A closed centrifugal device

Abstract

The present invention relates to a closed centrifugal device, comprising a tube body, wherein at least three openings are arranged on the tube body, one being a liquid inlet, one being a liquid outlet, and one being a vent. The closed centrifugal device provided by the present invention can perform centrifugal operation on a cell suspension in a closed state, thereby realizing rapid liquid inlet / outlet / liquid exchange, greatly reducing the risk of biological sample contamination, and at the same time meeting the regulatory requirement that the sample processing of Class III medical devices must be fully closed.

Description

Technical Field

[0001] The present invention relates to a closed centrifugal device, and more particularly to a device for separating cells and cell culture fluid in a closed state. Background Art

[0002] A centrifuge tube is defined as a tubular liquid container that can be used for centrifugation. It has been widely used in biological sciences, especially in the fields of biochemistry and molecular biology research. Centrifugation technology is mainly used for the separation and preparation of various biological samples. The biological sample suspension is placed in a centrifuge tube. Under high-speed rotation, due to the huge centrifugal force, the suspended tiny particles (such as cells, biological macromolecules, etc.) settle at a certain speed and accumulate at the bottom of the centrifuge tube, thereby being separated from the solution. Centrifuge tubes are an indispensable structural component of centrifugation technology. Centrifuge tubes of different specifications and styles can achieve different centrifugation purposes.

[0003] In biological experiments, cells often need to be washed, culture medium replaced, or other mixed solutions added, so centrifugation is often involved. In traditional centrifugation operations, experimenters usually pour liquids into centrifuge tubes or pour them out of centrifuge tubes into other containers; a standard centrifuge tube is a wide-mouth container, and the centrifuge tube cover is often opened / closed. Although the operation is completed on an ultra-clean workbench, the entire centrifugation process is in a completely open environment, and there is a risk of sample contamination; especially in the preparation of immune cells for clinical treatment (patients), high standards and strict requirements for zero contamination are required, and the above potential risks must be absolutely avoided.

[0004] Therefore, there is an urgent need for a centrifugal device that can be in a closed, sterile working state to meet the needs of the above applications.

[0005] On the market, Biosafe's online centrifugation system can achieve the centrifugation function under the above-mentioned closed working state, but the centrifugation effect can only achieve cell stratification, and cannot separate cells and their suspensions; and in many applications such as electrotransfection, the cell cleaning before electrotransfection requires the complete removal of the liquid, and the cells must form solid, non-dispersed clumps that can be separated from the liquid to facilitate the separation of cells and liquid.

[0006] In addition, cell enrichment is a necessary step to accurately control the cell density in the cell suspension. By controlling the cell density, the conductivity of the cell suspension can be controlled, and the electroporation efficiency and cell survival rate can be improved.

[0007] Specifically, we need to design a new closed centrifuge device that can achieve cell enrichment centrifugation effect under closed and sterile conditions.

[0008] The existing patent CN201721562489.7 provides an improved sampling device, which uses air pressure to ensure the sterile state of the bioreactor and the sampling device during multiple sampling or continuous sampling processes, while improving the efficiency of sampling during biological reaction tests; however, in the process of aseptic sampling, it is necessary to pressurize the bioreactor first and then pressurize from the position of the second tube, and the operation process is relatively complicated; in addition, the entire device includes a centrifuge tube, a first tube and a second tube, and the sealing cover of the centrifuge tube is provided with a first hole and a second hole, which is not an integrated tube body structure, and there is still a risk of contamination for the liquid in the tube.

[0009] Another patent CN201721017811.8 provides a sterile centrifuge tube set capable of keeping the centrifuge tube sterile, which as a whole comprises an outer tube and an inner tube; the entire invention aims to keep the inner tube sterile and airtight before and after centrifugation, thereby facilitating various aseptic operations after centrifugation; but the inner tube comprises a tube body, an inner tube plug, a sealing cap and an inner tube accessory, and is not an integrated tube body structure; during actual sampling, the sealing cap and the inner tube plug need to be opened / covered, and there is a risk of contamination for the liquid in the tube body.

[0010] The present invention aims at the application limitations of the above-mentioned prior art and proposes a novel closed centrifugal device which can be used in conjunction with the existing centrifuge and can achieve the cell enrichment centrifugal effect in a closed state. Summary of the invention

[0011] The present invention overcomes the above-mentioned deficiencies in the prior art and aims to provide a closed centrifugal device.

[0012] In order to achieve the above-mentioned purpose, the technical solution of the closed centrifugal device provided by the present invention is summarized as follows:

[0013] The closed centrifugal device of the present invention comprises a tube body, and at least three openings are arranged on the tube body, one is a liquid inlet, one is a liquid outlet, and one is a vent;

[0014] Preferably, the upper part and / or the lower part of the tube body is in a cone shape, and the middle part of the tube body is in a cylindrical shape;

[0015] Preferably, the upper and lower parts of the tube body are both in a cone shape;

[0016] Preferably, the liquid inlet, liquid outlet and vent are all arranged on the same side cone of the upper or lower part of the tube body;

[0017] Preferably, the liquid outlet is located at the tip of the upper or lower cone of the tube body;

[0018] Preferably, a barrier is provided inside the tube body to prevent the cell clusters / lumps near the tip of the bottom of the tube body, especially some sand-shaped, discrete cells, from sliding down along the tube wall to the liquid outlet when the liquid is discharged;

[0019] Preferably, the enclosure is arranged on the side wall of the cone at the lower part of the tube body;

[0020] Preferably, the width of the enclosure is about 0.2 cm-1 cm, and the length is about 1 cm-6 cm;

[0021] Preferably, the width of the enclosure is about 0.5 cm and the length is about 3 cm;

[0022] Preferably, valves are provided on the liquid inlet, liquid outlet and vent, respectively, to independently control the opening and closing of external pipelines sealedly connected to the liquid inlet, liquid outlet and vent;

[0023] Preferably, the valve can be selected from a stop valve, a hose clamp, etc.;

[0024] Preferably, the liquid inlet, liquid outlet and vent can be sealed and connected to an external pipeline;

[0025] Preferably, the sealed connection is connected via a pipe joint;

[0026] Preferably, the pipeline connector can be selected from a quick-connect connector, a needleless connector, etc.;

[0027] Preferably, the pipeline connector is a Luer connector;

[0028] Preferably, protective caps are provided at the liquid inlet, liquid outlet and vent, and the liquid inlet, liquid outlet and vent are respectively sealed and connected to the protective caps in a detachable manner such as threads or buckles;

[0029] Preferably, the tube body is provided with a protective cover;

[0030] Preferably, the tube body is embedded in the protective cover, and the tube walls of the two parts are in close contact;

[0031] Preferably, the inner cavity of the protective cover is substantially the same as the outer shape of the tube body;

[0032] Preferably, the height of the protective cover is greater than the height of the tube body, so that when the tube body is placed in the protective cover, the liquid inlet, liquid outlet and vent on the tube body and the protective cap thereof can be completely placed in the protective cover;

[0033] Preferably, the tube body is sealed and / or fixedly connected to the protective cover by a detachable manner such as thread and / or snap fastener;

[0034] The tube body of the present invention can be firstly split into at least two parts during preparation, which can be injection molded separately and then assembled into one by hot melting, welding and other processes;

[0035] Preferably, the liquid inlet, liquid outlet and vent, as well as the pipe joint, of the present invention can be injection molded together with the pipe body;

[0036] Preferably, a fourth opening is provided on the tube body, and the fourth opening can be used as a liquid suction port;

[0037] Preferably, a pipette is provided at the fourth opening;

[0038] Preferably, a pipette is arranged inside the tube body; one end of the pipette is sealed and connected to the fourth opening; preferably, the pipette is sealed and connected to the fourth opening by means of sealant, ultrasonic welding or the like, and the pipette is fixedly connected to the side wall of the tube body by means of an adhesive or the like;

[0039] Preferably, the other end of the pipette is located at the side wall of the lower cone inside the tube body, and preferably, the other end of the pipette is located at the junction of the lower edge of the cylinder in the middle of the tube body and the upper edge of the lower cone of the tube body.

[0040] Preferably, the fourth opening can be sealed and connected to a collecting device at one end outside the tube body; preferably, the fourth opening is sealed and connected to the collecting device through a liquid suction pipeline, and preferably, the collecting device is a negative pressure collecting device;

[0041] Preferably, the negative pressure collection device can be selected from a syringe, a pipette, a container with a negative pressure pump, etc.;

[0042] Preferably, the tube body is made of hard and / or soft material, and the protective cover is made of hard and / or soft material;

[0043] Preferably, the hard material is selected from non-deformable materials such as ceramics, glass, ABS, PC, PS, PA, POM, PMMMA, etc.;

[0044] Preferably, the soft material is selected from deformable materials such as silicone, rubber, PET, PE, PP, PVC, TPU, PU, ​​PPS, etc.;

[0045] Preferably, when the tube body is selected from a soft material, the suction and discharge of liquid can be achieved through the deformation of the tube body, and the vent can be omitted on the tube body.

[0046] The term "connector" mentioned above refers to the common pipe connectors in medical pipelines, which are designed to achieve a fast, safe, and leak-free biological operating environment. When the pipe connector does not have a built-in soft plug / rubber plug, it is called a "quick-connect connector", which means that the pipeline can be connected / disconnected without using tools (the common Luer connector is one of them); when the pipe connector has a built-in soft plug / rubber plug, it is called a "needleless connector": a) When the needleless connector is screwed into the pipeline, the core in the middle of one end of the connector will push up the built-in soft plug / rubber plug at the other end, and the pipeline is connected; b) When the needleless connector is screwed out of the pipeline, the soft plug / rubber plug returns to its original position, and the pipeline is sealed; in other words, if a needleless connector is used, there is no need to use a control valve.

[0047] A method or use of separating cells using a closed centrifugal device, comprising using the above-mentioned closed centrifugal device, wherein the centrifugal force generally ranges from 200 to 500 g, which is selected according to different application scenarios.

[0048] Preferably, the main types of the centrifuged cells include cells used for clinical treatment, such as PBMC, various immune cells such as resting or activated T cells, NK cells, DC cells, stem cells, etc.; various engineered cells, such as CHO-S, HEK293, etc.; and various biological cells.

[0049] Compared with the prior art, the present invention has the following beneficial technical effects:

[0050] The closed centrifugal device of the present invention can perform centrifugal operation on the cell suspension in a closed state, thereby realizing rapid liquid inlet / outlet / liquid exchange and greatly reducing the risk of sample contamination.

[0051] The closed centrifugal device of the present invention can achieve a cell enrichment centrifugation effect. Compared with a cell suspension in a cell stratification state, it can cleanly separate the liquid to be discarded from the cells, ensure that the electrotransfection buffer is not diluted in the electrotransfection application, and more accurately control the cell density to achieve high electrotransfection efficiency and high cell survival rate.

[0052] The term "cell stratification" mentioned above means: by adding separation liquid (referring to different liquids) for density gradient centrifugation or vortexing, liquid layers of different densities are formed after centrifugation / vortexing, and cells of different densities will be distributed in the liquid layers of corresponding density, thereby being separated; the stratified cells will be enriched to a certain extent, but because they are suspended in the liquid, they still have a certain degree of dispersion and cannot be separated from the liquid.

[0053] The term "cell enrichment" mentioned above means: highly enriching cells by centrifugation or the like, forming solid clumps in a liquid (referring to a single liquid), which do not disperse in the liquid and can be separated from the liquid.

[0054] The upper and lower parts of the tube body of the closed centrifugal device of the present invention are both cones, which can achieve: the cone of the upper part is used to discharge liquid, so that the liquid flows out from the tip of the cone and the minimum amount remains in the tube; the cone of the lower part is used to enrich cells by centrifugation, so that the cell enrichment degree is high and a solid cell mass is formed at the tip of the cone.

[0055] The liquid outlet of the closed centrifugal device of the present invention is arranged at the tip of the upper cone: in the liquid discharge state, in the process of increasing the inclination angle of the tube body, it is ensured that the cell fluid / buffer / other mixed liquid in the tube body is always gathered near the liquid outlet, so that the cell fluid / buffer / other mixed liquid can be completely discharged.

[0056] The enclosure provided in the lower cone of the tube body of the closed centrifuge of the present invention can be used to prevent cell clusters / lumps enriched near the tip of the cone, especially some sand-shaped, discrete cells, from sliding down along the tube wall to the liquid outlet in the liquid discharge state.

[0057] When the tube body of the closed centrifugal device of the present invention is made of soft material, liquid can be sucked in and discharged through the deformation of the tube body, the vent is omitted, the structure of the air filter element is eliminated, and the structure of the entire device is simplified.

[0058] The design of the tube body of the closed centrifuge device of the present invention can be used in conjunction with conventional, general-purpose commercial centrifuges on the market, without the need to customize / redesign a special centrifuge or a special centrifuge tube rack.

[0059] The closed centrifugal device of the present invention uses internal and external threads in a pipe joint: the liquid outlet end is an external thread, and the liquid inlet end is an internal thread, which always ensures that the liquid flows in the inner diameter direction, avoids liquid residue at the joint as much as possible, and reduces the risk of liquid contamination.

[0060] The protective cap and protective cover of the closed centrifugal device of the present invention play a multiple protection role and greatly reduce the risk of liquid contamination: during operation, human hands are prevented from touching each pipe joint as much as possible; after the pipe joint is removed, the protective cap must be installed; the height of the protective cover is greater than the height of the pipe body, and when the pipe body is placed in the protective cover, all liquids and / or air pipes on the pipe body can be completely placed in the protective cover and will not be exposed from the upper opening of the protective cover.

[0061] The core function of the closed centrifuge device of the present invention is to realize centrifugal operation in a closed environment, ensure the whole process of sterility, improve safety, and avoid the risk of contamination to the greatest extent. It meets the regulatory requirements that the sample processing of Class III medical devices must be fully closed. At the same time, as long as the application requires closed centrifugation, the closed centrifuge device of the present invention can be used. The device of the present invention can be used in the field of electrotransfection, and can also be used for cell processing in many other fields. For example, when the collection container is a cell culture bag, after completing the above-mentioned liquid discharge operation, cell culture can be directly carried out. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments. 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 work, among which:

[0063] Figure 1 , a schematic diagram of the overall structure of the closed centrifugal device with three openings according to the present invention;

[0064] Figure 2 , Figure 1 The enclosure in the top view of the AA' cross section;

[0065] Figure 3 , a schematic structural diagram of the closed centrifugal device with three openings in the liquid-intake state according to the present invention;

[0066] Figure 4 , a schematic structural diagram of the closed centrifugal device with three openings in the present invention in an initial state / centrifugal state;

[0067] Figure 5 , a schematic structural diagram of the closed centrifugal device with three openings in the liquid discharge state according to the present invention;

[0068] Figure 6 , another overall structural schematic diagram of the closed centrifugal device with three openings according to the present invention.

[0069] Figure 7 , a schematic diagram of the overall structure of the tube body (including external pipelines) of the closed centrifugal device with four openings according to the present invention;

[0070] Figure 8 , a schematic diagram of the closed centrifugal device with four openings according to the present invention in an initial state / centrifugal state;

[0071] Fig. 9 , a schematic structural diagram of the closed centrifugal device with four openings in the liquid discharge state according to the present invention.

[0072] Figure markings: 1-tube body, 2-liquid inlet, 3-liquid outlet, 4-vent, 5-valve a, 6-valve b, 7-valve c, 8-liquid inlet pipeline, 9-liquid outlet pipeline, 10-air filter, 11-Luer connector a, 12-Luer connector b, 13-Luer connector c, 14-enclosure, 15-sample container, 16-collection container, 17-protective cap a, 18-protective cap b, 19-protective cap c, 20-protective cover, 21-cell clumps / blocks, 22-sand-shaped, discrete cells, 23-needleless connector a, 24-needleless connector b, 25-needleless connector c, 26-needleless connector d, 27-pipette, 28-the fourth opening, 29-negative pressure collection device, 30-pipette pipeline, 31-protective cap d. DETAILED DESCRIPTION

[0073] The technical solutions in the embodiments of the invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the invention, not all of the embodiments. Based on the embodiments of the invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the invention.

[0074] Embodiment 1

[0075] The overall structure of the closed centrifugal device with three openings of the present invention is as follows: Figure 1 and Figure 4 and Figure 6 shown.

[0076] The device comprises a tube body (1), a liquid inlet (2), a liquid outlet (3), a vent (4), a valve a (5), a valve b (6), a valve c (7), a liquid inlet pipeline (8), a liquid outlet pipeline (9), an air filter element (10), a Luer connector a (11), a Luer connector b (12), a Luer connector c (13), a baffle (14), a sample container (15), a collection container (16), a protective cap a (17), a protective cap b (18), a protective cap c (19), and a protective cover (20);

[0077] The upper and lower parts of the tube body (1) are in a cone shape, and the middle part is in a cylindrical shape;

[0078] The liquid inlet (2), the liquid outlet (3) and the vent (4) are all arranged on the cone on the same side of the tube body (1), and the liquid outlet (3) is arranged at the tip of the upper part of the cone;

[0079] A baffle (14) is provided in the lower cone of the tube body (1). Figure 2As shown. In the liquid discharge state, the enclosure (14) is used to prevent the cell clusters / blocks (21) near the tip of the bottom of the tube body (1), especially some sand-shaped, discrete cells (22), from sliding down along the tube wall to the liquid outlet (3);

[0080] The liquid inlet (2), the liquid outlet (3) and the vent (4) are respectively provided with valves a (5), valves b (6) and valves c (7), which can independently control the opening and closing of the liquid inlet (2), the liquid outlet (3) and the vent (4); the valves a (5), valves b (6) and valves c (7) can be stop valves, or hose clamps, etc.;

[0081] The liquid inlet (2) is sealedly connected to the liquid inlet pipeline (8) via a Luer connector a (11), and the other end of the liquid inlet pipeline (8) is sealedly connected to a sample container (15);

[0082] The liquid outlet (3) is sealedly connected to the liquid outlet pipeline (9) via a Luer connector b (12), and the other end of the liquid outlet pipeline (9) is sealedly connected to a collection container (16);

[0083] The vent (4) is sealedly connected to the air filter (10) via a Luer connector c (13), and the air filter (10) is used to maintain the internal pressure balance of the tube body (1);

[0084] The protective cap a (17), the protective cap b (18) and the protective cap c (19) can be respectively sealed and connected to the liquid inlet (2), the liquid outlet (3) and the vent (4) by means of threads or the like;

[0085] The protective cover (20) can be fixedly connected to the pipe body (1) by means of threads or the like.

[0086] The liquid inlet process of the closed centrifugal device with three openings of the present invention is as follows: Figure 3 shown.

[0087] The tube body (1) is kept upright, the protective cap a (17) on the liquid inlet (2) is removed, the liquid inlet (2) is sealedly connected to the liquid inlet pipeline (8) through the Luer connector a (11), and the other end of the liquid inlet pipeline (8) is sealedly connected to the sample container (15); the protective cap c (19) on the vent (4) is removed, and the vent (4) is sealedly connected to the air filter element (10) through the Luer connector c (13); valves a (5) and c (7) are opened, and valve b (6) is kept in a closed state; cell fluid / buffer solution / other mixed solution etc. enters the tube body (1) through the liquid inlet (2) under the action of gravity or external force;

[0088] After the liquid is inletted, valve a (5) and valve c (7) are closed; the liquid inlet pipeline (8) is removed from the Luer connector a (11) of the liquid inlet (2); the liquid inlet (2), the liquid outlet (3) and the vent (4) are respectively covered with protective caps a (17), protective caps b (18) and protective caps c (19).

[0089] The centrifugal working process of the closed centrifugal device with three openings of the present invention is as follows: Figure 4 shown.

[0090] The protective cover (20) is fixedly connected to the tube body (1) by means of threads or the like; the entire centrifugal device is placed on a centrifugal tube rack of a centrifuge for centrifugation; suitable centrifugal parameters are selected so that obvious cell clusters / blocks (21) are formed near the tip of the cone; after the centrifugal operation is completed, the entire closed centrifugal device is removed from the centrifuge.

[0091] The working process of the liquid discharge of the closed centrifugal device with three openings of the present invention is as follows: Figure 4 and Figure 5 shown.

[0092] The protective cover (20) is removed from the tube body (1); the protective cap b (18) on the liquid outlet (3) is removed, and the liquid outlet (3) is sealedly connected to the liquid outlet pipeline (9) via the Luer connector b (12), and the other end of the liquid outlet pipeline (9) is sealedly connected to the collection container (16); the protective cap c (19) on the vent (4) is removed, and the vent (4) is sealedly connected to the air filter element (10) via the Luer connector c (13).

[0093] One end of the liquid outlet (3) is tilted downward, and the tube body (1) is tilted at a certain angle along the tube wall on the side where the baffle (14) is provided; the valve a (5) is kept in a closed state, and the valve b (6) is opened first, and then the valve c (7) is opened; the cell fluid / buffer solution / mixed liquid of the object to be transferred, etc., enters the collection container (16) through the liquid outlet (3) under the action of gravity or external force; at the same time, the tilt angle of the tube body (1) is gradually and slowly increased, and in this process, it is ensured that the cell fluid / buffer solution / other mixed liquid, etc. in the tube body (1) is always collected near the liquid outlet (3) until the cell fluid / buffer solution / other mixed liquid, etc. is completely discharged;

[0094] The baffle (14) is used to prevent the cell clusters / lumps (21) near the tip of the cone, especially some sand-shaped, discrete cells (22), from sliding down along the tube wall to the liquid outlet (3).

[0095] After the liquid is discharged, valve b (6) and valve c (7) are closed; the liquid discharge pipeline (9) is removed from the liquid outlet (3) through the Luer connector b (12); the liquid inlet (2), the liquid outlet (3) and the vent (4) are respectively installed with protective caps a (17), b (18) and c (19); and the protective cover (20) is fixedly connected to the pipe body (1) by means of threads or the like.

[0096] If multiple "liquid inlet → centrifugation → liquid outlet" are required, the above corresponding working process can be repeated cyclically.

[0097] Embodiment 2

[0098] Centrifugal operation using the enclosed centrifugal device of the present invention

[0099] Centrifugation enrichment was tested using Jurkat cells.

[0100] The cells were cultured in RPMI 1640 medium supplemented with 10% (v / v) fetal bovine serum at a density of 0.2-3×10 6 / mL. Before the test, cell counting and viability detection were performed. Viability detection was performed using APC-AnnexinⅤ / 7AAD staining and flow cytometry analysis. The total cell density was 2.32×10 6 / mL, the proportion of live cells of AnnexinⅤ- / 7AAD- was 96.5%.

[0101] Using the present invention Figure 1 150 mL of cell culture was collected in the centrifuge tube (hereinafter referred to as centrifuge tube 1) and the common open-cap centrifuge tube (hereinafter referred to as centrifuge tube 2) shown in the figure, and centrifuged at 200 g, 250 g, and 300 g for 3 min and 5 min, respectively. After the centrifugation, the centrifuge tube was inverted to discard the supernatant, and each group of cells was resuspended in 150 mL of culture medium, and each group of cells was taken for counting and viability analysis, and the results are shown in Table 1.

[0102] Table 1

[0103]

[0104] The results showed that under the same centrifugation conditions, there was no significant difference in cell density after resuspension in the two centrifuge tubes, indicating that there was no significant difference in the centrifugation enrichment effect. The device of the present invention did not cause a decrease in cell viability due to the longer operation time than the traditional centrifugation method, and there was no significant difference in cell viability between the groups.

[0105] Embodiment 3

[0106] The overall structure of the tube body of the closed centrifugal device with four openings of the present invention is as follows: Figure 7 and Figure 8 shown.

[0107] The device comprises a tube body (1), a liquid inlet (2), a liquid outlet (3), a vent (4), a fourth opening (28), a liquid inlet pipeline (8), a liquid outlet pipeline (9), an air filter (10), a sample container (15), a pipette (27), a pipette pipeline (30), a collection container (16), a negative pressure collection device (29), a needleless connector a (23), a needleless connector b (24), a needleless connector c (25), a needleless connector d (26), a protective cap a (17), a protective cap b (18), a protective cap c (19), a protective cap d (31), and a protective cover (20).

[0108] The liquid inlet (2), the liquid outlet (3), the vent (4), and the fourth opening (28) are all arranged on the same side cone at the upper part of the tube body (1), and the liquid outlet (3) is arranged at the tip of the cone;

[0109] The liquid inlet (2), the liquid outlet (3), and the vent (4) can be sealedly connected to the liquid inlet pipeline (8), the liquid outlet pipeline (9), and the air filter (10) through a needleless connector a (23), a needleless connector b (24), and a needleless connector c (25), respectively;

[0110] The air filter element (10) is used to filter the air entering the tube body (1) from the outside to maintain the pressure balance inside the tube body (1);

[0111] One end of the fourth opening (28) inside the tube body (1) is sealedly connected to the liquid pipette (27), and one end of the fourth opening (28) outside the tube body (1) can be sealedly connected to a negative pressure collection device (29) via a needleless connector d (26);

[0112] The negative pressure collection device (29) can be selected from a syringe, a pipette, a container with a negative pressure pump, etc.;

[0113] The end of the liquid pipette (27) is located at the junction of the lower edge of the middle cylinder of the tube body (1) and the upper edge of the cone at the lower part of the tube body (1);

[0114] The tube body (1) is embedded in the protective cover (20), and the tube walls of the two are in close contact;

[0115] The protective cover (20) is fixedly connected to the pipe body (1) by means of threads or the like;

[0116] The tube body (1) and the protective cover (20) can be prepared by mold injection molding, so that the tube body (1) can be embedded in the protective cover (20) during the installation process;

[0117] The entire workflow of the closed centrifugal device with four openings of the present invention is as follows: Figure 7-9 shown.

[0118] The cell fluid / buffer solution / other mixed solution in the sample container (15) is introduced into the tube body (1) through the liquid inlet pipeline (8), and the liquid inlet pipeline (8) is removed; the tube body (1) is embedded in the protective cover (20); the entire centrifugal device is placed in a centrifuge and centrifuged until obvious cell enrichment appears near the tip of the cone of the tube body (1); after the centrifugal operation is completed, the entire centrifugal device is removed from the centrifuge.

[0119] Next, one end of the fourth opening (28) outside the tube body (1) is sealed and connected to the negative pressure collection device (29) through the liquid suction pipeline (30); the tube body (1) is tilted so that the end of the liquid suction tube (27) contacts the cell fluid / buffer solution / other mixed liquid, etc.; the suction force generated by the negative pressure collection device (29) is used to quickly suck the cell fluid / buffer solution / other mixed liquid, etc. until the liquid is sucked clean, thereby achieving the purpose of separating cells in a closed state.

[0120] In the above-mentioned working process, similar to the operation in the first embodiment, after the liquid inlet pipeline (8), the liquid outlet pipeline (9), the air filter element (10) or the liquid suction pipeline (30) are removed from the entire centrifugal device, it is necessary to immediately install the protective cap a (17), the protective cap b (18), the protective cap c (19), and the protective cap d (31) on the liquid inlet (2), the liquid outlet (3), the vent (4), and the fourth opening (28).

[0121] If multiple "liquid inlet → centrifugation → liquid outlet" are required, the above corresponding working process can be repeated cyclically.

[0122] The above description is only a preferred embodiment of the present invention, but does not limit the present invention, and it cannot be determined that the embodiments of the present invention are limited to these embodiments. It should be pointed out that for ordinary technicians in this technical field, without departing from the technical principles and concepts of the present invention, several improvements or modifications can be made, which should be regarded as the protection scope of the present invention.

Claims

1. A closed centrifugal device, comprising a tube body, characterized in that: At least three openings are arranged on the tube body, one is a liquid inlet, one is a liquid outlet, and one is a vent. At least one enclosure is arranged inside the tube body. The upper and lower parts of the tube body are in a cone shape. The liquid outlet is located at the tip of the upper cone of the tube body, and the enclosure is arranged on the side wall of the lower cone of the tube body.

2. The closed centrifugal device according to claim 1, characterized in that: The tube body is provided with a fourth opening.

3. The closed centrifugal device according to claim 2, characterized in that: A liquid pipette is arranged at the fourth opening.

4. The closed centrifugal device according to claim 3, characterized in that: A liquid suction tube is arranged inside the tube body, and the liquid suction tube is sealed and connected to the fourth opening.

5. The closed centrifugal device according to claim 4, characterized in that: The fourth opening is sealedly connected to the collecting device through a liquid suction pipeline, and the collecting device is a negative pressure collecting device.

6. The closed centrifugal device according to claim 5, characterized in that: The vent is omitted on the tube body.

7. The closed centrifugal device according to any one of claims 1 to 5, characterized in that: The liquid inlet, liquid outlet and vent are sealed and connected to the external pipeline.

8. The closed centrifugal device according to claim 7, characterized in that: The sealed connection is achieved by a quick-connect joint.

9. The closed centrifugal device according to claim 7, characterized in that: The sealed connection is achieved by connecting via a needleless connector.

10. The closed centrifugal device according to any one of claims 1 to 6, characterized in that: The tube body is provided with a protective cover.

11. The closed centrifugal device according to claim 10, characterized in that: The tube body is fixedly connected to the protective cover.

12. A method for separating cells using a closed centrifugal device, characterized in that: The method comprises using the closed centrifugal device according to any one of claims 1 to 11.

13. The method according to claim 12, characterized in that The centrifugal force is 200-500g.

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