Membrane separation column for extracting exosome

Abstract

The membrane separation column comprises a column body, a pressing ring and a filter membrane, a liquid inlet and a liquid outlet which are oppositely arranged are formed in the column body, the pressing ring and the filter membrane are sequentially arranged in the column body from the liquid inlet to the liquid outlet, and the filter membrane is arranged in the column body. The filter membrane is formed by laminating two or more layers of nylon membranes and / or glass fiber membranes with the pore diameter range of not more than 3 microns. The exosome extraction device is convenient to prepare, simple and convenient to operate and high in exosome extraction efficiency, and is an ideal article in the field of modern biotechnology.

Description

Technical Field

[0001] The present invention relates to the field of biological extraction devices, and more particularly to a membrane separation column for extracting exosomes. Background Art

[0002] Exosomes are small bilayer lipid membrane vesicles with a diameter of 30-150 nm, secreted by living cells. They are widely present in various body fluids, including blood, saliva, urine, ascites, and amniotic fluid. They carry important bioinformatics molecules, including nucleic acids, proteins, lipids, enzymes, and metabolites, that are characteristic of the cells of origin. Exosome research is currently a hot topic in the biomedical field, and their extraction and identification are crucial for a deeper understanding of their functions and applications.

[0003] One common method for extracting exosomes is using a membrane separation column. Based on the affinity between exosomes and specific membrane materials, the exosomes are adsorbed onto the column, while impurities such as proteins are precipitated by centrifugation. Elution then allows the exosomes to be isolated and purified. This method is widely used by researchers due to its convenience, ease of use, and high throughput. However, its simplified impurity removal process can result in a high level of residual impurities, potentially contaminating subsequent experiments.

[0004] Therefore, it is highly desired to have a membrane separation column that is easy to operate and has a higher efficiency in extracting exosomes. Utility Model Content

[0005] The technical problem to be solved by the utility model is to provide a membrane separation column which is easy to operate and has higher exosome extraction efficiency.

[0006] The above technical problems are solved by the following technical solutions:

[0007] According to one aspect of the present invention, a membrane separation column for extracting exosomes is provided, comprising: a column body, a pressure ring, and a filter membrane. The column body is formed with a liquid inlet and a liquid outlet arranged opposite to each other. A pressure ring and a filter membrane are sequentially arranged in the column body from the liquid inlet to the liquid outlet. The filter membrane is formed by stacking two or more layers of nylon membrane and / or glass fiber membrane with a pore size range of no more than 3 μm.

[0008] The membrane separation column of the utility model is convenient to prepare, easy to operate, and has high efficiency in extracting exosomes, and is an ideal product in the field of modern biotechnology. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] The present invention will be described below with reference to the accompanying drawings, in which:

[0010] Figure 1 Schematic diagram of the structure of a membrane separation column according to the first embodiment of the present invention;

[0011] Figure 2 Schematic diagram of the structure of a membrane separation column according to a second embodiment of the present invention;

[0012] Figure 3 Schematic diagram of the structure of the liquid collecting pipe according to the present invention;

[0013] Figure 4 Schematic diagram of the structure of a membrane separation column according to a third embodiment of the present invention;

[0014] Figure 5 Schematic diagram of the structure of a membrane separation column according to a fourth embodiment of the present invention;

[0015] Figure 6 Schematic diagram of the structure of a membrane separation column according to a fifth embodiment of the present invention;

[0016] Among them: 1 - membrane separation column; 11 - column body; 111 - liquid inlet; 112 - liquid outlet; 12 - pressure ring; 13 - filter membrane; 14 - sieve plate; 15 - liquid collecting pipe; 151 - liquid collecting port; 16 - end cover; 161 - end cover connecting part. DETAILED DESCRIPTION

[0017] Some specific embodiments of the present invention will now be described for purposes of illustration and not limitation with reference to the accompanying drawings.

[0018] refer to Figure 1 , which shows a membrane separation column 1 according to a first embodiment of the present invention, comprising: a column body 11, a pressure ring 12 and a filter membrane 13, the column body 11 is formed with a liquid inlet 111 and a liquid outlet 112 which are relatively arranged, and a pressure ring 12 and a filter membrane 13 are sequentially arranged in the column body 11 from the liquid inlet 111 to the liquid outlet 112, the liquid outlet 112 is in a planar mesh shape, the pressure ring 12 and the filter membrane 13 are arranged above the liquid outlet 112, and the filter membrane 13 is formed by stacking two or more layers of polymer membranes and / or fiber membranes with a pore size range of not more than 3 μm.

[0019] refer to Figure 2 , which shows a membrane separation column 1 according to a second embodiment of the present invention, comprising: a column body 11, a pressure ring 12 and a filter membrane 13, the column body 11 is formed with a liquid inlet 111 and a liquid outlet 112 which are relatively arranged, and a pressure ring 12 and a filter membrane 13 are sequentially arranged in the column body 11 from the liquid inlet 111 to the liquid outlet 112, and the liquid outlet 112 is funnel-shaped, and the column body 11 also includes a sieve plate 14, the pressure ring 12 and the filter membrane 13 are arranged above the sieve plate 14, and the filter membrane 13 is formed by stacking two or more layers of polymer membranes and / or fiber membranes with a pore size range of not more than 3 μm.

[0020] Preferably, the positions of the pressing ring 12 , the filter membrane 13 and the sieve plate 14 in the column 11 are closer to the liquid outlet 112 than to the liquid inlet 111 .

[0021] The column 11, pressure ring 12, sieve plate 14, and liquid collection tube 15 in the above two embodiments are commercially available components, for example, from Shenzhen Biocomma. The filter membrane 13 is formed by laminating commercially available nylon membranes and / or glass fiber membranes, for example, from Maiborui Filtration Group.

[0022] In some embodiments, the filter membrane 13 is formed by laminating two layers of nylon membrane and / or glass fiber membrane.

[0023] In some embodiments, the filter membrane 13 is formed by laminating more than two layers of nylon membrane and / or glass fiber membrane. Preferably, the filter membrane 13 is formed by laminating 2-8 layers of nylon membrane and / or glass fiber membrane. More preferably, the filter membrane 13 is formed by laminating 3-6 layers of nylon membrane and / or glass fiber membrane.

[0024] Preferably, the pore size of the nylon membrane and the glass fiber membrane ranges from 0.22 μm to 2.7 μm. For example, the pore size of the nylon membrane and the glass fiber membrane can be 0.22 μm, 0.45 μm, 0.7 μm, 1.0 μm, 2.7 μm, etc.

[0025] When the filter membrane 13 is formed by stacking two or more layers of nylon membranes and / or glass fiber membranes with different pore sizes, preferably, the membrane layers are stacked in descending order of pore size from the liquid inlet 111 to the liquid outlet 112 .

[0026] For example, the filter membrane 13 may be the following AP membrane comprising membranes of different numbers of layers and different pore sizes, wherein the membranes of different pore sizes are stacked in descending order of pore size from the liquid inlet 111 to the liquid outlet 112:

[0027] A film: It is composed of a layer of 2.7μm film, a layer of 1.0μm film, two layers of 0.7μm film and a layer of 0.22μm film;

[0028] B film: It is composed of a layer of 2.7μm film and a layer of 0.22μm film;

[0029] C film: It is composed of three layers of 2.7μm film and one layer of 0.22μm film;

[0030] D film: It is composed of six layers of 2.7μm film and one layer of 0.22μm film;

[0031] E film: It is composed of a layer of 2.7μm film, a layer of 1.0μm film and two layers of 0.7μm film;

[0032] F film: It is composed of a layer of 1.0μm film and a layer of 0.22μm film;

[0033] G film: composed of two layers of 2.7μm film;

[0034] H film: composed of four layers of 2.7μm film;

[0035] I film: composed of two layers of 2.7μm film and two layers of 1.0μm film;

[0036] J film: composed of two layers of 1.0 μm film;

[0037] K film: composed of three layers of 1.0μm film;

[0038] L film: composed of four layers of 1.0μm film;

[0039] M film: composed of six layers of 1.0μm film;

[0040] N film: composed of eight layers of 1.0μm film;

[0041] O film: composed of two layers of 0.7μm film stacked together; or P film: composed of four layers of 0.7μm film stacked together.

[0042] Preferably, the filter membrane 13 is selected from the above A membrane, C membrane, E membrane, H membrane, I membrane, K membrane, L membrane, M membrane and P membrane.

[0043] Preferably, the filter membrane 13 is not functionalized.

[0044] Preferably, the filter membrane 13 is uncharged.

[0045] The membrane separation column 1 may further include a liquid collecting pipe 15, which is formed with a liquid collecting port 151 for the column body 11 to be inserted. Figure 3 shown.

[0046] Figure 4 The membrane separation column according to the third embodiment of the present invention is shown. Figure 1 In the structure shown, the membrane separation column 1 further includes a liquid collecting pipe 15 , and the membrane separation column 1 is inserted into the liquid collecting pipe 15 via a liquid collecting port 151 .

[0047] Figure 5 The membrane separation column according to the fourth embodiment of the present invention is shown. Figure 2 In the structure shown, the membrane separation column 1 further includes a liquid collecting pipe 15 , and the membrane separation column 1 is inserted into the liquid collecting pipe 15 via a liquid collecting port 151 .

[0048] Figure 6 A membrane separation column according to a fifth embodiment of the present invention is shown. Figure 6 As shown, compared to Figure 4In the structure shown, the membrane separation column 1 is further provided with an end cap 16, which is reversibly connected to the edge of the liquid inlet 111 via an end cap connection portion 161. The end cap 16 is used to open and close the liquid inlet 111. Specifically, the end cap connection portion 161 is a connecting piece or a connecting plate.

[0049] The membrane separation column provided in the embodiment of the present invention is used as follows: the column body 11 is inserted into the liquid collection tube 15, the end cap 16 is flipped to open the liquid inlet 111, the sample liquid is injected through the liquid inlet 111 and then centrifuged, and the filtered liquid flows out from the liquid outlet 112 into the liquid collection tube 15. The technician can collect or discard the liquid in the liquid collection tube as needed.

[0050] The biological sample may be pretreated or unpretreated before separation by the membrane separation column 1 .

[0051] In some embodiments, before separation by the membrane separation column 1 , the exosomes in the biological sample may be precipitated using a precipitant known to those skilled in the art.

[0052] The utility model can quickly extract exosomes from a variety of biological samples, such as plasma, serum, urine, cerebrospinal fluid, ascites, breast milk, amniotic fluid and other body fluids or cell culture fluids, and can be used for a variety of biological research and clinical testing.

[0053] The terms “include” and “comprising” used in the present invention encompass the case where the present invention further includes or comprises other elements not explicitly mentioned as well as the case where the present invention consists of the mentioned elements.

[0054] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs. When the definition of a term in this specification conflicts with the meaning commonly understood by those skilled in the art to which the present invention belongs, the definition described in this specification shall prevail.

[0055] Example

[0056] The following will further illustrate the concept, specific structure and technical effects of the present invention in conjunction with the embodiments, so that those skilled in the art can fully understand the purpose, features and effects of the present invention. Those skilled in the art will understand that the embodiments herein are for illustrative purposes only and the scope of the present invention is not limited thereto.

[0057] Examples 1-16

[0058] Examples 1-16 are examples of different membrane separation column structures according to the present invention.

[0059] Example 1

[0060] refer to Figure 6A membrane separation column 1 for extracting exosomes comprises: a column 11, a pressing ring 12, a filter membrane 13 and a liquid collection tube 15. The column 11 is formed with a liquid inlet 111 and a liquid outlet 112 which are relatively arranged. The liquid outlet 112 is in a planar mesh shape. The column 11 is sequentially provided with a pressing ring 12 and a filter membrane 13 from the liquid inlet 111 to the liquid outlet 112. The pressing ring 12 and the filter membrane 13 are arranged on the liquid outlet 112. The column 11 is inserted into the liquid collection tube 15 through the liquid collection port 151. The column 11 is also provided with an end cap 16, which is reversibly connected to the edge of the liquid inlet 111 through an end cap connecting portion 161. The filter membrane 13 is sequentially stacked with a layer of 2.7 μm membrane, a layer of 1.0 μm membrane, two layers of 0.7 μm membrane and a layer of 0.22 μm membrane (A membrane) from the liquid inlet 111 to the liquid outlet 112.

[0061] Example 2

[0062] A membrane separation column 1 is different from the membrane separation column in Example 1 in that the filter membrane 13 is composed of a layer of 2.7 μm membrane and a layer of 0.22 μm membrane (B membrane) stacked in sequence from the liquid inlet 111 to the liquid outlet 112.

[0063] Example 3

[0064] A membrane separation column 1 is different from the membrane separation column in Example 1 in that the filter membrane 13 is composed of three layers of 2.7 μm membranes and one layer of 0.22 μm membrane (C membrane) stacked in sequence from the liquid inlet 111 to the liquid outlet 112.

[0065] Example 4

[0066] A membrane separation column 1 is different from the membrane separation column in Example 1 in that the filter membrane 13 is composed of six layers of 2.7 μm membranes and one layer of 0.22 μm membrane (D membrane) stacked in sequence from the liquid inlet 111 to the liquid outlet 112.

[0067] Example 5

[0068] A membrane separation column 1 is different from the membrane separation column in Example 1 in that the filter membrane 13 is composed of a layer of 2.7 μm membrane, a layer of 1.0 μm membrane and two layers of 0.7 μm membrane (E membrane) stacked in sequence from the liquid inlet 111 to the liquid outlet 112.

[0069] Example 6

[0070] A membrane separation column 1 is different from the membrane separation column in Example 1 in that the filter membrane 13 is composed of a layer of 1.0 μm membrane and a layer of 0.22 μm membrane (F membrane) stacked in sequence from the liquid inlet 111 to the liquid outlet 112.

[0071] Example 7

[0072] A membrane separation column 1 is different from the membrane separation column in Example 1 in that the filter membrane 13 is a laminate of two 2.7 μm membranes (G membranes).

[0073] Example 8

[0074] A membrane separation column 1 is different from the membrane separation column in Example 1 in that the filter membrane 13 is a stack of four 2.7 μm membranes (H membranes).

[0075] Example 9

[0076] A membrane separation column 1 is different from the membrane separation column in Example 1 in that the filter membrane 13 is composed of two layers of 2.7 μm membrane and two layers of 1.0 μm membrane (I membrane) stacked in sequence from the liquid inlet 111 to the liquid outlet 112.

[0077] Example 10

[0078] A membrane separation column 1 is different from the membrane separation column in Example 1 in that the filter membrane 13 is a laminate of two 1.0 μm membranes (J membranes).

[0079] Example 11

[0080] A membrane separation column 1 is different from the membrane separation column in Example 1 in that the filter membrane 13 is a three-layer stacked 1.0 μm membrane (K membrane).

[0081] Example 12

[0082] A membrane separation column 1 is different from the membrane separation column in Example 1 in that the filter membrane 13 is a stack of four 1.0 μm membranes (L membranes).

[0083] Example 13

[0084] A membrane separation column 1 is different from the membrane separation column in Example 1 in that the filter membrane 13 is a stack of six 1.0 μm membranes (M membranes).

[0085] Example 14

[0086] A membrane separation column 1 is different from the membrane separation column in Example 1 in that the filter membrane 13 is a stack of eight 1.0 μm membranes (N membranes).

[0087] Example 15

[0088] A membrane separation column 1 is different from the membrane separation column in Example 1 in that the filter membrane 13 is a laminate of two 0.7 μm membranes (O membranes).

[0089] Example 16

[0090] A membrane separation column 1 is different from the membrane separation column in Example 1 in that the filter membrane 13 is a stack of four layers of 0.7 μm membranes (P membranes).

[0091] The column 11, the pressing ring 12, the sieve plate 14 and the liquid collecting pipe 15 were purchased from Shenzhen Comma Biotechnology Co., Ltd.

[0092] The 2.7 μm membrane was SPGFD015270N glass fiber membrane purchased from Maiborui Filtration Group;

[0093] The 1.0 μm membrane was SPGFB013100N glass fiber membrane purchased from Maiborui Filtration Group;

[0094] The 0.7 μm membrane was SPGFF021070N glass fiber membrane purchased from Maiborui Filtration Group; and

[0095] The 0.22 μm membrane was MSNY270022 nylon membrane purchased from Maiborui Filtration Group.

[0096] Examples 17-32

[0097] In Example 17-32, the AP membrane separation column in Example 1-16 was used in combination with a polymer precipitation method to extract exosomes, and the extraction steps included: mixing a human serum sample and a precipitant at a volume ratio of 1:2.5 at room temperature; loading the precipitated mixed solution into the column 11 through the liquid inlet 111 for centrifugation, and discarding the solution in the collection tube 15; loading a washing solution into the column 11 through the liquid inlet 111 for washing and centrifugation, and discarding the solution in the collection tube 15; and loading an eluent into the column 11 through the liquid inlet 111 for elution and centrifugation, and collecting the solution in the collection tube 15.

[0098] The CD63 and protein concentrations of the extracted exosomes were measured using a CD63 ELISA kit and a BCA protein quantification kit. The results showed that when the number of filter layers of the glass fiber / nylon membrane (with a pore size range of 0.22μm-2.7μm) was 2-8, the CD63 recovery rate was above 50%, and the protein residual rate did not exceed 4%. In particular, when the number of filter layers of the glass fiber / nylon membrane (i.e., membranes A, C, E, H, I, K, L, M, and P) was 3-6 (i.e., membranes A, C, E, H, I, K, L, M, and P), the CD63 recovery rate increased to over 70%.

[0099] The above descriptions of exemplary embodiments or examples of the present invention are not intended to limit the present invention. Various modifications and variations are possible for those skilled in the art. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention are intended to be included within the scope of the claims of this application.

Claims

1. A membrane separation column for extracting exosomes, characterized in that: include: A column, a pressure ring and a filter membrane, wherein the column is formed with a liquid inlet and a liquid outlet arranged opposite to each other, and the pressure ring and the filter membrane are sequentially arranged in the column from the liquid inlet to the liquid outlet, and the filter membrane is formed by stacking two or more layers of nylon membranes and / or glass fiber membranes with a pore size range of no more than 3μm.

2. The membrane separation column according to claim 1, characterized in that The liquid outlet is in a planar mesh shape, and the pressure ring and the filter membrane are arranged above the liquid outlet.

3. The membrane separation column according to claim 1, characterized in that The liquid outlet is funnel-shaped, and the column further includes a sieve plate. The pressure ring and the filter membrane are arranged above the sieve plate.

4. The membrane separation column according to claim 1, characterized in that The utility model further comprises a liquid collecting pipe, which is formed with a liquid collecting port for inserting the column.

5. The membrane separation column according to claim 1, characterized in that An end cover is also provided, which is flippably connected to the edge of the liquid inlet through an end cover connecting portion, and is used to open and close the liquid inlet.

6. The membrane separation column according to claim 1, characterized in that The filter membrane is formed by laminating 2 to 8 layers of nylon membranes and / or glass fiber membranes.

7. The membrane separation column according to claim 1, characterized in that The filter membrane is formed by laminating 3-6 layers of nylon membranes and / or glass fiber membranes.

8. The membrane separation column according to claim 1, characterized in that The filter membrane is formed by laminating nylon membranes and / or glass fiber membranes with a pore size range of 0.22 μm to 2.7 μm.

9. The membrane separation column according to claim 1, characterized in that The filter membrane is selected from the following AP membranes comprising membranes of different numbers of layers and different pore sizes, wherein the membranes of different pore sizes are stacked in descending order of pore size from the liquid inlet to the liquid outlet: A film: It is composed of a layer of 2.7μm film, a layer of 1.0μm film, two layers of 0.7μm film and a layer of 0.22μm film; B film: It is composed of a layer of 2.7μm film and a layer of 0.22μm film; C film: It is composed of three layers of 2.7μm film and one layer of 0.22μm film; D film: It is composed of six layers of 2.7μm film and one layer of 0.22μm film; E film: It is composed of a layer of 2.7μm film, a layer of 1.0μm film and two layers of 0.7μm film; F film: It is composed of a layer of 1.0μm film and a layer of 0.22μm film; G film: composed of two layers of 2.7μm film; H film: composed of four layers of 2.7μm film; I film: composed of two layers of 2.7μm film and two layers of 1.0μm film; J film: composed of two layers of 1.0 μm film; K film: composed of three layers of 1.0μm film; L film: composed of four layers of 1.0μm film; M film: composed of six layers of 1.0μm film; N film: composed of eight layers of 1.0μm film; O film: composed of two layers of 0.7μm film stacked together; and P film: It is composed of four layers of 0.7μm film stacked together.

10. The membrane separation column according to claim 9, characterized in that The filter membrane is selected from the group consisting of A membrane, C membrane, E membrane, H membrane, I membrane, K membrane, L membrane, M membrane and P membrane.

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