Organ-like inverted culture device, culture method and pathological analysis method

Abstract

The invention relates to the technical field of organoid culture, and discloses an organoid inverted culture device, a culture method and a pathological analysis method.The inverted culture device comprises a cell culture plate and an inverted culture small chamber, the cell culture plate comprises a plate body, and a plurality of rows of counter bores are evenly formed in the plate body; the inverted small culture chamber comprises supporting frames, a cylinder part, a supporting ring and an attachment film, the supporting ring is located above the cylinder part, the bottom end of the supporting ring is connected with the top end of the cylinder part through the three supporting frames, a through hole is formed in the center of the cylinder part, and the attachment film is arranged at the bottom end of the cylinder part; the outer diameter of the supporting ring is larger than the inner diameter of the counter bore, and the outer diameter of the cylinder part is smaller than the inner diameter of the counter bore. According to the method, the number of cells growing in a two-dimensional adherent mode in the organoid forming process can be effectively reduced, balling and growing of the organoid are facilitated, the organoid has the high multiplication capacity, and meanwhile the organoid has the immunohistochemical characteristics consistent with the tumor tissue from a patient.

Description

Technical Field

[0001] The present invention relates to the technical field of organoid culture, and in particular to an organoid inverted culture device, a culture method and a pathological analysis method. Background Art

[0002] Organoids are three-dimensional miniature organs formed in vitro by cells with self-renewal and self-organization capabilities, which are similar to real tissues and organs in the human body to a certain extent. Compared with two-dimensional cell culture, organoids have cells and spatial organizations similar to the corresponding organs, and have all the genetic characteristics of the human body. Therefore, they can be cultured in vitro for a long time and simulate the functions of real organs, and have broad application prospects in basic research and clinical diagnosis and treatment. At present, brain organoids, intestinal organoids, retinal organoids, lung organoids, heart organoids and various tumor organoids have been successfully established. The long-term expansion ability and physiological structure of organoids make them increasingly becoming research models in biomedicine and pharmacy. For example, organoids have been widely used in basic research, drug screening and gene editing. Compared with animal cancer models, which have a long reproduction cycle and cannot fully reflect the characteristics of the pathogenic process of cancer patients, tumor organoid models can obtain results in a short time. There are still various problems with the current tumor organoid culture methods. Therefore, exploring new and more conducive culture methods for tumor organoid growth can provide an efficient model for the transformation of basic research into clinical applications.

[0003] Esophageal cancer is a highly invasive malignant tumor, mainly including esophageal squamous cell carcinoma and esophageal adenocarcinoma. It is the sixth most common cause of cancer-related death in the world. Due to nutrition, hygiene, poor eating habits and other reasons, esophageal cancer has a long-term high incidence in my country. At present, the treatment of esophageal squamous cell carcinoma includes endoscopic treatment, surgery, chemotherapy, radiotherapy and comprehensive treatment. However, existing technical means cannot accurately predict the sensitivity of patients to treatment options. In the past decade of organoid research, a variety of culture methods have been developed to establish practical organoid experimental models. The simplest method is to use a non-sticky surface. This method prevents cell adhesion by hydrophobic treatment of the culture dish; in addition, there are rotating bioreactors, hanging drop methods, low-adhesion cell culture plates, and magnetic levitation methods.

[0004] So far, the widely used traditional organoid culture methods are as follows Figure 1 As shown, take an appropriate amount of matrix gel / cell suspension and mix it evenly in the center of the bottom of the well of the cell culture plate. After it solidifies, add sufficient organoid complete culture medium to the well for culture. During the culture process, esophageal squamous cell carcinoma organoids using this culture method often show two-dimensional cell adhesion growth, which leads to problems in organoid construction. In addition, most other organoid culture methods require special instruments and equipment, which greatly affects the popularization of organoid technology.

[0005] There are also some organoid culture devices and / or methods and / or pathological analysis methods in the prior art, such as the Chinese invention patent with application number CN202410237979.8 and name "Special serum-free culture medium for esophageal squamous cell carcinoma organoids", which culture and treat esophageal squamous cell carcinoma cells in a culture medium previously studied to obtain esophageal squamous cell carcinoma organoids. The esophageal squamous cell carcinoma organoids cultured by this method have a good morphology, can promote the rapid growth of esophageal squamous cell carcinoma organoids, greatly increase the number of organoids, and greatly improve cell survival rate. The culture medium contains pleiotropic protein (PTN) and midkine (MDK), which can maintain cell viability and promote cell proliferation. It has the advantages of short culture cycle, rapid proliferation, high cell number and cell survival rate, and long-term stable passage. Another example is the Chinese invention patent with application number CN202411141868.3 and name “A universal tumor organoid culture medium and its culture method”, which can be used to cultivate and maintain the growth and development of various organoids. It has the advantages of high efficiency and generality, enhanced cell growth, high biocompatibility, easy preparation and application, and can be widely used in laboratory culture and clinical applications of organoids. However, it mainly studies the culture medium, and its culture method for esophageal squamous cell carcinoma organoids or tumor organoids is only carried out on a specific culture medium, and does not involve research on organoid culture devices and / or pathological analysis methods. Another example is the Chinese invention patent with application number CN202411004587.3 and name “A cervical squamous cell carcinoma organoid culture medium, model and preparation method thereof”, which can solve the technical problem that the cervical squamous cell carcinoma organoid model in the prior art only has cervical cancer squamous cells and is quite different from the cervical cancer tissue of the normal human body. The new cervical squamous cell carcinoma organoids they cultured have both primary squamous cells of cervical cancer and primary fibroblasts, which are closer to normal human cervical cancer tissue. However, they mainly conducted relevant research on the culture medium, and also explored the organoid culture model and its preparation method. Summary of the invention

[0006] In order to overcome the shortcomings of the prior art, the purpose of the present invention is to provide an organoid inverted culture device, culture method and pathological analysis method, which can effectively reduce the number of adherent cells during organoid formation, facilitate organoid sphering and growth, and enable it to have a stronger proliferation ability, while having immunohistochemical characteristics consistent with patient-derived tumor tissue.

[0007] The present invention is implemented by the following technical scheme: an organoid inverted culture device, comprising: a cell culture plate and an inverted culture chamber, the cell culture plate comprising a plate body, and the plate body is evenly provided with a plurality of rows of countersunk holes; the inverted culture chamber comprises a support frame, a cylindrical portion, a support ring and an attachment film, the support ring is located above the cylindrical portion, the bottom end of the support ring and the top end of the cylindrical portion are connected by a plurality of support frames, a through hole is provided in the center of the cylindrical portion, and an attachment film is provided at the bottom end of the cylindrical portion; the outer diameter of the support ring is greater than the inner diameter of the countersunk hole, the outer diameter of the cylindrical portion is smaller than the inner diameter of the countersunk hole, and the sum of the heights of the cylindrical portion and the support frame is smaller than the height of the countersunk hole, so that the cylindrical portion and the support frame in the inverted culture chamber can be placed in the countersunk hole, and a certain space exists between the bottom and the outer side of the cylindrical portion and the inner wall of the countersunk hole for storing culture medium.

[0008] Furthermore, the attachment film is a PET film; the material of the cell culture plate is a medical-grade polystyrene material; the inverted culture chamber is a transparent plastic material; and there are three support frames.

[0009] An inverted culture method for esophageal squamous cell carcinoma organoids, using the inverted culture device for organoids, comprises the following steps:

[0010] Step 1: Place the cell culture plate in an incubator and preheat at 37°C for 1 hour to provide suitable temperature conditions for organoid growth;

[0011] Step 2: Prepare sufficient amount of esophageal squamous cell carcinoma single cells;

[0012] Step 3: Place the attached membrane in the inverted culture chamber facing upward in a 10 cm sterile culture dish;

[0013] Step 4: Use a pipette to immerse the bottom of the sterile pipette tip in ice-cold DPBS solution, and pipette up and down 6 times to pre-cool it; insert the matrix gel vertically into the ice box to keep it in liquid state, use the pre-cooled pipette tip to draw 5μl of matrix gel vertically and drop it in the center of the attachment membrane, and let it stand for 10 minutes to allow the isolation matrix gel to solidify;

[0014] Step 5: Place 2.8×10 4 The esophageal squamous cell carcinoma cells were transferred into a sterile EP tube and centrifuged at 500×g for 3 min at room temperature to precipitate the cells; the number of cells cultured in each well was 4×10 3 , the above cells were used to plate 6 wells to produce a sufficient number of organoids for subsequent experiments;

[0015] Step 6: Soak the bottom of the sterile pipette tip in ice-cold DPBS solution, pipette up and down 6 times to pre-cool; use the pre-cooled pipette tip to take 70 μl of matrix gel to resuspend the cells, take 10 μl of matrix gel / cell suspension and vertically drop it on the isolation matrix gel that has solidified in step 4, and let it stand for 10 minutes to allow the matrix gel / cell suspension to solidify;

[0016] Step 7: Take out the preheated cell culture plate and add 500 μl of preheated esophageal squamous cell carcinoma organoid complete culture medium to each well of the cell culture plate;

[0017] Step 8: Use sterile surgical forceps to gently invert the inverted culture chamber into the sink 12, so that the matrix gel / cell suspension is immersed in the complete medium of the organoid, and place it in a 37°C, 5% CO2 incubator for culture;

[0018] Step 9: Observe and photograph under a microscope every 3 days and replace with fresh organoid complete culture medium.

[0019] Further, preparing esophageal squamous cell carcinoma single cells comprises the following steps:

[0020] Step 1: Add 7 ml of basal culture medium to a sterile 15 ml centrifuge tube and pre-cool it in an ice box for 30 minutes; after the operation is completed, take a sufficient amount of esophageal squamous cell carcinoma tissue and store it in a 15 ml centrifuge tube, and transport it to the laboratory for subsequent processing in time;

[0021] Step 2: In a biosafety cabinet, use sterile tweezers to transfer the esophageal squamous cell carcinoma tissue to a 6 cm sterile culture dish; wash the specimen three times with basal culture medium to remove blood, blood vessels and necrotic tissue to reduce the chance of contamination and infection; use sterile dissecting scissors to cut the esophageal squamous cell carcinoma tissue into pieces with a diameter of less than 1 mm, which is conducive to promoting the efficiency of Dispase digestion;

[0022] Step 3: Transfer the chopped tissue fragments into an EP tube containing 1 ml of HBSS-DFCY solution, incubate at 37°C for 10 min, take out the EP tube every 2 min and flick the bottom to mix to promote Dispase digestion, until the chopped tissue fragments are evenly dispersed in the EP tube; then centrifuge at 188×g for 1 min at room temperature to precipitate the digested tissue fragments;

[0023] Step 4: Discard the supernatant and digest the precipitated tissue fragments with 1 ml of 0.25% trypsin-EDTA containing 0.5 U / ml DNase I, incubate at 37°C for 10 min, and then centrifuge at 188 × g for 3 min at room temperature to precipitate the digested tumor cells;

[0024] Step 5: Filter the cell suspension after trypsin-EDTA digestion through a cell mesh with a pore size of 100 μm into a 50 ml centrifuge tube containing 8 ml soybean trypsin inhibitor; remove the rubber plunger from the 10 ml syringe and use it to grind on the cell mesh 10 times to obtain more single tumor cells;

[0025] Step 6: Use a pipette to remove the filtrate from the 50 ml centrifuge tube and use this filtrate to wash the cell screen three times; centrifuge at 188 × g for 3 minutes at room temperature to precipitate single tumor cells;

[0026] Step 7: Gently resuspend the above cells in 1 ml of basal culture medium, take 10 μl of the cell suspension and drop it into a hemocytometer to count the number of cells.

[0027] Furthermore, the HBSS-DFCY solution includes 1.8 ml of HBSS-DF solution, 200 μl of Collagenase IV and 2 μl of Y-27632, and the HBSS-DF solution includes 1.6 ml of HBSS, 400 μl of Dispase and 4 μl of Fungizone.

[0028] Furthermore, the basic culture medium includes 500 ml of Advanced DMEM / F12, 5 ml of GlutaMAX, 5 ml of HEPES, 50 μl of Antibiotic-Antimycotic and 50 μl of Gentamicin.

[0029] Furthermore, the organoid complete culture medium includes 48 ml of basal culture medium, 5 μl of R-spondin-1, 5 μl of Noggin, 500 μl of N-2 supplement, 1 ml of B-27 supplement, 100 μl of N-Acetylcysteine, 5 μl of Recombinant human epidermal growth factor (EGF), 50 μl of Y-27632, 5 μl of Gentamicin and 500 μl of Antibiotic-Antimycotic.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] 1. The present invention provides an inverted organoid culture device, culture method and pathological analysis method, wherein the inverted culture chamber is made of transparent plastic material and can be matched and fitted with a cell culture plate; the attachment film has good transparency and optical clarity, and the growth state of the organoid thereon can be clearly observed through a microscope; in addition, the PET film has the characteristic of low adsorption rate, which can effectively reduce the loss of small molecule proteins and other compounds, and is more suitable for the present culture method.

[0032] 2. An inverted organoid culture device, culture method and pathological analysis method of the present invention culture organoids by inverted culture, using matrix gel pre-laid on the chamber PET membrane, which can isolate the contact between the matrix gel / cell suspension and the chamber PET membrane, eliminating the two-dimensional cell adhesion growth caused by gravity during the matrix gel solidification process; during inverted culture, the matrix gel / cell suspension can be immersed in the organoid complete culture medium so that it can fully absorb the nutrients in the complete culture medium; it can greatly improve the sphering rate of organoids, and make them have a strong proliferation ability, and at the same time have immunohistochemical characteristics consistent with the patient-derived tumor tissue.

[0033] 3. The present invention provides an inverted organoid culture device, culture method and pathological analysis method, wherein the HBSS-DFCY solution can digest tumor tissue and facilitate the preparation of single tumor cell samples with higher activity under the premise of effectively protecting tumor cells; the basal culture medium is conducive to ensuring the activity of recovered single tumor cells under the premise of effectively reducing contamination; the optimized complete culture medium can more effectively promote the growth of esophageal squamous cell carcinoma organoids.

[0034] 4. An inverted organoid culture device, culture method and pathological analysis method of the present invention can evaluate the proliferation ability and immunohistochemical characteristics of organoids. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 It is a widely used traditional tumor organoid culture method;

[0036] Figure 2 It is the tumor organoid culture method of the present invention;

[0037] Figure 3 This is the state and number of adherent cells in traditional esophageal squamous cell carcinoma organoid culture (red arrows indicate adherent cells);

[0038] Figure 4 is the state and number of adherent cells during culture of esophageal squamous cell carcinoma organoids of the present invention;

[0039] Figure 5 It is the sphere size during traditional esophageal squamous cell carcinoma organoid culture;

[0040] Figure 6 is the sphere size of the esophageal squamous cell carcinoma organoid cultured in the present invention;

[0041] Figure 7 is the proliferation capacity of traditional esophageal squamous cell carcinoma organoid culture (Ki-67 immunohistochemical staining);

[0042] Figure 8 is the proliferation capacity of the esophageal squamous cell carcinoma organoid cultured in the present invention (Ki-67 immunohistochemical staining);

[0043] Fig. 9 is the staining of esophageal squamous cell carcinoma markers CK5 / 6, TP63 and TP53 in the organoid cultured by the method of the present invention;

[0044] Fig.10 It is a schematic structural diagram of a cell culture plate of the present invention;

[0045] Fig.11 It is a schematic diagram of the structure of the inverted culture chamber of the present invention;

[0046] Fig.12 It is a schematic diagram of the inverted culture processing flow of the inverted culture chamber of the present invention.

[0047] In the figure: plate body 11; countersunk hole 12; support frame 21; cylindrical part 22; support ring 23; attachment film 24. DETAILED DESCRIPTION

[0048] The following is an explanation of the embodiments of the present invention by specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied in other different specific ways, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that the features in the following embodiments can be combined with each other without conflict.

[0049] The purpose of the present invention is to provide an organoid inverted culture device to address the defects of the prior art.

[0050] Embodiment 1

[0051] This embodiment provides an inverted organoid culture device, comprising: a cell culture plate and an inverted culture chamber.

[0052] Reference Fig.10 As shown, the cell culture plate includes a plate body 11, and a plurality of rows of countersunk holes 12 are evenly arranged on the plate body 11. Specifically, in this embodiment, the material of the cell culture plate is medical grade polystyrene material, which is sterilized before use. The distribution and size of each countersunk hole 12 are moderate, which is convenient for observing the growth state of the organoid under a microscope and recording its growth process.

[0053] Reference Fig.11As shown, the inverted culture chamber includes a support frame 21, a cylindrical portion 22, a support ring 23 and an attachment film 24, the support ring 23 is located above the cylindrical portion 22, the bottom end of the support ring 23 and the top end of the cylindrical portion 22 are connected by multiple support frames 21, a through hole is provided in the center of the cylindrical portion 22, and an attachment film 24 is provided at the bottom end of the cylindrical portion 22; the outer diameter of the support ring 23 is greater than the inner diameter of the counterbore 12, the outer diameter of the cylindrical portion 22 is smaller than the inner diameter of the counterbore 12, and the sum of the heights of the cylindrical portion 22 and the support frame 21 is smaller than the height of the counterbore 12, so that the cylindrical portion 22 and the support frame 21 in the inverted culture chamber can be placed in the counterbore 12, and there is a certain space between the bottom and outer side of the cylindrical portion 22 and the inner wall of the counterbore 12 for storing culture medium. Specifically in this embodiment, the inverted culture chamber is made of transparent plastic material, which is manufactured using 3D printing, and can better match and fit with the cell culture plate. The attachment film 24 is a PET film, which has good transparency and optical clarity, and the growth state of the organoids thereon can be clearly observed through a microscope; in addition, the PET film has the characteristics of a low adsorption rate, which can effectively reduce the loss of small molecule proteins and other compounds, and is more in line with the present culture method.

[0054] As an optimized technical solution in this embodiment, 24 countersunk holes 12 are arranged on the cell culture plate, with a total of 4 rows and 6 in each row. The outer diameter of the countersunk hole 12 is 16.28 mm, the inner diameter is 15.67 mm, and the inner height is 18.84 mm. The overall height of the inverted culture chamber is 15.50 mm; the thickness of the support ring 23 is 0.55 mm, and the outer diameter is 18.60 mm; the distance between the three support frames 21 is 15.45 mm, the outer diameter of the cylindrical part 22 is 9.10 mm, and the diameter of the central through hole is 7.50 mm; the overall diameter of the PET film is 7.50 mm, and the diameter of the pores of the PET film is 0.45 μm. When in use, the cylindrical part 22 and the support frame 21 in the inverted culture chamber can be placed in the countersunk hole 12, and there is a certain space between the bottom and the outer side of the cylindrical part 22 and the inner wall of the countersunk hole 12 for storing culture medium.

[0055] Embodiment 2

[0056] An inverted culture method for esophageal squamous cell carcinoma organoids is implemented using the above-mentioned inverted culture device for organoids, comprising the following steps:

[0057] Step 1: Place the cell culture plate in an incubator and preheat at 37°C for 1 hour to provide suitable temperature conditions for organoid growth;

[0058] Step 2: Add 7 ml of basal culture medium to a sterile 15 ml centrifuge tube and pre-cool it in an ice box for 30 minutes; after the operation is completed, take a sufficient amount of esophageal squamous cell carcinoma tissue and store it in the above 15 ml centrifuge tube, and transport it to the laboratory for subsequent processing in time;

[0059] Step 3: In a biosafety cabinet, use sterile tweezers to transfer the esophageal squamous cell carcinoma tissue to a 6 cm sterile culture dish; wash the specimen three times with basal culture medium to remove blood, blood vessels and necrotic tissue to reduce the chance of contamination and infection; use sterile dissecting scissors to cut the esophageal squamous cell carcinoma tissue into pieces with a diameter of less than 1 mm, which is conducive to promoting the efficiency of Dispase digestion;

[0060] Step 4: Transfer the chopped tissue fragments into an EP tube containing 1 ml of HBSS-DFCY solution, incubate at 37°C for 10 min, take out the EP tube every 2 min and flick the bottom to mix to promote Dispase digestion, until the chopped tissue fragments are evenly dispersed in the EP tube; then centrifuge at room temperature at 188×g for 1 min to precipitate the digested tissue fragments;

[0061] Step 5: Discard the supernatant and digest the precipitated tissue fragments with 1 ml of 0.25% trypsin-EDTA containing 0.5 U / ml DNase I, incubate at 37°C for 10 min, and then centrifuge at 188 × g for 3 min at room temperature to precipitate the digested tumor cells;

[0062] Step 6: Filter the cell suspension after trypsin-EDTA digestion through a cell sieve with a pore size of 100 μm into a 50 ml centrifuge tube containing 8 ml soybean trypsin inhibitor; remove the rubber plunger from the 10 ml syringe and use it to grind on the cell sieve 10 times to obtain more single tumor cells;

[0063] Step 7: Use a pipette to remove the filtrate from the 50 ml centrifuge tube and use this filtrate to wash the cell screen three times; centrifuge at 188 × g for 3 minutes at room temperature to precipitate single tumor cells;

[0064] Step 8: Gently resuspend the above cells with 1 ml of basal culture medium, take 10 μl of the cell suspension and drop it into a blood cell counting plate to count the number of cells; prepare a sufficient amount of esophageal squamous cell carcinoma single cells;

[0065] Step 9: Place the attached membrane 24 in the inverted culture chamber facing upward in a 10 cm sterile culture dish;

[0066] Step 10: Use a pipette to immerse the bottom of a sterile pipette tip in an ice-cold DPBS solution, and blow it up and down 6 times for precooling; insert the matrix gel vertically into an ice box to keep it in a liquid state, use a precooled pipette tip to absorb 5 μl of matrix gel and drop it vertically in the center of the attachment membrane 24, and let it stand for 10 minutes to allow the isolation matrix gel to solidify; pre-spreading the matrix gel on the attachment membrane 24 can isolate the contact between the matrix gel / cell suspension and the PET membrane, and eliminate the two-dimensional cell adhesion growth caused by gravity during the solidification process of the matrix gel / cell suspension;

[0067] Step 11: Place the 2.8×10 4 The esophageal squamous cell carcinoma cells were transferred into a sterile EP tube and centrifuged at 500×g for 3 min at room temperature to precipitate the cells; the number of cells cultured in each well 12 was 4×10 3 , the above cells were used to plate 6 wells to produce a sufficient number of organoids for subsequent experiments;

[0068] Step 12: Use a pipette to immerse the bottom of the sterile pipette tip in ice-cold DPBS solution, and blow it up and down 6 times for pre-cooling; use the pre-cooled sterile pipette tip to take 70μl of matrix gel, resuspend the cell pellet after centrifugation, and mix it thoroughly to form a matrix gel / cell suspension, take 10μl of matrix gel / cell suspension and drop it on the solidified 5μl isolation matrix gel, and let it stand for 10 minutes to allow the matrix gel / cell suspension to solidify; Steps 9, 10, and 12 are pre-treatment of the inverted culture chamber, and the flow diagram is shown as follows Fig.12 As shown;

[0069] Step 13: Take out the preheated cell culture plate, and add 500 μl of preheated esophageal squamous cell carcinoma organoid complete culture medium into each well 12 of the cell culture plate;

[0070] Step 14: Use sterile surgical forceps to gently invert the inverted culture chamber into the sink 12, so that the matrix gel / cell suspension is immersed in the complete medium of the organoid, and place it in a 37°C incubator containing 5% CO2 for culture; under the action of gravity, the inverted culture method can make the matrix gel / cell suspension completely immersed in the complete medium of the organoid, so that it can fully absorb the nutrients in the complete medium;

[0071] Step 15: Observe and photograph under a microscope every 3 days and replace with fresh organoid complete culture medium.

[0072] The schematic diagram of this culture method is as follows Figure 2 As shown in Figure 2, the adherent cell status and number of esophageal squamous cell carcinoma organoids on the sixth and ninth days are shown in Figure 2. Figure 4 As shown in Figure 2, the size and process of organoid formation are as follows: Figure 6 shown.

[0073] Carry out comparative tests, refer to Figure 1 The esophageal squamous cell carcinoma organoids were cultured in the traditional culture method shown in the figure. Take another identical cell culture plate, mix 15 μl of the matrix gel / cell suspension and spread it in the center of the bottom of the well 12 of the cell culture plate, and then add a sufficient amount of organoid complete culture medium to the well 12 for culture. The state and number of adherent cells of the esophageal squamous cell carcinoma organoids on the sixth and ninth days are shown in Figure 2. Figure 3 As shown in Figure 2, the size and process of organoid formation are as follows: Figure 5 shown.

[0074] It can be seen that the use of the organoid inverted culture device and culture method of the present invention, that is, pre-spreading 5 μl of matrix gel on the PET membrane of the small chamber for partition, then inoculating 10 μl of matrix gel / cell suspension on the solidified isolation matrix gel, and finally inverting the chamber for inverted culture, can greatly reduce the number of adherent cells of the organoid and significantly improve the organoid spheroidization rate.

[0075] In the traditional organoid culture method, during the solidification of the matrix gel / cell suspension, some cells are deposited due to gravity and finally grow in a two-dimensional form. On the one hand, the organoid spheroidization rate is reduced; on the other hand, the adherent cells will also rob the growth resources during the growth process, which is not conducive to the spheroidization of the organoid.

[0076] HBSS-DFCY solution was prepared using the reagents in Table 1, which included 1.8 ml of HBSS-DF solution, 200 μl of Collagenase IV, and 2 μl of Y-27632;

[0077] HBSS-DF solution includes HBSS, Dispase and Fungizone; the volume of HBSS (balanced salt solution) is 1.6 ml, the volume of Dispase (dispersase) is 400 μl, and its concentration is 50 U / ml, and the concentration of Fungizone (amphotericin B) is 4 μl, and its concentration is 250 μg / ml. The concentration of Collagenase IV is 200 mg / ml, and the concentration of Y-27632 is 10 mM. Take 1.8 ml of HBSS-DF solution to a 15 ml tube, add 200 μl of Collagenase IV and 2 μl of Y-27632 to mix to form HBSS-DFCY solution. HBSS-DFCY solution can digest tumor tissue and facilitate the preparation of single tumor cell samples with high activity under the premise of effectively protecting tumor cells.

[0078] Table 1

[0079]

[0080] The above solution is HBSS-DF. Take 1.8 ml of it and put it into a new 15 ml tube.

[0081]

[0082] As shown in Table 2, the formula of the basal culture medium for esophageal squamous cell carcinoma organoids includes: 500 ml of Advanced DMEM / F12, 5 ml of GlutaMAX, 5 ml of HEPES, 50 μl of Antibiotic-Antimycotic and 50 μl of Gentamicin, the concentrations of which are shown in Table 2. The basal culture medium is conducive to ensuring the activity of recovered single tumor cells while effectively reducing contamination.

[0083] Table 2

[0084]

[0085] As shown in Table 3, the formula of the complete culture medium for esophageal squamous cell carcinoma organoids includes: 48 ml of basal culture medium, 5 μl of R-spondin-1, 5 μl of Noggin, 500 μl of N-2 supplement, 1 ml of B-27 supplement, 100 μl of N-Acetylcysteine, 5 μl of Recombinant human epidermal growth factor (EGF), 50 μl of Y-27632, 5 μl of Gentamicin and 500 μl of Antibiotic-Antimycotic. This complete culture medium can more effectively promote the growth of esophageal squamous cell carcinoma organoids.

[0086] Table 3

[0087]

[0088] Embodiment 3

[0089] A method for pathological analysis of esophageal squamous cell carcinoma organoids, using the above-mentioned organoid inverted culture device and esophageal squamous cell carcinoma organoid culture method, processing the organoids when they are cultured to the ninth day, and performing an organoid pathological analysis operation, the method comprising the following steps:

[0090] Step 1: Discard the complete medium for organoids, add 500 μl of pre-cooled DPBS into well 12 of the cell culture plate, and pipette up and down to break up the matrix gel into small pieces.

[0091] Step 2: Centrifuge at 500×g for 3 min, then resuspend the matrix gel fragments with 1 ml DPBS. Centrifuge at 500×g for 3 min, discard the supernatant, resuspend the pellet in 1 ml 4% PFA, and store at 4°C overnight.

[0092] Step 3: The next day, centrifuge at 500×g for 3 minutes to remove as much supernatant as possible.

[0093] Step 4: Place the embedding gel in a beaker containing 100 ml of water and heat it in a microwave oven at the highest power for 1 min until the water begins to boil. Confirm that the embedding gel has liquefied and resuspend the organoid pellet in 50 μl of embedding gel.

[0094] Step 5: Transfer the embedding rack to a 4°C refrigerator and let it stand for 30 minutes. After the embedding gel solidifies, gently push out the gel and place it in an EP tube. Add 1 ml of 70% ethanol and store it at 4°C. After routine histological processing, embed it in paraffin for sectioning and staining.

[0095] Step 6: Using immunohistochemical staining technology, Ki-67 was used to evaluate the proliferation ability of organoid cells. The proliferation ability of esophageal squamous cell carcinoma organoids obtained by using the organoid inverted culture device and culture method of the present invention is as follows: Figure 8 As shown in Figure 2, the proliferation capacity of organoids cultured in the traditional way is as follows Figure 7 As shown, it can be seen that the Ki-67 positive ratio of the organoids cultured in the present invention is higher, indicating that its proliferation ability is stronger.

[0096] Step 7: Detect the immunohistochemical staining of esophageal squamous cell carcinoma organoid markers. Fig. 9 As shown, it can be found that the esophageal squamous cell carcinoma organoids constructed in the present invention maintain the immunohistochemical results consistent with those derived from human esophageal squamous cell carcinoma tissue, that is, CK5 / 6, TP63 and TP53 are all positively expressed, among which CK5 / 6 expression is mainly distributed in the cell membrane, and TP63 and TP53 are mainly distributed in the cell nucleus.

[0097] Table 4

[0098]

[0099] Table 4 shows the formula of embedding gel: dissolve 1g bacterial agar powder and 1.25g gelatin in 50ml pure water; let stand at room temperature for 30-60 minutes; sterilize at 121℃ for 20 minutes; and after the temperature drops, dispense into 15ml centrifuge tubes, 5ml per tube.

[0100] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment can also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

Claims

1. An inverted organoid culture device, characterized in that: include: Cell culture plates and inverted culture chambers, The cell culture plate comprises a plate body 11, and a plurality of rows of sink holes 12 are evenly arranged on the plate body 11; The inverted culture chamber comprises a support frame 21, a cylindrical portion 22, a support ring 23 and an attachment film 24, wherein the support ring 23 is located above the cylindrical portion 22, the bottom end of the support ring 23 is connected to the top end of the cylindrical portion 22 via a plurality of support frames 21, a through hole is provided in the center of the cylindrical portion 22, and an attachment film 24 is provided at the bottom end of the cylindrical portion 22; The outer diameter of the support ring 23 is larger than the inner diameter of the countersunk hole 12, the outer diameter of the cylindrical portion 22 is smaller than the inner diameter of the countersunk hole 12, and the sum of the heights of the cylindrical portion 22 and the support frame 21 is smaller than the height of the countersunk hole 12, so that the cylindrical portion 22 and the support frame 21 in the inverted culture chamber can be placed in the countersunk hole 12, and there is a certain space between the bottom and outer side of the cylindrical portion 22 and the inner wall of the countersunk hole 12 for storing culture medium.

2. The inverted organoid culture device according to claim 1, characterized in that: The attachment film 24 is a PET film; The material of the cell culture plate is medical grade polystyrene; The inverted culture chamber is made of transparent plastic material; There are three support frames 21 .

3. An inverted culture method for esophageal squamous cell carcinoma organoids, using the inverted culture device for organoids as claimed in claim 1 or 2, characterized in that: The following steps are involved: Step 1: Place the cell culture plate in an incubator and preheat it at 37°C to provide suitable temperature conditions for organoid growth; Step 2: Prepare sufficient amount of esophageal squamous cell carcinoma single cells; Step 3: Place the attached membrane 24 in the inverted culture chamber facing upward in a 10 cm sterile culture dish; Step 4: Use a pipette to immerse the bottom of a sterile pipette tip in ice-cold DPBS solution, and pipette up and down 6 times for precooling; insert the matrix gel vertically into an ice box to ensure that it is in liquid state, use the precooled pipette tip to absorb 5 μl of matrix gel and drop it vertically in the center of the attachment membrane 24, and let it stand for 10 minutes to allow the isolation matrix gel to solidify; Step 5: Place 2.8×10 4 The esophageal squamous cell carcinoma cells were transferred into a sterile EP tube and centrifuged at 500×g for 3 min at room temperature to precipitate the cells; the number of cells cultured in each well 12 was 4×10 3 , the above cells were used to plate 6 wells to produce a sufficient number of organoids for subsequent experiments; Step 6: Soak the bottom of the sterile pipette tip in ice-cold DPBS solution, pipette up and down 6 times to pre-cool; use the pre-cooled pipette tip to take 70 μl of matrix gel to resuspend the cells, take 10 μl of matrix gel / cell suspension and vertically drop it on the isolation matrix gel that has solidified in step 4, and let it stand for 10 minutes to allow the matrix gel / cell suspension to solidify; Step 7: Take out the preheated cell culture plate, and add 500 μl of preheated esophageal squamous cell carcinoma organoid complete culture medium into each well 12 of the cell culture plate; Step 8: Use sterile surgical forceps to gently invert the inverted culture chamber into the sink 12, so that the matrix gel / cell suspension is immersed in the complete medium of the organoid, and place it in a 37°C, 5% CO2 incubator for culture; Step 9: Observe and photograph under a microscope every 3 days and replace with fresh organoid complete culture medium.

4. The inverted culture method for esophageal squamous cell carcinoma organoids according to claim 3, characterized in that: Preparation of esophageal squamous cell carcinoma single cells, including the following steps: Step 1: Add 7 ml of basal culture medium to a sterile 15 ml centrifuge tube and pre-cool it in an ice box for 30 minutes; after the operation is completed, take a sufficient amount of esophageal squamous cell carcinoma tissue and store it in the above 15 ml centrifuge tube, and transport it to the laboratory for subsequent processing in time; Step 2: In a biosafety cabinet, use sterile tweezers to transfer the esophageal squamous cell carcinoma tissue to a 6 cm sterile culture dish; wash the specimen three times with basal culture medium to remove blood, blood vessels and necrotic tissue to reduce the chance of contamination and infection; use sterile dissecting scissors to cut the esophageal squamous cell carcinoma tissue into pieces with a diameter of less than 1 mm, which is conducive to promoting the efficiency of Dispase digestion; Step 3: Transfer the chopped tissue fragments into an EP tube containing 1 ml of HBSS-DFCY solution, incubate at 37°C for 10 min, take out the EP tube every 2 min and flick the bottom to mix to promote Dispase digestion, until the chopped tissue fragments are evenly dispersed in the EP tube; then centrifuge at 188×g for 1 min at room temperature to precipitate the digested tissue fragments; Step 4: Discard the supernatant and digest the precipitated tissue fragments with 1 ml of 0.25% trypsin-EDTA containing 0.5 U / ml DNase I, incubate at 37°C for 10 min, and then centrifuge at 188 × g for 3 min at room temperature to precipitate the digested tumor cells; Step 5: Filter the cell suspension after trypsin-EDTA digestion through a cell mesh with a pore size of 100 μm into a 50 ml centrifuge tube containing 8 ml soybean trypsin inhibitor; remove the rubber plunger from the 10 ml syringe and use it to grind on the cell mesh 10 times to obtain more single tumor cells; Step 6: Use a pipette to remove the filtrate from the 50 ml centrifuge tube and use this filtrate to wash the cell screen three times; centrifuge at 188 × g for 3 minutes at room temperature to precipitate single tumor cells; Step 7: Gently resuspend the above cells in 1 ml of basal culture medium, take 10 μl of the cell suspension and drop it into a hemocytometer to count the number of cells.

5. The inverted culture method for esophageal squamous cell carcinoma organoids according to claim 4, characterized in that: The HBSS-DFCY solution includes 1.8 ml of HBSS-DF solution, 200 μl of Collagenase IV and 2 μl of Y-27632, and the HBSS-DF solution includes 1.6 ml of HBSS, 400 μl of Dispase and 4 μl of Fungizone.

6. The inverted culture method for esophageal squamous cell carcinoma organoids according to claim 4, characterized in that: The basic culture medium includes 500 ml of Advanced DMEM / F12, 5 ml of GlutaMAX, 5 ml of HEPES, 50 μl of Antibiotic-Antimycotic and 50 μl of Gentamicin.

7. The inverted culture method for esophageal squamous cell carcinoma organoids according to claim 3 or 4, characterized in that: The organoid complete culture medium includes 48 ml of basal culture medium, 5 μl of R-spondin-1, 5 μl of Noggin, 500 μl of N-2 supplement, 1 ml of B-27 supplement, 100 μl of N-Acetylcysteine, 5 μl of Recombinant human epidermal growth factor (EGF), 50 μl of Y-27632, 5 μl of Gentamicin and 500 μl of Antibiotic-Antimycotic.

Images