Method for extracting migration body from in-vitro culture cells and application

Abstract

The invention discloses a method for extracting a migration body from in-vitro cultured cells. The method comprises the following steps: planting the cells at a dish bottom which is pre-coated with involved protein for 12-18 hours to obtain in-vitro cells; the in-vitro cells in the vessel are subjected to treatment including breakage of cell tail contraction filaments so as to form a cell mixture in which the cells are separated from the migration body; and transferring the cell mixture from the vessel to a test tube, carrying out density gradient centrifugation treatment on the cell mixture, and extracting the migration body from the cell mixture. According to the method, the NP-40 and the Trypsin-EDTA are used for treating the in-vitro cells, and the cell bodies and the migration bodies can be completely broken, so that the efficiency of extracting the migration bodies is improved.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a method and application for extracting migratory bodies from in vitro cultured cells. Background Technology

[0002] Migrasomes are monolayered membrane vesicles formed at the junctions of contractile filaments at the tail and tip of cells during directional cell migration. Migrasome formation depends on cell migration, and due to differences in formation mechanisms, migrasomes differ from other extracellular vesicles (VEs) such as exosomes and microvesicles in size, molecular composition, and biological function. Migrasomes are involved in various physiological and pathological processes, functionally transporting between tumor cells and between tumor cells and the tumor microenvironment, thereby influencing the occurrence, development, and metastasis of cancer. Early research results show that tumor-derived migrasomes can be released into the circulatory system, and their contents can reflect tumor cell characteristics, possessing the potential to replace tumor cells themselves as biomarkers in liquid biopsies.

[0003] One existing method for enriching migratory organisms relies primarily on physical properties (size, size, and density). It utilizes gradually increasing centrifugal force and the density differences of gradient sucrose solutions to separate migratory organisms from other EV components sequentially via ultracentrifugation and density gradient centrifugation. However, these methods require large sample volumes and suffer from drawbacks such as cumbersome operation, long processing time, high cost, low recovery rate, and lack of standardization. Furthermore, blood contains relatively few migratory organisms and a large number of cells, proteins, and inorganic ions. These substances can co-precipitate with the migratory organisms during traditional centrifugation enrichment, affecting the purity of the enriched migratory organisms and the accuracy of subsequent detection results.

[0004] Existing methods for extracting migratory bodies from in vitro cultured cells primarily involve digesting cells with Trypsin-EDTA, followed by collecting vesicles of specific sizes via density gradient centrifugation. However, existing techniques have several drawbacks: direct treatment of cells with Trypsin-EDTA can mediate a reduction in migratory body size or even their disappearance (e.g., ...). Figure 1 a) Secondly, because Trpsin-EDTA cannot break the contractile filaments, the cells and migratory bodies remain attached, thus reducing the extraction efficiency of the migratory bodies (e.g., Figure 1 b). Summary of the Invention

[0005] To address the technical problem of low migration body extraction efficiency in existing technologies, this invention provides a method for extracting migration bodies from in vitro cultured cells that can improve the extraction efficiency, as well as an application of NP-40 in the extraction of migration bodies from in vitro cultured cells.

[0006] According to a first aspect of the present invention, a method for extracting migratory bodies from in vitro cultured cells includes:

[0007] Cells were seeded on the bottom of a dish pre-coated with transport protein for 12-18 hours to obtain in vitro cells;

[0008] The in vitro cells in the vessel were treated with a process that included breaking down the contractile filaments of the cell tails to form a cell mixture in which the cells and the migratory bodies were separated.

[0009] The cell mixture was transferred from the dish to a test tube, and the cell mixture was subjected to density gradient centrifugation to extract the migratory bodies from the cell mixture.

[0010] Preferably, the treatment of in vitro cells, including breaking off the contractile filaments of the cell tails, includes:

[0011] In vitro cells were treated with a low concentration of NP-40 for 1-2 minutes to sever the contractile filaments connecting the migratory bodies to the cell bodies. The low concentration of NP-40 was then discarded. In vitro cells were then treated with Trypsin-EDTA for 2-3 minutes to accelerate the breakage of the contractile filaments.

[0012] Preferably, the low concentration of NP-40 is 0.006-0.008% NP-40.

[0013] Preferably, the low concentration of NP-40 is 0.007% NP-40.

[0014] Preferably, the cell mixture is subjected to density gradient centrifugation to extract the migratory bodies from the cell mixture, comprising:

[0015] The migratory bodies were collected by adding culture medium to the cell mixture and subjected to a first strong centrifugation at 4 degrees Celsius. The migratory bodies after the first strong centrifugation were subjected to density gradient centrifugation, and the migratory body liquid was aspirated. The aspirated migratory body liquid was subjected to a second strong centrifugation to obtain pure migratory bodies.

[0016] Preferably, the density gradient centrifugation of the migrated body after the first centrifugation to aspirate the migrated body liquid includes: preparing sucrose density gradient centrifuge solutions of 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, and 50%; resuspending the collected migrated body in a 10% density gradient centrifuge solution and adding it to the density gradient centrifuge solution; centrifuging at 150,000g for 4 hours in an ultracentrifuge so that the liquid after centrifugation will show migrated body bands; and aspirating the liquid containing the migrated body layer using a pipette tip.

[0017] Preferably, the second strong centrifugation of the aspirated migratory liquid includes: resuspending the migratory liquid in PBS and then performing a second strong centrifugation on the resuspended migratory liquid.

[0018] Preferably, both the first and second strong centrifugations are performed by centrifuging at 20,000g for 30 minutes at 4 degrees Celsius.

[0019] According to a second aspect of the invention, there is an application of NP-40 in the extraction of migratory bodies from in vitro cultured cells, wherein the NP-40 is used to break the contractile filaments of the cell tail.

[0020] Preferably, the NP-40 is 0.006-0.008% NP-40.

[0021] This invention utilizes NP-40 and Trypsin-EDTA to treat in vitro cells, which can completely break the cell body and the migratory body, thereby improving the efficiency of migratory body extraction. Attached Figure Description

[0022] Figure 1 This image illustrates the deficiencies in existing techniques for extracting migratory bodies from in vitro cultured cells using Trypsin-EDTA.

[0023] Figure 2 This is a schematic diagram illustrating the method and effects of the present invention for extracting migrants. Detailed Implementation

[0024] This invention provides a novel method for extracting migratory organisms, which can significantly improve the efficiency of extracting migratory organisms from in vitro cultured cells. The main features of this invention are: treating cells with a low concentration of 0.007% NP-40 for 1-2 minutes, followed by treatment with Trypsin-EDTA for 2-3 minutes; finally, collecting the cells and performing density gradient centrifugation.

[0025] NP-40 is commonly used as a component of cell lysis buffers for cell lysis. The inventors' contribution lies in the discovery that NP-40 used for cell lysis can mediate the breakage of contractile filaments during cell processing. After the contractile filaments break, the connection between the migratory bodies and the cells is severed, thereby improving the extraction efficiency of the migratory bodies.

[0026] A method for extracting migratory bodies from in vitro cultured cells according to the present invention includes:

[0027] Cells were seeded on the bottom of a dish pre-coated with transport protein for 12-18 hours to obtain in vitro cells;

[0028] The in vitro cells in the vessel were treated with a process that included breaking down the contractile filaments of the cell tails to form a cell mixture in which the cells and the migratory bodies were separated.

[0029] The cell mixture was transferred from the dish to a test tube, and the cell mixture was subjected to density gradient centrifugation to extract the migratory bodies from the cell mixture.

[0030] Figure 2 b illustrates the specific method for extracting migratory bodies from in vitro cultured cells according to the present invention, comprising: culturing in vitro cells in a dish for 12-18 hours to obtain in vitro cells; treating the in vitro cells cultured in the dish with NP-40 to sever the contractile filaments connecting the migratory bodies to the cell bodies; treating the in vitro cells treated with NP-40 with Trypsin-EDTA to weaken cell adhesion and reduce cell spreading area. During the process of reducing cell spreading area, the cells exert tension through the contractile filaments, thereby accelerating the breakage of the contractile filaments; transferring the mixture of in vitro cells and migratory bodies obtained by NP-40 and Trypsin-EDTA treatment to a test tube for strong centrifugation and density centrifugation to obtain purified migratory bodies.

[0031] The present invention describes a process for treating in vitro cells, including breaking the contractile filaments at the cell tails, which involves: treating in vitro cells with a low concentration of NP-40 for 1-2 minutes to sever the contractile filaments connecting the migratory body and the cell body; discarding the low concentration of NP-40; and then treating in vitro cells with Trypsin-EDTA for 2-3 minutes to accelerate the breaking of the contractile filaments.

[0032] The inventors discovered that the selection of NP-40 concentration is a key factor in achieving the objectives of this invention. Too low a concentration will not cut the shrinkage filaments, while too high a concentration will cause the migrating body to break down. Through repeated experiments, the inventors finally found that an NP-40 concentration of 0.006-0.008% is suitable for cutting the shrinkage filaments without causing the migrating body to break down. The optimal NP-40 concentration is 0.007%.

[0033] The present invention describes a density gradient centrifugation process for extracting migratory bodies from a cell mixture. This process includes: adding culture medium to the cell mixture, collecting the migratory bodies, and performing a first strong centrifugation on the collected migratory bodies at 4 degrees Celsius; performing density gradient centrifugation on the migratory bodies after the first centrifugation and aspirating the migratory body liquid; and performing a second strong centrifugation on the aspirated migratory body liquid to obtain purified migratory bodies.

[0034] The present invention describes a density gradient centrifugation method for aspirating the migrating liquid after the first centrifugation. This method involves preparing sucrose density gradient centrifuge solutions of 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, and 50%. The collected migrating liquid is resuspended in a 10% density gradient centrifuge solution and added to the density gradient centrifuge solution. The mixture is then centrifuged at 150,000 g for 4 hours in an ultracentrifuge to produce migrating bands in the centrifuged liquid. Finally, the liquid in the layer containing the migrating liquid is aspirated using a pipette tip.

[0035] The present invention includes a second strong centrifugation of the aspirated migration fluid, comprising: resuspending the migration fluid in PBS, and performing a second strong centrifugation on the resuspended migration fluid to obtain the migration fluid of cells in vitro.

[0036] The first and second strong centrifugations described in this invention are both performed by centrifuging at 20,000g for 30 minutes at 4 degrees Celsius using a centrifuge.

[0037] More specifically, the present invention relates to the step of extracting migratory bodies from cells cultured in vitro;

[0038] 1) Seed the cells in a dish pre-coated with transport protein for 12-18 hours;

[0039] 2) Discard the culture medium, rinse once with PBS, and add NP-40 for 1 minute;

[0040] 3) Discard NP-40 and add Trypsin-EDTA for 2-3 minutes;

[0041] 4) Add culture medium to collect the migratory bodies and centrifuge at 20,000g for 30 minutes in a four-degree centrifuge.

[0042] 5) Prepare sucrose density gradient centrifuge solutions of 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, and 50%. Resuspend the migratings from step four in a 10% density gradient centrifuge solution and add them to the density gradient centrifuge solutions.

[0043] 6) Centrifuge at 150,000g for 4 hours in an ultracentrifuge;

[0044] 7) After density gradient centrifugation, the liquid will show migratory bands. Use a pipette tip to collect the liquid in the layer containing the migratory bands.

[0045] 8) Resuspend the migratory bodies in PBS and centrifuge at 20,000g for 30 minutes in a four-degree centrifuge.

[0046] Furthermore, the present invention also provides an application of NP-40 in the extraction of migratory bodies from in vitro cultured cells, wherein NP-40 is used to break the contractile filaments at the cell tail, thereby improving the efficiency of extracting cell migratory bodies.

[0047] The concentration range of NP-40 used in this invention for cutting shrinkage yarn is 0.006-0.008%; the preferred concentration is 0.007%.

[0048] This invention utilizes NP-40 and Trypsin-EDTA to treat in vitro cells, which can completely break the cell body and the migratory body, thereby improving the efficiency of migratory body extraction.

[0049] The working principle of this invention: Treatment with a low concentration of NP-40 severs the contractile filaments connecting the migratory body and the cell body (e.g., Figure 2 (As shown in a). Trypsin-EDTA treatment weakens cell adhesion and reduces cell spreading area. During this reduction, cells exert tension through contractile filaments, accelerating their breakage (e.g., ...). Figure 2 (as shown in c&d).

[0050] This method effectively breaks down the contractile filaments connecting cells and migratory bodies, thus preventing changes in migratory body size and loss during Trypsin-EDTA treatment. Finally, by collecting cells and migratory bodies and performing density gradient centrifugation, the extraction efficiency of migratory bodies is improved. Compared to previous methods, this invention can extract more migratory bodies (e.g., ...). Figure 2 As shown in e), and simultaneously, scanning electron microscopy and transmission electron microscopy proved that the quality of the extracted migrants in this invention is high (e.g., Figure 2 (as shown in f&g).

[0051] This invention features a rapid and effective improvement in the extraction efficiency of migration bodies. It significantly advances research into the function of migration bodies. This invention can change our understanding of migration body extraction and improve extraction efficiency and accuracy, providing a solid foundation for further exploration of migration body functions.

[0052] Although the present invention has been described in detail above, it is not limited thereto, and those skilled in the art can make various modifications based on the principles of the present invention. Therefore, all modifications made in accordance with the principles of the present invention should be understood to fall within the protection scope of the present invention.

Claims

1. A method for extracting migratory bodies from in vitro cultured cells, comprising: Cells were seeded on the bottom of a dish pre-coated with transport protein for 12-18 hours to obtain in vitro cells; The in vitro cells in the vessel were treated with a process that included breaking down the contractile filaments of the cell tails to form a cell mixture in which the cells and the migratory bodies were separated. The cell mixture was transferred from the dish to a test tube, and the cell mixture was subjected to density gradient centrifugation to extract the migratory bodies from the cell mixture.

2. The method for extracting migratory bodies from in vitro cultured cells according to claim 1, wherein the treatment of the in vitro cells, including breaking down the cell tail contractile filaments, comprises: In vitro cells were treated with a low concentration of NP-40 for 1-2 minutes to sever the contractile filaments connecting the migratory bodies to the cell bodies. The low concentration of NP-40 was then discarded. In vitro cells were then treated with Trypsin-EDTA for 2-3 minutes to accelerate the breakage of the contractile filaments.

3. The method for extracting migratory bodies from in vitro cultured cells according to claim 3, wherein the low concentration of NP-40 is 0.006-0.008% NP-40.

4. The method for extracting migratory bodies from in vitro cultured cells according to claim 3, wherein the low concentration of NP-40 is 0.007% NP-40.

5. The method for extracting migratory bodies from in vitro cultured cells according to claim 3, wherein the cell mixture is subjected to density gradient centrifugation to extract the migratory bodies from the cell mixture comprises: The migratory bodies were collected by adding culture medium to the cell mixture and subjected to a first strong centrifugation at 4 degrees Celsius. After the first centrifugation, the migrating body was subjected to density gradient centrifugation, and the migrating body liquid was collected. The absorbed migrating liquid was subjected to a second strong centrifugation to obtain a pure migrating body.

6. The method for extracting migratory bodies from in vitro cultured cells according to claim 5, wherein density gradient centrifugation is performed on the migratory bodies after the first centrifugation to aspirate the migratory body liquid comprises: Prepare sucrose density gradient centrifuge solutions of 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, and 50%. Resuspend the collected migratings in a 10% density gradient centrifuge solution and add them to the density gradient centrifuge solutions. Centrifuge at 150,000 g for 4 hours in an ultracentrifuge to produce migratory bands in the centrifuged liquid; Use the nozzle to draw out the liquid in the layer containing the migrating body.

7. The method for extracting migratory bodies from in vitro cultured cells according to claim 6, wherein the aspirated migratory fluid is subjected to a second strong centrifugation, comprising: The resuspended migratory liquid was resuspended in PBS and then subjected to a second strong centrifugation.

8. A method for extracting migratory bodies from in vitro cultured cells according to claim 5 or 7, wherein the first and second strong centrifugations are performed by centrifuging at 20,000g for 30 minutes at 4 degrees Celsius.

9. An application of NP-40 in the extraction of migratory bodies from in vitro cultured cells, wherein the NP-40 is used to break the contractile filaments of the cell tail.

10. The application according to claim 9, wherein the NP-40 is 0.006-0.008% NP-40.

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