Novel method for pre-embedding organoid

Abstract

Compared with a traditional agarose embedding method, the novel method for pre-embedding the organoid has the advantages that the problem that a pre-embedding reagent is difficult to dissolve and easy to solidify can be solved, meanwhile, dehydration operation in later paraffin embedding is facilitated, more importantly, the organoid is not prone to being damaged by external factors in the subsequent paraffin embedding treatment process, and the stability of the organoid is improved. And the form integrity is ensured.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a pathological pre-embedding method for organoids. Background Art

[0002] Organoids are three-dimensional miniature cell clusters in vitro. From a professional perspective, organoids are miniature organs constructed in vitro with self-renewal and self-organization capabilities. They have similar spatial organization to real organs and can highly simulate the structure and function of corresponding organs in the body.

[0003] Tumor organoid models are primarily established rapidly from tissue samples obtained through surgical procedures and biopsies. Tumor organoids retain the complexity and genetic heterogeneity of the parental tumor tissue and can serve as an effective drug screening platform and a tool for clinical drug guidance. To verify whether the cultured organoids retain the basic characteristics of the parental tumor tissue, histopathological morphological analysis is an indispensable identification technique for organoid research. Histopathological identification is determined by organoid embedding technology, but in vitro cultured organoids are very small (10-500um) and composed of a single cell. They are easily damaged during handling, making it more difficult to prepare sections and directly affecting the initial culture plan and the time and experimental costs caused by repeated experiments. In addition, organoid histopathology testing also needs to ensure that the organoid's appearance structure, cell content, and cell arrangement hierarchy are all fully preserved, which shows the importance of organoid pre-embedding.

[0004] Currently, the agarose method is commonly used for pre-embedding of organoid pathological sections. However, this method is prone to solidification during the addition of agarose due to temperature issues. It also suffers from problems such as shrinkage during subsequent paraffin embedding and dehydration, as well as prolonged dehydration times due to the high water content of agarose. The most important issue is the difficulty in maintaining the integrity of the organoid's appearance and morphology during subsequent processing, as well as issues such as cell loss caused by organoid fragmentation. Therefore, in order to ensure the integrity of organoids during processing, it is urgent to find a new processing method to solve the pre-embedding problem during organoid histopathology testing. Summary of the Invention

[0005] To address the structural damage that can easily occur in existing organoid pre-embedding methods, this invention provides a pre-embedding method that can obtain intact organoid tissue sections, facilitating histopathological research. The steps are as follows: Aspirate the culture medium from the culture dish containing the organoid gel droplets and add 0.5-1 mL of 4% paraformaldehyde to the well plate to fix for 0.5 h.

[0006] After the organoids have been fixed, add 1 mL of PBS and wash twice. For the final wash, use a pipette to pipette through the matrix gel and organoid mixture, transfer it to a 1.5 mL or 15 mL centrifuge tube, and centrifuge at 300 g for 5 min to remove the supernatant.

[0007] Prepare an organoid histopathology pre-embedding reagent: 25% gelatin.

[0008] Prepare dye: either eosin or fuchsin.

[0009] Heat and dissolve the organoid tissue pathology pre-embedding reagent (40℃-50℃).

[0010] Stain the cell pellet with 0.1-1 μl of dye and gently resuspend the organoid pellet in 30-50 μl of dissolved pre-embedding reagent. Once the gelatin has cooled and solidified (the organoid pellet can be observed under a microscope), paraffin embedding and sectioning are possible.

[0011] The present invention provides a new method for pre-embedding organoids, which has the following advantages compared to the traditional agarose embedding method: The present invention can solve the problem that the pre-embedded reagent is difficult to dissolve and easy to solidify.

[0012] The pre-embedding reagent of the present invention is beneficial to the dehydration operation in the later paraffin embedding.

[0013] The present invention can prevent the organoid from being damaged by external factors during the subsequent paraffin embedding process, thereby ensuring the integrity of the organoid morphology. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] In order to make the purpose, technical solutions and beneficial effects of the present invention more clear, the present invention provides the following drawings for illustration: Figure 1 This is the pathological section result of the ileal organoid in Example 1 of the present invention; Figure 2 This is the pathological section result of the lung cancer organoid in Example 2 of the present invention; Figure 3 This is the pathological section result of intestinal organoids in comparative example 1 of the present invention; Figure 4 This is the pathological section result of lung cancer organoid in comparative example 2 of the present invention. DETAILED DESCRIPTION Example 1

[0015] Take a drop of ileal organoid gel, aspirate the culture medium from its culture plate, and add 0.5 mL of 4% paraformaldehyde to the plate for fixation for 0.5 h.

[0016] After the organoids have been fixed, add 1 mL of PBS and wash twice. For the final wash, use a pipette to pipette through the matrix gel and organoid mixture, transfer it to a 1.5 mL or 15 mL centrifuge tube, and centrifuge at 300 g for 5 min to remove the supernatant.

[0017] Prepare an organoid histopathology pre-embedding reagent: 25% gelatin.

[0018] Heat and dissolve the organoid tissue pathology pre-embedding reagent (40℃-50℃).

[0019] Stain the cell pellet with 0.5 μl of eosin dye and gently resuspend the organoid pellet in 30 μl of dissolved pre-embedding reagent. Once the gelatin has cooled and solidified (the organoid pellet can be observed under a microscope), paraffin embedding and sectioning are possible.

[0020] Example 1 Ileal organoid pathological section results are as follows Figure 1 As shown, the embedding reagent tightly wrapped the organoids, the organoid morphology was intact, and the vacuolar organoids were not ruptured. Example 2

[0021] Take a drop of lung cancer organoid gel, aspirate the culture medium from its culture plate, and add 1 mL of 4% paraformaldehyde to the plate for fixation for 0.5 h.

[0022] After the organoids have been fixed, add 1 mL of PBS and wash twice. For the final wash, use a pipette to pipette through the matrix gel and organoid mixture, transfer it to a 1.5 mL or 15 mL centrifuge tube, and centrifuge at 300 g for 5 min to remove the supernatant.

[0023] Prepare an organoid histopathology pre-embedding reagent: 25% gelatin.

[0024] Heat and dissolve the organoid tissue pathology pre-embedding reagent (40℃-50℃).

[0025] Stain the cell pellet with 0.1 μl of fuchsin dye and gently resuspend the organoid pellet in 50 μl of dissolved pre-embedding reagent. Once the gelatin has cooled and solidified (the organoid pellet can be observed under a microscope), paraffin embedding and sectioning are possible.

[0026] Example 2 Lung cancer organoid pathological section results are as follows Figure 2 As shown, the embedding reagent tightly wrapped the organoid, and the organoid morphology was intact and not broken.

[0027] Comparative Example 1 Take a drop of intestinal organoid gel, aspirate the culture medium from its culture plate, and add 0.5 mL of 4% paraformaldehyde to the plate for fixation for 0.5 h.

[0028] After the organoids have been fixed, add 1 mL of PBS and wash twice. For the final wash, use a pipette to pipette through the matrix gel and organoid mixture, transfer it to a 1.5 mL or 15 mL centrifuge tube, and centrifuge at 300 g for 5 min to remove the supernatant.

[0029] Prepare an organoid histopathology pre-embedding reagent: 5% agarose.

[0030] Heat and dissolve the organoid tissue pathology pre-embedding reagent (70℃-100℃).

[0031] Stain the cell pellet with 0.1 μl of eosin dye and gently resuspend the organoid pellet in 50 μl of dissolved pre-embedding reagent. Once the agarose cools and solidifies (organoid pellet can be observed under a microscope), paraffin embedding and sectioning are possible.

[0032] Comparative Example 1 Intestinal organoid pathological section results are as follows Figure 3 As shown, the organoid has a solid morphology that is intact and not ruptured, but there are obvious gaps around the organoid and in the embedding reagent; and the vacuolar organoid is ruptured.

[0033] Comparative Example 2 Take a drop of lung cancer organoid gel, aspirate the culture medium from its culture plate, and add 1 mL of 4% paraformaldehyde to the plate for fixation for 0.5 h.

[0034] After the organoids have been fixed, add 1 mL of PBS and wash twice. For the final wash, use a pipette to pipette through the matrix gel and organoid mixture, transfer it to a 1.5 mL or 15 mL centrifuge tube, and centrifuge at 300 g for 5 min to remove the supernatant.

[0035] Prepare an organoid histopathology pre-embedding reagent: 5% agarose.

[0036] Heat and dissolve the organoid tissue pathology pre-embedding reagent (70℃-100℃).

[0037] Stain the cell pellet with 0.5 μl of fuchsin dye and gently resuspend the organoid pellet in 50 μl of dissolved pre-embedding reagent. Once the agarose cools and solidifies (organoid pellet can be observed under a microscope), paraffin embedding and sectioning are possible.

[0038] Comparative Example 2 Lung Cancer Organoid Pathology Section Results Figure 4 As shown, there are obvious gaps around the organoid and in the embedding reagent.

[0039] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention shall be determined by the appended claims.

Claims

1. Organoid pathology pre-embedding reagent, characterized by: Use 25% gelatin.

2. Organoid pathology pre-embedding staining reagent, characterized by: Use eosin or fuchsin staining reagents.

3. A method for pre-embedding organoids for pathological purposes, characterized by: Aspirate the culture medium from the organoid gel droplet culture dish and add 0.5-1 mL of 4% paraformaldehyde to the wells of the plate for fixation for 0.5 h. After the organoids have settled, wash twice with 1 mL of PBS. Finally, pipette the Matrigel and organoid mixture through the wells, transfer it to a 1.5 mL or 15 mL centrifuge tube, and centrifuge at 300 g for 5 min to remove the supernatant.

4. The organoid pathological pre-embedding method according to claim 3, characterized in that: The organoid tissue pathology pre-embedding reagent can be dissolved by heating (40° C.-60° C.).

5. The organoid pathological pre-embedding method according to claim 3, characterized in that: Stain the cell pellet with 0.1-1 μl of dye, then gently resuspend the organoid pellet in 30-50 μl of dissolved pre-embedding reagent. Once the gelatin has cooled and solidified (the organoid pellet can be observed under a microscope), paraffin embedding and sectioning can be performed.

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