WO2022017482A1

WO2022017482A1 - Tissue slicing method and forming device for tissue slicing

Info

Publication number: WO2022017482A1
Application number: PCT/CN2021/108004
Authority: WO
Inventors: 江涛; 胡慧敏; 黄利杰
Original assignee: 北京市神经外科研究所
Priority date: 2020-07-23
Filing date: 2021-07-22
Publication date: 2022-01-27
Also published as: CN113970472A; CN113970472B

Abstract

A tissue slicing method and a forming device for tissue slicing. The method comprises: firstly, using a forming device with a flexible mold (101) and an embedding agent to embed a plurality of tissue blocks in the same clot, then taking out the clot from the flexible mold (1011), and further embedding and treating the clot, and finally carrying out slicing, such that multiple tissue blocks can be sliced at the same time. The method is particularly suitable for large-scale drug screening.

Description

Tissue sectioning method and forming device for tissue sectioning

technical field

The present disclosure relates to the field of biotechnology, and in particular, to a tissue sectioning method and a forming device for tissue sectioning.

Background technique

Active tissue blocks can better retain and reflect the original physiological/pathological state of biological individuals, therefore, active tissue blocks are increasingly used for drug screening. However, the active tissue block has problems such as difficulty in fluorescent labeling, proliferation activity detection and biomarker detection, etc. Therefore, the active tissue block usually needs to be sliced for study in the evaluation of drug effect.

In the related art, tissue is usually embedded with an embedding medium and then sectioned. However, in the existing tissue sectioning technology, only a single piece of tissue can be sectioned at one time, and the existing tissue sectioning technology is time-consuming and labor-intensive when performing large-scale drug screening.

SUMMARY OF THE INVENTION

The purpose of the present disclosure is to solve the problem of the small number of tissue blocks in a single slice in the existing tissue slice technology, and to provide a tissue slice method and a forming device for tissue slice.

In order to achieve the above object, the present disclosure provides a tissue sectioning method, the method comprising:

a. Transfer the tissue culture in at least one first culture well from the first culture plate to the first forming device to obtain a second forming device loaded with tissue culture, the tissue culture including tissue pieces and culture medium ; wherein, the first molding device comprises a second culture plate and a flexible mold arranged on the upper surface of the second culture plate, the flexible mold includes a bottom plate and a frame arranged on the bottom plate, and the bottom plate is provided with a frame There is at least one concave hole, the bottom plate is in contact with the upper surface of the second culture plate, and the concave hole on the bottom plate corresponds to the second culture hole on the second culture plate, and the tissue culture plate is placed. in the recessed hole;

b. Pre-processing the second forming device to remove the medium in the tissue culture to obtain a third forming device loaded with tissue blocks;

c. Add an embedding agent into the concave hole of the third molding device until the liquid level of the embedding agent is higher than the upper surface of the bottom plate, and solidify the embedding agent to obtain a clot-loaded a fourth shaping device, wherein the clot contains at least one tissue block in the recess;

d. taking out the clot from the fourth molding device, placing it in an embedding box, then adding an embedding agent into the embedding box, and solidifying the embedding agent to obtain an embedding block;

e. Slicing the embedded block to obtain tissue slices.

Optionally, when the embedding block is sliced in step e, the slicing direction is parallel to the arrangement direction of the tissue blocks in the embedding block.

Optionally, the flexible mold is made of a flexible material; preferably, the flexible material includes at least one of soft glue, silica gel, and TPE.

Optionally, the preprocessing of the second molding device in step b includes:

The second molding device is subjected to plate throwing and centrifugation, and the conditions of the plate throwing centrifugation include: the rotating speed is 100-2000 rpm, and the time is 1-10 min.

Optionally, in the first molding device, the shape of the bottom plate of the flexible mold matches the shape of the second culture plate, and the shape and number of the concave holes on the bottom plate are the same as those of the second culture plate. The shape and number of the second culture wells on the plate match;

The number of the second culture wells of the second culture plate is not less than the number of the first culture wells of the first culture plate.

Optionally, transferring the tissue culture in at least one first culture well from the first culture plate to the first forming device described in step a includes:

The tissue culture in at least one first culture hole of the first culture plate is correspondingly transferred into at least one concave hole of the first forming device.

Optionally, in step c, when an embedding agent is added into the concave hole of the third molding device, the liquid level of the embedding agent is 2-30 mm higher than the upper surface of the bottom plate;

The embedding agent includes at least one of tissue freezing embedding agent, paraffin embedding agent and plastic embedding agent.

Optionally, the method further includes:

In step d, after the clot is taken out from the fourth forming device, the clot is cut into sub-clots of a preset size, and then the sub-clots are placed in an embedding box for the process. Embedding; or,

In step e, before slicing the embedded block, the embedded block is cut into sub-embedded blocks of a preset size, and then the sub-embedded blocks are sliced.

Optionally, each of the sub-clots or each of the sub-embedded blocks contains m×n tissue blocks in the concave holes, wherein m and n are positive integers, and 1≤m≤30 , 1≤n≤60; preferably, m=3, n=6.

The present disclosure also provides a forming device for tissue slices, the forming device includes a second culture plate and a flexible mold disposed on the upper surface of the second culture plate, the flexible mold including a bottom plate and a base plate and a flexible mold disposed on the bottom plate At least one concave hole is provided on the bottom plate, the bottom plate is in contact with the upper surface of the second culture plate, and the concave hole on the bottom plate is in contact with the second culture hole on the second culture plate Corresponding coincidence.

Through the above technical solutions, in the tissue slicing method provided by the present disclosure, firstly, a forming device with a flexible mold and an embedding agent are used to embed a plurality of tissue blocks in the same clot, and then the clot is removed from the flexible mold. The clot is taken out, and the clot is further embedded, and finally sliced. Therefore, the method of the present disclosure can slice multiple tissue blocks at the same time, which has the advantages of saving time and effort, and is especially suitable for large-scale drug screening.

Other features and advantages of the present disclosure will be described in detail in the detailed description that follows.

Description of drawings

The accompanying drawings are used to provide a further understanding of the present disclosure, and constitute a part of the specification, and together with the following detailed description, are used to explain the present disclosure, but not to limit the present disclosure. In the attached image:

FIG. 1 is a schematic structural diagram of a molding device provided by an embodiment of the present disclosure;

2 is a schematic structural diagram of a flexible mold provided by an embodiment of the present disclosure;

FIG. 3 is a staining diagram of a frozen section provided by an embodiment of the present disclosure.

Description of reference numerals

1 Forming device 101 The second culture plate

102 Flexible mold 1011 The second culture hole

1021 Bottom plate 1022 Frame

1023 concave hole

detailed description

The specific embodiments of the present disclosure will be described in detail below with reference to the accompanying drawings. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present disclosure, but not to limit the present disclosure.

A first aspect of the present disclosure provides a tissue slicing method, the method comprising: a. transferring tissue culture in at least one first culture well from a first culture plate to a first forming device to obtain a tissue culture loaded with tissue culture The second molding device, the tissue culture includes tissue blocks and culture medium; wherein, the first molding device includes a second culture plate and a flexible mold arranged on the surface of the second culture plate, the flexible mold It includes a bottom plate and a frame arranged on the bottom plate, the bottom plate is provided with at least one concave hole, the bottom plate is in contact with the upper surface of the second culture plate, and the concave hole on the bottom plate is in contact with the first plate. The second culture holes on the two culture plates are correspondingly overlapped, and the tissue culture is placed in the concave hole; b. The second molding device is pretreated to remove the medium in the tissue culture to obtain a loading A third molding device with an organized block; c. Add an embedding agent into the concave hole of the third molding device until the liquid level of the embedding agent is higher than the upper surface of the bottom plate, and make the embedding agent The agent is solidified to obtain a fourth forming device loaded with clots, wherein the clots contain at least one tissue block in the concave hole; d. The clots are taken out from the fourth forming device, and placed In the embedding box, then adding an embedding agent into the embedding box, and making the embedding agent solidify to obtain an embedding block; e. Slicing the embedding block to obtain a tissue section.

Wherein, the method for solidifying the embedding agent in the above-mentioned technical scheme may be to place the molding device or the embedding box in a low temperature environment such as a refrigerator, or place the molding device or the embedding box on dry ice, or place the molding device or the embedding box on dry ice, or place the molding device or the embedding box on dry ice according to the embedding agent. The specific types and characteristics of the solidification treatment.

The tissue block involved in the above technical solution can be any tissue block in the field that needs to be subjected to slice research, for example, it can include in vitro cultured living tissue, normal tissue or tumor tissue-derived organoids, induced pluripotent stem cell-derived organoids. Organs, and the aforementioned tissue pieces after drug treatment during drug screening.

The first culture plate and the second culture plate involved in the above technical solution may be conventional in the field, for example, a 48-well culture plate, a 96-well culture plate, and the like.

In the above technical solution, the forming device includes a flexible mold, and the flexible mold includes a bottom plate with concave holes and a frame set on the bottom plate, the concave holes can be used to place tissue blocks, and the frame can make the embedding agent higher than the bottom plate at the liquid level The upper surface does not flow out, so multiple tissue pieces can be embedded in the same clot using the above-mentioned molding device. Meanwhile, the flexible mold is separable from the second culture plate, and the flexible mold is flexible, so the clot can be easily taken out from the flexible mold as a whole under the premise of maintaining the integrity of the clot. The removed clot can be sliced after embedding. It can be seen that the method of the present disclosure can simultaneously slice multiple tissue blocks, has the advantages of saving time and effort, and is especially suitable for large-scale drug screening.

Preferably, when the embedding block is sliced in step e, the slicing direction is parallel to the arrangement direction of the tissue blocks in the embedding block. In the above preferred case, the slicing direction is parallel to the arrangement direction of the tissue blocks in the embedding block, so as to ensure that all the tissue blocks in the clot can be cut in one slicing.

According to the present disclosure, the flexible mold may be made of a flexible material. The flexible material can be selected from a wide range, for example, the flexible material can include at least one of soft glue, silicone, and TPE.

According to the present disclosure, the pre-processing of the second molding device in step b may include, for example, performing a plate throwing and centrifugation on the second forming device. 2000rpm, the time can be 1-10min. Spin plate centrifugation allows tissue pieces from the tissue culture to settle at the bottom of the wells, facilitating removal of the medium from the tissue culture.

Optionally, in the first molding device, the shape of the bottom plate of the flexible mold matches the shape of the second culture plate, and the shape and number of the concave holes on the bottom plate are the same as those of the second culture plate. The shape and number of the second culture holes on the plate are matched; the number of the second culture holes of the second culture plate is not less than the number of the first culture holes of the first culture plate.

Optionally, transferring the tissue culture in the at least one first culture hole from the first culture plate to the first forming device in step a may include, for example: transferring at least one first culture plate of the first culture plate to the first forming device. The tissue culture in the culture hole is correspondingly transferred into at least one concave hole of the first forming device. Transfer the tissue culture in each first culture well to each concave well, to ensure that the tissue culture can be traced back to its position in the first culture plate according to the position of the concave hole where the tissue culture is located to avoid tissue culture. The numbering or identification of the numbering was scrambled during the transfer. Specifically, a multi-channel pipette can be used to transfer the tissue culture from the first culture well of the first culture plate to the well of the shaping device.

Preferably, in step c, when the embedding agent is added into the concave hole of the third molding device, the liquid level of the embedding agent may be 2-30 mm higher than the upper surface of the bottom plate. In this preferred case, the coagulum formed after the embedding medium is solidified is not easily broken and has a suitable thickness. The embedding agent may include at least one of a tissue freezing embedding agent, a paraffin embedding agent, and a plastic embedding agent.

According to the present disclosure, in order to meet the needs of different research scenarios, in step d, after the clot is taken out from the fourth forming device, the clot can also be cut into sub-clots of a preset size, and then The sub-clots are then placed in an embedding box for embedding. Alternatively, in step e, before slicing the embedded block, the embedded block can be cut into sub-embedded blocks of a preset size, and then the sub-embedded blocks are sliced.

The size of each sub-clot or each sub-embedding block can be set according to the needs of the research scene. For example, each sub-clot can contain m×n tissue blocks in the concave hole, Among them, m and n are positive integers, and 1≤m≤30, 1≤n≤60. Exemplarily, for microscope observation, the values of m and n may be m=3 and n=6 due to the limitation of the size of the microscope and the glass slide.

A second aspect of the present disclosure provides a shaping device for tissue sections. FIG. 1 is a schematic structural diagram of a molding device provided by an embodiment of the present disclosure, and FIG. 2 is a schematic structural diagram of a flexible mold provided by an embodiment of the present disclosure. As shown in FIG. 1 and FIG. 2 , the molding device 1 includes a second culture plate 101 and a flexible mold 102 disposed on the upper surface of the second culture plate 101 , the flexible mold 102 includes a bottom plate 1021 and a bottom plate 102 . The frame 1022 on the bottom plate 1021, the bottom plate 1021 is provided with at least one concave hole 1023, the bottom plate 1021 is in contact with the upper surface of the second culture plate 101, and the concave hole 1023 on the bottom plate 1021 is in contact with the first plate 1021. The second culture wells 1011 on the two culture plates 101 are correspondingly overlapped.

Alternatively, the flexible mold 102 may be made of a flexible material. The flexible material can be selected from a wide range, for example, the flexible material can include at least one of soft glue, silicone and TPE.

Optionally, in the first molding device 1, the shape of the bottom plate 1021 of the flexible mold 102 matches the shape of the second culture plate 101, and the shape and number of the concave holes 1023 on the bottom plate 1021 Matches the shape and number of the second culture holes 1011 on the second culture plate 101; the number of the second culture holes 1011 of the second culture plate 101 is not less than the first culture plate of the first culture plate. number of holes.

The present disclosure is further illustrated by the following examples, but the present disclosure is not limited thereby.

The raw materials, reagents, instruments and equipment involved in the embodiments of the present disclosure can be obtained through purchase unless otherwise specified.

For convenience of description, in the embodiments of the present disclosure, the tissue block is an active tissue block after drug treatment as an example for description.

Preparation Examples

This example is used to illustrate the preparation of the active tissue mass after drug treatment.

(1) Preparation of active tissue block

The active tissue blocks used for drug screening are screened through a special sieve cloth for cell tissue to obtain active tissue blocks with a diameter of 0.2 μm to 2 μm. The active tissue blocks obtained by the above screening were mixed with the corresponding medium, and inoculated in a 96-well culture plate using a multi-channel pipette or a continuous dispenser, so that the volumes of the active tissue blocks and the medium in each culture well were equal. .

(2) Treatment and culture of active tissue blocks with drugs

Add the test drug to the 96-well plate inoculated with the active tissue block obtained in step (1), one drug per well, and set parallel repeating wells for each drug (the number of repeating wells can range from 1 to 5), and set Control group without drug added (control group can be added without any substance, or can be added with the same volume of drug solvent as the drug test well). The above-mentioned 96-well plate with the test drug added is placed under the culture condition of active tissue for a certain period of time to obtain a 96-well plate containing the active tissue block treated with the drug.

Example 1

This example is used to illustrate the tissue sectioning method of the present disclosure.

(1) Spread a silica gel mold on the upper surface of a 96-well plate to obtain a first molding device. Among them, the length and width of the silicone mold are respectively equal to the length and width of the 96-well plate. The silicone mold consists of a bottom plate and a frame set on the bottom plate. The bottom plate has 96 concave holes, and the sizes and positions of the 96 concave holes are respectively Matches the size and position of the 96 wells of the 96-well plate; the border is 10mm above the top surface of the bottom plate. When the silica gel mold is spread on the upper surface of the 96-well plate, the 96 concave holes of the silica gel mold coincide with the 96 culture wells of the 96-well plate respectively.

(2) Use a multi-channel pipette to translate the active tissue block after the drug treatment obtained in the preparation example, together with the remaining drug and culture medium, into the concave hole of the first forming device, to obtain the first tissue block loaded with the active tissue block. Second molding device.

(3) The second molding device is placed on a plate throwing centrifuge to carry out plate throwing centrifugation, the centrifuge rotating speed is 1000rpm, and the centrifugation time is 5min. After the centrifugation was completed, the medium was removed to obtain a third shaped device loaded with tissue pieces.

(4) Add a cryosection embedding agent OCT (purchased from Sakura, USA) into each concave hole of the third molding device until the liquid level of the cryosection embedding agent is 8 mm higher than the upper surface of the bottom plate, and then the third molding The device was placed on dry ice, and after the OCT was completely solidified to form a clot, a fourth molding device loaded with the clot was obtained.

(5) Separate the silicone mold of the fourth molding device from the 96-well plate, then take out the clot in the silicone mold, and use a mini handheld cutting machine to cut the clot into rectangles with 3 holes/columns and 3 holes/rows clot. The cut sub-clots were placed in a suitable size embedding box, and the frozen section embedding agent OCT (purchased from Sakura, USA) was added, and then the embedding box was placed on dry ice, and the OCT was completely solidified to form an embedding block. .

(6) Transfer the embedded block obtained in step 5 to a cryostat at a low temperature (not higher than -10°C), fix it on the sample holder, and perform frozen sectioning, the thickness of the section is 5-50 μm, and the sectioning direction is the same as that of the molding device. The upper surfaces of the base plates are parallel. After sectioning, the sections were adhered to cryosection universal slides and stored at low temperature.

Test Example

This example is used to verify the rationality of the tissue sectioning method of the present disclosure.

The cryosection slides prepared in Example 1 were stained with HE, and then placed under a microscope for observation. The results are shown in FIG. 3 .

It can be seen from Figure 3 that the frozen section prepared in Example 1 contains 9 active tissues. It is illustrated that the method of the present disclosure can slice multiple tissue blocks at the same time.

The preferred embodiments of the present disclosure have been described above in detail with reference to the accompanying drawings. However, the present disclosure is not limited to the specific details of the above-mentioned embodiments. Various simple modifications can be made to the technical solutions of the present disclosure within the scope of the technical concept of the present disclosure. These simple modifications all fall within the protection scope of the present disclosure.

In addition, it should be noted that the various specific technical features described in the above-mentioned specific embodiments can be combined in any suitable manner unless they are inconsistent. In order to avoid unnecessary repetition, the present disclosure provides The combination method will not be specified otherwise.

In addition, the various embodiments of the present disclosure can also be arbitrarily combined, as long as they do not violate the spirit of the present disclosure, they should also be regarded as the contents disclosed in the present disclosure.

Claims

A tissue sectioning method, characterized in that the method comprises:

a. Transfer the tissue culture in at least one first culture well from the first culture plate to the first forming device to obtain a second forming device loaded with tissue culture, the tissue culture including tissue pieces and culture medium ; wherein, the first molding device comprises a second culture plate and a flexible mold arranged on the upper surface of the second culture plate, the flexible mold includes a bottom plate and a frame arranged on the bottom plate, and the bottom plate is provided with a frame There is at least one concave hole, the bottom plate is in contact with the upper surface of the second culture plate, and the concave hole on the bottom plate corresponds to the second culture hole on the second culture plate, and the tissue culture plate is placed. in the recessed hole;

b. Pre-processing the second forming device to remove the medium in the tissue culture to obtain a third forming device loaded with tissue blocks;

c. Add an embedding agent into the concave hole of the third molding device until the liquid level of the embedding agent is higher than the upper surface of the bottom plate, and solidify the embedding agent to obtain a clot-loaded a fourth shaping device, wherein the clot contains at least one tissue block in the recess;

d. taking out the clot from the fourth molding device, placing it in an embedding box, then adding an embedding agent into the embedding box, and solidifying the embedding agent to obtain an embedding block;

e. Slicing the embedded block to obtain tissue slices.
The method according to claim 1, wherein when slicing the embedded block in step e, the slicing direction is parallel to the arrangement direction of the tissue block in the embedded block.
The method of claim 1, wherein the flexible mold is made of a flexible material;

Preferably, the flexible material includes at least one of soft glue, silica gel and TPE.
The method according to claim 1, wherein the preprocessing of the second molding device in step b comprises:

The second molding device is subjected to plate throwing and centrifugation, and the conditions of the plate throwing centrifugation include: the rotating speed is 100-2000 rpm, and the time is 1-10 min.
The method according to claim 1, wherein, in the first forming device, the shape of the bottom plate of the flexible mold matches the shape of the second culture plate, and the concave holes on the bottom plate The shape and quantity match the shape and quantity of the second culture wells on the second culture plate;

The number of the second culture wells of the second culture plate is not less than the number of the first culture wells of the first culture plate.
The method according to claim 5, characterized in that, in step a, transferring the tissue culture in at least one first culture well from the first culture plate to the first forming device, comprising:

The tissue culture in at least one first culture hole of the first culture plate is correspondingly transferred into at least one concave hole of the first forming device.
The method according to claim 1, wherein in step c, when an embedding agent is added into the concave hole of the third molding device, the liquid level of the embedding agent is higher than the upper surface of the bottom plate 2～30mm;

The embedding agent includes at least one of tissue freezing embedding agent, paraffin embedding agent, and plastic embedding agent.
The method according to any one of claims 1 to 7, wherein the method further comprises:

In step d, after the clot is taken out from the fourth forming device, the clot is cut into sub-clots of a preset size, and then the sub-clots are placed in an embedding box for the process. Embedding; or,

In step e, before slicing the embedded block, the embedded block is cut into sub-embedded blocks of a preset size, and then the sub-embedded blocks are sliced.
The method according to claim 8, wherein each of the sub-clots or each of the sub-embedded blocks contains m×n tissue blocks in the concave holes, wherein m and n are A positive integer, and 1≤m≤30, 1≤n≤60; preferably, m=3, n=6.
A molding device for tissue slices, characterized in that the molding device comprises a second culture plate and a flexible mold disposed on the upper surface of the second culture plate, the flexible mold comprising a bottom plate and a base plate and a flexible mold disposed on the bottom plate At least one concave hole is provided on the bottom plate, the bottom plate is in contact with the upper surface of the second culture plate, and the concave hole on the bottom plate is in contact with the second culture hole on the second culture plate Corresponding coincidence.