Method for tissue slice collection and transfer and tissue slice detection method

Abstract

The invention belongs to the field of biological medicine, and particularly relates to a method for collecting and transferring tissue slices and a tissue slice detection method. The method for collecting and transferring the tissue slices comprises the steps that the tissue slices are collected through a collecting assembly, the collecting assembly comprises at least one collecting cover, the collecting cover is provided with a main body part and a filling part located in a groove of the main body part, the filling part is filled with an organic silicon material, and the organic silicon material is used for collecting the tissue slices; a punching sampling device is adopted to enable a first filling part to be separated from a collecting assembly, and the first filling part comprises an organic silicon material connected with a tissue slice. Therefore, non-destructive and pollution-free collection and transfer of the tissue slices can be realized, and the analysis accuracy of the tissue slices can be improved.

Description

Technical Field

[0001] The present application relates to the field of biomedicine, and specifically relates to a method for collecting and transferring tissue slices and a method for detecting tissue slices. Background Art

[0002] The existing methods for collecting and transferring laser microdissected tissue slices are mainly as follows: (1) relying on gravity to collect tissue slices, and the cut tissue slices fall into the tube cover by gravity. This method is very easy to lose the target tissue slices in a smaller area, and it is difficult to observe whether the tissue slices collected after cutting are consistent with the selected target area; (2) relying on reverse gravity laser pressure to eject the cut tissue slices. This method is also very easy to lose the target tissue slices in a smaller area, and it is difficult to observe whether the tissue slices collected after cutting are consistent with the selected target area; (3) immersing the collection cover in an ultrasonic water tank for ultrasonication. The ultrasonic heating process is prone to leakage of lysis solution and entry of ultrasonic solution into the collection cover to contaminate the tissue. In addition, since the collection cover itself has a certain thickness, the ultrasound needs to pass through the collection cover material with a certain thickness to contact the tissue slice, which will affect the ultrasonic effect of the tissue slice and result in poor tissue slice lysis effect.

[0003] Therefore, current methods of tissue section collection and transfer urgently need further improvement. Summary of the Invention

[0004] The present application aims to solve one of the technical problems in the related art at least to a certain extent.

[0005] In one aspect, the present application provides a method for collecting and transferring tissue slices, comprising: using a collection assembly to collect tissue slices, wherein the collection assembly includes at least one collection cap, the collection cap having a main body and a filling portion located within a groove of the main body, the filling portion being filled with an organosilicon material, the organosilicon material being used to collect the tissue slices; and using a punching sampling device to detach a first filling portion from the collection assembly, the first filling portion including the organosilicon material connected to the tissue slices. This facilitates the non-destructive and contamination-free collection and transfer of tissue slices, and improves the accuracy of tissue slice analysis.

[0006] According to some embodiments of the present application, using a punch sampling device to detach the first filling portion from the collection assembly includes: causing the punch sampling device to perform punch sampling on a side of the filling portion away from the main body. This allows the first filling portion to be removed from the filling portion in a simple manner to facilitate transfer of the tissue slice.

[0007] According to some embodiments of the present application, the surface of the organosilicon material facing away from the main body has a raised structure, which is used to collect the tissue slices. This reduces the probability of contamination of the tissue slices during collection and is less likely to cause loss of tissue slices from smaller target areas.

[0008] According to some embodiments of the present application, the height of the protruding structure is 30 micrometers to 400 micrometers, thereby alleviating the problem of easy loss of tissue slices and reducing the probability of contamination of tissue slices during the tissue slice collection process.

[0009] According to some embodiments of the present application, the method further includes introducing the first filling portion into a lysis solution and performing ultrasonic treatment. Thus, after the tissue slice in the first filling portion is lysed, it is convenient to analyze the properties of the tissue slice in terms of NGS, proteomics, metabolomics, etc.

[0010] According to some embodiments of the present application, the collection assembly further comprises a tube body, which is connected to the collection cap via a first connection portion, and the lysis solution is located within the tube body. Thus, tissue slices collected by the organosilicon material are placed within the tube body for lysis, and the collection assembly can achieve both the effect of collecting and lysing tissue slices.

[0011] According to some embodiments of the present application, the organosilicon material includes polysiloxane. Therefore, the organosilicon material is extremely chemically stable, non-biotoxic, and has no effect on mass spectrometry. Using the organosilicon material to receive sample tissue sections is less likely to cause sample contamination.

[0012] According to some embodiments of the present application, the organosilicon material is obtained by reacting an organosilicon prepolymer and a curing agent, thereby facilitating obtaining the organosilicon material through a low-cost and simple method.

[0013] According to some embodiments of the present application, the organosilicon prepolymer includes polydimethylsiloxane.

[0014] According to some embodiments of the present application, the curing agent includes at least one of an aldehyde curing agent, a borate curing agent, and an isocyanate curing agent.

[0015] According to some embodiments of the present application, the mass ratio of the organosilicon prepolymer to the curing agent is (0.5-20): 1. This facilitates obtaining the organosilicon material through a low-cost and simple method.

[0016] According to some embodiments of the present application, the punch sampling device includes a punch sampler, thereby facilitating better sampling.

[0017] In a second aspect of the present application, a tissue slice detection method is proposed, wherein the tissue slices obtained by the aforementioned method for collecting and transferring tissue slices are used, thereby facilitating improved analysis accuracy of the tissue slices. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The above and / or additional aspects and advantages of the present application will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0019] Figure 1 This is a schematic structural diagram of a collection component according to an embodiment of the present application;

[0020] Figure 2 A cross-sectional view of a collection assembly according to an embodiment of the present application;

[0021] Figure 3 A cross-sectional view of a collection assembly according to another embodiment of the present application;

[0022] Figure 4 This is a schematic structural diagram of a collection component according to another embodiment of the present application;

[0023] Figure 5 A photograph of the collection assembly for one embodiment of the present application;

[0024] Figure 6 This is a schematic structural diagram of a collection component according to another embodiment of the present application;

[0025] Figure 7 This is a schematic structural diagram of a collection component according to another embodiment of the present application;

[0026] Figure 8 A schematic diagram of a collection component collecting a sample according to an embodiment of the present application;

[0027] Figure 9 This is a photograph of the laser-cut tissue section of Example 1;

[0028] Figure 10 This is a photograph of the tissue section before transfer in Example 1;

[0029] Figure 11 This is a photograph of the tissue section after transfer in Example 1.

[0030] Reference numerals:

[0031] Collection cover 100; main body 110; groove 111; filling part 120; tube body 200; first connecting part 300; second connecting part 400; third connecting part 500; tissue sample 1; membrane 2; slide 3; laser 4. DETAILED DESCRIPTION

[0032] The following describes in detail embodiments of the present application. Examples of the embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain the present application and are not to be construed as limiting the present application.

[0033] In the description of this application, the terms "upper" and "lower" etc. indicate orientations or positional relationships based on the orientations or positional relationships shown in the accompanying drawings. They are only for the convenience of describing this application and do not require that this application must be constructed and operated in a specific orientation. Therefore, they should not be understood as limitations on this application.

[0034] In the description of this specification, the description with reference to the term "some embodiments" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment are included in at least one embodiment of the present application. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in a suitable manner in any one or more embodiments or examples. In addition, those skilled in the art can combine and combine the different embodiments or examples described in this specification and the features of different embodiments or examples, unless otherwise clearly defined. In addition, it should be noted that in this specification, the terms "first" and "second" are used for descriptive purposes only and cannot be understood as indicating or implying relative importance or implicitly indicating the number of technical features indicated. In the description of this application, the meaning of "multiple" is two or more, unless otherwise clearly defined.

[0035] Unless otherwise defined, all technical and scientific terms used in this application have the same meanings as those commonly understood by technicians in the technical field to which this application belongs; the terms used in this application are only for the purpose of describing specific embodiments and are not intended to limit this application; unless otherwise specified, the numerical values of the various parameters mentioned in this application can be measured using various measurement methods commonly used in the art (for example, they can be tested according to the methods given in the embodiments of this application).

[0036] The terms "include" and "have" in the description and claims of this application and any variations thereof are open expressions, that is, including the contents specified in this application but not excluding other contents.

[0037] In one aspect of the present application, a method for collecting and transferring tissue sections is provided, with reference to Figure 1-Figure 3The invention relates to a method for collecting tissue slices using a collection assembly, wherein the collection assembly includes at least one collection cap 100 having a main body 110 and a filling portion 120 located within a groove 111 of the main body. The filling portion 120 is filled with an organic silicon material for collecting tissue slices. A punching sampling device is used to detach the first filling portion 120 from the collection assembly. The first filling portion 120 comprises an organic silicon material connected to the tissue slice. This facilitates the non-destructive and non-contamination collection and transfer of tissue slices, thereby improving the accuracy of tissue slice analysis.

[0038] The following explains the principle by which the aforementioned method can achieve the aforementioned technical effects:

[0039] When using the collection assembly to collect laser microdissected tissue slices, the tissue can be prepared on a membrane, and the collection cover 100 is located on the side of the membrane away from the tissue. Since the chemical properties of the silicone material are stable, the biological toxicity is low, and it has no effect on the mass spectrometer, the silicone material is used to adhere the tissue slices to achieve the collection of the tissue slices, which is not easy to cause contamination to the tissue slices. In addition, the silicone material is a soft material, and the punching sampling device can be used to easily transfer the silicone material loaded with tissue slices from the filling part 120, thereby achieving lossless transfer of the tissue slices.

[0040] The specific material of the membrane is not particularly limited in the present application. Those skilled in the art can select a conventional membrane in the field according to actual needs, and only need to prepare the required biological tissue sample on the membrane.

[0041] In some embodiments, the organosilicon material includes polysiloxane. The chemical properties of the organosilicon material are extremely stable, non-biotoxic, and have no effect on mass spectrometry. Using the organosilicon material to receive the sample tissue slice is not likely to cause contamination to the tissue slice.

[0042] In some embodiments, reference Figure 1-Figure 3 The surface of the side of the organosilicon material facing away from the main body 110 has a raised structure, and the raised structure is used to collect tissue slices. The raised structure in the organosilicon material is used to collect tissue slices. The raised structure has a certain contact area with the diaphragm, and the raised structure can achieve the collection of tissue slices by adhering to the diaphragm. Therefore, during the collection process, the collection cover 100 can avoid direct contact with the tissue slices, reducing the probability of contamination of the tissue slices during the tissue slice collection process. In addition, when using this collection component to collect tissue, it is less likely to cause the problem of tissue slices in smaller target areas to be lost, which is further conducive to achieving non-destructive and pollution-free collection and transfer of tissue slices, and is conducive to improving the analysis accuracy of tissue slices.

[0043] In some embodiments, reference Figure 1-Figure 3The protruding structure can be a part of a sphere or an ellipsoid, and the sphere can be regular or irregular. As long as the surface of the side of the silicone material away from the main body 110 has a protruding structure, the protruding structure can be in close contact with the membrane when collecting tissue slices.

[0044] In some embodiments, the height of the protrusion structure is 30 μm to 400 μm.

[0045] As specific examples, the height of the protruding structures may be 30 μm, 40 μm, 50 μm, 80 μm, 100 μm, 150 μm, 200 μm, 250 μm, 300 μm, 350 μm, or 400 μm.

[0046] By controlling the height of the protrusion within the aforementioned range, the protrusion structure has a larger contact area with the diaphragm, which is conducive to close contact between the protrusion structure and the diaphragm to collect tissue slices and alleviate the problem of easy loss of tissue slices. In addition, it is also conducive to reducing the contact between the collection cover 100 and the tissue slices, reducing the probability of contamination of the tissue slices during the tissue slice collection process.

[0047] In some embodiments, using a punch sampling device to separate the first filling portion 120 from the collection assembly includes: causing the punch sampling device to perform punch sampling from a side of the filling portion 120 away from the main body 110. This facilitates removal of the first filling portion 120 from the filling portion 120 to transfer the tissue slice.

[0048] Specifically, a punching sampling device is extended toward the filling portion 120 of the collection cover 100, and the front end of the punching sampling device gradually extends to the inner wall surface of the groove 111 of the main body. The punching sampling device is pulled out of the collection cover 100, and the silicone material loaded with tissue slices will pop out in the form of a cylinder to achieve the transfer of the tissue slices. Alternatively, the silicone material loaded with tissue slices in the form of a cylinder is taken out by tweezers to achieve the transfer of the tissue slices. It is understandable that: in the process of separating the punching sampling device and the collection cover 100, by controlling the speed at which the punching sampling device is pulled out of the collection cover 100, it is helpful to alleviate the silicone material loaded with tissue slices from adhering to the punching sampling device, so as to better achieve the transfer of the tissue slices.

[0049] In some embodiments, the punch sampling device comprises a punch sampler.

[0050] In some embodiments, after removing the first filling section 120, the first filling section 120 is placed in a lysis solution and subjected to ultrasonic treatment. Thus, the tissue slice in the first filling section 120 is lysed, facilitating analysis of NGS, proteomics, metabolomics, and other properties of the tissue slice.

[0051] Furthermore, during the ultrasonic process, the first filling portion 120 is arranged so that the side with the tissue slice faces the ultrasonic direction of the ultrasonic device, which is conducive to the lysis of the tissue slice. As an example, if the ultrasonic direction of the ultrasonic device is from bottom to top, the first filling portion 120 is arranged so that the side with the tissue slice faces downward.

[0052] In some embodiments, reference Figure 4 and Figure 5 The collection assembly further includes a tube body 200, which is connected to the collection cover 100 via a first connection portion 300, and a lysis solution is located in the tube body 200. Thus, the tissue slices collected by the organosilicon material are placed in the tube body 200 for lysis, and the collection assembly can achieve both the effect of collecting tissue slices and the effect of lysing the tissue slices.

[0053] In some embodiments, reference Figure 4 and Figure 5 The tube body 200 and the main body 110 can be part of the same PCR tube or centrifuge tube. In some embodiments, the centrifuge tube can be a 0.2 mL centrifuge tube, a 0.5 mL centrifuge tube, a 1.5 mL centrifuge tube, or a 2 mL centrifuge tube.

[0054] In some embodiments, the volume of the tube body 200 may be 0.2 mL, the diameter of the groove 111 of the main body 110 may be 4.3 mm, and the height of the protruding structure may be 80 μm to 140 μm.

[0055] In other embodiments, the volume of the tube body 200 may be 0.5 mL, the diameter of the groove 111 of the main body 110 may be 6.1 mm, and the height of the protruding structure may be 210 μm to 340 μm.

[0056] In other embodiments, the volume of the tube body 200 may be 1.5 mL, the diameter of the groove 111 of the main body 110 may be 8.7 mm, and the height of the protruding structure may be 50 μm to 190 μm.

[0057] In other embodiments, the volume of the tube body 200 may be 2 mL, the diameter of the groove 111 of the main body 110 may be 8.7 mm, and the height of the protruding structure may be 50 μm to 190 μm.

[0058] In some embodiments, reference Figure 4 and Figure 5 The number of the collecting cover 100 may be one, and the collecting assembly may include a tube body 200 , and the tube body 200 and the collecting cover 100 may be connected via a first connecting portion 300 .

[0059] In other embodiments, reference Figure 6 and Figure 7The collecting assembly may include a plurality of collecting covers 100 , and any two adjacent collecting covers 100 may be connected via a second connecting portion 400 .

[0060] In other embodiments, reference Figure 7 The collection assembly may include multiple collection caps 100 and multiple tubes 200. Any two adjacent collection caps 100 may be connected via a second connection portion 400, and any two adjacent tubes 200 may be connected via a third connection portion 500. In some embodiments of the present application, the number of collection caps and the number of tubes may be the same, for example, 8 each. Thus, tissue slices collected by the multiple collection caps can be placed in corresponding tubes for subsequent research.

[0061] For ease of understanding, as an example, the method for preparing the aforementioned collecting component may include the following steps:

[0062] S100: Provide the main body

[0063] In some embodiments, reference Figure 2 and 3 The main body 110 is a cover structure having a groove 111. In some embodiments, the main body 110 can be provided by providing a PCR tube or a centrifuge tube. In addition to the main body 110, the PCR tube or the centrifuge tube can also include a tube body 200 corresponding to the main body 110, such as Figure 4 or Figure 5 shown.

[0064] S200: Filling the groove 111 with an organic silicon material so that the height of the organic silicon material is higher than the depth of the groove 111 and forming a convex structure on the side of the organic silicon material away from the groove 111

[0065] In some embodiments, the organosilicon material can be obtained by reacting an organosilicon prepolymer and a curing agent, or by mixing existing organosilicon silica gels.

[0066] In some embodiments, the silicone prepolymer includes polydimethylsiloxane.

[0067] In some embodiments, the silicone prepolymer includes at least one of monovinyl terminated dimethyl (siloxane and polysiloxane) (CAS: 68083-19-2), vinyl MQ resin mixture (CAS: 68988-89-6), tetrakis(trimethylsiloxy)silane (CAS: 3555-47-3), and the like.

[0068] In some embodiments, the curing agent includes at least one of an aldehyde curing agent, a borate curing agent, and an isocyanate curing agent.

[0069] In some embodiments, the curing agent includes at least one of dimethyl methyl hydrogen (siloxanes and polysiloxanes) (CAS: 68037-59-2), monovinyl terminated dimethyl (siloxanes and polysiloxanes) (CAS: 68083-19-2), vinyl MQ resin mixture (CAS: 68988-89-6), and the like.

[0070] In some embodiments, the mass ratio of the organosilicon prepolymer to the curing agent is (0.5-20):1.

[0071] As a specific example, the mass ratio of the silicone prepolymer to the curing agent can be 0.5:1, 1:1, 3:1, 5:1, 10:1, 12:1, 15:1 or 20:1.

[0072] By controlling the mass ratio of the organosilicon prepolymer to the curing agent within the aforementioned range, the reaction effect of the organosilicon prepolymer and the curing agent is improved.

[0073] In some specific embodiments, the organosilicon material and the curing agent can both be a mixture. The organosilicon material can be a mixture consisting of monovinyl-terminated dimethyl (siloxanes and polysiloxanes) (CAS: 68083-19-2), a vinyl MQ resin mixture (CAS: 68988-89-6), tetrakis(trimethylsiloxy)silane (CAS: 3555-47-3), and ethylbenzene (CAS: 100-41-4). The curing agent can be a mixture consisting of dimethyl methyl hydrogen (siloxanes and polysiloxanes) (CAS: 68037-59-2), monovinyl-terminated dimethyl (siloxanes and polysiloxanes) (CAS: 68083-19-2), and a vinyl MQ resin mixture (CAS: 68988-89-6). In some specific embodiments, the mass ratio of the organosilicon material to the curing agent can be (1-10):1.

[0074] In some embodiments, the curing temperature of the organosilicon prepolymer and curing agent can be 15°C, 25°C, 50°C, 80°C, 90°C, 120°C, 150°C, etc. These curing temperatures facilitate the curing of the organosilicon prepolymer and curing agent and are unlikely to exceed the temperature tolerance of the organosilicon material. In actual operation, those skilled in the art can adjust the curing temperature based on the specific composition of the organosilicon prepolymer and curing agent. As long as the mixture does not exceed its temperature tolerance, the curing time of the mixture can be shortened by increasing the curing temperature.

[0075] In some embodiments, bubbles in the mixture of the silicone prepolymer and the curing agent can be removed by standing or using a vacuum device. After that, the mixture of the silicone prepolymer and the curing agent can be filled into the groove 111 of the main body 110 to form a slightly convex structure in the center. The collecting cover 100 is placed at room temperature or in an oven until the mixture of the silicone prepolymer and the curing agent is completely cured. The cured collecting cover 100 is as shown in FIG. Figure 5 shown.

[0076] In a second aspect of the present application, a tissue slice detection method is proposed, wherein the tissue slices obtained by the aforementioned method for collecting and transferring tissue slices are used, thereby facilitating improved analysis accuracy of the tissue slices.

[0077] The following examples of the present application are described in detail. It should be noted that the following examples are illustrative and are intended only to explain the present application and are not to be construed as limiting the present application. In addition, unless otherwise expressly stated, all reagents used in the following examples are commercially available or can be synthesized according to methods described herein or known methods. Reaction conditions not listed are also readily available to those skilled in the art.

[0078] Example 1

[0079] The method for tissue section collection and transfer includes the following steps:

[0080] (1) placing polysiloxane in the groove of the collection cap of a PCR tube, with the height of the polysiloxane slightly higher than the depth of the groove, so that the polysiloxane forms a protrusion structure on the side away from the groove, and the height of the protrusion structure is 100 μm, to obtain a collection assembly;

[0081] (2) The collection assembly is fixed by a collection holder placed on a laser microdissection system (LCM), refer to Figure 8 The collection area is parallel to the stage. The slide 3, the tissue sample 1 prepared on the membrane, and the membrane 2 form a sandwich structure and are fixed to the stage. The tissue sample 1 is located between the slide 3 and the membrane 2. The silicone material on the collection cover 100 is pressed down to tightly bond with the membrane 2. After the target area is selected, the laser 4 in the laser microdissection system (LCM) directly cuts the target area of the tissue sample from the bottom through the slide 3, and 25 tissue slices of 20 microns x 20 microns are cut. Figure 9 As shown, the collecting cover 100 is raised, and the sample tissue slices are collected on the organic silicon material of the collecting component. The tissue slices on the organic silicon material are as shown in FIG. Figure 10 As shown;

[0082] (3) The silicone material loaded with tissue slices is sampled using a punch sampler, and the cylindrical silicone material loaded with tissue slices is taken out using tweezers. The tissue slices on the cylindrical silicone material are observed, such as Figure 11 As shown, the tissue slices in the smaller target area are not lost and the tissue slices are not contaminated. Thus, the non-destructive and non-contamination collection and transfer of tissue slices is achieved, and the tissue slices have a higher analysis accuracy.

[0083] Although the embodiments of the present application have been shown and described above, it can be understood that the above embodiments are exemplary and cannot be understood as limitations on the present application. Ordinary technicians in this field can change, modify, replace and modify the above embodiments within the scope of the present application.

Claims

1. A method for collecting and transferring tissue sections, characterized in that: include: A collection assembly is used to collect tissue slices, wherein the collection assembly includes at least one collection cover, the collection cover having a main body and a filling portion located in a groove of the main body, the filling portion being filled with an organic silicon material, and the organic silicon material is used to collect the tissue slices; A punch sampling device is used to detach a first filling portion from the collecting assembly, wherein the first filling portion includes the organic silicon material connected to the tissue section.

2. The method according to claim 1, characterized in that The use of a punching sampling device to separate the first filling portion from the collecting assembly includes: allowing the punching sampling device to perform punching sampling from a side of the filling portion away from the main body.

3. The method according to claim 1, characterized in that A surface of the organosilicon material on one side facing away from the main body has a convex structure, and the convex structure is used for collecting the tissue slices.

4. The method according to claim 3, characterized in that The height of the protruding structure is 30 micrometers to 400 micrometers.

5. The method according to claim 1, wherein Also includes: The first filling part is introduced into the lysate and subjected to ultrasonic treatment.

6. The method according to claim 5, characterized in that The collection assembly further includes a tube body, the tube body is connected to the collection cover via a first connection portion, and the lysate is located in the tube body.

7. The method according to claim 1, characterized in that The organic silicon material includes polysiloxane.

8. The method according to claim 1 or 7, characterized in that The organosilicon material is obtained by reacting an organosilicon prepolymer and a curing agent, and the organosilicon material includes at least one of the following conditions: The organosilicon prepolymer includes polydimethylsiloxane; The curing agent includes at least one of an aldehyde curing agent, a borate curing agent and an isocyanate curing agent; The mass ratio of the organosilicon prepolymer to the curing agent is (0.5-20):

1.

9. The method according to claim 1, characterized in that The punch sampling device comprises a punch sampler.

10. A tissue section detection method, characterized in that: The tissue sections are collected and transferred using the method according to any one of claims 1 to 9.

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