support rods (11) to synchronously deflect, the support rods (11) pull the bottom of the tube rack (9) to be close to the support base (3), until the tube rack (9) drives the centrifuge tube (21) to be parallel to the central axis of the support base (3), the support base (3) is taken out from the centrifuge (1), and then the centrifuge tube (21) can be vertically pulled out upwards.

Images