Cell disruption method and application thereof

Abstract

The invention relates to a cell disruption method and application thereof, the cell disruption method comprises the following steps: a cell suspension passes back through a filter membrane under the action of external force to obtain disrupted cells, and the aperture of the filter membrane is smaller than the cell diameter and larger than the cell nucleus diameter. According to the invention, the cells are broken by a squeezing and filtering method, when the pressure is relatively large or cell walls and cell membranes are relatively fragile, part of the cells are squeezed and broken when passing through a filter membrane, and the aim of breaking most of the cells can be achieved by repeating for many times. The method is low in noise, cell components can be removed or collected through sequential combination of differential centrifugation, and the method can be operated aseptically, is free of chemical additives, does not pollute cells, and has wide application prospects.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, in particular to a method for disrupting cells and application thereof. Background Art

[0002] Cells are the basic structural and functional units of organisms. It is known that all organisms except viruses are composed of cells, but the life activities of viruses must also be manifested in cells. Generally speaking, most microorganisms such as bacteria and protozoa are composed of one cell, that is, single-cell organisms, while higher plants and higher animals are multicellular organisms. Cells can be divided into two categories: prokaryotic cells and eukaryotic cells. Cells are mainly composed of nuclei and cytoplasm, with cell membranes on the surface. Higher plants have cell walls outside the cell membrane, and organelles such as chloroplasts, vacuoles and mitochondria in the cytoplasm.

[0003] Cell disruption technology refers to the use of external force to destroy the cell membrane and cell wall to release the cell contents including the target product components. When cell disruption is used to separate and purify a certain protein, the protein must first be released from the tissue or cell and kept in its original natural state without losing activity. Appropriate methods need to be used to disrupt the tissue and cells.

[0004] Cell disruption methods can be divided into two categories: mechanical and non-mechanical methods. Mechanical methods include high-pressure homogenization, oscillating bead beating, high-speed stirring bead grinding, ultrasonic, impact, etc. Non-mechanical methods include osmotic pressure impact, freeze-thaw, enzyme, chemical, etc. Existing physical disruption methods such as ultrasound, high-speed impact, and grinding have high requirements for equipment, high noise, and sometimes difficult aseptic operation; chemical disruption methods, chemical reagents will contaminate the disruption suspension, and the subsequent separation process is complicated.

[0005] CN104531531A discloses a bacterial cell crushing method, the steps of which are as follows: a) freezing: collecting bacterial fermentation liquid and pumping it into a water separation plate of a flake ice machine, and the bacterial fermentation liquid on the water separation plate is sprinkled on the ice surface in the flake ice machine to form an ice layer; b) melting and crushing: the bacterial liquid ice flakes are scraped off the ice surface by an ice knife and fall into an ice bin, first frozen at -5 to -90°C for 1 to 10 hours, and then melted at 10 to 60°C to obtain crushed bacterial cell liquid.

[0006] CN107557352A discloses a method and application of ultrasonic solid phase cell disruption, wherein the cell sample is pre-treated, the ultrasonic power and ultrasonic treatment time are accurately set, and the pre-treated cell sample is ultrasonically disrupted. According to the characteristics of the cells, accurate ultrasonic conditions are set, especially the ultrasonic power and ultrasonic treatment time are accurately determined, which overcomes the defects of high cost, sharp temperature rise and easy inactivation of products in the cell disruption process.

[0007] In summary, how to disrupt cells quickly and efficiently and avoid cell contamination has become one of the urgent problems to be solved in this field. Summary of the invention

[0008] In order to solve the above technical problems, the present invention provides a method for crushing cells and its application. The cells are crushed by squeezing and filtration. The pore size of the filter membrane is smaller than the cell diameter and larger than the cell nucleus diameter. When the pressure is high, or the cell wall and cell membrane are relatively fragile, some cells will be crushed when passing through the filter membrane, forming cell fragments passing through the grid. This situation is repeated several times to achieve the purpose of crushing most of the cells.

[0009] To achieve this object, the present invention adopts the following technical solutions:

[0010] In a first aspect, the present invention provides a method for disrupting cells, comprising: passing a cell suspension back and forth through a filter membrane under the action of an external force to obtain the disrupted cells, wherein the pore size of the filter membrane is smaller than the cell diameter and larger than the cell nucleus diameter.

[0011] The present invention breaks cells by squeezing and filtering, does not use ultrasonic breaking, has low noise, has no chemical additives, does not need subsequent separation of chemical substances, does not pollute cells, and has broad application prospects.

[0012] Preferably, the number of times of passing through the filter membrane is 5-100 times (e.g., 5 times, 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times or 100 times, etc.).

[0013] Preferably, the pore size of the filter membrane is 0.2-50 μm (for example, 0.2 μm, 0.5 μm, 1 μm, 2 μm, 5 μm, 10 μm, 20 μm, 30 μm, 40 μm or 50 μm, etc.).

[0014] Preferably, the material of the filter membrane includes any one of a metal filter mesh, filter paper or filter cloth.

[0015] Preferably, the cells include animal cells and / or plant cells.

[0016] Preferably, the animal cell comprises any one of T cells, B cells, CHO cells, HeLa cells or K562 cells, or a combination of at least two of them.

[0017] Preferably, the concentration of the cell suspension is 2×10 6 -5×10 7 / mL (e.g. 2×10 6 / mL, 3×10 6 / mL, 5×10 6 / mL, 1×10 7 / mL, 2×10 7 / mL, 3×10 7 / mL, 4×10 7 / mL, 5×10 7 / mL, etc.).

[0018] Preferably, the filter membrane is disposed on a cell disruption device.

[0019] Preferably, the cell disruption device comprises a first cell fluid container provided with a feed inlet, a second cell fluid container provided with a feed outlet, a first pipeline, a second pipeline and a filter membrane clamp equipped with a filter membrane.

[0020] The cell disruption device provided by the present invention has a structure as follows: Figure 1 As shown, it can be sterilized and reused, or it can be designed as a disposable consumable, which is packaged as a whole and stored aseptically, and the aseptic characteristics of the cell disruption device can prevent cells from being contaminated. In addition, the various parts of the cell disruption device provided by the present invention can be connected by detachable pipelines, which is convenient for cleaning, sterilization and reuse; it can also be integrally non-detachable to reduce the possibility of leakage and facilitate aseptic operation.

[0021] It is understandable that for the cell disruption device provided by the present invention, if a detachable connection is adopted, a leakage-proof design can also be added, such as installing a silicone ring pad in the middle of the filter membrane clamp, and the filter membrane clamp is clamped and reinforced in a special way, such as anti-slip spirals, anti-slip screws, etc., and the pipe connection is clamped with a clamp, or an anti-slip tower connector is used, etc., all of which are within the protection scope of the present invention.

[0022] Preferably, the first pipe and the second pipe are respectively connected to the filter membrane clamp, and the first pipe and the second pipe are respectively perpendicular to the filter membrane.

[0023] Preferably, the first cell fluid container is connected to the first pipeline, and the second cell fluid container is connected to the second pipeline.

[0024] Preferably, the feed port and the discharge port are respectively connected to a device with a pressurizing effect.

[0025] The liquid flow power source of the cell disruption device provided by the present invention can be manual squeezing through a syringe-type structure, squeezing a pipeline through a peristaltic pump, or using an electric mechanical squeezing of the syringe-type structure.

[0026] Preferably, the method for disrupting cells specifically comprises the following steps:

[0027] (a) adding a cell suspension into a first cell liquid container;

[0028] (b) pressurizing the feed port to allow the cell suspension to flow from the first cell liquid container through the first pipe, the filter membrane, and the second pipe into the second cell liquid container;

[0029] (c) pressurizing the discharge port to allow the cell suspension to flow from the second cell liquid container through the second pipe, the filter membrane and the first pipe into the first cell liquid container;

[0030] (d) Repeat steps (b) and (c) to collect the disrupted cell suspension.

[0031] Preferably, the pressurizing device comprises a peristaltic pump.

[0032] Preferably, the feed port and the discharge port are connected to a forward and reverse peristaltic pump.

[0033] Preferably, the flow rate of the peristaltic pump is 20-30 mL / min (for example, 20 mL / min, 21 mL / min, 22 mL / min, 23 mL / min, 24 mL / min, 25 mL / min, 26 mL / min, 27 mL / min, 28 mL / min, 29 mL / min or 30 mL / min, etc.).

[0034] Preferably, the first cell fluid container, the second cell fluid container, the first pipe and the second pipe are all straight cylindrical, the bottom surface of the first cell fluid container is connected to the bottom surface of the first pipe, and the bottom surface of the second cell fluid container is connected to the bottom surface of the second pipe.

[0035] Preferably, the bottom diameters of the first pipe and the second pipe are independently 2-20 mm (e.g., 2 mm, 4 mm, 6 mm, 8 mm, 10 mm, 12 mm, 14 mm, 16 mm, 18 mm or 20 mm, etc.).

[0036] Preferably, the bottom diameters of the first cell fluid container and the second cell fluid container are independently 5-30 mm (e.g., 5 mm, 7 mm, 10 mm, 13 mm, 15 mm, 17 mm, 20 mm, 22 mm, 25 mm, 27 mm or 30 mm, etc.).

[0037] In a second aspect, the present invention provides the use of the method for disrupting cells as described in the first aspect in separating cell nuclei and / or cell organelles.

[0038] Preferably, the cell organelles include any one of mitochondria, lysosomes, peroxisomes, liposomes, endoplasmic reticulum, Golgi bodies or ribosomes, or a combination of at least two of them.

[0039] In a third aspect, the present invention provides a method for isolating cell nuclei, comprising: disrupting the cell suspension using the method for disrupting cells described in the first aspect, centrifuging at 800-1200g (e.g., 800g, 850g, 900g, 950g, 1000g, 1050g, 1100g, 1150g or 1200g, etc.) for 8-12min (e.g., 8min, 8.5min, 9min, 9.5min, 10min, 10.5min, 11min, 11.5min or 12min, etc.), collecting the precipitate, and obtaining the cell nucleus.

[0040] In a fourth aspect, the present invention provides a method for separating organelles, characterized in that the method for separating organelles comprises the following steps:

[0041] (a) isolating cell nuclei from a cell suspension using the method for isolating cell nuclei described in the third aspect;

[0042] (b) taking the supernatant obtained in step (a), performing a first centrifugation, collecting the precipitate, and obtaining mitochondria, lysosomes, and peroxisomes;

[0043] (c) taking the supernatant obtained in step (b), performing a second centrifugation, collecting the precipitate, and obtaining liposomes, endoplasmic reticulum and Golgi bodies;

[0044] (d) taking the supernatant obtained in step (c), performing a third centrifugation, collecting the precipitate, and obtaining ribosomes.

[0045] The present invention removes or collects certain components through the sequential combination of differential centrifugation, thereby achieving the purpose of collecting cell components.

[0046] Preferably, the first centrifugation speed is 18000-22000g (for example, 18000g, 18500g, 19000g, 19500g, 20000g, 20500g, 21000g, 21500g or 22000g, etc.), and the time is 15-25min (for example, 15min, 16min, 17min, 18min, 19min, 20min, 21min, 22min, 23min, 24min or 25min, etc.).

[0047] Preferably, the second centrifugation speed is 75000-85000g (e.g., 75000g, 76000g, 77000g, 78000g, 79000g, 80000g, 81000g, 82000g, 83000g, 84000g or 85000g, etc.), and the time is 45-75min (e.g., 45min, 50min, 55min, 60min, 65min, 70min or 75min, etc.).

[0048] Preferably, the third centrifugation speed is 140000-160000g (for example, 140000g, 142000g, 144000g, 146000g, 148000g, 150000g, 152000g, 154000g, 156000g, 158000g or 160000g), and the time is 2.5-3.5h (for example, 2.5h, 2.6h, 2.7h, 2.8h, 2.9h, 3h, 3.1h, 3.2h, 3.3h, 3.4h or 3.5h, etc.).

[0049] Other specific point values ​​within the above numerical ranges can be selected and will not be described in detail here.

[0050] Compared with the prior art, the present invention has the following beneficial effects:

[0051] The present invention breaks cells by squeezing and filtering. When cells pass through a mesh with a pore size smaller than the cell diameter, a filtering effect is generated, the mobile phase flows through the mesh, and the cells are left on one side of the mesh; but when the pressure is high, or the cell wall and cell membrane are relatively fragile, some cells will be crushed, forming cell fragments that pass through the mesh. This situation is repeated several times to achieve the purpose of breaking most cells. The method has low noise, no chemical additives, and can be operated aseptically without contaminating cells, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 The structure diagram of the cell disruption device, wherein: 1 is a feed port, 2 is a first cell fluid container, 3 is a discharge port, 4 is a second cell fluid container, 5 is a first pipeline, 6 is a second pipeline, 7 is a filter membrane, and 8 is a filter membrane clamp;

[0053] Figure 2 This is a graph showing the flow cytometry results before cell disruption in Example 5;

[0054] Figure 3 This is a graph showing the results of flow cytometry detection after cell disruption in Example 5. DETAILED DESCRIPTION

[0055] To further illustrate the technical means and effects of the present invention, the present invention is further described below in conjunction with the embodiments and drawings. It should be understood that the specific implementation methods described herein are only used to explain the present invention, rather than to limit the present invention.

[0056] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0057] In the description of the present invention, unless otherwise clearly specified and limited, the terms "connected", "connected", and "fixed" should be understood in a broad sense, for example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be a direct connection or an indirect connection through an intermediate medium, it can be the internal connection of two elements or the interaction relationship between two elements. For ordinary technicians in this field, the specific meanings of the above terms in the present invention can be understood according to specific circumstances.

[0058] In the description of this embodiment, the terms "upper", "lower", "left", "right" and other directions or positional relationships are based on the directions or positional relationships shown in the drawings, and are only for the convenience of description and simplification of operation, rather than indicating or implying that the device or element referred to must have a specific direction, be constructed and operate in a specific direction, and therefore cannot be understood as limiting the present invention. In addition, the terms "first" and "second" are only used to distinguish in the description and have no special meaning.

[0059] Reagents used in the following examples:

[0060] K562 cells: purchased from ATCC cell bank, product number is CCL-243.

[0061] Preparation Example 1

[0062] In this preparation example, a cell disruption device is prepared, which includes a first cell fluid container 2 provided with a feed port 1, a second cell fluid container 4 provided with a feed port 3, a first pipe 5, a second pipe 6 and a filter membrane clamp 8 equipped with a filter membrane 7, wherein the pore size of the filter membrane 7 is 1.3 μm; the first pipe 5 and the second pipe 6 are respectively connected to the filter membrane clamp 8, and the first pipe 5 and the second pipe 6 are respectively perpendicular to the filter membrane 7; the first cell fluid container 2, the second cell fluid container 4, the first pipe 5 and the second pipe 6 are all straight cylindrical, the bottom diameters of the first cell fluid container 2 and the second cell fluid container 4 are both 15 mm, the bottom diameters of the first pipe 5 and the second pipe 6 are both 6 mm, the bottom surface of the first cell fluid container 2 is connected to the bottom surface of the first pipe 5, and the bottom surface of the second cell fluid container 4 is connected to the bottom surface of the second pipe 6.

[0063] Preparation Example 2

[0064] This preparation example prepares a cell disruption device, which is different from Preparation Example 1 only in that the pore size of the filter membrane 7 is 1 μm, the bottom diameters of the first cell fluid container 2 and the second cell fluid container 4 are both 20 mm, and the bottom diameters of the first pipe 5 and the second pipe 6 are both 4 mm.

[0065] Preparation Example 3

[0066] This preparation example prepares a cell disruption device, which is different from Preparation Example 1 only in that the pore size of the filter membrane 7 is 1.5 μm, the bottom diameters of the first cell fluid container 2 and the second cell fluid container 4 are both 12 mm, and the bottom diameters of the first pipe 5 and the second pipe 6 are both 8 mm.

[0067] Preparation Example 4

[0068] This preparation example prepares a cell disruption device, which is different from the preparation example 1 only in that the bottom diameters of the first cell liquid container 2 and the second cell liquid container 4 are both 5 mm.

[0069] Preparation Example 5

[0070] This preparation example prepares a cell disruption device, which is different from the preparation example 1 only in that the bottom diameters of the first cell liquid container 2 and the second cell liquid container 4 are both 30 mm.

[0071] Preparation Example 6

[0072] This preparation example prepares a cell disruption device, which is different from the preparation example 1 only in that the bottom diameters of the first pipe 5 and the second pipe 6 are both 2 mm.

[0073] Preparation Example 7

[0074] This preparation example prepares a cell disruption device, which is different from the preparation example 1 only in that the bottom diameters of the first pipe 5 and the second pipe 6 are both 20 mm.

[0075] Preparation Example 8

[0076] This preparation example prepares a cell disruption device, which is different from the preparation example 1 only in that the pore size of the filter membrane 7 is 10 μm.

[0077] Preparation Example 9

[0078] This preparation example prepares a cell disruption device, which is different from Preparation Example 1 only in that the pore size of the filter membrane 7 is 0.15 μm.

[0079] Example 1

[0080] This embodiment provides a method for disrupting cells and separating cell nuclei and organelles, comprising the following steps:

[0081] Adjust the K562 cell concentration to 2 × 10 cells / mL using sterile PBS solution. 7 / mL, and repeatedly squeezed and filtered 80 times with 1.3μm filter cloth. Centrifuge the cell suspension at 1000g for 10min, collect the precipitate to obtain the cell nucleus; take the supernatant and centrifuge it at 20000g for 20min, collect the precipitate to obtain mitochondria, lysosomes, and peroxisomes; take the supernatant and centrifuge it at 80000g for 60min, collect the precipitate to obtain liposomes, endoplasmic reticulum, and Golgi bodies; take the supernatant and centrifuge it at 150000g for 3h, collect the precipitate to obtain ribosomes.

[0082] Example 2

[0083] This embodiment provides a method for disrupting cells and separating cell nuclei and organelles, comprising the following steps:

[0084] Adjust the K562 cell concentration to 2 × 10 cells / mL using sterile PBS solution. 6 / mL, and repeatedly squeezed and filtered 50 times with 1μm filter cloth. Centrifuge the cell suspension at 800g for 12min, collect the precipitate to obtain the cell nucleus; take the supernatant and centrifuge it at 22000g for 15min, collect the precipitate to obtain mitochondria, lysosomes, and peroxisomes; take the supernatant and centrifuge it at 75000g for 75min, collect the precipitate to obtain liposomes, endoplasmic reticulum, and Golgi bodies; take the supernatant and centrifuge it at 160000g for 2.5h, collect the precipitate to obtain ribosomes.

[0085] Example 3

[0086] This embodiment provides a method for disrupting cells and separating cell nuclei and organelles, comprising the following steps:

[0087] Adjust the K562 cell concentration to 5 × 10 cells / mL using sterile PBS solution. 7 / mL, and repeatedly squeezed and filtered 100 times with 1.5μm filter cloth. Centrifuge the cell suspension at 1200g for 8min, collect the precipitate to obtain the cell nucleus; take the supernatant and centrifuge it at 18000g for 25min, collect the precipitate to obtain mitochondria, lysosomes, and peroxisomes; take the supernatant and centrifuge it at 85000g for 45min, collect the precipitate to obtain liposomes, endoplasmic reticulum, and Golgi bodies; take the supernatant and centrifuge it at 140000g for 3.5h, collect the precipitate to obtain ribosomes.

[0088] Example 4

[0089] This embodiment provides a method for disrupting cells and separating cell nuclei and organelles, comprising the following steps:

[0090] 8.1×10 6K562 cells with a concentration of 100 / mL were repeatedly squeezed and filtered through a 4.5μm filter cloth for 20 times, and the obtained cell suspension was repeatedly squeezed and filtered through a 1.2μm filter cloth for 20 times. The cell suspension was centrifuged at 1000g for 10 minutes, and the precipitate was collected to obtain the cell nucleus; the supernatant was centrifuged at 20000g for 20 minutes, and the precipitate was collected to obtain mitochondria, lysosomes, and peroxisomes; the supernatant was centrifuged at 80000g for 60 minutes, and the precipitate was collected to obtain liposomes, endoplasmic reticulum, and Golgi bodies; the supernatant was centrifuged at 150000g for 3 hours, and the precipitate was collected to obtain ribosomes.

[0091] Example 5

[0092] This embodiment provides a method for disrupting cells and separating cell nuclei and organelles, using the cell disruption device provided in Preparation Example 1 for disruption. The method for disrupting cells includes the following steps:

[0093] (a) 1×10 7 Add a K562 cell suspension with a concentration of 100 cells / mL into the first cell liquid container 2, connect the feed port 1 and the discharge port 3 to a forward and reverse peristaltic pump, and the flow rate of the peristaltic pump is 25 mL / min;

[0094] (b) pressurizing the feed port 1 so that the cell suspension enters the second cell liquid container 4 from the first cell liquid container 2 through the first pipe 5, the filter membrane 7 and the second pipe 6;

[0095] (c) pressurizing the discharge port 3 so that the cell suspension enters the first cell liquid container 2 from the second cell liquid container 4 through the second pipe 6, the filter membrane 7 and the first pipe 5;

[0096] (d) repeating steps (b) and (c) for a total of 50 times of passing through the filter membrane, and collecting the disrupted cell suspension;

[0097] (e) The cell suspension was centrifuged at 1000 g for 10 min, and the precipitate was collected to obtain the cell nucleus; the supernatant was centrifuged at 20000 g for 20 min, and the precipitate was collected to obtain the mitochondria, lysosomes, and peroxisomes; the supernatant was centrifuged at 80000 g for 60 min, and the precipitate was collected to obtain the liposomes, endoplasmic reticulum, and Golgi bodies; the supernatant was centrifuged at 150000 g for 3 h, and the precipitate was collected to obtain the ribosomes.

[0098] Example 6

[0099] This example provides a method for disrupting cells and separating cell nuclei and organelles, using the cell disruption device provided in Preparation Example 2 for disruption. The method for disrupting cells includes the following steps:

[0100] (a) 2×10 6Add a K562 cell suspension with a concentration of 100 cells / mL into the first cell liquid container 2, connect the feed port 1 and the discharge port 3 to a forward and reverse peristaltic pump, and the flow rate of the peristaltic pump is 30 mL / min;

[0101] (b) pressurizing the feed port 1 so that the cell suspension enters the second cell liquid container 4 from the first cell liquid container 2 through the first pipe 5, the filter membrane 7 and the second pipe 6;

[0102] (c) pressurizing the discharge port 3 so that the cell suspension enters the first cell liquid container 2 from the second cell liquid container 4 through the second pipe 6, the filter membrane 7 and the first pipe 5;

[0103] (d) repeating steps (b) and (c) for a total of 75 times of passing through the filter membrane, and collecting the disrupted cell suspension;

[0104] (e) The cell suspension was centrifuged at 800 g for 12 min, and the precipitate was collected to obtain the cell nucleus; the supernatant was centrifuged at 22,000 g for 15 min, and the precipitate was collected to obtain the mitochondria, lysosomes, and peroxisomes; the supernatant was centrifuged at 75,000 g for 75 min, and the precipitate was collected to obtain the liposomes, endoplasmic reticulum, and Golgi bodies; the supernatant was centrifuged at 160,000 g for 2.5 h, and the precipitate was collected to obtain the ribosomes.

[0105] Example 7

[0106] This example provides a method for disrupting cells and separating cell nuclei and organelles, using the cell disruption device provided in Preparation Example 3 to disrupt cells. The method for disrupting cells includes the following steps:

[0107] (a) 5×10 7 Add a K562 cell suspension with a concentration of 100 cells / mL into the first cell liquid container 2, connect the feed port 1 and the discharge port 3 to a forward and reverse peristaltic pump, and the flow rate of the peristaltic pump is 20 mL / min;

[0108] (b) pressurizing the feed port 1 so that the cell suspension enters the second cell liquid container 4 from the first cell liquid container 2 through the first pipe 5, the filter membrane 7 and the second pipe 6;

[0109] (c) pressurizing the discharge port 3 so that the cell suspension enters the first cell liquid container 2 from the second cell liquid container 4 through the second pipe 6, the filter membrane 7 and the first pipe 5;

[0110] (d) repeating steps (b) and (c) for a total of 60 times of passing through the filter membrane, and collecting the disrupted cell suspension;

[0111] (e) The cell suspension was centrifuged at 1200 g for 8 min, and the precipitate was collected to obtain the cell nucleus; the supernatant was centrifuged at 18000 g for 25 min, and the precipitate was collected to obtain the mitochondria, lysosomes, and peroxisomes; the supernatant was centrifuged at 85000 g for 45 min, and the precipitate was collected to obtain the liposomes, endoplasmic reticulum, and Golgi bodies; the supernatant was centrifuged at 140000 g for 3.5 h, and the precipitate was collected to obtain the ribosomes.

[0112] Example 8

[0113] This example provides a method for disrupting cells and separating cell nuclei and organelles. The only difference from Example 5 is that the cell disruption device provided in Preparation Example 4 is used for disruption.

[0114] Example 9

[0115] This example provides a method for disrupting cells and separating cell nuclei and organelles. The only difference from Example 5 is that the cell disruption device provided in Preparation Example 5 is used for disruption.

[0116] Example 10

[0117] This example provides a method for disrupting cells and separating cell nuclei and organelles. The only difference from Example 5 is that the cell disruption device provided in Preparation Example 6 is used for disruption.

[0118] Embodiment 11

[0119] This example provides a method for disrupting cells and separating cell nuclei and organelles. The only difference from Example 5 is that the cell disruption device provided in Preparation Example 7 is used for disruption.

[0120] Example 12

[0121] This example provides a method for disrupting cells and separating cell nuclei and organelles. The only difference from Example 5 is that the cell disruption device provided in Preparation Example 8 is used for disruption.

[0122] Example 13

[0123] This example provides a method for disrupting cells and separating cell nuclei and organelles. The only difference from Example 5 is that the cell disruption device provided in Preparation Example 9 is used for disruption.

[0124] Embodiment 14

[0125] This embodiment provides a method for disrupting cells and separating cell nuclei and organelles, which is different from Embodiment 5 only in that the flow rate of the peristaltic pump is 18 mL / min.

[0126] Embodiment 15

[0127] This embodiment provides a method for disrupting cells and separating cell nuclei and organelles, which is different from Embodiment 5 only in that the flow rate of the peristaltic pump is 32 mL / min.

[0128] Example 16

[0129] This example provides a method for disrupting cells and separating cell nuclei and organelles. The only difference from Example 5 is that the cell concentration in step (a) is 1.5×10 6 Pieces / mL.

[0130] Embodiment 17

[0131] This example provides a method for disrupting cells and separating cell nuclei and organelles. The only difference from Example 5 is that the cell concentration in step (a) is 5.5×10 7 Pieces / mL.

[0132] Embodiment 18

[0133] This example provides a method for disrupting cells and separating cell nuclei and organelles, which is different from Example 5 only in that the filter membrane is passed a total of 30 times in step (d).

[0134] Embodiment 19

[0135] This embodiment provides a method for disrupting cells and separating cell nuclei and organelles, which is different from Embodiment 5 only in that the filter membrane is passed through a total of 100 times in step (d).

[0136] Test Example 1

[0137] This test uses the method provided in Example 1-19 to disrupt cells, and the statistical results of cell disruption rate are shown in Table 1. The method for measuring the disruption rate is as follows: the cell concentration is adjusted to 1×10 6 The cells / mL were directly detected by flow cytometry, with the horizontal axis adjusted to FSC-H and the vertical axis adjusted to SSC-H, and the proportions of cell clusters and debris clusters were compared. Figure 2 These are the cells before disruption in Example 5, and the range of cell fragments is circled according to the lower left corner. Figure 3 The cells after disruption in Example 5 were Figure 3 The circled cell fragments show that the fragmentation rate is 94.65%.

[0138] Table 1

[0139]

[0140]

[0141] From Table 1, we can conclude that:

[0142] (1) Comparing Examples 1-4 with Examples 5-7, it can be seen that the cell disruption efficiency can be improved by placing the filter membrane on the cell disruption device and coordinating the specific container size, peristaltic pump flow rate, cell suspension concentration, filter membrane pore size and filtration times;

[0143] (2) Comparing Example 5 with Examples 8-11, it can be seen that the cross-sectional sizes of the first pipe 5, the second pipe 6, the first cell fluid container 2, and the second cell fluid container 4 affect the cell disruption efficiency;

[0144] (3) Comparing Example 5 with Examples 12-13, it can be seen that a filter membrane pore size that is too large will lead to a decrease in the cell disruption rate, and a filter membrane pore size that is too small has no obvious effect on the cell disruption rate, but the peristaltic pump needs to increase the power to maintain a certain flow rate, consuming more energy;

[0145] (4) Comparing Example 5 with Examples 14-15, it can be seen that too large or too small a peristaltic pump flow rate will lead to a decrease in cell disruption rate, and under the same filtration times, a peristaltic pump flow rate that is too small requires more filtration time;

[0146] (5) Comparing Example 5 with Examples 16-17, it can be seen that a too low concentration of the cell suspension leads to a decrease in the cell disruption rate, while a too high concentration of the cell suspension has no significant effect on the cell disruption rate;

[0147] (6) Comparing Example 5 with Examples 18-19, it can be seen that too few filtration times lead to a decrease in the cell disruption rate, while too many filtration times have no obvious effect on the cell disruption rate, but require the peristaltic pump to work for more time and consume more energy.

[0148] Test Example 2

[0149] This test uses the method provided in Examples 1-19 to disrupt the cells. The lysosome concentration test results collected after cell disruption are shown in Table 2. The detection method uses the human lysosome-associated membrane protein 2 antibody (HLAMP-2Ab) ELISA kit purchased from Jining Biology. First, use the purchased human lysosomes (Thermo Fisher) for detection and make a standard curve. Then use the lysosomal product obtained by crushing and centrifugation for detection, and obtain the lysosome amount according to the standard curve, divide it by the corresponding number of cells, and calculate the concentration unit as ng / 10 7 Characterization data of cells, indicating that every 1×10 7 The amount of lysosomes that can be extracted from each cell.

[0150] Table 2

[0151]

[0152] Comparing Table 2 with Table 1, it can be concluded that the amount of lysosomes that can be extracted by crushing is highly correlated with the crushing rate, and the change pattern is basically the same. This shows that in the extraction of lysosomes, as long as it is within the centrifugal speed range, the centrifugal speed has little effect on the extraction, and it is mainly affected by the different crushing methods and the different crushing rates.

[0153] In summary, the present invention breaks cells by squeezing and filtering, the pore size of the filter membrane is smaller than the cell diameter and larger than the cell nucleus diameter, when the pressure is large, or the cell wall and cell membrane are relatively fragile, some cells will be crushed, forming cell fragments passing through the grid, and this situation is repeated several times to achieve the purpose of breaking most cells. The method has low noise, and the cell components can be removed or collected by the sequential combination of differential centrifugation. The method can be operated aseptically, has no chemical additives, does not pollute cells, and has broad application prospects.

[0154] The applicant declares that the above is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention shall fall within the protection scope and disclosure scope of the present invention.

Claims

1. A method for disrupting cells, characterized in that: The method for disrupting cells comprises: passing the cell suspension back and forth through a filter membrane under the action of an external force to obtain the disrupted cells, wherein the pore size of the filter membrane is smaller than the cell diameter and larger than the cell nucleus diameter.

2. The method for disrupting cells according to claim 1, characterized in that: The number of times of passing through the filter membrane is 5-100 times; Preferably, the pore size of the filter membrane is 0.2-50 μm; Preferably, the material of the filter membrane includes any one of a metal filter mesh, filter paper or filter cloth; Preferably, the cells include animal cells and / or plant cells; Preferably, the animal cell comprises any one or a combination of at least two of T cells, B cells, CHO cells, HeLa cells or K562 cells; Preferably, the concentration of the cell suspension is 2×10 6 -5×10 7 Pieces / mL.

3. The method for disrupting cells according to claim 1 or 2, characterized in that: The filter membrane is arranged on a cell disruption device; Preferably, the cell disruption device comprises a first cell fluid container (2) provided with a feed port (1), a second cell fluid container (4) provided with a discharge port (3), a first pipeline (5), a second pipeline (6) and a filter membrane holder (8) equipped with a filter membrane (7); Preferably, the first pipe (5) and the second pipe (6) are respectively connected to the filter membrane clamp (8), and the first pipe (5) and the second pipe (6) are respectively perpendicular to the filter membrane (7); Preferably, the first cell fluid container (2) is connected to the first pipeline (5), and the second cell fluid container (4) is connected to the second pipeline (6); Preferably, the feed port (1) and the discharge port (3) are respectively connected to a device having a pressurizing effect.

4. The method for disrupting cells according to any one of claims 1 to 3, characterized in that: The method for disrupting cells specifically comprises the following steps: (a) adding a cell suspension into a first cell liquid container (2); (b) pressurizing the feed port (1) so that the cell suspension flows from the first cell liquid container (2) through the first pipe (5), the filter membrane (7) and the second pipe (6) into the second cell liquid container (4); (c) pressurizing the discharge port (3) so that the cell suspension enters the first cell liquid container (2) from the second cell liquid container (4) through the second pipe (6), the filter membrane (7) and the first pipe (5); (d) Repeat steps (b) and (c) to collect the disrupted cell suspension.

5. The method for disrupting cells according to claim 3 or 4, characterized in that: The pressurizing device comprises a peristaltic pump; Preferably, the feed port (1) and the discharge port (3) are connected to a forward and reverse peristaltic pump; Preferably, the flow rate of the peristaltic pump is 20-30 mL / min; Preferably, the first cell fluid container (2), the second cell fluid container (4), the first pipe (5) and the second pipe (6) are all in the shape of straight cylinders, the bottom surface of the first cell fluid container (2) is connected to the bottom surface of the first pipe (5), and the bottom surface of the second cell fluid container (4) is connected to the bottom surface of the second pipe (6); Preferably, the bottom diameters of the first pipe (5) and the second pipe (6) are independently 2-20 mm; Preferably, the bottom diameters of the first cell fluid container (2) and the second cell fluid container (4) are independently 5-30 mm.

6. Use of the method for disrupting cells as claimed in any one of claims 1 to 5 in separating cell nuclei and / or cell organelles.

7. The use according to claim 6, characterized in that: The cell organelles include any one of mitochondria, lysosomes, peroxisomes, liposomes, endoplasmic reticulum, Golgi bodies or ribosomes, or a combination of at least two of them.

8. A method for isolating cell nuclei, characterized in that: The method for isolating cell nuclei comprises: disrupting a cell suspension using the method for disrupting cells according to any one of claims 1 to 5, centrifuging at 800-1200 g for 8-12 minutes, collecting the precipitate, and obtaining the cell nuclei.

9. A method for separating organelles, characterized in that: The method for separating organelles comprises the following steps: (a) isolating cell nuclei from a cell suspension using the method for isolating cell nuclei according to claim 8; (b) taking the supernatant obtained in step (a), performing a first centrifugation, collecting the precipitate, and obtaining mitochondria, lysosomes, and peroxisomes; (c) taking the supernatant obtained in step (b), performing a second centrifugation, collecting the precipitate, and obtaining liposomes, endoplasmic reticulum and Golgi bodies; (d) taking the supernatant obtained in step (c), performing a third centrifugation, collecting the precipitate, and obtaining ribosomes.

10. The method for separating organelles according to claim 9, characterized in that: The first centrifugation is performed at a speed of 18000-22000 g for 15-25 min; Preferably, the second centrifugation is performed at a speed of 75000-85000 g and for a time of 45-75 min; Preferably, the third centrifugation has a rotation speed of 140,000-160,000 g and a time of 2.5-3.5 h.

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